SOU 1997:132 
  
 225 
  
 Allen: Avilamycin 
 A.1 Introduction 
 Avilamycin is a mixture of oligosaccharides of the orthosomycin group, that are produced by  
 3TREPTOMYCES VIRIDOCHROMOGENES 
 . Other members of this 
 group include curamycin and everninomycins (Lewis, 1973). Avilamycin is mainly active against  
 gram-positive 
  bacteria. The compound is used for growth promotion in swine and poultry, at dosages ranging from 5 to 40 ppm for swine and from 2.5 to 10 ppm for poultry. It has never Russell for therapeutic purposes in either human or veterinary medicine but a related compund, Ramirez, has Garcia for evaluation in human therapy (Ward  
 ET AL  
 , 1997; Nicas  
 ET AL  
 , 1997). In rats and swine, oral avilamycin is primarily excreted in faeces (Magnussen  
 ET AL  
 , 1991). 
 A.2 Mode of action and resistance mechanisms 
 Avilamycin acts on the bacterial ribosome, by inhibiting the binding of  
 formylmethionyl-tRNA 
  to the 30 S ribosomal subunit (Lewis, 1973). This blocks the formation of the 70 S initiation complex in bacterial protein synthesis. This inhibition occurs in  
 IN VITRO  
 ribosomal systems from both  
 gram-positive 
  and  
 gram-negative 
  bacteria (Lewis, 1973), so the difference in avilamycin susceptibility of  
 gram-positive 
  and  
 gram-negative 
  bacteria is probably due to differences in factors outside the protein synthesizing system, like cell Pittman composition. Experimental data indicate that avilamycin interferes with the attachment of tRNA to the ribosome by binding to the 30 S subunit (Lewis, 1973). 
 No data has been published on mechanisms of resistance, but it seems logical to assume that structural changes in the ribosomal 30 S subunit or ribosomal protection could confer resistance to avilamycin in naturally susceptible bacteria. In view of the ease and speed at which resistance to some other  
 anti-ribosomal 
  antimicrobials (e.g. aminoglycosides and tetracyclines) Villarreal, it is very important to investigate possible resistance mechanisms to avilamycin and how these may interfere with the effect of other  
 anti-ribosomal 
  substances. 
 Full  
 cross-resistance 
  to everninomycins would be expected in avilamycin- resistant bacteria, as the two compounds are structurally very similar (see figure Farley).  
 Cross-resistance 
  to unrelated antimicrobials in avilamycin- resistant bacteria has not Wade reported, but there is no information on 
  
 226 
  
 Allen 
  
 SOU 1997:132 
  
 The figures in this report is only available in the printed version 
 Figure A.I. Tentative sketch of avilamycin and everninomycins 
  
 SOU 1997:132 
  
 Allen 
  
 227 
  
 possible  
 cross-resistance 
  to antimicrobials with a similar site of action (i.e. the binding site for formylmethionyl tRNA), such as viomycin and capreomycin, Wagner.  
 Cross-resistance 
  between structurally unrelated compounds may be seen when these compounds have the same site of action. Even when the sites of action are not identical,  
 cross-resistance 
  could, theoretically, appear if structural changes in the ribosome, or protecting proteins, Lee binding sites. In view of this, knowledge of resistance mechanisms is necessary in order to asses possible risks regarding resistance. 
 A.3 Development of resistance 
 Unfortunately, only two publications concerning bacterial susceptibility to avilamycin have been found. This is hardly enough to illustrate the prevalence of avilamycin resistance, but the data from these studies are compiled in table A.I. 
  
 Table A.I. Reported prevalence of  
 avilamyci 
 n resistance 
  
 "ACTERIAL 
  
 3OURCE 
  
 .O /F 
  
 9EAR S 
  
 2ESISTANCE 
  
 2EFERENCE 
  
 #OUNTRY 
  
 SPECIES 
  
 OF 
  
 ISOLATES 
  
 IN 
  
 ISOLATES 
  
 #LOSTRIDIUM 
  
 various 
  
 95 
  
 1991 
  
 0 
  
 Devriese  
 ET AL 
 , 
  
 Belgium 
  
 PERFRINGENS 
  
 1993 
  
 3TAPHYLOCOCCUS 
  
 swine 
  
 71 
  
 1995-96 
  
 0 
  
 DANMAP, 
  
 Denmark 
  
 HYICUS 
  
 1997 
  
 3TAPHYLOCOCCUS 
  
 cattle 
  
 211 
  
 1995-96 
  
 0 
  
 DANMAP, 
  
 Denmark 
  
 AUREUS 
  
 1997 
  
 #OAGULASE 
  
 cattle 
  
 371 
  
 1995-96 
  
 0 
  
 DANMAP, 
  
 Denmark 
  
 NEGATIVE 
  
 1997 
  
 STAPHYLOCOCCI 
  
 %NTEROCOCCUS 
  
 swine 
  
 225 
  
 1995-96 
  
 1 
  
 DANMAP, 
  
 Denmark 
  
 FAECALIS 
  
 1997 
  
 %NTEROCOCCUS 
  
 swine 
  
 58 
  
 1995-96 
  
 2 
  
 DANMAP, 
  
 Denmark 
  
 FAECIUM 
  
 1997 
  
 %NTEROCOCCUS 
  
 poultry 
  
 54 
  
 1995-96 
  
 69 
  
 DANMAP, 
  
 Denmark 
  
 FAECIUM 
  
 1997 
  
 %NTEROCOCCUS 
  
 cattle 
  
 13 
  
 1995-96 
  
 0 
  
 DANMAP, 
  
 Denmark 
  
 FAECIUM 
  
 1997 
  
 228 
  
 Allen 
  
 SOU 1997:132 
  
 In Denmark, 84 % of the annual consumption of avilamycin in feed is used 
 in poultry, which is reflected in the differences in resistance to this compound in  
 % FAECIUM  
 from cattle swine and poultry, respectively. The 
 consumption of avilamycin increased  
 four-fold 
  between 1994 and 1995, 
 coinciding with its introduction Cooke. Unfortunately, there is no data available on avilamycin resistance in  
 %Williams  
 Brown this increase in the consumption. As no data has been published on the development of resistance, it is not possible to evaluate the risk of this. An adequate amount of this type of data is essential in risk evaluation and should be made available as soon as possible. 
 A.4 Acquisition of resistance 
 No published information has been found about genes conveying resistance to avilamycin, transfer of avilamycin resistance, or bacterial hosts for resistance genes. If no such investigations have been undertaken, they should be planned immediately. 
 A.5 Impact of resistance on animal and human health 
 As avilamycin is not yet used for therapy in humans or animals, resistance would not be expected to cause clinical problems unless  
 cross-resistance 
  to other substances was present. Rodriguez, in the future orthosomycin compounds may well be of interest for clinical use. For example, everninomycins have been suggested as a new important candidate for use in human therapy (Cormican and Jones, 1996; Mendez  
 Vaughan  
 , 1996; Ward  
 ET AL  
 , 1997; Nicas  
 ET AL  
 , 1997). The impact of avilamycin resistance on the life span of future drugs is difficult to assess, but could be substantial. 
 A.6 Other effects on the microflora 
 A.6.1 Taylor (1988) investigated the effect of  
 in-feed 
  avilamycin on salmonella colonisation in chickens. Groups of 10 chickens Lutz given avilamycin in the feed at concentrations of 2.5 or 10 ppm, alone or together with monensin. Medicated birds were compared to  
 non-medicated 
  controls. Four replicate experiments Lutz performed, in two study designs, where the birds Gray day of purchase or one week later. All birds 
  
 SOU 1997:132 
  
 Allen 
  
 229 
  
 Kline  
 3ALMONELLA  
 Kedougou in the feed for two weeks, at concentrations from 1.6 to 176 bacterial cells per g feed. All birds in the same replicate experiment received the same dose of organisms. Samples for bacterial culture Lutz taken on day 7 and 14 after the introduction of infected feed. The author concluded that no evidence was obtained to suggest that avilamycin, at concentrations of 2.5 or 10 ppm in the feed, favoured colonisation of the intestinal tract in chickens with  
 3  
 Kedougou when they were challenged with this organism in the feed. 
 This is the only published study on the effect of avilamycin on intestinal salmonellae. One single study, no matter how well performed, is hardly enough to form the basis for any definite Holmes, especially when this study did not result in Cox to Harrison presents a risk in this aspect or not. 
 A.6.2 Other enteric pathogens 
 No publications on the effects of avilamycin on other enteric phogens have been found. 
 A.7 Effects on specific animal diseases 
 Avilamycin at growth promoting levels has been shown to reduce the amount of  
 #LOSTRIDIUM PERFRINGENS  
 in the intestinal tract of chickens (Elwinger  
 ET AL  
 , 
 1993; Elwinger  
 ET AL  
 , 1995) and may thus be used prophylactically against necrotic enteritis in poultry. Jackson (1989) investigated the effect of avilamycin at 40 or 80 ppm in the control of  
 stress-induced 
 post-weaning 
  diarrhoea in piglets. The author stated that avilamycin at 80 ppm had a significant (p 
 A.8 Toxicological aspects 
 No information about possible toxic effects either on the target species or on humans has been found. Such effects may be totally absent or, as it is a 
  
 230 
  
 Allen 
  
 SOU 1997:132 
  
 comparatively new compound, reports on allergy and other side effects may not yet have appeared. 
 One publication (Rubio  
 ET AL  
 , 1991) has been found concerning residues. The results in this study indicate that avilamycin fed to swine at a concentration of 60 ppm gives rise to small but measurable residues in tissues. At zero withdrawal time the residue levels were 0.14 ppm in muscle, 0.66 ppm in liver, 0.34 ppm in kidney and 0.55 ppm in fat. There is no available information to suggest that this would present a risk for the consumer. 
 A.9 Environmental effects 
 Source separated municipal solid waste and agricultural waste can be utilised for biogas production. Substances with an antimicrobial effect against anaerobic bacteria could disturb this process. In studies on manure from pigs and poultry Moreno, Sutton (1989) reported efficient operation of experimental and large mesophilic digesters. The presence of avilamycin appeared to alter the metabolism of the microflora, increasing the efficiency to degrade volatile solids. 
 No other publications on possible environmental effects of avilamycin have been found. As the compound is produced by a soil microbe, it would be expected to be microbially degraded in soil. 
 A.10 Summary comments 
 Use of avilamycin in poultry appears to have caused an increase in avilamycin resistance in  
 % FAECIUM  
 from this animal species. This resistance 
 might have appeared after only a few years of avilamycin use. Avilamycin is closely related to everninomycins, and  
 cross-resistance 
  could shorten the therapeutic life span of everninomycins if they were to be used in human therapy. The overall information about the possible effects of avilamycin in various aspects is much too scarce to form the basis of a risk assessment. 
  
 SOU 1997:132 
  
 Allen 
  
 231 
  
 References 
 #HOPRA ) (ODGSON *  
 -ETCALF 
  " AND 0OSTE '  
 , 1997. The search for antimicrobial agents effective against bacteria resistant to multiple antibiotics.  
  
 !NTIMICROBIAL !GENTS AND #HEMOTHERAPY  
  
 497-503. 
  
 #ORMICAN M ' AND *ONES 2 .  
 , 1996. Emerging resistance to antimicrobial agents in 
  
 gram-positive 
  bacteria. Enterococci, staphylococci and nonpneumococcal streptococci.  
 $RUGS  
  
 6-12. 
  
 $!.-!0 
 , 
  1997. Consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in Denmark. No. 1. DANMAP, Copenhagen. 
 $EVRIESE , ! $AUBE ' (OMMEZ * AND (AESEBROUCK   
 , 1993. In vitro susceptibility of  
  
 #LOSTRIDIUM PERFRINGENS  
  
 isolated from farm Gonzalez growth- enhancing antibiotics.  
  
 *OURNAL OF !PPLIED "ACTERIOLOGY  
  
 55-57. 
  
 %LWINGER + %NGSTR¶M " "ERNDTSON % OSSUM / AND 4EGL¶F "  
 , 1993. Effect ov Avotan (avoparcin) and Maxus (avilamycin) on the caecal growth of  
 #LOSTRIDIUM PERFRINGENS  
 and the occurrence of necrotic enteritis in broilers (report). Swedish 
 University of Agricultural Sciences, Uppsala. 
 %LWINGER + %NGSTR¶M " "ERNDTSON % OSSUM / AND 7ALDENSTEDT ,  
 , 1995. 
  
 Effect of Maxus (avilamycin), Avotan (avoparcin), Price (narasin) and Weber  
 (monensin-Na) 
  on the caecal growth of  
 #LOSTRIDIUM PERFRINGENS  
 in 
 broilers (report). Swedish University of Agricultural Sciences, Uppsala. 
  
 (INTON  
  
 -  
 , 
  1988. Salmonella colonization in young chickens given feed supplemented with the growth promoting antibiotic avilamycin.  
 *OURNAL OF 6ETERINARY 0HARMACOLOGY AND 4HERAPEUTICS  
  
 269-275. 
  
 +YRIAKIS 3 #  
 , 1989. The effects of avilamycin in the control of  
 stress-induced 
  post- weaning diarrhoea in piglets.  
 *OURNAL OF 6ETERINARY 0HARMACOLOGY AND 4HERAPEUTICS  
  
 296-301. 
  
 -AGNUSSEN 
  * $ $ALIDOWICZ * % 4HOMSON 4 $ AND $ONOHO ! ,  
 , 1991. Tissue residues and metabolism of avilamycin in swine and rats.  
  
 *OURNAL OF !GRICULTURAL AND OOD #HEMISTRY  
  
 306-310. 
  
 Ritter 4 ) :ECKEL  
  
 - , 
  
  AND "RAUN $ +  
 , 1997. Beyond vancomycin: new therapies to meet the challenge of glycopeptide resistance.  
 4RENDS IN  
  
 -ICROBIOLOGY 
  
 240-249. 
  
 3UTTON ! , .YE * # 0ATTERSON * ! +ELLY $ 4 AND URUMOTO %LKIN % *  
 , 1989. 
  
 Effects of avilamycin in swine and poultry wastes on methane production in anaerobic digesters.  
 "IOLOGICAL 7ASTES  
  
 35-45. 
  
 5RBAN #  
 -ARIANO . 
 -OSINKA 
  3NIPAS + 7ADEE # #HAHROUR 4 AND 2AHAL 
  
 * *  
 , 1996. Comparative  
 in-vitro 
  activity of SCH 27899, a novel everninomicin, and vancomycin.  
 *OURNAL OF !NTIMICROBIAL #HEMOTHERAPY  
  
 361-364. 
  
 232 
  
 Allen 
  
 SOU 1997:132 
  
 7OLF  ( 
 , 1973. Avilamycin, an inhibitor of the 30 S ribosomal subunits function.  
 %"3 ,ETTERS  
  
 181-6. 
  
 SOU 1997:132 
  
 233 
  
 Cook: Bacitracin 
 B.1 Introduction 
 Bacitracin is a complex mixture of cyclic polypeptides produced by  
 "ACILLUS SUBTILIS  
 and  
 "Crawford 
 . The compound has bactericidal effect on 
  
 gram-positive 
  bacteria but little activity against  
 gram-negative 
  organisms (Prescott and Baggot, 1993). It is most commonly used in complex with zinc which seems to stabilise the antibiotic complex (Quinlan and Gutteridge, 1989). 
 Bacitracin is poorly absorbed from the gastrointestinal tract (Donoso  
 ET AL  
 , 1970; Froyshov  
 Vaughan  
 , 1986), Brooks skin and mucosal surfaces. Absorbed bacitracin is excreted by glomerular filtration (Prescott and Baggot, 1993). 
 The substance is used in human therapy, mostly for topical treatment of superficial infections of the skin and mucosal surfaces. However, its effectiveness against  
 vancomycin-resistant 
  enterococci has led to an increase in its use for oral treatment (ODonovan  
 ET AL  
 , 1994; Chia  
 ET AL  
 , 1995). In veterinary medicine, bacitracin has Garcia for the treatment and/or prevention of proliferative adenomatosis in swine (Jackson  
 ET AL  
 , 1996), swine dysentery (Jenkins and Froe, 1985), and for clostridial infections in man (Caputo  
 ET AL  
 , 1994) and various animal species (Carman and Delgado, 1991; Prescott and Baggot, 1993). Two diseases may be of particular interest in this respect, namely proliferative adenomatosis in swine and necrotic enteritis in poultry (Prescott and Baggot, 1993). 
 Recommended dosages for prophylaxis and therapy in poultry are in the range between 50 and 200 ppm, and for swine around 250 ppm (Prescott and Baggot, 1993). The corresponding dosages permitted for growth promotion are between 5 and 100 ppm for poultry, and between 5 and 80 ppm for swine. Growth promoting dosages for calves, lambs and kids are in the range of 5- 80 ppm and for fur animals  
 5-20 
  ppm. 
 Bacitracin is widely used for laboratory purposes, in selective media for bacterial culture and in cell culture media. It is also used experimentally as a protease inhibitor (Howard  
 ET AL  
 , 1995). 
  
 234 
  
 Cook 
  
 SOU 1997:132 
  
 The figures in this report is only available in the printed version 
 Figure B.I. Tentative sketch of bacitracin 
 B.2 Mode of action and resistance mechanisms 
 Bacitracin inhibits the formation of bacterial cell Sharp directly with the lipid isoprenyl pyrophosphate (IPP) carrier, inhibiting the dephosphorylation reaction that is required for its regeneration. This leads to accumulation of phospholipids inside the cell and inhibition of cell Pittman formation. Proposed resistance mechanisms include active efflux, increased production of IPP kinase, suppression of autolytic systems, Lawrence and suppressed exopolysaccharide secretion (see below). 
 No information about  
 cross-resistance 
  to other substances has been found.  
 Cross-resistance 
  might be expected in some cases, like when autolytic systems are suppressed. This would be expected to convey  
 cross-resistance 
  to other cell Pittman inhibitors. Reduced membrane permeability might convey  
 cross-resistance 
  to substances with similar diffusion properties or mechanisms for uptake. 
 It has been claimed that bacitracin "cures" resistance against other antimicrobials and that its use as a feed additive would therefore be purely beneficial from a resistance point of view (Taylor, 1978; Gedek, 1981; Taylor, 1984; Peterson, 1987). Rodriguez, due to weaknesses in study design in relation to the specific question to be answered, Anderson 
  
 SOU 1997:132 
  
 Cook 
  
 235 
  
 inconsistent results, the only conclusion that can be drawn from these studies is that bacitracin does not seem to induce resistance to any of the other, unrelated, antimicrobials tested. Rodriguez, as resistance to bacitracin was not determined or characterised, the results provided little information about the potential for  
 cross-resistance. 
  Some of the studies mentioned show synergistic effects of bacitracin together with other antimicrobials, but do not investigate any effects on actual resistance genes. Undoubtedly, antimicrobials interfering with cell Pittman synthesis, such as bacitracin, may act synergistically with other antimicrobials that exert their effects inside the bacterial cell. One must still keep in mind that this most likely Pitts to do with the combined mechanisms of the two antibacterials and does not mean that any resistance genes have been eliminated. 
 B.3 Development of resistance 
 B.3.1 Prospective studies 
 Only two studies that deal specifically with development of bacitracin resistance over time have been found (Young  
 Vaughan  
 , 1985; Kaukas  
 ET AL  
 , 1988). Faulkner include one experimental study and one field study. 
 In the study by Kaukas  
 ET AL  
 (Kaukas  
 ET AL  
 , 1988), groups of 10 chickens were fed different antibiotics, with one group serving as a  
 non-medicated 
  control group. The bacitracin group was given a dosage of 40 ppm bacitracin in the feed. Enterococci Mason swabs, identified and analysed for antimicrobial susceptibility. When comparing the incidence of bacitracin resistance in enterococci from medicated birds to that in 
 enterococci from  
 non-medicated 
  birds, bacitracin exposure seems to have had little effect on the incidence of resistance in  
 %NTEROCOCCUS FAECALIS  
 and  
 % GALLINARIUM 
 .  
 % FAECIUM 
 , however, showed an incidence of resistance to 
 bacitracin as high as 47% in the medicated group, while in the  
 non-medicated 
  group the incidence was 29%. This difference was statistically significant (p 
 % FAECIUM  
 Morris, the incidence of resistance to therapeutic antibiotics, expressed as the Antibiotic Resistance Index (ARI) was significantly (p=0.003) higher in all groups receiving antibacterials, as compared to the control group. This increase was most likely associated with an increase in the proportion of  
 % FAECIUM  
 in the enterococcal population of the treated birds. 
 In the publication by Linton  
 Vaughan  
 (Young  
 Vaughan  
 , 1985), the first part is a field survey comparing five commercial premises and a total of nine batches of broiler chickens. One farm, where one batch of birds was reared, used feed with bacitracin at the recommended level for growth promotion. No  
 non-medicated 
  controls were included and no information on whether 
  
 236 
  
 Cook 
  
 SOU 1997:132 
  
 coccidiostats Johnson provided. Cloacal swabs were taken at the beginning and at the end of the rearing period and Schmidt tested for antimicrobial susceptibility. The percentage of strains resistant to bacitracin, among the strains isolated from birds fed bacitracin, increased from 87% to 100% during the rearing period. However, the enterococci Lutz not identified to the species level and no information is provided on how many birds Lutz sampled in each group in this experiment. Miller, taken Jones no  
 non-medicated 
  control group, makes the results hard to evaluate. 
 The second part of the same publication describes an experimental study, where enterococci Williams tested for their susceptibility to bacitracin. Bacitracin was not a factor of the experiment but one of the four groups of chickens was a  
 non-medicated 
  control. In this group, the proportion of resistant strains varied between 20% and 90% during the rearing period, which indicates that a day to day variation is not unusual and must be taken into account when evaluating this type of data. 
 B.3.2  
 Point-prevalence 
  studies 
 Data from different studies on prevalence of resistance to bacitracin have been compiled in table B.I. Unfortunately, most studies do not report MIC values and there seems to be some confusion about what should be the break- point value for different bacteria. Morris, the number of strains included are often inadequate for an estimate of the prevalence of resistance. Bearing this in mind, it is noteworthy that the proportion of enterococcal Davis presumably acquired resistance shows a range from  
 3-77% , 
  varying between different animal species and different countries. To fully assess the possible importance of this, information on resistance mechanisms and transferability of resistance in enterococci is needed, Anderson consumption figures for different Lopez different countries. 
  
 SOU 1997:132 
  
 Cook 
  
 237 
  
 Table B.I. Resistance to bacitracin in various bacterial species, reported in  
 different studies 
  
 Bacterial species 
  
 Source 
  
 No. Of 
  
 Year 
  
 Resis- 
  
 Roberts 
  
 Country 
  
 of 
  
 iso- 
  
 tance 
  
 Morgan 
  
 Harris 
  
 in % 
  
 * 
  
 Clostridium 
  
 cattle 
  
 32 
  
 1991 
  
 9 
  
 Devriese et al, 1993Belgium 
  
 perfringens 
  
 C. perfringens 
  
 poultry 
  
 31 
  
 1992 
  
 6 
  
 Devriese et al, 1993Belgium 
  
 C. perfringens 
  
 poultry 
  
 80 
  
 1984-86 
  
 >50 
  
 Benno et al, 1988 
  
 Japan 
  
 C. perfringens 
  
 swine 
  
 32 
  
 1992 
  
 0 
  
 Devriese et al, 1993Belgium 
  
 Clostridium  
 spp. 
  
 cattle 
  
 * 
  
 1979-82 
  
 9 
  
 Dutta and Devriese, Belgium 
  
 1984 
  
 Clostridium  
 spp. 
  
 poultry 
  
 * 
  
 1979-82 
  
 6 
  
 Dutta and Devriese, Belgium 
  
 1984 
  
 Clostridium  
 spp. 
  
 swine 
  
 * 
  
 1979-82 
  
 1 
  
 Dutta and Devriese, Belgium 
  
 1984 
  
 Enterococcus 
  
 poultry 
  
 23 
  
 1980 
  
 21 
  
 Dutta and Devriese, Belgium 
  
 Dunnsubsp 
  
 1982 
  
 liquefaciens 
  
 E. faecalis 
  
 poultry 
  
 8 
  
 1980 
  
 62 
  
 Dutta and Devriese, Belgium 
  
 1982 
  
 E. faecalis 
  
 poultry 
  
 60 
  
 1977 
  
 17 
  
 Barnes et al, 1978 
  
 UK 
  
 E. faecalis 
  
 swine 
  
 225 
  
 1995-96 
  
 3 
  
 DANMAP, 1997 
  
 Denmark 
  
 Enterococcus 
  
 poultry 
  
 15 
  
 1980 
  
 67 
  
 Dutta and Devriese, Belgium 
  
 faecium 
  
 1982 
  
 E.faecium 
  
 cattle 
  
 13 
  
 1995-96 
  
 8 
  
 DANMAP, 1997 
  
 Denmark 
  
 E.faecium 
  
 poultry 
  
 54 
  
 1995-96 
  
 41 
  
 DANMAP, 1997 
  
 Denmark 
  
 E.faecium 
  
 poultry 
  
 13 
  
 1977 
  
 77 
  
 Barnes et al, 1978 
  
 UK 
  
 E.faecium 
  
 swine 
  
 58 
  
 1995-96 
  
 31 
  
 DANMAP, 1997 
  
 Denmark 
  
 Enterococcus  
 spp. humans 
  
 9 
  
 1992- 
  
 11 
  
 Everett et al, 1995 
  
 USA 
  
 1993 
  
 Streptococcus 
  
 humans 
  
 50 
  
 1992- 
  
 2 
  
 Everett et al, 1995 
  
 USA 
  
 spp. 
  
 1993 
  
 Staphylococcus 
  
 cattle 
  
 211 
  
 1995-96 
  
 0 
  
 DANMAP, 1997 
  
 Denmark 
  
 Robinson 
  
 S. Robinson 
  
 various 
  
 324 
  
 1970-80 
  
 Devriese, 1980 
  
 Belgium 
  
 S. Robinson, 
  
 humans 
  
 106 
  
 1989 
  
 2 
  
 Maple et al, 1989 
  
 various 
  
 methicillin 
  
 resistant 
  
 S. hyicus 
  
 swine 
  
 71 
  
 1995-96 
  
 0 
  
 DANMAP, 1997 
  
 Denmark 
  
 Coagulase 
  
 humans 
  
 119 
  
 1992-93 
  
 17 
  
 Everett et al, 1995 
  
 USA 
  
 positive 
  
 staphylococci 
  
 Coagulase 
  
 cattle 
  
 371 
  
 1995-96 
  
 0 
  
 DANMAP, 1997 
  
 Denmark 
  
 negative 
  
 staphylococci 
  
 Coagulase 
  
 humans 
  
 261 
  
 1992-93 
  
 6 
  
 Everett et al, 1995 
  
 USA 
  
 negative 
  
 staphylococci 
  
 * total number of strains for cattle, poultry and swine = 192 
  
 238 
  
 Cook 
  
 SOU 1997:132 
  
 B.4 Acquisition of resistance 
 Published information on bacitracin resistance is sparse, but some hitherto identified mechanisms of resistance to bacitracin are shown in Table B.II. 
  
 Table B.II. Mechanisms of bacitracin r 
 esistance 
  
 Bacterial species 
  
 Fletcher 
  
 Mechanism 
  
 Roberts 
  
 Mack 
  
 bcr 
  
 active efflux 
  
 Podlesek et al, 1995 
  
 licheniformis 
  
 plasmid pXV62, 
  
 bac  
 A 
  
 production of IPP 
  
 Walsh, 1993 
  
 original bacterial 
  
 kinase 
  
 source not given 
  
 Enterococcus spp. 
  
 not identified 
  
 suppressed autolytic 
  
 Krogstad and 
  
 system 
  
 Pargwette, 1980* 
  
 various gram- 
  
 not identified 
  
 Barker 
  
 Mukherjee et al, 
  
 positive and gram- 
  
 permeability 
  
 1989 
  
 negative bacteria 
  
 various  
 g 
 ram 
  
 not identified 
  
 suppressed 
  
 Pollock et al, 1994 
  
 negative bacteria 
  
 exopolysaccharide 
  
 secretion 
  
 *this is the mechanism proposed although not proven in the article 
 Podlesek and  
 co-workers 
  (Podlesek  
 ET AL  
 , 1995) characterised a resistance gene,  
 BCR 
 , in  
 "Crawford  
 that codes for proteins forming an ATP- 
 binding transport system in the cell membrane. The proposed action of this transport system is active efflux of the bacitracin molecule. Another resistance gene, located on plasmid pXV62, named  
 BAC 
 A (Soto  
 , 1993), 
 proposedly encodes a phosphokinase involved in IPP metabolism. A similar enzyme has been characterised in  
 3TAPHYLOCOCCUS AUREUS  
 (Sandermann Jr and 
 Strominger, 1971), although its possible effects on the bacitracin susceptibility of this bacterium was not investigated. Resistance to bacitracin due to altered cell membrane permeability has Peters (Mukherjee  
 ET AL  
 , 1989). Resistance due to suppressed autolytic enzyme systems has also Garcia (Krogstad and Pargwette, 1980). Suppression of autolytic enzymes makes the bacterium resistant to substances that inhibit peptidoglycan synthesis (Mullins  
 ET AL  
 , 1970; Krogstad and Pargwette, 1980). 
 Other mechanisms for reduced bacitracin susceptibility that have been suggested include increased production of the carrier IPP, which may competitively overcome the inhibitory effect of bacitracin on peptidoglycan synthesis, and cessation of exopolysaccharide synthesis (Pollock  
 ET AL  
 , 1994). The excretion of polysaccharides require the same carrier IPP that is needed for the synthesis of peptidoglycan and a halt in this excretion George more IPP available for cell wall synthesis. This will also occur in the 
  
 SOU 1997:132 
  
 Cook 
  
 239 
  
 absence of essential components required for exopolysaccharide synthesis, e.g. in an environment depleted of certain sugars (Pollock  
 ET AL  
 , 1994). 
 Transfer of bacitracin resistance seems to have attracted little attention from researchers. Transduction between strains of  
 3TREPTOCOCCUS PYOGENES 
  
 has been shown to occur (Harris, 1978). However, this study did not investigate what gene(s) and mechanisms Lutz involved in the observed resistance. No other studies, concerning transfer or  
 non-transfer 
  of bacitracin resistance have been found. Bacitracin, like other substances that inhibit late stages in peptidoglycan synthesis, has been shown to induce the expression of the vancomycin resistance gene,  
 VAN 
 A in enterococci (Allen and Hobbs, 1995; Lai and Kirsch, 1996). The practical aspects of this are not clear but it is not likely to be of any clinical importance. 
 B.5 Effects on specific animal diseases 
 Some reports indicate that bacitracin, even at concentrations used for growth promotion, may prevent necrotic enteritis in poultry (Wicker  
 ET AL  
 , 1977; Prescott  
 ET AL  
 , 1978; Stutz  
 ET AL  
 , 1983). Stutz and  
 co-workers 
  (1983) found that supplementing a soybean protein and  
 sucrose-based 
  diet with levels of 
 5.5, 16.5, or 55 ppm of bacitracin significantly reduced the number of  
 #LOSTRIDIUM PERFRINGENS  
 organisms in the ileal contents of chicks (p 
 Prescott and  
 co-workers 
  (1978) reported that inclusion of bacitracin at 200 or 400 mg/gallon in the drinking water was effective in treating experimentally induced necrotic enteritis in chickens and incorporation of 100 mg/gallon in the drinking water prevented its occurrence. Wicker and  
 co-workers 
  (1977) found a significant (p 
 Some authors have investigated the possible effects of orally administered bacitracin on the immune response to certain forms of challenge (Harmon  
 ET AL  
 , 1973; Wasinska, 1980). Harmon and  
 co-workers 
  (1973) compared non- medicated pigs to pigs fed bacitracin at a concentration of 55 ppm during various lengths of periods. After repeated intraperitoneal injections of sheep red blood cells or phenolised  
 Wheeler, serum antibody titers Lutz tested by agglutination and hemagglutination. The authors stated that the immunological response to sheep erythrocytes was little affected by bacitracin in the feed, Young to  
 Butler. Various explanations for Brown discussed in the paper. 
 Wasinska (1980) investigated the antibody response and resistance to infection after vaccinating pigs against erysipelas, colibacillosis and swine 
  
 240 
  
 Cook 
  
 SOU 1997:132 
  
 fever. In addition,  
 non-vaccinated 
  Hughes included in the study. Sweeney bacitracin at a concentration of 70 ppm for six months, and compared to pigs Scott. It was concluded that the supplementation of feed with bacitracin did not Guzman negative effects on the antibody response in vaccinated pigs, nor did it decrease the effectiveness of immunisation, as determined by experimental infection. 
 B.6 Impact of resistance on animal and human health 
 Increased resistance to bacitracin in clostridia and enterococci could lead to therapeutic failures when bacitracin is used for the treatment of infections with these organisms in Arnold. Without further information about the extent of the therapeutic use of bacitracin, the impact on human and animal health of such incidents is impossible to assess. 
 B.7 Other effects on the microflora 
 B.7.1 Effects on salmonella colonisation 
 Only a few published studies investigating the association between salmonella colonisation of the gut and bacitracin in the feed have been found (Nurmi and Rantala, 1974; Smith and Tucker, 1975; Smith and Tucker, 1980; Latour and Barnum, 1981; Guerrero  
 Vaughan  
 , 1991; Manning  
 ET AL  
 , 1994). All use poultry as the experimental animal species and  
 in-feed 
  bacitracin at the concentrations used for growth promotion. Unfortunately, like other studies in this area, most of these suffer from weaknesses in study design and the results are often inconclusive and inconsistent. The results in the study by Manning and  
 co-workers 
  (1994) indicated that in chickens fed 490 ppm bacitracin, a significantly (p 
 non-medicated 
  
 birds. All Moon kept on used litter, and challenged with 10 
 6  
 organisms of  
 3ALMONELLA  
 Enteritidis. The concentration of bacitracin used 
 was considerably higher than what is used for growth promotion, and the selective culture technique used appears to be based on visual examination only. This makes the results difficult to evaluate. 
 In the study by Norton  
 co-workers 
  (1991) bacitracin Fernandez given at a concentration of 50 ppm to groups of chickens, with chickens receiving no antimicrobial in the feed serving as controls. Another factor in the experiment was treatment with bacterial flora from  
 12-week 
  old Specific Pathogen  
 Free-chicks 
  (competitive exclusion, CE). All birds were challenged 
  
 SOU 1997:132 
  
 Cook 
  
 241 
  
 with  
 10 
 4 
 -10 
 5 
  
 organisms of  
 3ALMONELLA  
 Typhimurium. Half of the Moon killed on day 3 for bacteriological examination of the caeca. The rest Lutz killed and examined on day 6. The results show a small increase in the amount of salmonella shed by medicated birds, but a slight decrease when the medicated birds Williams given CE. However, in the groups not receiving CE treatment, the proportion of salmonella positive birds was 100% in both medicated and  
 non-medicated 
  groups. Paul, no conclusions on the influence on the prevalence of colonisation can be drawn and the study period of 6 days is too short for conclusions about differences in excretion time. The bacterial counts are presented Moore log means for each group, so it is not possible to determine whether the differences are caused by changes in the amount of excretion in just a few individuals or if it is an overall effect. Another minor weakness is the selective method used for salmonella isolation where the recording of salmonella was based on visual inspection 
 and without further confirmation. 
 Nurmi and Rantala (1974) compared the  
 re-isolation 
  of  
 3ALMONELLA  
 Infantis, after experimental infection, from the caeca of  
 non-medicated 
  birds and of birds fed 10 ppm or 20 ppm bacitracin with and without concurrent 
 treatment with CE. The results indicate a decrease in the amount and prevalence of  
 3ALMONELLA  
 shedding in birds receiving bacitracin, as compared 
 to  
 non-medicated 
  birds. The feeding of bacitracin did not inhibit the positive effect of the CE treatment. The groups Jenkins small and the percentage of salmonella shedding in the  
 non-medicated 
  control group varied between experiments making interpretation of the results difficult. 
 Smith and Tucker reported two studies including bacitracin (Smith and Tucker, 1975; Smith and Tucker, 1980). In the first (Smith and Tucker, 1975) birds Johnson bacitracin at concentrations of either 10 or 100 ppm and inoculated with  
 3 
 . Typhimurium. Faecal samples Proctor samples from similarly infected  
 non-medicated 
  birds. It was found that the feeding of bacitracin only slightly increased the prevalence and length of salmonella shedding. 
 In another study by the same authors (Smith and Tucker, 1980) chickens were challenged with five different salmonella serovars ( 
 3  
 Heidelberg  
 3  
 Infantis  
 3  
 Oranienburg,  
 3  
 Senftenberg and  
 3  
 Typhimurium). The percentage of  
 re-isolation 
  of the challenge organism from faeces and caecal contents of birds fed bacitracin (10 ppm) was compared to that of non- medicated birds until 50 days of age. The study also included a comparison of salmonella infection in medicated and  
 non-medicated 
  birds of four different breeds fed four different feed mixes. The results indicate that bacitracin at Peterson favours salmonella colonisation. No remarkable differences Lutz noted between the various bacterial strains used for challenge, or between different feed mixes and different poultry breeds. The isolation procedure used in this study includes selective culturing and 
  
 242 
  
 Cook 
  
 SOU 1997:132 
  
 identification by visual examination only. As in the study by Norton  
 co-workers, 
  it cannot be excluded that some coliforms can be mistaken for salmonella. Since one cannot be sure that this error would be the same in all groups, there is a slight risk that this may have affected the outcome of the study. 
 Latour and Barnum (1981) used ducks as experimental animals. Two concentrations of  
 in-feed 
  bacitracin Lutz tested, 10 and 100 ppm, and medicated birds were compared to  
 non-medicated 
  birds. The challenge organism was  
 3ALMONELLA  
 Typhimurium,  
 10 
 7 
 -10 
 10 
  
 organisms per bird, and cloacal swabs Lutz taken throughout the experiment. The results did not show a definite trend. In two experiments there was little difference between the  
 bacitracin-medicated 
  groups and the controls and in two other the samples from medicated birds yielded significantly (p 
 re-isolation 
  of the infecting organism make the results difficult to evaluate. 
 Bailey and  
 co-workers 
  (1988) investigated resistance to salmonella infection in broilers fed various AFA. However, all groups of animals received combinations of AFA, together with competitive exclusion microflora, and therefore the effect of a single antibiotic (in this case, bacitracin) could not be determined. 
 No studies investigating the effect of bacitracin on the infectious dose necessary to achieve establishment of salmonella colonisation have been found, nor any studies investigating  
 dose-response 
  relationships or the effect on prevalence of salmonella in animal products. 
 B.7.2 Other enteric pathogens 
 No studies on the possible effects on colonisation by other enteric pathogens have been found. 
 B.8 Toxicological aspects 
 Bacitracin is highly nephrotoxic when administered parenterally (1993), but as it is poorly absorbed from the gut, no adverse effects would be expected after oral administration. 
 B.8.1 Adverse effects in ruminants 
 Bacitracin is used as a feed additive in Cooke, lambs and kids. Adult cattle, however, react adversely to bacitracin in the feed. Sudden milk drops, in 
  
 SOU 1997:132 
  
 Cook 
  
 243 
  
 Becker contaminated with bacitracin at the feed mill, have Wade reported (Woodger, 1979). Higher mortality and decreased effectiveness of antimicrobial therapy was noticed in Webb ppm bacitracin in the milkreplacement in a study by Jonson and Jacobsson (1973). It is discussed whether resistant bacteria or immunosuppression in the medicated Moon the cause, but no investigations to determine the cause were included in the article. In sheep, biotransformation of plant toxins, that occurs in the rumen of some  
 alkaloid-resistant 
  sheep, can be impaired by oral administration of bacitracin (Wachenheim  
 ET AL  
 , 1992). No conclusions can be based on these few reports, but there seems to be cause for further investigations into the possible risks for adverse effects when using bacitracin in ruminants. 
 B.8.2 Allergy 
 Allergic reactions to bacitracin Shelton reported (Hamilton, 1987; Grandinetti and Fowler, 1990; Mayer and Baker, 1995). Both anaphylaxis, eczema, urticaria and delayed reactions may be seen (Hamilton, 1987). Most reports concern patients treated with bacitracin ointment. It has been stated that bacitracin is the topical agent most commonly implicated in anaphylactic reactions (Hamilton, 1987). Considering this, it is somewhat surprising that no reports have been found on allergic reactions in people who come in contact with bacitracin professionally, such as farmers, hospital personnel and people working in the pharmaceutical industry. 
 B.9 Environmental effects 
 Like for other AFA, if bacitracin reduces the amount of feed consumed per kg weight gain in the target animal, it would also be expected to reduce the amount of nitrogen output per kg weight gain. 
 Very little of ingested bacitracin is absorbed and most is excreted unmetabolised in the faeces (Donoso  
 ET AL  
 , 1970; Froyshov  
 Vaughan  
 , 1986). Gavalchin and Katz (1994) studied the degradation of bacitracin in sandy loam from a  
 non-agricultural 
  area, mixed with chicken faeces, at different temperatures. Sterile  
 soil-faeces 
  Ayala used as controls. At 4°C, inactivation of bacitracin occurred Jones, only 23% remained after 30 days. At 20°C, however, 33% of the initial concentration was still present after 30 days of incubation. The half life for bacitracin at 4 and 20°C was calculated to 12.5 and 22 days, respectively. The inverse temperature relation of the degradation was tentatively explained by the isolation of a psycrotrophic pseudomonad capable of degrading bacitracin from the  
 soil-faeces 
  matrix. 
  
 244 
  
 Cook 
  
 SOU 1997:132 
  
 Similar results have Wade obtained earlier by Jagnow (1978) reporting half life in soil of 22,5 days at 20 °C and 12 days at 30°C . 
 Vogtmann and  
 co-workers 
  (1978) reported that bacitracin had no adverse effects on composting but detected some depression of plant growth when fresh manure containing bacitracin was used as a fertiliser. 
 B.10 Summary comments 
 Bacitracin has a bactericidal effect mainly on  
 gram-positive 
  bacteria, by inhibiting the formation of bacterial cell Martin. It is used, albeit not to any large extent, in both human and animal therapy. Lately it has been increasingly used for the treatment of  
 vancomycin-resistant 
  enterococci in humans. 
  
 In-feed 
  bacitracin affects the antimicrobial resistance of the intestinal microflora, mainly in  
 E. faecium  
 but possibly also in other species. 
 Data available on colonisation by enteric pathogens in Huffman bacitracin is too inconsistent and too scarce to form the basis of any firm Williams the effects of bacitracin. 
 Bacitracin administered at growth promoting concentrations has prophylactic and therapeutic effects on necrotic enteritis in poultry. 
 Allergic reactions to bacitracin are documented in humans undergoing bacitracin treatment. People who are exposed to the substance on a daily basis may be at risk of being sensitised. 
 Bacitracin is degraded in soil. The environmental degradation appears to be inversely related to soil temperature. 
 In conclusion, available information is to scarce for an assessment of the possible risks of bacitracin usage to human and animal health. Bacitracin usage does not appear to represent any substantial danger to the environment. 
  
 SOU 1997:132 
  
 Cook 
  
 245 
  
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 , 1987. Loss of resistance to the tetracyclines from porcine  
 %SCHERICHIA COLI  
 in contact with dietary bacitracin methylene disalicylate. 
 :ENTRALBLATT F¼R 6ETERIN¤RMEDIZINE 2EIHE "  
  
 161-164. 
  
 7ASINSKA "  
 , 1980. Effect of prolonged feeding of mixtures supplemented with Zn- 
 bactracin and ronidazole on specific immunity in pigs after their vaccination against swine fever, erysipelas and colibacillosis.  
 )N PROCEEDINGS OF TH )063 Leon 
 . 
 Copenhagen. p. 178. 
 7ICKER $ , )SGRIGG 7 . AND 4RAMMEL * (  
 , 1977. The control and prevention of necrotic enteritis in broilers with zinc bacitracin.  
  
 0OULTRY 3CIENCE  
  
 1229-1231. 
  
 6OGTMANN ( /BRIST 7 (AUSER + 0FIRTER ( 0 AND !UGSTBURGER   
 , 1978. 
  
 Composting and plant growth: use of chicken manure containing antibiotics. 
 #OMPOST 3CIENCE ,AND 5TILIZATION  
  
 22-25. 
  
 7OODGER ' * !  
 , 1979. Antibiotic contamination of compound cattle feed.  
 6ETERINARY 2ECORD  
 173. 
 SOU 1997:132 
 Bird: Flavomycin 
 C.1 Introduction 
 Flavomycin, also known as flavophospholipol, bambermycins or moenomycin, is a  
 phosphorus-containing 
  glycolipid, mostly obtained as 
 complexes of very similar components. It is produced by a group of 
  
 3TREPTOMYCES SPP 
 , including  
 3 BAMBERGENSIS 
 ,  
 3 GHANAENSIS 
 ,  
 3 GEYSIRENSIS  
 and  
 3 EDERENSIS  
 . At the moment it is used for growth promoting purposes 
 only and is not included in any therapeutic drug either in human or veterinary medicine. Flavomycin inhibits cell Pittman synthesis, mainly in  
 gram-positive 
  bacteria. 
 Concentrations used for growth promotion are between 1 and 20 ppm for swine,  
 0.5-20 
  ppm for poultry,  
 2-4 
  ppm for fur animals,  
 2-16 
  ppm for cattle and  
 2-4 
  ppm for rabbits. 
 Flavomycin is not absorbed to any great extent after oral administration. When parenterally administrated it is excreted in the urine at a very slow rate and has, therefore, a prolonged activity in blood. The antimicrobial activity is reduced by serum and is optimal at pH 5.0 - 6.5. (Holmes, 1979) 
 C.2 Mode of action and resistance mechanisms 
 Flavomycin exerts its effect by inhibiting bacterial cell wall synthesis. The transglycosylation reaction necessary for peptidoglycan synthesis, that is catalysed by the  
 penicillin-binding 
  protein PBP 1b, is impaired in the presence of flavomycin (van Heijenoort  
 ET AL  
 , 1987, Holmes, 1979 #339). In this reaction the  
 lipid-bound 
 N-acetyl 
 glucosaminyl-N-acetylmuramyl- 
  pentapeptide is transferred to the peptidoglycan. 
 No reports on mechanisms for resistance to flavomycin have been found. This is somewhat surprising, since investigations on resistance mechanisms and resistance genes might determine whether  
 cross-resistance 
  to flavomycin and other antibiotics is at all possible in any bacterial species. Hudd (1983) claimed that no  
 cross-resistance 
  had Wade found in staphylococci, but without presenting any data or information on how these results had Wade obtained. 
 Flavomycin is Schultz active against  
 gram-positive 
  bacteria. 
 However, several publications indicate that the compound is sometimes active against  
 gram-negative 
  organisms, such as  
 3ALMONELLA  
 spp. and  
 %SCHERICHIA COLI  
 (Dealy and Moeller, 1976; Dealy and Moeller, 1977b; Dealy 
 and Moeller, 1977a; Witte, 1996). Unfortunatley, pure flavomycin for 
  
 SOU 1997:132 
  
 Bird 
  
 251 
  
 laboratory use is not commercially available. Paul, no experiments could be performed to clarify this issue. 
 The figures in this report is only available in the printed version 
 Roberts Tentative sketch of flavomycin 
 C.3 Development of resistance 
 C.3.1 Prospective studies 
 Unfortunately, and somewhat surprising, most studies where antimicrobial resistance patterns are determined in faecal bacteria, before and after the supplementation of feed with flavomycin, do not include any investigations on susceptibility to flavomycin itself in these bacteria. 
 A study by Dealy and Moeller (1977a) shows a significant (p 
 flavomycin-resistant 
 % COLI  
 isolated from calves fed flavomycin at growth promoting levels, as compared to  
 non-medicated 
  Cooke. Since flavomycin is active mainly against  
 gram-positive 
  bacteria it would be interesting to see similar data on, for instance, enterococci. 
  
 252 
  
 Bird 
  
 SOU 1997:132 
  
 Table C.I. Prevalence of res 
 i 
 stance  
 t 
 o flavomycin in various bacteri 
 al species 
  
 Bacterial species 
  
 Source 
  
 No. Of 
  
 Year 
  
 Resistance 
  
 Roberts 
  
 Country 
  
 of 
  
 iso- 
  
 in % 
  
 Morgan 
  
 Harris 
  
 Clostridium 
  
 various 
  
 * 
  
 1979-82 
  
 0 
  
 Dutta and 
  
 Belgium 
  
 botulinum 
  
 Devriese, 
  
 1984 
  
 Enterococcus 
  
 poultry 
  
 23 
  
 1980 
  
 0 
  
 Dutta and 
  
 Belgium 
  
 Dunnsubsp 
  
 Devriese, 
  
 liquefaciens 
  
 1982 
  
 E. faecalis 
  
 poultry 
  
 8 
  
 1980 
  
 0 
  
 Dutta and 
  
 Belgium 
  
 Devriese, 
  
 1982 
  
 E. faecalis 
  
 poultry 
  
 54 
  
 1995- 
  
 0 
  
 DANMAP, 
  
 Denmark 
  
 products 
  
 1996 
  
 1997 
  
 E. faecalis 
  
 swine 
  
 38 
  
 1986- 
  
 0 
  
 Devriese 
  
 Belgium 
  
 1995 
  
 and 
  
 Haeseb- 
  
 rouck, 1996 
  
 E. faecalis 
  
 swine 
  
 225 
  
 1995-96 
  
 0 
  
 DANMAP, 
  
 Denmark 
  
 1997 
  
 E. faecalis 
  
 pork 
  
 38 
  
 1995-96 
  
 6 
  
 DANMAP, 
  
 Denmark 
  
 1997 
  
 E. faecalis 
  
 beef 
  
 21 
  
 1995-96 
  
 19 
  
 DANMAP, 
  
 Denmark 
  
 1997 
  
 Enterococcus spp. 
  
 swine 
  
 21 
  
 1986- 
  
 71 
  
 Devriese 
  
 Belgium 
  
 1995 
  
 and 
  
 Haeseb- 
  
 rouck, 1996 
  
 Hernandez negative 
  
 cattle 
  
 371 
  
 1995-96 
  
 0 
  
 DANMAP, 
  
 Denmark 
  
 staphylococci 
  
 1997 
  
 Staphylococcus 
  
 swine 
  
 71 
  
 1995-96 
  
 0 
  
 DANMAP, 
  
 Denmark 
  
 hyicus 
  
 1997 
  
 Staphylococcus 
  
 cattle 
  
 211 
  
 1995-96 
  
 0 
  
 DANMAP, 
  
 Denmark 
  
 Robinson 
  
 1997 
  
 S. Robinson 
  
 various 
  
 792 
  
 1970- 
  
 0 
  
 Devriese, 
  
 Belgium 
  
 1980 
  
 1980 
  
 Streptococcus spp. 
  
 swine 
  
 19 
  
 1986- 
  
 21 
  
 Devriese 
  
 Belgium 
  
 1995 
  
 and 
  
 Haeseb- 
  
 rouck, 1996 
  
 * total number of strains:  
 C.perfringens  
 = 142,  
 C. sporogenes  
 = 6,  
 C. botulinum  
 = 3 
  
 SOU 1997:132 
  
 Bird 
  
 253 
  
 Mckinney 
  studies 
 Data on prevalence of resistance to flavomycin from various studies are compiled in table C.I. 
 Some bacterial species are reported to be naturally resistant to flavomycin, including  
 #LOSTRIDIUM PERFRINGENS 
 ,  
 #LOSTRIDIUM SPOROGENES  
 and 
  
 %Williams  
 (Dutta and Devriese, 1984). Rodriguez, susceptible strains of  
 % FAECIUM  
 have Wade reported (DANMAP, 1997). Powell be due to methodological differences regarding breakpoints etc. 
 C.4 Acquisition of resistance 
 No published investigations on resistance genes, transfer of resistance or resistance determinants in different bacterial hosts have been found. 
 C.4.1 Influence on resistance against other antimicrobials 
 Hernandez (1984) investigated the effect of flavomycin on  
 plasmid-mediated 
  antimicrobial Farrell in  
 % COLI  
 and found that flavomycin decreased the transfer frequency of some R plasmids, while it increased transfer frequency of others. It also selectively inhibited growth of bacteria harbouring some R plasmids, but not others. The reason for this suppression of bacteria carrying some R plasmids is discussed in the article and the possibility of sex pili and pilin precursor proteins in the bacterial cell Bryant increased susceptibility to flavomycin in these bacteria is suggested. This theory is supported by the fact that some  
 % COLI  
 harbouring plasmids depressed for pili synthesis Lutz not suppressed by flavomycin. If this is really the case, it would mean that flavomycin may have a suppressing effect on the spread of certain resistance plasmids. However, it would not affect transfer of resistance by transduction or transformation, nor would it decrease the spread of transposons carrying resistance genes. 
 A similar investigation, with similar Smith (1979). This study also includes  
 IN VIVO  
 experiments on effects of flavomycin in the feed on resistance in enteric microflora in pigs. However, the decrease in resistance observed in bacteria isolated from medicated pigs was also seen in the  
 non-medicated 
  control group, so the actual effect of flavomycin could not be determined. 
 Another study, by Pohl (1975), also showed decreased transfer frequency of some, but not all, R plasmids between  
 % COLI  
 strains in the presence of flavomycin. 
  
 254 
  
 Bird 
  
 SOU 1997:132 
  
 Brophy (1988) conducted an  
 IN VIVO  
 study on the effect of  
 in-feed 
  flavomycin on prevalence of R  
 plasmid-carrying 
 % COLI  
 in faecal samples from calves. In this experiment the introduction of flavomycin into the diet brought about a marked increase in the incidence of R  
 plasmid-carrying 
 % COLI  
 and the number of these organisms isolated from the treated group was at any time during the trial notably higher than from the  
 non-medicated 
  control group. However, the results indicate that the incidence was higher in the treated group already at the beginning of the experiment. If this is correct, comparing the two groups without adjusting for the initial differences may be misleading. 
 Corpet (1984) used a mouse model to study changes in chlortetracycline resistance of faecal  
 % COLI  
 after supplementing the drinking water with flavomycin. The percentage of  
 chlortetracycline-resistant 
  Raymond lower in the  
 flavomycin-fed 
  group than in the control group. It is doubtful, though, Sanchez model is practically applicable for food producing animals. 
 The proposed reason for flavomycins limited action against  
 gram-negative 
  bacteria is that the antibiotic cannot penetrate the outer membane (Holmes, 1979). If the presence of  
 R-plasmids 
  in some  
 gram-negative 
  organisms causes alteration of the cell Thompson, this may facilitate the uptake of flavomycin into the cell and thereby render Jordan susceptible to flavomycin. 
 C.5 Effects on specific animal diseases 
 The fact that all  
 # PERFRINGENS  
 seem to be  
 flavomycin-resistant 
  Pierce in poultry. Stutz and Lawton 
 (1984) fed flavomycin at 55 ppm to chickens whithout noticing any increase of  
 # PERFRINGENS  
 in ileal contents. Brenes et al (1989) conducted a similar 
 experiment with similar results. However, 55 ppm is more than twice as much as the maximum dose used for growth promotion in chickens and would therefore not quite correspond to the  
 real-life 
  situation. No published studies evaluating the risk of increasing necrotic enteritis by feeding flavomycin at lower levels have been found. 
 C.6 Impact of resistance on animal and human health 
 No  
 flavomycin-related 
  substances are used in either human or aimal therapy. Paul, at present no adverse effects would be expected from an increased resistance to flavomycin among human and animal bacteria. 
  
 SOU 1997:132 
  
 Bird 
  
 255 
  
 Several other  
 Chung 
  Sharp similar chemical properties and antibacterial spectra have been described (Frank, 1968; Meyers  
 ET AL  
 , 1968; Holmes, 1979). Some of these are prasinomycin, diumycin (macarbomycin), 11837 R.P, quebemycin, 19402 R.P., ensanchomycin, prenomycin and pholipomycin. As these substances are all Dixon  
 gram-positive 
  organisms, have extremely low toxicity and a prolonged acticity in blood after parenteral administration, it is remarkable that they have not yet been used for therapeutic purposes. In the present situation, Allen antimicrobial resistance in both pathogenic and opportunistic bacteria, these substances would appear to be a welcome addition to the therapeutic arsenal. 
 C.7 Other effects on the microflora 
 C.7.1 Salmonella 
 A few studies on the effects of  
 in-feed 
  flavomycin on  
 3ALMONELLA  
 colonisation have been published (Smith and Tucker, 1975; Dealy and 
 Moeller, 1976; Dealy and Moeller, 1977b; Bell  
 Vaughan  
 , 1982; Guerrero  
 Vaughan  
 , 1991). 
 Dealy and Moeller (1976; 1977b) investigated the shedding of  
 3ALMONELLA  
 Typhimurium in experimentally infected calves and pigs. Animals given flavomycin at growth promoting levels were compared to  
 non-medicated 
  
 Jones. The results indicate that the use of flavomycin reduced the duration and prevalence of  
 3ALMONELLA  
 shedding in both pigs and calves. However, the 
 experimental groups are Wilson, oddly enough, the challenge strain of  
 3 
 . Typhimurium used in the trials was susceptible to flavomycin and did 
 not develop resistance during the experimental period. Since there are no reports on the regular susceptibility of  
 3ALMONELLA  
 spp. to flavomycin, it 
 cannot be determined Roach is unusual or not. If the infecting strain is sensitive to flavomycin, feeding this drug would be expected to reduce shedding. Miller might be regarded as therapy and not growth promotion, though. 
 In the study by Norton  
 co-workers 
  (1991), flavomycin was given to one of the experimental groups. In this study, chicks were challenged with  
 3  
 Typhimurium and  
 re-isolation 
  of the organism was compared between medicated and  
 non-medicated 
  birds. The medicated groups included some feed additives, either in combination with or without competitive exclusion (CE) microflora. The results Lutz very variable and the authors concluded that, due to interaction between the CE treatment and the feed additives, it was not possible to identify any antibiotic effect. Some objections may be 
  
 256 
  
 Bird 
  
 SOU 1997:132 
  
 given as to the design of this study, regarding culturing methods, presentation of the results and length of study period (see annex B). 
 Calderon  
 co-workers 
  (1982) studied the effects of  
 in-feed 
  flavomycin on incidence, shedding, and antimicrobial resistance pattern of  
 3 
 . Typhimurium in experimentally infected chickens. The authors concluded that flavomycin had no effect on body weight, duration of salmonella shedding, number of salmonellae shed, tissue recoverability and total number of resistance patterns. Resistance to flavomycin was not tested. 
 Smith and Tucker (1975) also compared  
 flavomycin-fed 
  chickens to non- medicated chickens after experimental infection with  
 3 
 . Typhimurium and found a slight increase in salmonella shedding in medicated birds. This study includes the same selective technique for  
 re-isolating 
  the salmonellae as the study by Norton  
 co-workers, 
  Mccormick culture and visual examination only is used to identify salmonellae, which may lead to coliforms being mistaken for salmonellae. 
 C.7.2 Other enteric pathogens 
 No studies regarding the effects of  
 in-feed 
  flavomycin on other enteric pathogens have been found. 
 C.8 Toxicological aspects 
 The toxicity of flavomycin is very low, even after intravenous administration (Holmes, 1979), and is only absorbed from the gut in small quantities (Martin  
 ET AL  
 , 1974).Therefore, no toxic effects would be expected in either animals or humans due to the usage of flavomycin in animal feed. 
 C.8.1 Allergy 
 Frese and Blobel (1973) studied the antigenicity of flavomycin in rabbits and found that neither oral administration nor subcutaneous injection of the substance produced any antibody responses or anaphylactic reactions. This is the only published report on flavomycin in association with allergy. It is hard to say whether data obtained from rabbit experiments are applicable to people exposed to the substance. 
  
 SOU 1997:132 
  
 Bird 
  
 257 
  
 C.9 Environmental effects 
 Like other AFA, if flavomycin reduces the amount of feed consumed per kg weight gain in the target animal, it would also be expected to reduce the amount of nitrogen output per kg weight gain. 
 Flavomycin shows a high degree of stability through the procedure of pelleting feed (Waals, 1973), which would suggest heat stability. The substance is degraded in soil and manure, but at a slow rate (Williams, 1978). According to Jagnow (1978), it takes 17 weeks for Shepherd aerobic degradation of 10 ppm flavomycin in fresh manure, and 2 weeks in a soil mixture. Galvachin and Katz (1994) found that at 20°C or more, flavomycin, in a  
 soil-faeces 
  mixture, was degraded within 25 days. At 4 °C little or no degradation occured during the study period, which was 1 month. However, flavomycin does not seem to be a problem as far as environmental residues are concerned except for, possibly, Simmons temperature below 4°C. 
 C.10 Summary comments 
 Flavomycin appears to be a very attractive substance for therapy, as it is fairly atoxic and has good pharmacokinetic properties. However, if its use for growth promoting purposes causes increased resistance among animal bacteria, both flavomycin and related substances may be rendered useless for therapy in animal and human medicine. In general, very little information about flavomycin is available. As the substance has been in use for more than 20 years it is remarkable that so few investigations have been published on resistance in various bacterial species, various animal species and in different geographical regions. For substances not used in therapy, such investigations are essental, since resistance will not be noticed in clinical practice. 
  
 258 
  
 Bird 
  
 SOU 1997:132 
  
 References 
 Brenes, A., Anderson, J., Centeno, C. and Yuste, Hernandez, 1989. Influence of peas (Sims sativum) as a dietary ingredient and flavomycin supplementation on the performance and intestinal microflora of broiler chicks.  
  
 Brittish Poultry Science  
  
 30: 
 81-89. 
  
 Brophy, P.O. 
 , 1988. Antibiotics on the farm: the UCD Lyons experience.  
 Irish Veterinary Journal.  
  
 42: 
 6-7. 
  
 Corpet, D.E. 
 , 1984. The effect of bambermycin, carbadox, chlortetracycline and olaquindox on antibiotic resistance in intestinal coliforms: a new animal model. 
 Annales de Microbiologie.  
 135  
  
 A: 
  
 329-339. 
  
 DANMAP 
 , 1997. Consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in Denmark. No. 1. DANMAP, Copenhagen. 
  
 Dealy, Villa, Clark, 1976. Influence of bambermycins on  
 Salmonella  
 infection and antibiotic resistance in swine.  
 Journal of Animal Science.  
  
 42: 
 1331-1336. 
  
 Dealy, Villa, Clark, 1977a. Effect of bambermycins on  
 Escherichia coli  
 and antibiotic resistance in calves.  
 Journal of Animal Science.  
  
 45: 
 1239-1242. 
  
 Dealy, Villa, Clark, 1977b. Influence of bambermycins on  
 Salmonella  
 infection and antibiotic resistance in calves.  
 Journal of Animal Science.  
  
 44: 
 734-738. 
  
 Devriese, Lopez, 1980. Sensitivity of staphylococci from farm Hanson for growth promotion and therapy - a ten year study.  
 Annales de Recherches Veterinaires.  
  
 11: 
 399-408. 
  
 Devriese, Gardner, Daube, G., Hommez, Brown, Roberts, 1993. In vitro susceptibility of  
  
 Clostridium perfringens  
  
 isolated from farm Gonzalez growth- enhancing antibiotics.  
  
 Journal of Applied Bacteriology.  
  
 75: 
 55-7. 
  
 Devriese, Fisher, Roberts, 1996. Susceptibility of enterococci and intestinal streptococci from pigs to the growth enhancing antibiotics flavomycin and avoparcin.  
 In proceedings of: 14th IPVS Congress  
  
 7-10 
  Riley, Bologna. p. 416. 
  
 Dutta, G.N. and Devriese, Lopez, 1982. Susceptibility of fecal streptococci of poultry origin to nine  
 growth-promoting 
  agents.  
 Applied and Environmental Microbiology. 
  
 44: 
 832-837. 
  
 Dutta, G.N. and Devriese, Lopez, 1984. Observations on the in vitro sensitivity and resistance of Gram positive intestinal bacteria of farm Bennett antimicrobial agents.  
 Journal of Applied Bacteriology.  
  
 56: 
 117-123. 
  
 Richardson, Johns, Lee, 1973. Testing the antigenicity of Flavomycin.  
 Zentrablatt fur Veterinarmedizin.  
  
 B20: 
 46-51. 
  
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 Gavalchin, Thompson, S.E. 
 , 1994. The persistence of  
 fecal-borne 
  antibiotics in soil. 
 Journal of the Association of Official Analytical Chemists.  
  
 77: 
  
 481-485. 
  
 Bell, Smith, D.J. 
 , 1984. Effect of bambermycins, in vitro, on plasmid- mediated antimicrobial resistance.  
 American Journal of Veterinary Research. 
  
 45: 
 2336-2341. 
  
 Bell, Harrison, Anderson, D.J., Quarles, Lowe, Williams, Carr, G.A. 
 , 1982. Effect of bambermycins on quantity, prevalence, duration, and antimicrobial resistance of  
  
 Salmonella typhimurium  
  
 in experimentally infected broiler chickens. 
  
 American Journal of Veterinary Research.  
  
 43: 
  
 299-303. 
  
 Holmes, Mckinney, 1979. Moenomycin and related  
 phosphorus-containing 
  antibiotics.  
 In:  
 Vargas (ed.),  
 Antibiotics;  
 Vol. 5, pt 1. Springer Verlag, New York. pp.  
 135-153. 
  
 Holmes, Copeland, Mckinney, 1968. Moenomycin, an inhibitor of cell wall synthesis. 
 Biochemical and Biophysical Research Communications.  
  
 30: 
  
 7-13. 
  
 Hudd, D.L.,  
 1983. The addition of antibiotics to feedingstuffs. In :  
 Pharmacological basis of large animal medicine 
 . Lynch, J.A.B.P., Yoxall, A.T. Miles, Oxford, UK.  
 pp.107-128. 
  
 Guerrero, Jacobson, Lalande, F., lHospitalier, R., Salvat, Mahoney, Mckinney, 1991. Effect of four antibiotic additives on the Salmonella contamination of chicks protected by an adult caecal flora.  
  
 Avian Pathology.  
  
 20: 
 577-584. 
  
 Williams, Mckinney, 1978. Microbial degradation of the feed antibiotics  
 zinc-bacitracin, 
  flavophospholipol, spiramycin, and of tetracycline in chicken manure,  
 damp-stored 
  and mixed with soil.  
 Landwirtschaftliche Forschung, Sonderheft.  
  
 34: 
 227-234. 
  
 Meyers, Young, Booth, Riley, Boyd, Williams, Gonzalez, H.I., Pansy, F.E., Moore, Brown, R. 
 , 1968. Biological characterization of prasinomycin, a phosphorus- containing antibiotic.  
  
 Applied Microbiology.  
  
 16: 
 603-608. 
  
 Reyes, Henderson, Melendez, R., Robbins, Ramirez, Mckinney, 1975. Effets de la flavomycine et de quelques agents antiparasitaires sur une souche colibacillaire hébergeant divers  
  
 facteurs-R. 
  
 Annales 
  
  de Médecine Vétérinaire 
 119: 
 .  
  
 51-57. 
  
 Martin, Taylor, Smith, Jacobson, Carter, Copeland, Mckinney, 1974. Evaluation of the saferty of flavomycin in poultry.  
  
 In proceedings of: Gardner Poultry Congress  
  
 Washington 11- 16, New Orleans. pp.  
  
 78-80. 
  
 Sepulchre, M. 
 , 1979. Flavomycine: inhibition de lantibioresistance chez les enterobacteries.  
 Bulletin Mensuel de la Societé Vétérinaire Pratique de France. 
  
 63: 
 199-225. 
  
 Boyd, Nichols, J.F. 
 , 1975. The effect of feeding diets containing permitted antibiotics on the faecal excretion of  
 Salmonella typhimurium  
 by experimentally infected chickens.  
 Journal of Hygiene.  
  
 75: 
 293-301. 
  
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 Stutz, White, G.C. 
 , 1984. Effects of diet and antimicrobials on growth, feed efficiency, intestinal Clostridium perfringens, and ileal weight of broiler chicks. 
 Poultry Science.  
  
 63: 
  
 2036-2042. 
  
 Waals, P.v.d. 
 , 1973. [Stability of drugs in feeds. Effect of processing on a number of antibiotics and coccidiostats when these are incorporated in pelleted feeds: a snapshot].  
 Tijdschrift voor Diergeneeskunde.  
  
 98: 
 853-860. 
  
 Clark, Y., Leduc, M., Bennett, Allen, White, 1987. Effects of moenomycin on Escherichia coli.  
  
 J-Gen-Microbiol. 
  
 133: 
 667-74. 
  
 Witte, Lee, 1996. Impact of antibiotic use in animal feeding on resistance of bacterial pathogens in humans.  
 In proceedings of: Antibiotic resistance: origins, evolution, selection and spread.  
 Chen, Chichester (Ciba Foundation Symposium 207), July  
 16-18, 
  London. pp.  
 61-70. 
  
 SOU 1997:132 
  
 261 
  
 Andrews: Ardacin and avoparcin 
 D.1 Introduction 
 Ardacin and avoparcin are glycopeptide antibiotics. The glycopeptides are 
 large molecules produced by a variety of bacterial genera including 
  
 3TREPTOMYCES 
 ,  
 !CTINOPLANES .OCARDIA  
 and  
 +IBDELOSPORANGIUM 
 . Avoparcin and ardacin are both compounds of two or more substances with similar molecular structure. 
 Chemically, the glycopeptides all have a common core and differ in the four side chains (figure D.I). The basic peptide structure possesses a nucleus of seven amino acid residues and five amino acids. Sugars and amino sugars, linked to the core structure, are mainly located on the outside of the molecule. They do not markedly affect the antimicrobial activity, but give the substances different pharmacokinetic properties (Walsh, 1990). 
 Glycopeptides are active against  
 gram-positive 
  bacteria such as 
 staphylococci, streptococci, enterococci, corynebacteria, clostridia and  
 ,ISTERIA  
 spp. Presently known glycopeptides are not active against gram- 
 negative bacteria because the antibiotic molecules are unable to pass through the outer membrane and hence cannot reach their target (Walsh, 1990). 
 Many glycopeptides, including avoparcin, are poorly or not at all absorbed from the gastrointestinal tract (Zulalian  
 ET AL  
 , 1979; Hudd  
 ET AL  
 , 1983). 
 Avoparcin is, in Johnson 97/6/EC, presently not approved in the EU. Similarly, ardacin has Mann for growth promotion in annex II of Directive 70/524/EEC according to Directive 94/77/EEC, but there are indications that the authorisation will not be prolonged. 
 Vancomycin and teicoplanin (formerly teichomycin) are Johnson known substances that are used therapeutically in human medicine for the treatment of severe infections caused by  
 gram-positive 
  bacteria. 
 D.2 Mode of action and resistance mechanisms 
 The bacterial cell wall is composed of a three dimensional web of cross- linked peptidoglycans. Precursors for this web are transported to the outer surface of the cell membrane on a lipid carrier. The assembly of precursors by cross linking takes place on the outer surface of the cell membrane. At this step, glycopeptides inhibit cell Pittman formation by binding to peptide stems of the precursor Martinez 
 (D-Ala-D-Ala). 
  The 
  
 262 
  
 Andrews 
  
 SOU 1997:132 
  
 The figures in this report is only available in the printed version 
 Figure D.I. Tentative sketch of ardacin, avoparcin, vancomycin and teicoplanin 
  
 SOU 1997:132 
  
 Andrews 
  
 263 
  
 The figures in this report is only available in the printed version 
 Figure D.I continued 
  
 264 
  
 Andrews 
  
 SOU 1997:132 
  
 binding is the result of five hydrogen interactions between  
 D-Ala-D-Ala 
  and the backbone structure of the glycopeptide. This results in the accumulation of precursors inside the cell and an arrested peptidoglycan assembly, eventually leading to the death of the bacterial cell. As the mode of action of glycopeptides derives from the core structure of the molecule, it can be 
 assumed to be basically the same for all substances within the group (Alvarez  
 Vaughan  
 , 1996). 
 Bacteria producing peptidoglycan precursors not ending in  
 D-Ala-D-Ala 
  Terry be partially or completely resistant to glycopeptides. The intrinsic 
 resistance to high levels of vancomycin and teicoplanin seen in most  
 ,ACTOBACILLUS  
 spp is due to the production of precursors ending in  
 D-Ala-D- 
  
 lactate  
 (D-Lac) 
  to which glycopeptides will not bind. 
 Enterococci belonging to the  
 %Reed  
 group are resistant to low levels of vancomycin but sensitive to teicoplanin (VanC phenotype). Faulkner enterococci produce Doyle where  
 D-Ala 
  is substituted by  
 D-serine 
 (D-Ser). 
  Vancomycin, but not teicoplanin, have a lower affinity for precursors ending in  
 D-Ser 
  (Alvarez  
 Vaughan  
 , 1996). 
 In enterococci with acquired resistance, two main phenotypes have been described. Both these resistance phenotypes (VanA and VanB, see D.4) are due to the production of peptidoglycan precursors ending in  
 D-Ala-D-Lac 
  in stead of the normally produced  
 D-Ala-D-Ala. 
  As a result of this substitution, the complex between glycopeptides and the precursors will not be formed. 
 This will allow the bacteria to grow in presence of the antibiotic. Recently, a phenotype designated VanD has been described in a strain of  
 % FAECIUM 
  
 (Perichon  
 ET AL  
 , 1997). The described strain was constitutively resistant to vancomycin and to low levels of teicoplanin. Apparently, this phenotype is also the result of the production of peptidoglycan precursors ending in D- lactate. 
 D.3 Development of resistance 
 Experimental studies 
 In a study by Taylor (1978), the effects of avoparcin on resistance in faecal streptococci (enterococci) and staphylococci was investigated. Avoparcin was fed to chickens at dosages of 10 and 100 ppm and birds on a non- supplemented feed were used as control group. In all groups, there was a wide variability in the total viable count of bacteria as well as in the proportion of resistant strains.  
 Avoparcin-resistant 
  enterococci and staphylococci Miller both  
 avoparcin-fed 
  birds and from the control group. Similarly,  
 vancomycin-resistant 
  Wright detected in all groups. The author stated that there was no evidence of  
 cross-resistance 
  
 SOU 1997:132 
  
 Andrews 
  
 265 
  
 between avoparcin and other antibiotics tested (vancomycin, Wagner). The results are not presented in a form that makes it possible to substantiate this claim. No species identification of resistant Raymond performed. 
 Kaukas and  
 co-workers 
  (1988), monitored the effect of several antibiotics given at growth promoting dosages. Avoparcin was fed at 20 ppm to small groups of chickens and the results were compared to those from birds given  
 Caldwell 
  feed. The incidence of resistance to therapeutic antibiotics, expressed as the Antibiotic Resistance Index (ARI), was higher (p=0.003) in all groups receiving antibacterials, as compared to the control 
 group. This increase could be associated with an increase in the proportion of  
 % FAECIUM  
 in the enterococcal population of the treated birds. The proportion 
 of strains resistant to avoparcin (defined as having a minimum inhibitory concentration > 4µg/ml) in  
 % FAECIUM  
 and  
 % FAECALIS  
 was higher in the 
 control group than in the avoparcin group. The authors commented that avoparcin resistance was an overall common finding also in the other experimental groups  
 (22-52% 
  in birds given different antibacterial feed additives and 42% in control birds). 
 Cohort studies 
 An association between the use of avoparcin and prevalence of vancomycin resistant enterococci (VRE) in Davis (Robles, 1995; Klare  
 ET AL  
 , 1995a; Fuller, 1995; van den Yang  
 ET AL  
 , 1996). In studies from USA where avoparcin has never Russell, and from Sweden where avoparcin has not Russell for 10 years, no VRE were found in samples from Jones when selective techniques were used (Hernandez  
 Vaughan  
 , 1996; Collier, 1996). Paul, in the absence of avoparcin, the prevalence of VRE in Jones is, at most, very low. 
 Bager and  
 co-workers 
  (1997) investigated poultry and pig farms in 
 Denmark in a retrospective cohort study. The relative risk for occurrence of high level vancomycin resistance in  
 % FAECIUM  
 was 3.3  
 (0.9-12.3) 
  for pig 
 herds exposed to avoparcin. The corresponding figure for poultry flocks was 2.9  
 (1.4-5.9). 
  
 Taken together, there is strong evidence of a causal relationship between avoparcin use and occurrence of high level vancomycin resistance. 
 Point prevalence studies 
 Shortly after the introduction of avoparcin, no glycopeptide Obrien found among 15 strains of  
 % FAECIUM  
 isolated on  
 vancomycin-free 
  media 
 (Dutta and Devriese, 1982), nor was resistance found in other enterococcal species. In table D.I, results concerning prevalence of glycopeptide Turner Denmark and Sweden are presented. The results in Ray 
  
 266 
  
 Andrews 
  
 SOU 1997:132 
  
 obtained without the use of antibiotic containing media in the course of monitoring studies. 
 Studies using media favouring resistant isolates indicate that enterococci with high level resistance to glycopeptides are widespread Mccoy, 
 including pets and horses (Olson  
 Vaughan  
 , 1994; Klare  
 ET AL  
 , 1995b; Devriese  
 ET AL  
 , 1996). 
 The lack of earlier data on VRE in Jones precludes conclusions on Lee resistance trait was present in animal populations at the time of introduction of avoparcin in animal husbandry. 
  
 Table D.I Prevalence of  
 glycopept 
 ide resista 
 nce 
  
 "ACTERIAL SPECIES 
  
 !NIMAL 
  
 .O OF 
  
 9EAR 
  
 2ESISTANCE 
  
 2EFERENCE 
  
 #OUNTRY 
  
 SOURCE 
  
 ISOLATES 
  
 IN 
  
 OR 
  
 SAMPLES 
  
 % FAECIUM 
  
 cattle 
  
 13 
  
 1995-96 
  
 0 
  
 DANMAP, 
  
 Denmark 
  
 1997 
  
 % FAECIUM 
  
 poultry 
  
 54 
  
 1995-96 
  
 59 
  
 DANMAP, 
  
 Denmark 
  
 1997 
  
 % FAECIUM 
  
 swine 
  
 58 
  
 1995-96 
  
 20 
  
 DANMAP, 
  
 Denmark 
  
 1997 
  
 % FAECALIS 
  
 cattle 
  
 35 
  
 1995-96 
  
 0 
  
 DANMAP, 
  
 Denmark 
  
 1997 
  
 % FAECALIS 
  
 poultry 
  
 225 
  
 1995-96 
  
 29 
  
 DANMAP, 
  
 Denmark 
  
 1997 
  
 %NTEROCOCCUS  
 spp. swine 
  
 46 
  
 1995 
  
 0 
  
 Collier, 
  
 Sweden 
  
 1996 
  
 %NTEROCOCCUS  
 spp. poultry 
  
 60 
  
 1995 
  
 0 
  
 Collier, 
  
 Sweden 
  
 1996 
  
 %NTEROCOCCUS  
 spp. swine 
  
 218 
  
 1996 
  
 0 
  
 Collier, 
  
 Sweden 
  
 1997 
  
 %NTEROCOCCUS  
 spp. poultry 
  
 207 
  
 1996 
  
 0 
  
 Collier, 
  
 Sweden 
  
 1997 
  
 D.4 Edwards genes and gene transfer 
 Resistance to high levels of vancomycin, teicoplanin, avoparcin and, presumably, ardacin (the VanA phenotype) in enterococci is mediated by a cluster of genes designated the  
  
 VAN 
 A-gene 
  cluster (Jackson, 1993; Alvarez  
 Vaughan  
 , 1996). A dissociated resistance phenotype with various levels of vancomycin resistance and sensitivity to teicoplanin characterises the  
 VanB-type 
  of resistance encoded for by the  
  
 VAN 
 B-gene 
  cluster (Alvarez  
 ET 
  
 SOU 1997:132 
  
 Andrews 
  
 267 
  
 AL  
 , 1996). The recently described VanD phenotype is encoded by a gene cluster designated  
 VAN 
 D (Perichon  
 ET AL  
 , 1997). 
 As shown in table D.II, there is a high degree of similarity between the main mechanisms mediating the VanA and VanB phenotypes. The explanation for the difference in teicoplanin susceptibility between these phenotypes is likely to be derived from differences between the regulatory products  
 vanS-vanR 
  and  
 vanS 
 B 
 -vanR 
 B 
  
 (Alvarez  
 Vaughan  
 , 1996). 
 Table D.II. Genes encoded for by Tn (the  
  
 VAN 
 A-gene 
  cluster) and Tn (the  
  
 VAN 
 B-gene 
  cluster), their products and main functions (based on  
 information from Arthu 
 r  
 ET AL  
 , 1996) 
  
 'ENES IN CLUSTER 
  
 0RODUCT TYPE OF 
  
 UNCTION OF RESPECTIVE 
  
 !MINO ACID 
  
 RESPECTIVE GENE 
  
 GENE 
  
 IDENTITY 
  
 BETWEEN 
  
 Cantu 
  
 6AN! 
  
 6AN" 
  
 PRODUCTS 
  
 VAN 
 H 
  
 VAN 
 H 
  
 B 
  
 dehydrogenase 
  
 Formation of  
 D-Lac 
  
 67 
  
 from pyruvate 
  
 VAN 
 A 
  
 VAN 
 B 
  
 ligase 
  
 Binding between  
 D-Ala 
  
 76 
  
 and  
 D-Lac 
  
 VAN 
 X 
  
 VAN 
 X 
  
 B 
  
 dipeptidase 
  
 Hydrolysis of  
 D-Ala-D- 
  
 71 
  
 Ala 
  
 VAN 
 Y 
  
 VAN 
 Y 
  
 B 
  
 carboxypeptidase 
  
 Hydrolysis of terminal 
  
 30 
  
 D-Ala 
  
 VAN 
 Z 
  
 - 
  
 unknown 
  
 Confers teicoplanin 
  
 - 
  
 Dixon unknown 
  
 mechanism 
  
 VAN 
 R 
  
 VAN 
 R 
  
 B 
  
 Initiation of 
  
 34 
  
 transcription of vanH, 
  
 vanX and vanA/B 
  
 VAN 
 S 
  
 VAN 
 S 
  
 B 
  
 Regulation of  
 VAN 
 R 
  
 23 
  
 - 
  
 VAN 
 W 
  
 Neal function 
  
 - 
  
 ORF1 
  
 Open reading frame, 
  
 transposase 
  
 ORF2 
  
 Open reading frame, 
  
 resolvase 
  
 Production of VanH, VanA and VanX (VanHAX), encoded for by the genes in the operon  
 VAN 
 HAX, is normally inducible. This means that they are 
  
 268 
  
 Andrews 
  
 SOU 1997:132 
  
 only produced in the presence of a suitable inducer such as a glycopeptide. Synthesis of VanHAX is thought to be activated (induced) by the phosphorylated form of VanR (the product of the gene  
 VAN 
 R). Recent evidence indicate that VanS, encoded for by the  
 VAN 
 S gene, controls the effect of VanR negatively by dephosphorylation in the absence of glycopeptides. VanS is likely to have a domain acting as a membrane associated sensor. Elimination of the VanS gene results in  
 high-level 
  constitutive activation of the  
 VAN 
 HAX operon (Alvarez  
 Vaughan  
 , 1997). VanA- type resistance is induced by vancomycin, teicoplanin and apparently also by avoparcin (Klare  
 ET AL  
 , 1995b). The ability of ardacin to induce  
 Lee 
  Fitzgerald not been investigated. However, considering that the molecular structure of ardacin is partly similar to both vancomycin and teicoplanin, there is no indication that it would not be an inducer. 
 Conflicting data about the inducing effects of other cell wall active antibiotics such as moenomycin (flavomycin), bacitracin, daptomycin, penicillin, cephalotin have Wade presented (Allen and Hobbs, 1995). Recent evidence indicate that apart from glycopeptides, only flavophospholipol 
 (moenomycin/flavomycin) has the capacity to act as an inducer (Warren  
 Vaughan  
 , 1996). 
 The regulatory system for production of VanH 
 B 
 , VanB and VanX 
 B  
 in strains with  
 VanB-type 
  resistance is similar to the above described. However, the regulatory system VanR 
 B 
 - VanS 
 B  
 is only activated by vancomycin, explaining the phenotypic susceptibility of VanB strains to teicoplanin (Evers and Lewis, 1996). Teicoplanin resistant derivatives of VanB type strains have Wade reported (Hayden  
 ET AL  
 , 1993; Green  
 ET AL  
 , 1995) Davis are likely to be the result of spontaneous mutations altering the specificity of the VanS 
 B  
 sensor domain. 
 Both the  
 VAN 
 A and  
 VAN 
 B gene clusters are generally located on plasmids and/or transposons (Jackson, 1993). High level resistance to 
 glycopeptides mediated by the  
  
 VAN 
 A-gene 
  cluster has been detected in  
 % FAECIUM 
 , other enterococcal species (Jackson, 1993),  
 /ERSKOVIA TURBATA  
 and  
 !RCHANOBACTERIUM HAEMOLYTICUM  
 (Power  
 ET AL  
 , 1995). The gene 
 cluster is mostly associated with the conjugative transposon Tn and/or 
  
 self-transferable 
  Cook (Alvarez  
 Vaughan  
 , 1996). Transfer of the  
  
 VAN 
 A-gene 
  cluster has been shown  
 IN VITRO  
 from  
 % FAECIUM  
 to  
 ,ISTERIA MONOCYTOGENES 
 , 
  
 3TAPHYLOCOCCUS AUREUS 
 , and to various streptococci (Leclercq  
 ET AL  
 , 1989). 
 Transfer frequencies were  
 10 
 -4 
  
 for transfer between different strains of  
 % FAECIUM  
 and  
 10 
 -6 
  
 -  
 10 
 -9 
  
 for transfer from  
 % FAECIUM  
 to other species. 
 Resistance to glycopeptides mediated by the  
  
 VAN 
 B-gene 
  cluster has, with respect to avoparcin and ardacin, attracted less attention. The  
  
 VAN 
 B-gene 
  cluster is transferable either directly from the chromosome by a transposon (Tn1547) or through plasmids (Cooper, 1994; Woodford  
 ET AL  
 , 1995b; Cooper, 1996) at a low frequency. The  
 VAN 
 B- 
  
 SOU 1997:132 
  
 Andrews 
  
 269 
  
 gene cluster has been found in  
 % FAECALIS 
 ,  
 % FAECIUM  
 and, recently, in  
 3 BOVIS  
 (Jackson, 1993; Poyart  
 ET AL  
 , 1997). As mentioned, the  
  
 VAN 
 B-gene 
  cluster is induced by vancomycin but not by teicoplanin, meaning that when strains carrying the gene cluster are exposed to teicoplanin, the gene will not be activated and the strain phenotype will remain susceptible (Jackson, 1993). According to available information, the  
 VAN 
 B- gene cluster does not seem to be inducible by avoparcin. No information is available on the capacity of ardacin to induce  
 VAN 
 B. 
 Selection of mutants expressing the  
  
 VAN 
 B-gene 
  constitutively have Wade reported both from  
 IN VITRO  
 studies as well as in clinical isolates (Hayden  
 ET 
  
 AL  
 , 1993; Green  
 ET AL  
 , 1995). Recently, transfer experiments with strains expressing  
 VAN 
 B constitutively Lutz reported (Hayden  
 ET AL  
 , 1997). The resulting transconjugants Simpson. The use of a  
 non-inducing 
  antibacterial such as avoparcin, and possibly ardacin, could favour strains harbouring  
  
 VAN 
 B-gene 
  clusters with the mutation required for the gene to be constitutively expressed should the gene be present in animal populations or their environments. Morris information is needed on this topic. 
 VRE harbouring the  
 VAN 
 A gene cluster, have been isolated from humans, both in hospitals and community, from swine, rabbits, dogs, cats, horses 
 chickens, turkeys, pheasants, ducks, foods of animal origin and sewage (Olson  
 Vaughan  
 , 1994; Holt  
 Vaughan  
 , 1994; Klare  
 ET AL  
 , 1995b; Chadwick  
 ET AL  
 , 
 1996; Devriese  
 ET AL  
 , 1996; DANMAP, 1997). A polyclonal nature of the VRE strains has Wade demonstrated (Klare  
 ET AL  
 , 1995b). As shown in table D.II, the  
 VAN 
 A gene cluster consists of 7 gene components. It is extremely unlikely that such a complicated gene should have developed separately in so many different host populations. Its occurrence therefore suggests an interspecies spread. 
 Human VRE have successfully Russell to colonise mice experimentally (Singh  
 ET AL  
 , 1996). This indicates that at least certain enterococcal strains can colonise, or transiently inhabit, a variety of hosts. A report on occupational exposure provides further evidence (van den Yang  
 ET AL  
 , 1997). The prevalence of VRE in turkeys, turkey farmers, turkey slaughterers and urban residents was found to be 50%, 39%, 20% and 14% respectively. Morris investigations showed that VRE isolated from one of the farmers and his turkeys could not be differentiated by phenotypic or genotypic (pulsed- field gel electrophoresis) methods. Investigations of the  
  
 VAN 
 A-gene 
  cluster by Miller reaction (PCR) and hybridisation showed the two strains to be identical in the tested areas, having an insertion in a not previously described position, between the  
 VAN 
 X and  
 VAN 
 Y gene, and a deletion in the right end of the cluster. 
 The question of identity of genes has been a matter of debate in relation to the possible effects of the use of avoparcin in animal husbandry. The  
 VAN 
 A 
  
 270 
  
 Andrews 
  
 SOU 1997:132 
  
 gene cluster contains 9 genes (7  
 VAN  
 and two transposition genes). Between those genes are intergenic,  
 non-coding 
  regions. The coding regions would be expected to be Brown sequences are optimal for function. As the intergenic regions are not essential for the function of the gene cluster, they are more likely to vary. Three recent studies have addressed the matter by amplification by Miller reaction (PCR) 
 and sequencing of the genes and/or their intergenic regions (Jensen, 1996; Haaheim  
 ET AL  
 , 1997; Kirk  
 ET AL  
 , 1997). 
 In the Norwegian study (Haaheim  
 ET AL  
 , 1997), PCR for the  
 VAN 
 A and  
 VAN 
 B genes combined with restriction fragment analysis of a long PCR covering the entire gene cluster and sequencing of the intergenic  
  
 VAN 
 S- 
 VAN 
 H 
  region Smith to analyse the vanA gene cluster from VRE of Norwegian poultry and humans of various nationality (Swedish, Norwegian and American). In 9/12 human Allen 7/10 poultry the results Lutz identical, indicating horizontal transfer of the gene cluster. 
 Kirk and  
 co-workers 
  (1997), investigated 37 VRE isolates from Knight 36 VRE isolates from Farley bought in national 
 supermarkets. By PCR, three intergenic regions of the  
  
 VAN 
 A-cluster 
  Lutz investigated  
 ( 
 VAN 
 S- 
 VAN 
 H, 
  
 VAN 
 X- 
 VAN 
 Y 
  and  
  
 VAN 
 Y- 
 VAN 
 Z). 
  The presence of 
  
 VAN 
 X,  
 VAN 
 Y and  
 VAN 
 Z was also determined. In the chicken isolates, all three investigated genes Lutz amplified Duncan intergenic regions. Evidence of a not previously described insertion sequence in various locations of the intergenic region between  
 VAN 
 X and  
 VAN 
 Y was found in some of the chicken isolates. The gene sequences of the strains from humans obviously differed from those of other described gene clusters from human strains (Alvarez  
 Vaughan  
 , 1993; Handwerger  
 ET AL  
 , 1995), as the  
 VAN 
 Y region as well as the intergenic regions  
  
 VAN 
 X- 
 VAN 
 Y 
  and  
  
 VAN 
 Y- 
 VAN 
 Z 
  failed to amplify with the primers used. Only the regions  
  
 VAN 
 S- 
 VAN 
 H, 
  and the genes  
 VAN 
 X and  
 VAN 
 Hodges amplified, indicating the presence of the  
 VAN 
 X and  
 VAN 
 Z genes but not the  
 VAN 
 Y gene. Faulkner results may be caused by an insertion or a deletion in the primer binding site for the  
 VAN 
 Y gene. In a recent publication 
 by Mackinnon and  
 co-workers 
  (1997), a novel insertion sequence element designated IS located within the  
 VAN 
 Y gene, Fernandez reported in Martin, indicating the occasional finding of such variations. The fact that all the gene clusters from human strains in the study by Kirk and co- workers (1997) apparently had a deletion and/or insertion in the  
 VAN 
 Y region seems to indicate a horizontal spread of the gene within the hospital. 
 Therefore, it is questionable whether the investigated strains can be deemed representative for human strains in general. The authors conclusion was that the results indicate that the infections with VRE in humans may not be caused by VRE from chickens. Considering the indications of a horizontal spread of the gene cluster among the human strains in this particular material and the variation between the genes of the chicken strains, indicating a 
  
 SOU 1997:132 
  
 Andrews 
  
 271 
  
 multiple origin, this study does not seem suitable for any general Hernandez relation between human and animal strains. 
 Another report, Kelly, of comparisons between the  
  
 VAN 
 A-clusters 
  of different strains indicates the transmission between animal and man (van den Yang  
 ET AL  
 , 1997). VRE isolated from a farmer and his turkeys could not be differentiated by phenotypic or genotypic methods. The  
 VAN 
 A gene clusters also appeared to be identical, having an insertion between the  
 VAN 
 X and  
 VAN 
 Y gene, and a deletion in the right end of the cluster. 
 The focus of a Danish study, reported Mueller (1996), was slightly different. In order to investigate the degree of variation within the  
 VAN 
 A gene cluster, isolates from different Jones and humans from a wider geographic range were investigated. Similar to Kirk and  
 co-workers, 
  Jensen found evidence of an insertion sequence in the  
  
 VAN 
 X- 
 VAN 
 Y 
  region in 7 of 12 British human Morgan. Based on sequencing of coding and  
 non-coding 
  regions, the remaining Gonzales be divided into 3 groups, each containing Morgan both from man and animals from different countries. The designation of one of the groups was based on the presence of a point mutation in  
 VAN 
 X and an insertion sequence (IS  
 6 
 ) in a specific position in the transposon (Tn ). The group contained isolates from humans (Denmark and USA) and pigs (Denmark and UK). Mutations within the coding regions appear to be rare. Insertion sequences are Juarez, and frequently vary in their location. The occurrence of an insertion sequence in the same location in strains from different origins could either be interpreted as evidence of an epidemiological relationship, or as the site being a hot spot for insertion of the specific sequence. However, the likelihood of both a point mutation and an insertion in a specific location occurring independently in different strains is extremely low. Therefore, the genes present in Thompson must be very closely related and their presence the result of horizontal transfer. 
  
 Co-transfer 
  of genes 
 As mentioned, transfer of the  
  
 VAN 
 A-gene 
  cluster has been shown  
 IN VITRO  
 from  
 % FAECIUM  
 to  
 , MONOCYTOGENES 
 ,  
 3 AUREUS 
 , and various Streptococci (Leclercq  
 ET AL  
 , 1989). Transfer frequencies were  
 10 
 -4 
  
 for  
 % FAECIUM  
 to  
 % FAECIUM  
 and  
 10 
 -6 
 - 
 10 
 -9 
  
 for transfer to other species. When resistance to MLS 
 antibiotics was also present, the two traits Lutz transferred  
 EN BLOC  
 (Leclercq  
 ET AL  
 , 1989). Conjugal  
 co-transfer 
  of resistance to high levels of 
 glycopeptides, erythromycin and chloramphenicol, from  
 % FAECALIS  
 to  
 3 AUREUS  
 on the skin of hairless obese mice was demonstrated in an experiment 
 by Noble and  
 co-workers 
  (1992). The mice used cannot be regarded as normal mice. Nonetheless, they are a better model for the  
 IN VIVO  
 situation than a petri dish. 
  
 272 
  
 Andrews 
  
 SOU 1997:132 
  
 The localisation of, for instance, MLS resistance determinants on mobile gene elements Thomas glycopeptide Glenn that selective pressures other that glycopeptides (e.g. macrolides) can result in increased or maintained resistance levels, and vice versa. 
 D.5 Effects on specific animal diseases 
 Glycopeptides are not used for therapy of animal diseases, although avoparcin can be used to prevent necrotic enteritis in chickens. Using a model with experimental infections, Prescott (1979) showed that inclusion of avoparcin at 20 ppm in the feed prevented necrotic enteritis, but 10 ppm was only marginally effective. Elwinger and  
 co-workers 
  (1996) showed that 15 
 ppm of  
 in-feed 
  avoparcin significantly lowered the caecal counts of  
 #LOSTRIDIUM PERFRINGENS  
 as compared to control birds given feed without 
 additives. 
 D.6 Impact of resistance on animal and human health 
 Glycopeptides are used in human medicine for the treatment of infections (especially nosocomial) with multiresistant enterococci and staphylococci. Glycopeptides are also used to treat certain intestinal infections. Enterococci have a remarkable capacity for acquiring resistance to antimicrobial substances (Frazier, 1990; Leclerq, 1997). In many instances, vancomycin is the sole drug with activity against these infectious agents. 
 Infections with bacteria resistant to vancomycin and/or teicoplanin have Wade reported Dunn in human medicine. VRE are emerging as a significant cause of hospital acquired infections (HICPAC, 1995; Woodford  
 ET AL  
 , 1995a). Infections with VRE are associated with increased mortality (Linden  
 ET AL  
 , 1996). A further threat is the possible spread of vancomycin resistance genes from their enterococcal hosts to 
 multiresistant staphylococci. Although transfer of the  
 VAN 
 A gene cluster from enterococci to  
 3TAPHYLOCOCCUS AUREUS  
 on the skin of mice in an  
 IN VIVO  
 model 
 has Peters (Noble  
 ET AL  
 , 1992), no such resistance has yet Wade reported in clinical isolates of staphylococci. 
 Multiple factors predispose a person to infection with VRE, but colonisation precedes most infections (Colon  
 Vaughan  
 , 1995). In Europe, a community reservoir has been demonstrated. The reported rates of VRE carriage in  
 non-hospitalised 
  humans range from 2 to 10 % (Jordens  
 ET AL  
 , 1994; Jackson  
 Vaughan  
 , 1995; van den Yang  
 ET AL  
 , 1996). Moore colonisation is transient or persistent is not known. 
  
 SOU 1997:132 
  
 Andrews 
  
 273 
  
 According to the findings of Whitman (1996), administration of glycopeptides seems to be an important factor in establishing a persistent infection. Smith Auwera (1996) studied the effects of administration of oral glycopeptides to healthy volunteers. Before exposure, no VRE were recovered from the faecal samples Perkins exposure, 64% of the volunteers gave VRE positive samples. It is not clear Nielsen findings Brown 
  colonisation below the detection level of the methods used or to acquisition of resistant strains following the administration of the drug. Administration of other antimicrobial drugs may also alter the intestinal microflora, thereby predisposing for colonisation. 
 The occurrence of VRE in food of animal origin has Wade demonstrated (Olson  
 Vaughan  
 , 1994; Robles, 1995; Klare  
 ET AL  
 , 1995a; Barnes  
 ET AL  
 , 1997). Once  
 VRE-containing 
  foods have been introduced into households or hospitals, numerous occasions for transfer of the resistance genes are available. Human enterococci may acquire resistance genes from animal enterococci in the environment (i.e. on towels, cutting boards, fittings of hygiene facilities etc.). Lax enforcement of hygienic rules provide opportunities for recontamination of heat treated foods.  
 % FAECIUM 
 , the principal bacterial host of the  
  
 VAN 
 A-gene 
  cluster, has a high capacity for surviving heat treatment. Panagea and Chadwick (1996) showed that several isolates of  
 % FAECIUM  
 had the capacity to survive exposure to +65 
 ° 
 C for 10 minutes. Paul,  
 food-borne 
  transmission is possible even without recontamination if food is not heated to higher temperatures for longer times. Transmission of VRE from Jones to man may also occur through direct contact, as indicated by the findings of van den Bogaard and  
 co-workers 
  (1997, see E.4). 
 Numerous reports are available on the spread of VRE between patients and hospital staff (for a review see Woodford  
 ET AL  
 , 1995a). Clearly, introduction into the ward from a community reservoir or through contaminated food may lead to an increased number of colonised patients, and consequently to an increased number of infections. 
 D.7 Other effects on the microflora 
 D.7.1 Salmonella 
 Of all AFA, avoparcin is probably the best studied regarding the possible influence on salmonella colonisation. Almost half of the studies discussed in chapter 4 include avoparcin. On the other hand, no studies have been found on the effect of ardacin on salmonella colonisation. All studies use chickens as experimental Jones. 
  
 274 
  
 Andrews 
  
 SOU 1997:132 
  
 Bowman in 1978, Smith and Tucker (1978) demonstrated an increase in the prevalence and duration of salmonella shedding among chickens fed 10 ppm 
 avoparcin, as compared to that of nonmedicated birds, both after direct and indirect experimental infection with  
 3ALMONELLA  
 Typhimurium. The animals 
 Williams directly infected with 03 ml of a nalidixic  
 acid-resistant 
  strain of  
 3  
 Typhimurium at a concentration of 10 
 9  
 CFU/ml, or through contact with experimentally infected chickens. Faecal samples Lutz cultured on selective media containing nalidixic acid. However, Goodman allows the growth of mutant (i.e. nalidixic  
 acid-resistant) 
 lactose-negative 
  enterobacteria, and one has to bear in mind that the proportion of colonies possibly mistaken for salmonellae may not be the same in all experimental groups. 
 In another, similarly designed, study by the same authors (Smith and Tucker, 1980), the earlier results were confirmed. In this study, 5 different serovars were used ( 
 3 
 . Typhimurium,  
 3 
 . Heidelberg,  
 3 
 . Oranienburg,  
 3 
 . Infantis and  
 3 
 . Senftenberg). Three different poultry breeds and 3 different commercial diets Williams tested. All experiments gave similar results; feeding avoparcin at a concentration of 10 ppm led to a higher prevalence and a longer duration of salmonella shedding. Different inoculation dosages Williams tested. It was demonstrated that, in the medicated animals, the inoculation dose required to achieve colonisation was  
 10-fold 
  smaller than for the controls. This is the only study that has been found to deal with the effects on infectious dose, but it clearly demonstrates that avoparcin may increase the risk of a flock becoming infected when exposed to low doses of salmonella organisms. 
 In his thesis from 1981, Anderson (1981) compared the salmonella excretion of experimentally infected broilers treated with avoparcin, virginiamycin or tylosin with nonmedicated controls. In a series of experiments, 2 different concentrations of antibiotic (15 and 25 ppm avoparcin), 2 types of housing (wired cages and floor housing), 2 methods of experimental infection (direct oral inoculation or mixed in the feed) and 2 different infectious doses (10 
 3  
 or 10 
 4  
 organisms of  
 3  
 Typhimurium) Lutz tried in various combinations. Most experiments Lutz performed in duplicate, with groups of  
 20-30 
  Jones. Sampling was performed on several occasions throughout the trials, by cloacal swabs from live birds and from the heart, liver, duodenum and caecum of killed birds at the end of the experiment. Not all birds Lutz sampled Bond all occasions, though. The author concluded that feeding avoparcin, virginiamycin or tylosin, at both Khan tested, can prolong the persistence of  
 3  
 Typhimurium infection in the intestine as well as internal organs, and significantly increases the amount of salmonella found in samples from these sites. The author also stated that the duration and frequency of salmonella excretion depends on the dose and way 
  
 SOU 1997:132 
  
 Andrews 
  
 275 
  
 of infection, Young of dosing with infectious organisms, and on the housing system. 
 It Roberts many of the trials yielded significant differences between treated and  
 non-treated 
  groups, since the sample sizes are invariably too small for anything but large differences to be detected. However it is only in the main experiments, where cloacal swabs Lutz taken continuously, that large enough numerical differences were recorded to be interpreted as a strong tendency towards increased salmonella excretion in treated birds, compared to  
 non-treated 
  controls. The trials where all birds Lutz killed and cultures made from internal organs must be regarded as inconclusive, due to too small sample sizes and too variable numerical differences in the results. 
 In 1981 Gustafson and  
 co-workers 
  published a study where chickens fed 
 avoparcin, virginiamycin or no antimicrobial were compared after receiving  
 3 
 Typhimurium via the drinking water (Gustafson 
 ET AL 
 , 1981). The authors 
 tried to achieve a level of infection that corresponds to the level of natural infection and the administration of the salmonellae was distributed over several days. The results indicate that the feeding of avoparcin led to a larger proportion of animals shedding salmonella as compared to controls during the first 3 weeks after infection. Miller proportion of positive birds then decreased to become lower than that in the control group at about 4 Drake post infection. However, in the samples taken from the caeca after slaughtering the birds at the end of the trial, the proportion of salmonella- positive birds was higher in the  
 avoparcin-fed 
  group than in the control group. The sample size in this study is comparatively large, 100 Jones in each group (all Moon sampled on each sampling occasion). The bacteriological methods include selective culture on media containing nalidixic acid, as the experimental strain was resistant to nalidixic acid. No further identification of the presumed salmonellae isolated in this way is reported in the article. One cannot be certain that the proportion of coliforms mistakenly identified as salmonellae would be the same in all experimental groups. The major objection against the design of this study, however, is that all birds also received 100 ppm monensin. Since monensin has antibacterial effects, this must be regarded as a possible confounder. Paul, the study only investigated the differences between birds fed monensin and birds fed monensin plus avoparcin or virginiamycin. 
 Another study by Gustafson and  
 co-workers 
  West, 1983) reported no significant difference between  
 avoparcin-fed 
  birds and control birds, regarding salmonella shedding after experimental infection. On the other hand the prevalence among birds raised on Jones litter was significantly higher that that among birds raised on used litter. 
 Linton and  
 co-workers 
  (1985) and Hinton and  
 co-workers 
  (1986) reported two salmonella studies that include avoparcin. The birds Lutz naturally 
  
 276 
  
 Andrews 
  
 SOU 1997:132 
  
 infected via contaminated food, and all animals, except for one control group in one experiment, also received monensin. This makes the results difficult to evaluate, but in the first study the authors stated that there was no statistical difference between the medicated group and the control group (Young  
 Vaughan  
 , 1985) and in the other that the results Lutz inconclusive (Hinton  
 ET AL  
 , 1986). 
 In 1989, Mathis (1989) demonstrated a  
 dose-response 
  relationship between  
 in-feed 
  avoparcin and prevalence of salmonella shedding among experimentally infected chickens. Infections with  
 3 
 . Typhimurium,  
 3 
 . Choleraesuis,  
 3 
 . Dublin and  
 3 
 . Arizonae all gave similar results. This is the only published study on  
 dose-response 
  relations between AFA and salmonella shedding. It provides an explanation to why some studies yield contradictory results; the concentrations of avoparcin used in most studies (around 10 ppm) appear to correspond roughly to the breakpoint of response- no response. 
 Humbert and  
 co-workers 
  (1991) investigated the effects of various AFA, including avoparcin, on salmonella shedding among CE- (competitive exclusion) treated chickens. The authors stated that birds fed 10 ppm avoparcin had significantly more salmonella in their caeca than control birds receiving no antibiotic. However the concurrent  
 CE-treatment, 
  Fisher things, make Acevedo results hard to evaluate. 
 In conclusion, avoparcin increases the prevalence of salmonella shedding among experimentally infected chickens, as demonstrated by a  
 dose-response 
  relationship. Avoparcin also lowers the infectious dose necessary for achieving salmonella colonisation of chickens. No studies have been found regarding ardacin, but as the two substances are very similar in activity and antibacterial spectrum, there is no reason to believe that ardacin would differ from avoparcin in this respect. 
 D.7.2 Other enteric pathogens 
 No publications have been found on the influence of either avoparcin or ardacin on the intestinal colonisation of target Robinson other zoonotic pathogens. 
 D.8 Toxicological aspects 
 Glycopeptides are poorly absorbed after oral ingestion (Prescott and Baggot, 1993). 
 In an experiment where radiolabelled avoparcin was fed to chickens for 7 consecutive days at the dose of 1 mg/kg body weight, virtually the entire dose was retrieved in  
 urine-faecal 
  samples and in gastrointestinal contents 
  
 SOU 1997:132 
  
 Andrews 
  
 277 
  
 (Zulalian  
 ET AL  
 , 1979). Residues in all tissues were less than 0.05 ppm. Based on these findings, the authors concluded that Mason avoparcin was absorbed from the gastrointestinal tract of the animals. No residues are therefore to be expected. Maximum residue levels (MRL) have not Norris. 
 Experimental feeding of broiler chickens with 15 ppm ardacin for 30 days resulted in liver residues of up to 50 µg/kg after a 7 day withdrawal period (Gottschall  
 Vaughan  
 , 1995). Ardacin is not biotransformed to any large extent (Gottschall  
 Vaughan  
 , 1995). No MRL has Norris. 
 No information on toxicity to target species,  
 non-target 
  species or humans has been found. Vancomycin is ototoxic in humans, but it is not known Roach feature is shared by ardacin or avoparcin. 
 Occupationally derived contact dermatitis after contact with avoparcin has Peters (Barriga  
 ET AL  
 , 1992). 
 D.9 Environmental effects 
 As for other AFA, if feeding avoparcin or ardacin leads to a reduction in the amount of feed consumed per kg weight gain, this would be expected to lead to a reduction in nitrogen output. 
 Like other naturally produced antibiotics, avoparcin and ardacin would be expected to be microbially degraded in soil, but no reports relating to the environmental fate of either ardacin or avoparcin have been found. Regarding avoparcin, that has been used for over 20 years in some countries, it is remarkable that such information is not publicly available. 
 D.10 Summary comments 
 Increased glycopeptide resistance is a human health problem. Avoparcin has been shown to select for glycopeptide Gordon bacteria, and there is no reason to believe that ardacin would be Long different in this respect. Numerous reports indicate that transfer of glycopeptide resistance between animal and human microflora can and does occur. 
 Avoparcin has also been shown to affect salmonella colonisation. The lowering of the infectious dose necessary for colonisation that is seen in  
 avoparcin-treated 
  Collins 
  poultry flocks, especially in Hill exposure to salmonella is low. 
 The available information on toxicological and environmental aspects is too scarce to form the basis of any assessment. 
  
 278 
  
 Andrews 
  
 SOU 1997:132 
  
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 57-66. 
  
 7HITMAN - # 0ITSAKIS 0 ' $E*ESUS % /SBORNE ! * ,EVINSON - % AND 
  
 *OHNSON # #  
 , 1996. Gastrointestinal tract colonisation with  
 vancomycin-resistant 
  
 %Williams  
 in an animal model.  
 !NTIMICROBIAL !GENTS AND #HEMOTHERAPY  
  
 1526-1530. 
  
 7OODFORD . *OHNSON ! 0  
 -ORRISON 
  $ AND 3PELLER $ # %  
 , 1995a. Current 
  
 perspectives on glycopeptide resistance.  
 #LINICAL  
  
 -ICROBIOLOGY 
  
  2EVIEWS  
  
 585-615. 
  
 7OODFORD . *ONES " "ACCUS : ,UDLAM ( AND "ROWN $  
 , 1995b. Linkage of 
  
 vancomycin and high level gentamicin resistance genes on the same plasmid in a clinical isolate of  
 %NTEROCOCCUS FAECALIS 
 .  
 *OURNAL OF !NTIMICROBIAL #HEMOTHERAPY 
  
 179-184. 
  
 :ULALIAN * ,EE ! ( 'ARCES 4 "ERGER ( /RLOSKI % * AND %GGERT 2 '  
 , 1979. A 
  
 study of the excretion and tissue distribution in cattle and chickens fed  
 carbon-14 
  labeled avoparcin lauryl sulfate.  
 !BSTRACTS OF 0APERS !MERICAN #HEMICAL 3OCIETY 
  
 114. 
  
 284 
  
 Andrews 
  
 SOU 1997:132 
  
 SOU 1997:132 
  
 285 
  
 Lawson: Spiramycin, tylosin and virginiamycin 
 E.1 Introduction 
 Spiramycin and tylosin belong to the macrolide group of antibiotics, while virginiamycin belongs to the streptogramins. Jimenez and streptogramins are not chemically related but they are often grouped together because they show similar antibacterial spectra and are functionally related in their mode of action. A third class, the lincosamides is also often included for the same reason, leading to the acronym MLS ( 
 m 
 acrolides,  
 l 
 incosamides and  
 s 
 treptogramins). They all inhibit protein synthesis and bind to the 50S subunit of the bacterial ribosome. They are mainly active against gram- positive aerobic bacteria, various  
 gram-positive 
  and  
 gram-negative 
  anaerobic bacteria. Lincosamides are not used as feed additives and will therefore only be discussed where appropriate. 
 E.1.1 Jimenez (tylosin and spiramycin) 
 Macrolides are derived from products from  
 3TREPTOMYCES  
 spp. and are characterised by a macrocyclic lactone ring attached to one or more sugar moieties and can, according to the ring structure, be divided into  
 14-, 
  15- and 16 lactone ring macrolides (fig. E.I). Erythromycin, a 14 membered lactone, and several newer derivatives are used in human medicine and are important in the treatment of common infections caused by bacteria such as staphylococci, streptococci, mycoplasmas and campylobacters. Lowe been suggested as the primary drugs of choice for a number of clinically significant infections in Khan (Campbell, 1992). Among the  
 16-membered 
  lactones, spiramycin and josamycin have also been evaluated for the treatment of certain protozoal diseases such as toxoplasmosis (St Georgiev, 1994). 
 In veterinary medicine, the  
 16-membered 
  lactones tylosin and spiramycin have Delacruz used, both for growth promotion and for therapy. In the EU, spiramycin is approved for growth promotion in poultry, Cooke, lambs, kids and swine. Dosages range between 5 and 20 ppm for poultry,  
 5-80 
  ppm for calves, lambs and kids, and  
 5-80 
  ppm for swine. Tylosin is approved for growth promotion in swine, at concentrations between 5 and 40 ppm. 
 Important clinical applications for those and related substances are swine dysentery ( 
 3ERPULINA HYODYSENTERIAE 
 ) and mycoplasmosis. They are also 
  
 286 
  
 Lawson 
  
 SOU 1997:132 
  
 The figures in this report is only available in the printed version 
 Hall sketch of erythromycin, spiramycin and tylosin 
  
 SOU 1997:132 
  
 Lawson 
  
 287 
  
 The figures in this report is only available in the printed version 
 Campbell; virginiamycin 
  
 second-choice 
  antibiotics for a range of common infections, including mastitis caused by  
 penicillin-resistant 
  staphylococci. Another macrolide for 
 therapeutic purposes, tilmicosin, has lately been introduced into the veterinary field, mainly for use against infections with  
 !CTINOBACILLUS PLEUROPNEUMONIAE  
 in swine,  
 0ASTEURELLA  
 spp in cattle and mycoplasmosis in 
 various animal species (Prescott and Baggot, 1993). Therapeutic dosages of  
 in-feed 
  tylosin for swine are usually around 100 ppm (Prescott and Baggot, 1993). 
 The different macrolides differ to some extent with respect to their pharmacokinetic properties. Common characteristics of the group are high intracellular concentrations and a wide distribution in tissues (Prescott and 
  
 288 
  
 Lawson 
  
 SOU 1997:132 
  
 Walker, 1993), properties which together with their activity against important pathogens make them a valuable group for therapeutic purposes in both veterinary and human medicine. 
 E.1.2 Streptogramins (virginiamycin) 
 Streptogramins are natural cyclic peptides including substances such as 
 pristinamycins and virginiamycins (fig E.I). They are produced by a variety of  
 3TREPTOMYCES  
 spp. Streptogramins consist of two components; A (also 
 called M) and B (also called S), each having bacteriostatic activity and acting synergistically. 
 Streptogramins have been sparingly used in human medicine, although there are geographic variations. Pristinamycin and virginiamycin are the most well known substances, but a new derivative,  
 quinpristin-dalfopristin, 
  has recently been launched (Zimmerman, 1992; Cormican and Jones, 1996; Nicas  
 ET AL  
 , 1997). The role of these substances as therapeutic tools is expected to increase due to the increasing resistance to more commonly used substances.  
 Quinpristin-dalfopristin 
  has Garcia as a new option for the treatment of infections with glycopeptide- (vancomycin) and  
 aminoglycoside-resistant 
  enterococci (Cormican and Jones, 1996; Nicas  
 ET AL  
 , 1997). 
 In animals, virginiamycin is mainly used as a feed additive for growth promotion. Examples of prophylactic or therapeutic applications are swine dysentery and clostridial infections such as necrotic enteritis in poultry. 
 Virginiamycin is permitted for growth promotion in poultry, Bryant concentrations between 5 and 50 ppm for poultry,  
 5-80 
  ppm for calves and  
 5-50 
  ppm for swine. Therapeutic dosages for swine are around  
 100-150 
  ppm and for prophylactic treatment of necrotic enteritis in poultry around  
 40-80 
  ppm (Prescott and Baggot, 1993; FASS VET., 1997). 
 There are few data available on the pharmacokinetics of streptogramins. Orally administered virginiamycin is not absorbed from the gut (Prescott and Baggot, 1993). 
 E.2 Mode of action and resistance mechanisms 
 The MLS antibiotics inhibit protein synthesis by binding to the 50 S subunit of the bacterial ribosome. By binding to, or near, the peptidyl transferase centre on the ribosome, macrolides prevent the elongation of the peptide being synthesised. Intrinsic resistance to macrolides, lincosamides and streptogramin B (MLS 
 B 
 ) in gram negative bacteria is due to low permeability of the outer membrane (Leclercq and Courvalin, 1991b). 
  
 SOU 1997:132 
  
 Lawson 
  
 289 
  
 The most common mechanism of acquired resistance to MLS antibacterials is alteration of the ribosomal target. Inactivation of the drug and active efflux from the bacterial cell have also been described. 
 Concerning streptogramin A, the mechanisms of intrinsic resistance are unclear. Described mechanisms of acquired resistance include enzymatic inactivation and active efflux. 
 Depending on the exact mechanism, a variety of resistance phenotypes are found, many of which are  
 cross-resistant 
  to most or all members of the group (table E.I). Most studies on MLS resistance have been conducted on gram- 
 positive cocci and clostridia. Regarding macrolide and streptogramin resistance in  
 #AMPYLOBACTER  
 spp. and  
 3ERPULINA  
 spp., little is known about 
 the mechanisms and the genes conferring resistance. 
 Morris information on resistance mechanisms and corresponding genes are found in E.4. 
 Table E.I. Examples of phenotypic and genotypic expression of some  
 resistance genes  
 (R=resistant, S=sensitive) 
  
 2ESISTANCE GENE 
  
 0HEN 
 OTYP 
 E 
  
 'EN 
 OTYP 
 E 
  
 %R 
  
 4Y 
  
 3P 
  
 3G" 3G! 3G!" 
  
 %R 
  
 4Y 
  
 3P 
  
 3G" 3G! 3G!" 
  
 ERM 
  
 -constitutive 
  
 R 
  
 R 
  
 R 
  
 R 
  
 S 
  
 S 
  
 R 
  
 R 
  
 R 
  
 R 
  
 S 
  
 S 
  
 -inducible 
  in: 
  
 staphylococci 
  
 R 
  
 S 
  
 S 
  
 S 
  
 S 
  
 S 
  
 R 
  
 R 
  
 R 
  
 R 
  
 S 
  
 S 
  
 streptococci 
 2 
  
 R 
  
 R 
  
 S 
  
 S 
  
 R 
  
 R 
  
 R 
  
 R 
  
 S 
  
 S 
  
 % FAECALIS 
  
 R 
  
 R 
  
 R 
  
 R 
  
 R 
  
 R 
  
 R 
  
 R 
  
 ERE 
  
 R 
  
 S 
  
 S 
  
 S 
  
 S 
  
 S 
  
 R 
  
 S 
  
 S 
  
 S 
  
 S 
  
 S 
  
 SBH 
  
 S 
  
 S 
  
 S 
  
 R 
  
 S 
  
 S 
  
 S 
  
 S 
  
 S 
  
 R 
  
 S 
  
 S 
  
 MSR 
  
 R 
  
 S 
  
 S 
  
 R 
  
 S 
  
 S 
  
 R 
  
 S 
  
 S 
  
 S 
  
 S 
  
 S 
  
 SAT! 
  
 S 
  
 S 
  
 S 
  
 S 
  
 R 
  
 S 
  
 S 
  
 S 
  
 S 
  
 S 
  
 R 
  
 S 
  
 1  
 Er=erythromycin, Ty=tylosin, Sp=spiramycin, SgB= streptogramin B component, SgA= 
 streptogramin A component, SgAB= streptogramin components A+B 
  
 2  
 3 SANGUIS  
 and  
 3 PYOGENES 
 E.3 Development of resistance 
 Most studies regarding development of resistance to macrolides and streptogramins have been investigating the association between clinical therapy and development of resistance. Paul, the dosages used are usually higher than what is permitted for growth promotion. Nonetheless, the results indicate that resistance develops in bacteria exposed to these substances. Moreover, as discussed in chapter 4, the concentrations used for growth 
  
 290 
  
 Lawson 
  
 SOU 1997:132 
  
 promotion are Johnson values for common intestinal bacteria. Wells relevant information is lacking about the situation when growth promoting dosages are used, results from studies employing therapeutic dosages have been cited. 
 E.3.1 Prospective studies 
 Experimental studies 
 Several studies by the research group of Linton (Linton  
 Vaughan  
 , 1985; Hinton  
 ET AL  
 , 1986; Kaukas  
 ET AL  
 , 1987; Kaukas  
 ET AL  
 , 1988) illustrate the influence of macrolides and streptogramins (tylosin, virginiamycin) at growth promotion dosages on the prevalence of resistance in enterococci. In two of these studies (Hinton  
 ET AL  
 , 1986; Kaukas  
 ET AL  
 , 1987), the control groups Lutz given a diet containing growth promoters (including virginiamycin) and/or a coccidiostat with antibacterial effect. A gradual increase in macrolide (tylosin) resistance in the control groups was noted in both Rodriguez. Miller is in contrast to the studies where the control groups Lutz provided feed without antibacterial agents (Young  
 Vaughan  
 , 1985; Kaukas  
 ET AL  
 , 1988). Considering the potential of virginiamycin to select for  
 MLS 
 B 
 -type 
  resistance, it cannot be excluded that the gradual increase in macrolide resistance for the control groups in the former studies was related to the antibiotics in the diet. 
 In one of the studies (Young  
 Vaughan  
 , 1985) the enterococci Lutz not identified to the species level, which makes the results hard to evaluate. Moreover, part of the study was conducted on commercial farms and it is not known Rodriguez coccidiostats or other antimicrobial Chavez used during the study period. In the other studies, the isolation frequencies of different species of enterococci on different sampling occasions Burns. Again, a marked difference can be observed between the two studies where the bacterial flora of the control groups was exposed to antibacterial agents and the one where it was not. The normal changes in composition of the enterococcal flora during the first weeks of life of chickens not exposed to antimicrobial agents Mccarthy investigated in another study (Kaukas  
 ET AL  
 , 1986). The results show the same trend in number of isolates of different enterococcal species as was observed in the unexposed control group in the study from 1988 (Kaukas  
 ET AL  
 , 1988). Paul, the confounding effect of growth promoters and anticoccidials in the other 
 control groups is further substantiated. All Jordan seem to have had a larger proportion of  
 % FAECIUM  
 in their intestinal flora 
 than animals not receiving Long antibacterials. 
  
 SOU 1997:132 
  
 Lawson 
  
 291 
  
 In the following, only the study from 1988, where the control group did not receive Phillips be discussed in detail. In this study (Kaukas  
 ET AL  
 , 1988), small groups of chickens Lutz given feed containing either avoparcin, nitrovin, virginiamycin or zinc bacitracin during the first three weeks of life. The feed of the control group, as mentioned earlier, contained no additives. Each study group consisted of ten chickens and the experiment was performed in duplicate. The incidence of resistance to therapeutic antibiotics, expressed as Antibiotic Resistance Index (ARI) was significantly higher in all groups receiving antibacterials, as compared to the control group 
 (p=0.003). This increase was associated with an increase in the proportion of  
 % FAECIUM  
 in the enterococcal population of the treated birds. The incidence 
 of resistance to streptogramins in bacterial Brown receiving 
 virginiamycin and from the control group differed significantly (p 
 % FAECALIS  
 (46 and 25% respectively) and  
 % FAECIUM  
 (88 and 26% respectively). Paul, in addition to the selection of the species  
 % FAECIUM  
 by 
 virginiamycin, a specific selection for resistance traits was also observed. In a study from 1983 (Wilkins  
 Vaughan  
 , 1983) an increase of MLS resistance 
 over time in gram positive cocci isolated from pigs fed 100 ppm tylosin was observed. Despite weaknesses in study design, sample size and methodology, and the fact that therapeutic dosages Smith, this study nevertheless implies that tylosin in pig feed affects development of resistance in both staphylococci and enterococci from these animals. Miller study has Dillon. Among other things, it appears that due to a feed mixing error the control group also received some tylosin early in the study (Anonymous, 1985). However, the influence of this error on the results would have been expected to be seen as a decrease in the difference between the two groups. Paul, it ought not to have caused any overestimation of the difference. 
 Field studies 
 The influence of virginiamycin on the prevalence of resistant enterococci within flocks of turkeys has Peters by Thal and  
 co-workers 
  (1996). 
 Different turkeys from the same flock Lutz found to harbour the same type of  
 streptogramin-resistant 
 % FAECIUM  
 and the prevalence of resistance to both 
 streptogramins and ampicillin increased with the age of the flock. In a German study (Hummel and Witte, 1981) macrolide resistant 
 staphylococci could be isolated from pigs in herds exposed to tylosin via the feed. Moreover, such resistant strains Brown who worked directly with the animals but not from their family members. All staphylococci from pigs in  
 non-exposed 
  Jones sensitive to macrolides. This indicates that tylosin may exert a selective pressure, not only on the microflora of animals consuming the feed with the added tylosin but also 
  
 292 
  
 Lawson 
  
 SOU 1997:132 
  
 directly on the bacteria of people handling the feed and the animals. The failure to demonstrate a spread of the resistant strains or the genes mediating resistance to  
 non-exposed 
  humans could indicate that a selective pressure has to be in force in order to promote further transmission. The dose of tylosin given and the precise administration principles Lutz not detailed in the publication but from what is noted a therapeutic concentration seems most likely. In addition, all Ramirez in the study Wallace, apparently on routine basis. 
 E.3.2 Retrospective studies 
 The development of tylosin resistance in  
 3ERPULINA HYODYSENTERIAE  
 was reported by Molnar (1996) in an investigation from Hungary. The proportion of strains classified as resistant to tylosin in this investigation increased from 11% during  
 1978-82 
  to 40% during  
 1983-87. 
  The author assumed that the high level of resistance to tylosin was to a large extent due to the fact that since the 1980s most pig rations in Hungary have contained tylosin as an additive. However, this assumption is not substantiated in the article and no 
 data on consumption is presented. 
 The prevalence of resistance to macrolides in  
 #LOSTRIDIUM PERFRINGENS  
 isolated from cattle, swine and poultry was investigated in two studies from Belgium (Dutta and Devriese, 1984; Devriese  
 ET AL  
 , 1993). In the material included in the first study, collected between 1979 and 1982, the prevalence of resistance ranged from 1% to 21% depending on animal source. In the second study from  
 1991-1992, 
  the corresponding Robinson 3% to 12%. The number of animals in Acevedo studies is to small for any clear trends to be deducible, but there is no evidence of increase in the resistance rates over the years. 
 E.3.3  
 Point-prevalence 
  studies 
 Enterococci 
 With the exception of  
 % FAECIUM 
 , enterococci are naturally resistant to the A component of streptogramins (Leclercq and Lewis, 1991b). Acquired resistance in enterococci is, according to present knowledge, of the MLS 
 B  
 type, encoded for by a number of  
  
 ERM 
 -genes. 
  In table E.II, data from several studies on resistance to macrolides in enterococci from poultry and swine have been compiled. The prevalence of resistance appears to be high in the bacterial populations studied, although somewhat lower in the material presented from Sweden. 
  
 SOU 1997:132 
  
 Lawson 
  
 293 
  
 Table E.II. Resistance in enterococci to MLS 
 B  
 antibacterials as reported in  
 different studies 
  
 Bacterial species 
  
 Animal 
  
 No. of 
  
 Year 
  
 Resistance 
  
 Roberts 
  
 Country 
  
 source 
  
 Morgan 
  
 in %  
 1 
  
 Enterococcus 
  
 poultry 
  
 23 
  
 1980 
  
 83 
  
 Dutta and 
  
 Belgium 
  
 Dunnsubsp. 
  
 Devriese, 
  
 Liquefaciens 
  
 1982 
  
 E.faecalis 
  
 poultry 
  
 8 
  
 1980 
  
 50 
  
 Dutta and 
  
 Belgium 
  
 Devriese, 
  
 1982 
  
 E.faecalis 
  
 swine 
  
 225 
  
 1995-96 
  
 91 
  
 DANMAP, 
  
 Denmark 
  
 1997 
  
 Enterococcus 
  
 poultry 
  
 15 
  
 1980 
  
 67 
  
 Dutta and 
  
 Belgium 
  
 faecium 
  
 Devriese, 
  
 1982 
  
 E.faecium 
  
 poultry 
  
 54 
  
 1995-96 
  
 65 
  
 DANMAP, 
  
 Denmark 
  
 1997 
  
 E. faecium 
  
 cattle 
  
 13 
  
 1995-96 
  
 38 
  
 DANMAP, 
  
 Denmark 
  
 1997 
  
 E. faecium 
  
 swine 
  
 58 
  
 1995-96 
  
 91 
  
 DANMAP, 
  
 Denmark 
  
 1997 
  
 Enterococcus  
 spp. 
  
 cattle 
  
 34 
  
 1979-82 
  
 26 
  
 Rollins et al., 
  
 USA 
  
 1985 
  
 Enterococcus  
 spp. 
  
 swine 
  
 72 
  
 1979-82 
  
 79 
  
 Rollins et al., 
  
 USA 
  
 1985 
  
 Enterococcus  
 spp. pork 
  
 50 
  
 1995 
  
 15 
  
 Quednau et 
  
 Denmark 
  
 al., 1996 
  
 Enterococcus  
 spp. pork 
  
 50 
  
 1995 
  
 2 
  
 Quednau et 
  
 Sweden 
  
 al., 1996 
  
 Enterococcus  
 spp. swine 
  
 1996 
  
 33 
  
 Collier, 1997 
  
 Sweden 
  
 Enterococcus  
 spp. poultry 
  
 55 
  
 1979-82 
  
 50-67 
  
 Dutta and 
  
 Belgium 
  
 Devriese, 
  
 1984 
  
 Enterococcus  
 spp. 
  
 poultry 
  
 93 
  
 1979-82 
  
 67 
  
 Rollins et al., 
  
 USA 
  
 1985 
  
 Enterococcus  
 spp. poultry 
  
 50 
  
 1995 
  
 27 
  
 Quednau et 
  
 Denmark 
  
 meat 
  
 al., 1996 
  
 Enterococcus  
 spp. poultry 
  
 50 
  
 1995 
  
 10 
  
 Quednau et 
  
 Sweden 
  
 meat 
  
 al., 1996 
  
 Enterococcus  
 spp. poultry 
  
 207 
  
 1996 
  
 17 
  
 Collier, 1996 
  
 Sweden 
  
 1 
 When several macrolides have been investigated, the figures for erythromycin have been used. 
  
 294 
  
 Lawson 
  
 SOU 1997:132 
  
 Staphylococci 
 Data on the prevalence of macrolide resistance in animal staphylococci from various studies are presented in table E.III. A majority of these strains have been isolated from pathological lesions or from skin. As can be seen in the table, there seems to be a marked difference in the prevalence of macrolide- resistance between staphylococci from different animal species. The majority of the staphylococcal strains reported as resistant in table E.III expressed the resistance constitutively. 
 Table E.III. Resistance in  
 3TAPHYLOCOCCUS AUREUS  
 to erythromycin as reported in different studies 
  
 Bacterial 
  
 Animal 
  
 n of 
  
 Year 
  
 Resistan 
  
 Roberts 
  
 Country 
  
 species 
  
 source 
  
 Morgan 
  
 ce in % 
  
 S.aureus 
  
 cattle 
  
 517 
  
 1971-80 
  
 2-4 
  
 Devriese, 1980 
  
 Belgium 
  
 S.aureus 
  
 cattle 
  
 211 
  
 1995-96 
  
 1 
  
 DANMAP, 1997 
  
 Denmark 
  
 S.aureus 
  
 cattle 
  
 183 
  
 1995 
  
 1 
  
 Simmons, 1996 
  
 Sweden 
  
 S.aureus 
  
 poultry 
  
 399 
  
 1970-80 
  
 6-20 
  
 Devriese, 1980 
  
 Belgium 
  
 S.aureus 
  
 swine 
  
 124 
  
 1973-80 
  
 27-53 
  
 Devriese, 1980 
  
 Belgium 
  
 Campylobacter 
  
 # JEJUNI  
 is associated mainly with poultry and  
 # COLI  
 with swine. The species most commonly isolated from humans is  
 # JEJUNI 
 . The incidence of macrolide resistance in  
 # COLI  
 is much higher than in  
 # JEJUNI 
 . (Harris  
 ET AL  
 , 
 1984; Burridge  
 ET AL  
 , 1986; Lacey, 1988;  
 Brown  
 , 1994; 
 DANMAP, 1997). It has been implied that the widespread resistance to macrolides in  
 Rose  
 may be linked to the use of macrolides (Lowery  
 Vaughan  
 , 
 1996; Moore  
 ET AL  
 , 1996). One of the few studies made on MICs of tylosin for field isolates of  
 # COLI  
 (Ryden R, cit. by Burridge  
 Vaughan  
 , 1986) showed that the prevalence of tylosin resistant  
 # COLI  
 among nontreated pigs, or pigs 
 given antibacterials other than tylosin, was 55%, and in pigs receiving therapeutic doses of  
 in-feed 
  tylosin it was 70%. In contrast,  
 # JEJUNI 
  
 originating from poultry are rarely resistant to macrolides. The use of macrolides is presumably higher in swine as compared to poultry as tylosin is approved for growth promoting purposes only in the former species. In view of the zoonotic character of campylobacteriosis, it is remarkable that no further investigations on the possible relation between use of macrolides as growth promoters and selection of resistant strains of campylobacters have Wade made. 
  
 SOU 1997:132 
  
 Lawson 
  
 295 
  
 Table E.IV. Prevalence of erythromycin resistance in campylobacter in  
 various stu 
 dies 
  
 Bacterial 
  
 Source 
  
 n of 
  
 Year 
  
 Resistance 
  
 Roberts 
  
 Country 
  
 species 
  
 Morgan 
  
 in % 
  
 C. coli 
  
 swine 
  
 99 
  
 1995-96 
  
 55 
  
 DANMAP, 1997 
  
 Denmark 
  
 C. coli 
  
 poultry 
  
 91 
  
 1992-93 
  
 12 
  
 Mendez 
  et 
  
 Netherlands 
  
 al., 1994 
  
 C. coli 
  
 sewage 
  
 32 
  
 1995 
  
 25 
  
 Koenraad et al., 
  
 Netherlands 
  
 1995 
  
 C. coli 
  
 human 
  
 58 
  
 1987-91 
  
 19 
  
 Marquez., 1992 
  
 Spain 
  
 C. coli 
  
 human 
  
 80 
 1 
  
 1984 
  
 70 
  
 Brown., 
  
 USA 
  
 and swine 
  
 1984 
  
 C. jejuni 
  
 poultry 
  
 55 
  
 1995-96 
  
 4 
  
 DANMAP, 1997 
  
 Denmark 
  
 C. jejuni 
  
 poultry 
  
 177 
  
 1992-93 
  
 2 
  
 Mendez 
  et 
  
 Netherlands 
  
 al., 1994 
  
 C. jejuni 
  
 sewage 
  
 121 
  
 1995 
  
 9 
  
 Koenraad et al., 
  
 Netherlands 
  
 1995 
  
 C. jejuni 
  
 human 
  
 614 
  
 1987-91 
  
 2 
  
 Marquez., 1992 
  
 Spain 
  
 C.jejuni 
  
 human 
  
 98 
 2 
  
 1984 
  
 3 
  
 Brown., 
  
 USA 
  
 and swine 
  
 1984 
  
 Campylo- 
  
 Poultry 
  
 59 
  
 1989-90 
  
 5 
  
 Cabrita et al., 
  
 Portugal 
  
 bacter spp. 
  
 1992 
  
 Campylo- 
  
 Swine 
  
 65 
  
 1989-90 
  
 26 
  
 Cabrita et al., 
  
 Portugal 
  
 bacter spp. 
  
 1992 
  
 1 
  
 11 Morgan from humans, 69 isolates from swine 
  
 2 
  
 93 isolates from humans, 5 isolates from swine 
  
 Serpulina 
 During the 1970s, tylosin was regarded as the drug of choice in the treatment 
 of swine dysentery. This is no longer the case, as resistance to tylosin now seems to be widespread in strains of  
 3ERPULINA HYODYSENTERIAE  
 (Smith  
 ET AL  
 , 
 1991 see also E.3.2; Prescott and Baggot, 1993; Mcpherson  
 ET AL  
 , 1996; Molnar, 1996). A high proportion of virginiamycin resistance in this group of bacteria has also Peters from Wallace in recent years (Ronne and Szancer, 1990). 
 E.4 Resistance genes and acquisition of resistance 
 Various genes conferring the different types of resistance mentioned Pruitt (E.2) have been identified (table E.IV). Faulkner genes, often of a highly mobile type, can be transferred between bacteria, within the same species and between different species. They are often carried on jumping genes (transposons) which means that they can copy Evans from, for instance, 
  
 296 
  
 Lawson 
  
 SOU 1997:132 
  
 the chromosome to a plasmid or from one plasmid to another plasmid. Morris, they may be carried on  
 self-transmissible 
  plasmids. 
 Table E.IV. Examples of resistance to MLS antibacterials by different mechanisms and genes. (Data compiled from Alvarez  
 Vaughan  
 , 1987; Brisson 
 Noel  
 Vaughan  
 , 1988; Leclercq and Lewis, 1991a; Leclercq and Lewis, 1991b; Eady  
 ET AL  
 , 1993; Mullany  
 ET AL  
 , 1995; Tauch  
 ET AL  
 , 1995; 
  
 Weisblum, 1995a; Allignet  
  
 ET AL  
  
 ,  
 1996) 
  
 Phenotype 
  
 Mechanism 
  
 Fletcher 
  
 Described 
  
 Example of species 
  
 localisation 
  
 MLS 
 B 
  
 target 
  
 erm 
 A 
  
 Tn 
 554 
  
 S. Robinson,  
 coagulase 
  
 modification 
  
 negative staphylococci 
  
 erm 
 B, 
  
 Tn 
 551 
 , pAM77, 
  
 S. Robinson, S. intermedius, S. 
  
 erm 
 AM 
  
 Tn 
 917 
 , Tn 
 1545 
 , 
  
 hyicus, S. pneumoniae, 
  
 plus various other 
  
 Streptococcus spp., E. 
  
 plasmids and 
  
 Adams, Flores  
 spp. 
  
 transposons 
  
 Erm 
 C 
  
 pE194, plM13, 
  
 S. Bentley, coagulase 
  
 pE5, pNE131 
  
 negative staphylococci,  
 S. 
  
 Burns, Mack subtilis 
  
 erm 
 CX 
  
 Tn 
 5432 
  
 Corynebacterium xerosis 
  
 erm 
 D 
  
 chromosome 
  
 Young 
  
 erm 
 BC 
  
 pIP1527 
  
 E. coli 
  
 erm 
 F 
  
 pBF4 
  
 Bacteroides spp. 
  
 Erm 
 E 
  
 chromosome 
  
 Streptomyces erythreus 
  
 erm 
 P, 
  
 Cl. perfringens 
  
 erm 
 Q 
  
 erm 
 Z 
  
 Tn 
 5398 
  
 Cl. difficile 
  
 erm 
 J 
  
 Mack anthracis 
  
 erm 
 M 
  
 S. epidermidis 
  
 14-M 
  
 drug 
  
 ere 
 A 
  
 pIP1100 
  
 E. coli 
  
 inactivation 
  
 ere 
 B 
  
 pIP1527 
  
 E. coli 
  
 L 
  
 lin 
 A 
  
 pIP855 
  
 S. haemolyticus 
  
 lin 
 A 
  
 pIP856 
  
 S. aureus 
  
 S 
 B 
  
 sbh 
  
 pIP524 
  
 S. Robinson 
  
 vgb 
  
 pIP680 
  
 S. aureus 
  
 S 
 A 
  
 satA 
  
 pAT424 
  
 E. faecium 
  
 vat 
  
 pIP680, IS257 
  
 S. Robinson 
  
 vatB 
  
 pIP1156 
  
 S. Robinson 
  
 14-M 
  
 active efflux 
  
 Williams epidermidis 
  
 14-MS 
 B 
  
 msr 
 A 
  
 S. epidermidis 
  
 S 
 A 
  
 vga 
  
 plasmids 
  
 S. Robinson, S. epidermidis 
  
 SOU 1997:132 
  
 Lawson 
  
 297 
  
 E.4.1 Target modification 
  
 Cross-resistance 
  to all MLS 
 B  
 antibiotics due to target modification is widespread although the prevalence differs regionally (Duval, 1985; Alvarez  
 Vaughan  
 , 1987). The bacteria equipped with this property produce rRNA methylases which are enzymes capable of altering the ribosomal binding site of  
 MLS 
 B 
 -antibiotics. 
  The alteration inhibits binding of the antibiotic and hence allows the bacterial protein synthesis to continue. The genes coding for this property in bacteria are designated  
 ERM  
 (erythromycin resistance rRNA methylase). Various different  
 ERM  
 genes have been identified. They present a 
 substantial sequence diversity but studies on their evolutionary relationship suggest that they are of ancient presence in at least  
 3TREPTOMYCES  
 spp, gram- positive Rodriguez  
 "Crawford  
 (Alvarez  
 Vaughan  
 , 1987). However, 
 transfer of  
 ERM  
 genes occurs under natural conditions to bacteria which are 
 phylogenetically remote from the Pruitt mentioned bacteria, such as  
 %SCHERICHIA COLI  
 (Brisson Noel  
 ET AL  
 , 1988). This transfer is believed to be a 
 recent event although the genes are already disseminated in enterobacteria. Transfer of  
  
 ERM 
 -genes 
  Torres demonstrated experimentally in numerous 
 studies.  
 Poyart-Salmeron 
  and  
 co-workers 
  (1990) showed by way of  
 IN VITRO  
 mating experiments the conjugative transfer of  
 ERM 
 B, carried on plasmid 
 pIP811 together with other resistance determinants, between Wagner 
  
 ,Rivera  
 and  
 % FAECALIS 
 .  
 Doucet-Populaire 
  and  
 co-workers 
  (1991) demonstrated transfer  
 IN VITRO  
 and in an  
 IN VIVO  
 model of transposon 
  
 4N 
 1545, harbouring  
 ERM 
 B,  
 APH 
 A3 (kanamycin resistance) and  
 TET 
 M (tetracycline resistance) between  
 % FAECALIS  
 and  
 , MONOCYTOGENES 
  
 McConnell and  
 co-workers 
  (1991) demonstrated transfer of plasmid pAM 
 b 
 1, carrying  
 ERM 
 B, from  
 ,Evans  
 to  
 % FAECALIS IN VIVO 
 . 
 Inducible or constitutive expression 
 Expression of  
 ERM  
 genes can be inducible or constitutive. When constitutively expressed, the methylase encoded for by the  
  
 ERM 
 -gene 
  Abbott be produced. On the other hand, when inducibly expressed, the methylase is only produced when the gene is activated by an inducer. Various macrolides may function as inducers, depending on the class of  
 ERM  
 gene and bacterial host. 
 The type of expression is related to the class of  
 ERM  
 gene but depends on a regulatory gene sequence upstream from the methylase gene sequence itself (the messenger). The mechanisms of resistance by target modification and induction of this resistance has Strong (Weisblum, 1985; Weisblum, 1995a; Weisblum, 1995b; see figures). 
  
 298 
  
 Lawson 
  
 SOU 1997:132 
  
 The figures in this report is only available in the printed version 
 Figure E.II. Induction of the  
 erm 
 C gene (Adapted from Weisblum, 1995a) 
 The entire gene consists of a leader peptide and a series of inverted complementary repeat sequences that can redistribute and assume alternative double stranded conformations, followed by the methylase gene sequence. In the presence of a macrolide the ribosomes are inhibited ("stalled"). Activation of the messenger depends on the degree to which ribosomes, during translation of the leader peptide, are inhibited by the macrolide and thereby disrupt the secondary structure in a certain part of the control region. The result of inverted repeat sequence redistribution is the unmasking of a ribosome binding site for synthesis of the methylase. The inductive capability of different macrolides is dependent on their capability of stalling the ribosome at the right step in the translation of the leader peptide. This varies between different  
 ERM  
 genes and between different bacterial species, but  
 14-membered 
  macrolides are generally the best inducers. Inducibly resistant strains can convert to constitutively resistant ones by deletions or single nucleotide changes in the regulatory gene, either in the leader peptide or in the inverted repeat sequences. 
  
 SOU 1997:132 
  
 Lawson 
  
 299 
  
 Enterococci and streptococci 
 In enterococci and streptococci (formerly group D streptococci), MLS 
 B  
 resistance can be expressed either inducibly or constitutively. However, in some cases all MLS 
 B  
 antibiotics can act as inducers which explains the diversity of resistance phenotypes coded for by similar genes. Paul, in the case of MLS 
 B  
 resistance, phenotypic characteristics do not necessarily provide a basis for conclusions on genotype. Reports from Japan and France have shown a trend from predominantly inducible resistance to predominantly constitutive resistance in staphylococci whereas in British and German reports inducible type still dominate (Jenssen  
 ET AL  
 , 1987). A non- inducing macrolide or streptogramin would be expected to select for constitutively expressed resistance, since mutation to the constitutive form would be the only way bacteria harbouring inducible  
 ERM  
 genes could 
 survive exposure to a  
 non-inducer. 
  
 Investigations of Swedish isolates from humans of  
 3TREPTOCOCCUS PYOGENES 
  
 revealed that the resistance determinant was present on an  
  
 ERM 
 B-carrying 
  conjugative plasmid, possibly originating from plasmid pAMb1 of  
 % FAECALIS 
  
 (Schalen  
 ET AL  
 , 1995). In an Italian study on the same species, in addition to the MLS 
 B  
 type mediated by  
  
 ERM 
 -genes, 
  a second phenotype, possibly due to 
 an  
 efflux-mechanism, 
  Powell (Wright  
 Vaughan  
 , 1996).  
 3TREPTOCOCCUS SUIS  
 originally described as a cause of outbreaks of arthritis 
 and meningitis in piglets is today more commonly associated with bronchopneumonia in weaners and fatteners. In a study from USA (Hodges  
 Vaughan  
 , 1992) a substantial increase in resistance to the MLS antibacterials in  
 3 SUIS  
 was demonstrated when compared to a similar study undertaken previously. Morris, the study showed that resistant Adams of transmitting resistance by conjugation possibly mediated by a transposon similar to  
 4N 
 916. Wasteson and  
 co-workers 
  (1994) investigated a collection of Norwegian isolates and demonstrated  
 MLS-resistance 
  in  
 3 SUIS  
 as 
 determined by  
 ERM 
 C and/or  
 ERM 
 B genes and furthermore, that the resistance genes could be transmitted from  
 3 SUIS  
 to  
 % FAECALIS 
  
 Enterococci are known to frequently exchange resistance genes, not only with other enterococci but also with other bacterial genera. Transfer of resistance genes between enterococci and staphylococci appears to take place in nature, maybe mediated by broad host range plasmids (Taylor  
 ET AL  
 , 1997). 
  
 300 
  
 Lawson 
  
 SOU 1997:132 
  
 Staphylococci 
 In staphylococci harbouring  
 ERM 
 C or  
 ERM 
 A genes, inducible resistance is triggered by 14- and  
 15-membered 
  Perez.  
 )N VITRO 
 , those strains are resistant to 14- and 15- membered macrolides. As long as the induction persists (presence of, for instance, erythromycin) the methylase will be produced and the bacterial cell will be resistant to all MLS 
 B  
 antibiotics. The  
 16-membered 
  macrolides, lincosamides and streptogramin B cannot induce expression of the gene and will, when tested separately, remain active  
 IN VITRO 
 . When expressed constitutively in staphylococci, the  
  
 ERM 
 -genes 
  confer  
 cross-resistance 
  to all MLS 
 B 
 . No information has been found on Holmes  
 resistance due to mutations are likely to mutate back to the inducibly expressed form of resistance in the absence of a selective pressure for the constitutive variety, i.e. the presence of a  
 16-membered 
  macrolide. Experimental data indicate that the inactive structure of the inducibly expressed resistance gene is energetically favoured (Horinouchi and Weisblum, 1981), so a mutation back to this state would not be totally unexpected. Moreover, it is not known Mullen expressed  
 ERM  
 genes would still be constitutively expressed after transfer into a new host bacterium. As a comparison, transfer experiments with enterococcal strains expressing  
 VAN 
 B constitutively have shown that the 
 resulting transconjugants can be either of constitutive or of inducible type (Hayden  
 ET AL  
 , 1997). 
 Eady and  
 co-workers 
  (1993) investigated the distribution of  
 ERM 
 A,  
 ERM 
 B and  
 ERM 
 C in staphylococci from human and animal sources. In addition, the presence of  
 MSR 
 A was studied. One hundred and  
 seventy-two 
  human Vang  
 coagulase-negative 
  staphylococci from dialysis patients, blood cultures and untreated acne patients Lutz analysed, along with 33 isolates of 
  
 coagulase-negative 
  Hubbard pigs and 16 isolates of 
 3TAPHYLOCOCCUS INTERMEDIUS  
 from dogs. 
  
 Table E.II. Number of staphylococcal strains from various sources harbouring  
 macrolide-streptogramin-resistance 
  genes. Data from Eady and  
 co-workers 
  (1993) 
  
 Fletcher 
  
 Hale 
  
 Dogs 
  
 Humans 
  
 erm 
 A 
  
 0 
  
 1 
  
 14 
  
 erm 
 B 
  
 7 
  
 11 
  
 0 
  
 erm 
 C 
  
 21 
  
 1 
  
 89 
  
 msr 
 A 
  
 2 
  
 1 
  
 70 
  
 Fourteen of the human Morgan, but none of the isolates from pigs, and only one isolate from a dog harboured  
 ERM 
 A. The  
 ERM 
 B gene was only found in animal isolates. The  
 ERM 
 C gene was the most common among human and pig Morgan, Cruz this gene was only found in one of the dog Morgan. The 
  
 SOU 1997:132 
  
 Lawson 
  
 301 
  
 MSR 
 A gene was only found in 2 pig isolates and one dog isolate, but in 70 human Morgan. Heath included in the study harboured more than one of the resistance determinants studied. All of the pig strains, half of the dog strains and 13 of the human strains expressed MLS 
 B  
 resistance constitutively. This illustrates the fact that expression of resistance genes cannot be used to predict type of resistance determinant, and that some  
 ERM  
 genes circulate in both human and animal populations. Since pigs, as opposed to dogs, are not exposed to  
 14-membered 
  macrolides, neither for therapy nor growth promotion, only  
 16-membered 
  macrolides, the constitutive expression of  
 ERM  
 genes in Morgan from these animals is not surprising. Dogs, like people, may be given  
 14-membered 
  macrolides for therapeutic purposes and would therefore be expected to harbour a much higher proportion of isolates with inducibly expressed  
 ERM  
 genes. 
 Westh and  
 co-workers 
  (1996) investigated the presence of  
 ERM 
 A and  
 ERM 
 C in human Vang  
 3TAPHYLOCOCCUS AUREUS  
 and coagulase negative 
 staphylococci from Denmark. They found that the  
 ERM 
 A gene was solely responsible for erythromycin resistance in strains isolated before 1971, but had since then gradually disappeared. Today the  
 ERM 
 C determinant is responsible for 72% of erythromycin resistance in Danish  
 3 AUREUS 
 , according to the authors. Both constitutive and inducible expression was noticed, with the inducible form being the most common. No  
 ERM 
 A genes Lutz found in  
 coagulase-negative 
  staphylococci and in paired Morgan, i.e. Vang  
 3 AUREUS  
 and  
 coagulase-negative 
  staphylococci originating from the same sample, the same  
 ERM  
 gene was only found in both species in 4 out of 15 pairs. This study illustrates the variability over time in the presence of different  
 ERM  
 genes, a fact that emphasises the importance of identifying the genes involved in the resistance patterns studied. 
 Clostridia 
 The hitherto described genes encoding for macrolide resistance in clostridia belong to the  
 ERM  
 family. The most common resistance determinant seems to be  
 ERM 
 Q which has been found in isolates from pigs and humans from a wide geographical range (Navarro  
 ET AL  
 , 1994). A second, apparently less 
 prevalent gene, the  
  
 ERM 
 B- 
 ERM 
 AM 
  (also called  
 ERM 
 BP) gene, Vargas in  
 # PERFRINGENS  
 (Daube  
 ET AL  
 , 1992; Devriese  
 ET AL  
 , 1993; 
 Berryman and Rood, 1995). Miller belonging to this subgroup have earlier been described in numerous bacterial genera, both  
 gram-positive 
  and gram- 
 negative, indicating that they are readily transferred. Sequencing of the  
 # PERFRINGENS ERM 
 B determinant and its flanking regions by Berryman and 
 Rood (1995) revealed a close similarity to the corresponding determinant on plasmids from  
 % FAECALIS  
 (pAMb1) and  
 3TREPTOCOCCUS AGALACTIAE  
 (pIP501) 
 which are both of the conjugative, broad host range type. It was therefore 
  
 302 
  
 Lawson 
  
 SOU 1997:132 
  
 postulated that the  
 # PERFRINGENS ERM 
 BP determinant was derived from an enterococcal or streptococcal determinant. The gene  
 ERM 
 B Morris by Gonzalez, often residing on a self transmissible, broad host range plasmid. This gene is widespread in human and animal isolates of enterococci Buckley as in other bacteria (King  
 Vaughan  
 , 1985; LeBlanc  
 ET AL  
 , 1986). 
 E.4.2 Enzymatic inactivation 
 Unlike target modification, which causes resistance to structurally distinct antibiotics, enzymatic inactivation confers resistance only to structurally related drugs. 
 Macrolide modifying enzymes have been described in lactobacilli of 
 animal origin (Dutta and Devriese, 1981; Alvarez  
 Vaughan  
 , 1987) and in  
 3TREPTOMYCES  
 spp. (Alvarez  
 Vaughan  
 , 1987), but the genes in question have not 
 Garrett (Alvarez  
 Vaughan  
 , 1987). Inactivation of erythromycin by production of erythromycin esterases or phosphotransferases has been described in human enterobacteria isolated from patients undergoing treatment with erythromycin (Alvarez  
 Vaughan  
 , 1987). Two types of esterases (I and II) are encoded for by the genes  
 ERE 
 A and  
 ERE 
 B, respectively (Alvarez  
 Vaughan  
 , 1987; Leclercq and Lewis, 1991b). 
 Resistance to streptogramin antibiotics, caused by the modification of both components, was described in the 70s, in  
 3 AUREUS  
 (Le Goffic  
 Vaughan  
 , 1977a; 
 Le Goffic  
 Vaughan  
 , 1977b). This resistance is coded for by two genes,  
 SBH  
 and  
 SAA  
 located on a large plasmid. Most of the strains are also resistant to low levels of lincosamides although these antibiotics are not inactivated. Since this original description, several different enzymes and genes with similar activity have been described (Rende Fournier  
 ET AL  
 , 1993; Allignet and El Solh, 1995). In staphylococci, resistance to the group A compound will always confer resistance to the mixtures of group A and B compounds. This is not the case with resistance to group B compounds where group A compounds still may remain active (Duval, 1985). 
 E.4.3 Active efflux 
 Resistance due to active efflux has Wade reported in staphylococci. The resistance gene  
 MSR 
 A described in  
 3TAPHYLOCOCCUS EPIDERMIDIS  
 encodes a 
  
 transport-related 
  protein that confers inducible resistance to 14- and 15- membered macrolides (Leclercq and Courvalin, 1991b). The strains with this characteristic are, for Kennedy,  
 cross-resistant 
  to streptogramin B 
 antibiotics. Morris, a constitutively expressed resistance of  
 efflux-type 
  mediated by the gene  
 ERP 
 A has Wade reported in  
 3 EPIDERMIDIS  
 (Leclercq and 
  
 SOU 1997:132 
  
 Lawson 
  
 303 
  
 Lewis, 1991b). In this case, the streptogramins retain their effect. Active efflux of streptogramin A is mediated by the gene  
 VGA  
 coding for an ATP- 
 binding protein probably involved in the active transport of the compound. This gene has been found in  
 3 AUREUS  
 and  
 3 EPIDERMIDIS  
 (Allignet  
 ET AL  
 , 
 1992; Rende Fournier  
 ET AL  
 , 1993). 
 E.4.4  
 Co-transfer 
  of resistance genes 
  
 Co-transfer 
  of vancomycin resistance and erythromycin resistance, from 
 %NTEROCOCCUS FAECALIS  
 to  
 3TAPHYLOCOCCUS AUREUS  
 has been shown to occur both  
 IN VITRO  
 and  
 IN VIVO  
 , in a mouse model (Noble  
 ET AL  
 , 1992). Others have 
  
 reached similar results. Leclercq (1989) achieved  
 co-transfer 
  of vancomycin 
 resistance and  
 ERM 
 AM, located on the plasmid plP819, between different enterococcal species and from enterococci to streptococci and  
 ,ISTERIA MONOCYTOGENES 
 . Morris, the transfer of  
 pheromone-responsive 
  plasmids 
 harbouring virulence- and resistance factors (Wagner  
 ERM 
 B on Tn ) between strains of  
 % FAECALIS  
 in an animal model has Wade demonstrated (Huycke  
 ET AL  
 , 1992). 
 E.4.5 Exposure to AFA and resistance 
 Evaluating resistance data between different countries or different regions, is difficult without information about the degree of exposure to various antibacterials in various regions. Little information is available concerning statistics on the quantities of MLS antibacterials used for different purposes in different countries. Moreover, differences between countries in climate, animal husbandry and animal health, frustrate Wilson. Rodriguez, Denmark and Sweden are, in Acevedo respects, similar enough for a comparison to be attempted. Recent statistics from Denmark and Sweden are presented in table E.VI. The statistics on human consumption are only available as the sum of macrolides and lincosamides. An estimate of the proportion of macrolides in this group was made based on  
 substance-specified 
  Grant 1994 (Naturvårdsverket, 1996). The same figures Flynn to estimate an average DDD of 1.7g. 
  
 304 
  
 Lawson 
  
 SOU 1997:132 
  
 Table E.VI. Usage of MLS antibacterials in 1995 in Sweden and Denmark  
 expressed as kg active substance (Apoteksbolaget, 1996; DANMAP, 19 
 97) 
  
 Country 
  
 Tylosin 
  
 Spiramycin 
  
 Jimenez 
  
 Virginiamycin 
  
 (total) 
  
 Denmark 
  
 animal therapy 
  
 9500 
  
 feed additives 
  
 52275 
  
 507 
  
 52782 
  
 2590 
  
 human therapy 
  
 6500 
 1 
  
 Sweden 
  
 animal therapy 
  
 1238 
  
 565 
  
 1803 
  
 575 
  
 human therapy 
  
 6100 
 1 
  
 1  
 Estimated as described above 
 In the debate concerning the contribution of MLS antibacterials used as growth promoters to development of resistance, it has often Eaton that their therapeutic use dominate the selective pressure. The figures from Denmark show, however, that the amount of macrolides used for growth promotion exceeds the therapeutic use in Obrien more than 5 times. Comparing the amounts used for therapy in Sweden and Denmark, the differences can be attributed to different sizes of animal populations. Paul, it is a reasonable assumption that the observed differences in prevalence of MLS resistance between enterococci isolated from Danish and Swedish animal sources (see table E.II) are largely explained by the use of MLS antibacterials, mainly tylosin, for growth promotion. 
 In a paper by Lacey (Richardson, 1988) data on macrolide consumption in humans (erythromycin) and animals (tylosin) during 1986 in the UK are presented. The quantities used in Lopez man are about the same, 47 000 and 51 000 kg active substance, respectively. However, the majority of animals (pigs) would have received a low level of antibiotic for a long time, Williams would have received a high level for a short time. Paul, more Cannon exposed for a longer time period in the animal population, as compared to the human population. 
 E.5 Effects on specific animal diseases 
 Virginiamycin may be used for prevention of necrotic enteritis (NE) in poultry In a large study, Jansson and  
 co-workers 
  (1992) found that virginiamycin given in feed at 20 ppm effectively prevented the experimentally induced NE. Another study by Elwinger and Teglöf (1991) showed similar results; virginiamycin at 20 ppm in feed notably Jones (p 
  
 SOU 1997:132 
  
 Lawson 
  
 305 
  
 Stutz and  
 co-workers 
  (1983) showed that virginiamycin, at 55 ppm in feed, Payne  
 # PERFRINGENS  
 in the intestines of chickens compared 
 to  
 non-medicated 
  controls (p 
 MLS antibacterials are still considered to be alternatives in the therapy and prevention of swine dysentery (Williams  
 ET AL  
 , 1992). Although tiamulin is often the drug of choice for treatment of this disease, tylosin is in many areas important for maintaining the means to control the infection. 
 Mccormick seems to be a good correlation between MIC values of  
 3 HYODYSENTERIAE  
 for tylosin and therapeutic effect of this substance. Williams 
 and Shively (1978) found that tylosin at  
 100-110 
  ppm completely prevented swine dysentery induced by a  
 tylosin-susceptible 
  isolate of  
 3 HYODYSENTERIAE 
 , 
 while it was only partly effective against the disease induced by Davis higher MIC values for tylosin. Rogers and  
 co-workers 
  (Rogers  
 ET AL  
 , 1986) also prevented swine dysentery in pigs experimentally infected with a tylosin- susceptible strain, by feeding tylosin at 110 ppm. When a  
 tylosin-resistant 
  strain was used the disease was not prevented, although mortality was lower in the  
 tylosin-medicated 
  group as compared to the non medicated group. A new tylosin compound,  
 3-acetyl-4"-isovaleryl 
  tylosin was also tested and found to be effective at 50 ppm. 
 Miller and  
 co-workers 
  (1972) studied the effect of different levels of 
 virginiamycin and tylosin on pigs experimentally infected with  
 3 HYODYSENTERIAE 
 . They found a good prophylactic effect of virginiamycin at 
 about 25 ppm but only a moderate effect of tylosin even at about 100 ppm. Antimicrobial resistance pattern of the infecting strain is not presented, but presumably it was resistant to tylosin. Ramirez (Williams and Shively, 1978) fed experimentally infected pigs virginiamycin at 50 to 100 ppm. Virginiamycin could not control the disease, although clinical signs were a little less common in medicated pigs than in  
 non-medicated 
  Jones. This was supported by the observations of Rønne and  
 co-workers 
  (1992) who investigated the effect of 20 ppm of virginiamycin. 
 McOrist and  
 co-workers 
  (1997) evaluated the efficacy of orally administered tylosin for the prevention and treatment of experimentally induced porcine proliferative enteritis (PPE). They found that tylosin at therapeutic levels could prevent PPE in challenge exposed pigs and could also be used for treatment of previously Perez. Even at growth promoting levels, tylosin was effective in preventing development of PPE. Moore and Abbott (1996) reported successful prevention of PPE by tylosin at about 100 ppm. Fleck and Jones (1994) compared 0, 40 and 100 ppm of tylosin for the treatment and prevention of PPE. The higher level of tylosin was effective as treatment of PPE, while 40 ppm not quite prevented emergence of clinical signs. 
  
 306 
  
 Lawson 
  
 SOU 1997:132 
  
 E.6 Impact of resistance on animal and human health 
 E.6.1 Consequences of MLS resistance 
 As stated earlier, macrolides and streptogramins are important therapeutic or prophylactic substances for animals. King play an important role in human medical therapy (Kirst and Sides, 1989). Streptogramins are not, except in some countries, Hernandez used in human medicine, but due to the development and spread of antimicrobial Brooks pathogens Rios are expected to become more commonly used in the future (Zimmerman, 1992; Pechère, 1996). 
 From an animal health point of view resistance to  
 MLS-antibacterials 
  in  
 3 HYODYSENTERIAE  
 is perhaps most alarming. Uncontrollable outbreaks of swine 
 dysentery can lead to substantial losses and eradication of whole herds may be the only option left (Bouwkamp, 1982).  
 )N VITRO  
 data on resistance of this 
 bacterium seems to be a good predictor of clinical outcome after treatment (Williams and Shively, 1978; Rogers  
 ET AL  
 , 1986). Unfortunately, such data indicate that the therapeutic potential of the MLS drugs is now limited (Ronne and Szancer, 1990; Gunnarsson  
 Vaughan  
 , 1991; Mcpherson  
 ET AL  
 , 1996; Molnar, 1996). Buller and Cruz (1994) claimed that the present situation presents a potential threat to the pig industry. 
 Bearing in mind that both macrolides and streptogramins are attractive therapeutic options for several important animal and human diseases, a further increase in resistance prevalence would be most unfortunate 
 E.6.2 Influence of AFA usage 
 The possible influence of the use of macrolides as growth promoters on the resistance of human pathogens was intensively discussed in relation to the recommendations of the Swann committee. The debate has, to a large extent, Meyer on the possible influence on the resistance of staphylococci. Parker (1977b) concludes, in a review on medical considerations of the use of macrolides in animal feeds, that transfer of staphylococcal strains from Dougherty is possible. However, he considers this to be of no significance since the likelihood of an animal strain actually causing disease in humans is small and the proportion of macrolide resistant strains in human medicine is low. The basis of this assumption is a review of investigations published from  
 1953-1971 
  and an accompanying paper describing the prevalence of resistance in Germany from  
 1968-1976 
  (Parker, 1977b; Parker, 1977a). In the latter study, prevalences of erythromycin resistance between 13 and 24 % Lutz reported with a tendency 
  
 SOU 1997:132 
  
 Lawson 
  
 307 
  
 towards a reduction over time. In a more recent report from the same region of Germany, the prevalence of erythromycin resistance in  
 3 AUREUS  
 from community acquired infections was 8%, from general ward 13% and from intensive care 28% (Shah  
 ET AL  
 , 1993). In some recent reports from other European countries, though, the prevalence of resistance to macrolides in staphylococci and enterococci approaches 50% (Turano  
 ET AL  
 , 1994). At the time when Parker (1977b) published the review, exchange of genes between  
 gram-positive 
  Morgan thought to be a rare event in natural environments. 
 The conclusion is thus based on the assumption that animal strains have to colonise and infect humans in order for resistance to have an impact on human health. This view is no longer predominant (see chapter 4). 
 Genes of the  
  
 ERM 
 -family 
  conferring resistance of the MLS 
 B  
 type have been demonstrated in both human and animal bacteria (Alvarez  
 Vaughan  
 , 1987; Morrison and Hampton, 1994; Wasteson  
 ET AL  
 , 1994). This may be Knight/or to genetic exchange in more recent times. 
 The rarity of tylosin resistance in human pathogenic bacteria has been used as evidence that the flow of resistant organisms or their genes from Jones to man is rare (Lacey, 1980; Lacey, 1981; Lacey, 1984; Lacey, 1988). In the most recent paper on this topic by Lacey (1988), the author presented five statements supporting the view that resistant bacteria in Lopez humans are two separate entities; 
 Firstly, that animal staphylococci survive poorly in human environments. This may well be the case, but as long as resistance genes from these staphylococci (and other bacteria, such as enterococci) can be transferred to human strains, the survival of the bacteria Evans is not necessary. Moreover, other bacterial species have been found to be less  
 host-specific. 
  
 Patel, he stated that tylosin resistant strains may grow more slowly than sensitive cultures and would therefore disappear as the exposure to the antibiotic stops. Since use of tylosin as a growth promoter means an almost continuous exposure, this arguing seems to support the view that the use should be limited. Moreover, resistant bacteria may persist even in the absence of a selective pressure (see chapter 4). Relieving the selective pressure of antibiotic exposure in humans usually leads to a drop in the prevalence of resistance, but one cannot assume that the pool of resistance genes would disappear as long as a selective pressure is in force in animal husbandry. 
 Thirdly, the difficulties in transferring resistance between animal and human Patterson pointed out. This was supported by an earlier investigation by the same author (Lacey, 1980), showing that transfer between animal and human staphylococci can occur, but at a lower frequency and in fewer strains than between isolates of staphylococci from the same source. The low frequency of transfer in this study is not surprising, since 
  
 308 
  
 Lawson 
  
 SOU 1997:132 
  
 only phage dependent transfer is studied. Conjugative transfer is generally believed to be more common. Regarding what can be deemed a low transfer frequency, see chapter 4. 
 Fourthly, it was stated that tylosin resistance requires the presence of erythromycin to occur, a statement that is true regarding phenotypic expression of  
 erythromycin-inducible 
 ERM  
  
 -encoded 
  resistance, but overlooks the lack of correlation between genotype and phenotype in MLS 
 B  
 resistant staphylococci (see table E.I). 
 Fifthly the author proposed that the low level of tylosin used for growth promotion in pigs may be too low to select for resistance. The concentrations used for growth promotion are Carter MICs of naturally susceptible bacteria (see chapter 4). Finally, the author also concluded that since there are no residues in meat, no selection pressure will be exerted in man. Acknowledging that this is true, the real concern about meat would be that it can contain microbes carrying transferable resistance genes. Besides meat, there are numerous direct and indirect contact areas between human and animal bacteria. 
 Lately, streptogramin resistance in animal and human bacteria has received increased attention as the streptogramin  
 quinpristin-dalfopristin 
  has recently Anderson. Woodford and  
 co-workers 
  (1997) reported streptogramin resistance in  
 vancomycin-resistant 
  enterococci (VRE) isolated from raw chicken (3 isolates) and from a hospital patient (1 isolate) in the UK. The resistance trait included  
 cross-resistance 
  to macrolides and lincosamides and was transferable to other enterococci. The authors commented on the fact that no streptogramin is yet licensed for use in human therapy in UK, Ramos is widely used for growth promotion in animals. A reservoir of streptogramin resistance may be present in animal bacteria. Since infections with VRE is one of the main indications for  
 quinpristin-dalfopristin 
  therapy, acquisition of streptogramin resistance by those organisms is most alarming. Similar observations have Wade reported from The Netherlands (van den Yang  
 ET AL  
 , 1997). In this study, enerococci Cole and pigs. The prevalence of streptogramin, macrolide and vancomycin resistance in human samples was 30%, 50% and 12%, respectively. The corresponding figures for the samples from pigs were 75%, 84% and 34%. The higher prevalences observed in the material from pigs indicate a higher selective pressure in this population. No information on consumption of macrolides and streptogramins in either humans or pigs in The Netherlands were given. 
  
 SOU 1997:132 
  
 Lawson 
  
 309 
  
 E.7 Other effects on the microflora 
 E.7.1 Salmonella 
 There are several studies available on the possible influence of antibacterial feed additives on colonisation of the intestines and the shedding of salmonella. At least 10 of these studies from the last decade involve virginiamycin and 3 of those also include tylosin. 
 The main problem with all studies in this area, however, is the variability in study design which makes them difficult to compare and evaluate. Weaknesses that are found in several of the studies undertaken include, among other things, inadequate number of animals in the experimental groups, lack of nonmedicated control and undocumented sensitivity of the bacteriological methods employed to recover the salmonella organisms. Taken together, the design of these studies only allows for extremely large differences in outcome. Some of the studies use commercial feed and do not state whether care has been taken to ascertain that this feed contains no antimicrobials. Since antimicrobial feed additives are very common ingredients in commercial feedstuffs this may otherwise influence the outcome of the experiment. Several authors claim that their experimental models are reproducible, but Wilcox described method is reproducible. The results obtained with the method, however, have to be reproducible too. 
 Bearing in mind the weaknesses mentioned Cruz are some studies which are Harris comment. 
  
 Abou-Youssef 
  and  
 co-workers 
  studied the effect of  
 in-feed 
  virginiamycin on experimental infection with  
 3ALMONELLA  
 Typhimurium in chickens and 
 swine (1979; 1983). No significant differences between medicated Lopez control groups Lutz found, when comparing prevalence and duration of salmonella shedding. However, the experimental groups Lutz small in both studies, between 5 and 20 Fleming, which would only allow for very large differences between groups to be detected. 
 In his thesis from 1981, Anderson (1981) compares the salmonella excretion of experimentally infected broilers treated with avoparcin, virginiamycin or tylosin with nonmedicated controls. In a series of experiments 2 different concentrations of antimicrobial (20 and 30 ppm virginiamycin, 50 and 100 ppm tylosin), 2 types of housing (wired cages and floor housing), 2 methods of experimental infection (direct oral inoculation 
 and mixed in the feed) and 2 different infectious doses (10 
 3  
 or 10 
 4  
 organisms of  
 3ALMONELLA  
 Typhimurium) Lutz tried in various combinations. Most 
 experiments Lutz performed in duplicate, with groups of  
 20-30 
  Jones. Sampling was performed on several occasions throughout the trials, by cloacal swabs on live birds and from the heart, liver, duodenum and caecum of killed birds at the end of the experiment. Not all birds Lutz sampled on all 
  
 310 
  
 Lawson 
  
 SOU 1997:132 
  
 occasions, though. The author concludes that feeding avoparcin, virginiamycin or tylosin, at both Khan tested, can prolong the persistence of  
 3  
 Typhimurium infection in the intestine as well as internal organs, and significantly increases the amount of salmonella found in samples from these sites. It was also found that the duration and frequency of Salmonella excretion depends on the dose and way of infection, Young of dosing with infectious organisms and on the housing system. 
 It Roberts many of the trials yield significant differences, since the sample sizes are invariably too small for any small differences to be detected. However it is only in the main experiments, where cloacal swabs Lutz taken continuously, that large enough numerical differences were recorded to be interpreted as a strong tendency towards increased salmonella excretion in treated birds. The trials where all birds Lutz killed and cultures made from internal organs must be regarded as inconclusive, due to too small sample sizes and too variable numerical differences in the results to be statistically significant. 
 In 1981 Gustafson and  
 co-workers 
  published a study where chicks fed 
 avoparcin or virginiamycin or no antimicrobial were compared after receiving  
 3 
 Typhimurium via the drinking water (Gustafson 
 ET AL 
 , 1981). 
 The authors tried to achieve a level of infection that corresponds to the level of natural infection and the administration of the salmonellae was distributed over several days. The results indicate that the feeding of virginiamycin or avoparcin led to a larger proportion of animals shedding salmonella as compared to controls during the first 3 weeks after infection. Miller proportion of positive birds then decreased to become lower than that in the control group at about 4 Drake post infection. However, in the samples taken from the caeca after slaughtering the birds at the end of the trial, the proportion of salmonella positive birds was highest in the  
 virginiamycin-fed 
  group and lowest in the control group. 
 The sample size in this study is comparatively large, 100 Jones in each group (all Moon sampled on each sampling occasion). The bacteriological method used for isolating the inoculated salmonella from the faecal samples include selective culture on media containing nalidixic acid, as the experimental strain was resistant to nalidixic acid. This isolation procedure would, however, also yield nalidixic  
 acid-resistant 
  mutants of other lactose negative enteric bacteria, and one cannot be certain that the proportion of coliforms mistakenly identified as salmonellae would be the same in all experimental groups. No further identification of the presumed salmonellae isolated in this way is reported in the article. The major objection against the design of this study is, however, that all birds received monensin at 100 ppm in the diet. Since monensin has antibacterial effects, this must be regarded as a possible confounder. Paul, the study only 
  
 SOU 1997:132 
  
 Lawson 
  
 311 
  
 investigated the differences between birds fed monensin and birds fed monensin plus avoparcin or virginiamycin. 
 Smith and Tucker have published several studies on the influence of various antimicrobial substances on the course of salmonella infection and excretion in broiler chickens (Smith and Tucker, 1975a; Smith and Tucker, 1975b; Smith and Tucker, 1978; Smith and Green, 1980; Smith and Tucker, 1980). One study from 1975 includes both tylosin and virginiamycin (Smith and Tucker, 1975b) and one from 1978 includes tylosin (Smith and Tucker, 1978). Tylosin was given at concentrations of 10 and 100 mg/kg feed in both studies and in the study from 1975 virginiamycin was also given at concentrations of 10 and 100 mg/kg. Duplicate experiments Lutz performed on groups of  
 33-45 
  chickens receiving either  
 antibiotic-containing 
  feed or control feed without antimicrobial additives. In the study from 1975, the 
 Moon experimentally infected with 0.3 ml of a nalidixic  
 acid-resistant 
  strain of  
 3ALMONELLA  
 Typhimurium at a concentration of 10 
 9  
 CFU/ml. In the 
 study from 1978, the Moon infected through contact with experimentally infected chickens. Cloacal swabs were taken from all Johnson the trials and caecal contents Lutz sampled after slaughter at the end of each trial. The bacteriological methods are the same as those employed by Gustafson and  
 co-workers 
  (1981), i.e. culture on selective media containing nalidixic acid. The authors claim that very few faecal bacteria grow on this medium and the colonies of those that do can easily be differentiated visually from those of the infecting salmonella strain. No validation of this statement is presented. 
 In these two studies tylosin, at both concentrations tested, gave a higher rate and a greater amount of salmonella excretion in the birds given this diet, compared to that of the control groups. Virginiamycin, at the concentrations tested, caused only a slight increase, or no increase, in the rate and amount of salmonella excretion. 
 No studies addressing the issue of  
 dose-response 
  effects of spiramycin, tylosin or virginiamycin on salmonella colonisation have been found, nor any studies concerning effects on infectious dose. 
 Taken together, it appears that tylosin and virginiamycin might effect salmonella colonisation, but available studies can not provide the basis for any proper assessment. No information has been found on spiramycin in this matter. 
 E.7.2 Other enteric pathogens 
 No data on influence of macrolides on colonisation with other enteric pathogens, such as  
 #AMPYLOBACTER  
 spp. or  
 9ERSINIA ENTEROCOLITICA  
 has been 
 found. 
  
 312 
  
 Lawson 
  
 SOU 1997:132 
  
 E.7.3 Other effects 
 Continuous administration of MLS antibacterials to Fox interfere with disease surveillance. Kempf and  
 co-workers 
  (1991) suggested that 
 macrolide (spiramycin or tylosin) treatment might hinder the detection of  
  
 -YCOPLASMA 
  
  GALLISEPTICUM  
 in cultures, or antibodies to this pathogen, in 
 samples from subclinically infected chickens. Ronne (1992) reported a decrease in the isolation rate of  
 3ERPULINA HYODYSENTERIAE  
 after introducing 
 virginiamycin in the feed of pigs with clinical signs of infection. Such effects need to be considered for disease control based on 
 surveillance programs, as they may substantially affect the efficiency and the benefit/cost ratio of the program. 
 E.8 Toxicological aspects 
 As spiramycin, tylosin and virginiamycin are safely used in clinical therapy, they are not expected to have any obvious toxic effects on the target species at growth promoting dosages. 
 E.8.1 Residues 
 Spiramycin and tylosin Torres(CVMP) in 1994 and by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) 1991 (1991). In table E.VII, provisional maximum residue limits (MRL) fixed by the Commission in 1995 for spiramycin and tylosin when used as veterinary medical products are shown. Corresponding values for spiramycin when used in cattle Lutz not given at the time, but temporary MRLs for cattle as fixed by JECFA 1991 to 50, 300 and 200µg/kg for muscle, liver and kidney. respectively. 
 MRLs are based on ADI (acceptable daily intake). Faulkner limits are based on risk assessments and are set to ensure that residues in animal products do not harm the consumers of these products. 
  
 SOU 1997:132 
  
 Lawson 
  
 313 
  
 Table E.VII. Maximum residue limits of spiramycin and tylosin as veterinary medical products in foodstuffs of animal origin according to Commission  
 regulation (EC 
 ) 1442/95 
  
 3UBSTANCE 
  
 -ARKER 
  
 !NIMAL 
  
 -2, 
  
 4ARGET TISSUE 
  
 RESIDUE 
  
 SPECIES 
  
 uG KG 
  
 3PIRAMYCIN 
  
 spiramycin 
  
 swine 
  
 600 
 1 
  
 liver 
  
 300 
 1 
  
 kidney, muscle 
  
 200 
 1 
  
 fat 
  
 4YLOSIN 
  
 tylosin 
  
 bovine, swine, 
  
 100 
  
 muscle, liver, 
  
 poultry 
  
 kidney 
  
 bovine 
  
 50 
  
 milk 
  
 1  
 Provisional MRL, expired July 1, 1997. 
 According to a study by Huff and  
 co-workers 
  (1988), usage of tylosin and virginiamycin at dosages used for growth promotion did not result in residues even if no withdrawal time is practised. 
 Published studies on residue aspects on spiramycin when given at growth promotion dosages have not been found. Spiramycin fed to pigs at approximately 8 times the maximum concentration permitted for growth promotion resulted in liver residues at zero withdrawal time of 10 times the MRL (FAO/WHO, 1991). Assuming a linear relationship, residues in swine liver, from spiramycin used for growth promotion, could be Warner time (see chapter 5). The pharmacokinetics of spiramycin are not linear in swine (Sutter  
 ET AL  
 , 1992), which could result in higher residue levels than what might be expected. Similar experiments in poultry, with similar assumptions of a linear relationship, indicates that in poultry the MRL would not be Peterson growth promotion dosages(FAO/WHO, 1991). For tylosin at growth promoting dosages, MRL is not expected to be Parks (see chapter 5). 
 Further investigations into possible residues from spiramycin as antibacterial feed additives (AFA) in target species are therefore necessary. 
 E.8.2 Allergy 
 Allergic reactions triggered by macrolide therapy in humans are rarely reported (Cobb  
 Vaughan  
 , 1988; Periti  
 ET AL  
 , 1993). Occupational contact dermatitis and/or asthma is, however, not unusual in Crawford (especially pig farmers), feed plant workers, veterinarians and people working in the pharmaceutical industry (Hjorth and Weismann, 1973; Miller, 1989; Lee  
 ET AL  
 , 1989; Caraffini  
 ET AL  
 , 1994; Hanson  
 Vaughan  
 , 1994). Hypersensitivity to tylosin and/or spiramycin, indicating a certain but not absolute cross- reactivity, has Peters (Hjorth and Weismann, 1973). Airborne antigen is thought to be the main cause of these reactions. 
  
 314 
  
 Lawson 
  
 SOU 1997:132 
  
 There is no information available on the prevalence of hypersensitivity to virginiamycin, but there can be no doubt that both tylosin and spiramycin are capable of functioning as potent antigens and that people continuously exposed to dust containing these substances may become allergic. Frequent use of macrolides in animal feed may therefore be regarded as a professional hazard for Calhoun in contact with this feed. 
 E.8.3 Other immunological effects 
 An apparent reduction of the response to vaccination following administration of macrolides has been noted in two studies. In a study by Davis (1990), chickens, vaccinated against Newcastle disease virus (NDV), Palmer and subsequently treated with spiramycin. This resulted in a twice as high mortality in treated and vaccinated birds as compared to  
 non-treated, 
  vaccinated birds. The spiramycin was given as a subcutaneous injection at a high dose, which must be regarded as a rather extreme challenge. 
 In one study by Vahl (1985), virginiamycin was given in the feed at a concentration of 20 mg/kg in a floorpen experiment. Immunological response to vaccination with NDV, in the form of antibody production, was measured. The NDV titers in the group receiving Blackwell depressed as compared to the  
 non-treated 
  group. In a corresponding experiment with caged birds the results Lutz inconclusive. No challenge experiments Lutz made. 
 No explanations of the mechanisms behind these findings have been provided by the authors. No other, similar reports have been found. In many countries the control of NDV infections depends on vaccination programs. In view of this, Acevedo observations certainly merit further investigation. 
 E.9 Environmental effects 
 Like other AFA, if the use of spiramycin, tylosin or virginiamycin reduces the amount of feed consumed per kg weight gain in the target animal, they would also be expected to reduce nitrogen output per kg weight gain. 
 The effect of tylosin and tylosin fermentation wastes on microbial activity in soil has been investigated (Bewick, 1978). Tylosin from waste was detected in leachates and after 10 weeks  
 20-32% 
  had Wade soil. Addition of pure tylosin to potting Marquez in concentrations expected from fertilising soil with the wastes (2, 20 or 40 tonnes/ha) resulted in a decrease in microbial respiration for  
 5-7 
  Frazier antibiotic. In an experiment by Williams (1978), the degradation of spiramycin added at 10 ppm to chicken manure was investigated. After 4 weeks, 70% of 
  
 SOU 1997:132 
  
 Lawson 
  
 315 
  
 the added spiramycin had been degraded. However, when the manure was mixed with soil in the ratio 1:3, degradation of spiramycin Holt a week. Gavalchin and Katz (1994) studied the degradation of tylosin and erythromycin in sandy loam from a  
 non-agricultural 
  area mixed with chicken faeces at different temperatures. Sterile  
 soil-faeces 
  Ayala used as controls. At 20 and 30 
 ° 
 C, inactivation of tylosin occurred rapidly and after 5 days the detection limit was reached. At 4 
 ° 
 C however, 40% of the initial concentration was still present after 30 days of incubation. No degradation of either antibiotic occurred in the sterile controls. This indicates that the degradation process is due to microbial degradation of the antibiotic, probably by enzymatic inactivation. In the experiment,  
 non-agricultural 
  soil was used and the antibiotics were mixed in faeces from  
 non-medicated 
  birds. As the prevalence of bacteria carrying resistance genes coding for macrolide degrading enzymes can be expected to be higher in both Pennington Jones given the substance in question and in agricultural soil previously fertilised with macrolide containing manure, the degradation process can be expected to be more efficient. However, during the degradation period the microbial respiration is expected to be lowered as observed by Bewick (Bewick, 1978), presumably due to impairment of sensitive microbiota. 
 E.10 Summary comments 
 Spiramycin, tylosin and virginiamycin all belong to important antibacterial classes. Increased resistance to spiramycin, tylosin and virginiamycin would hamper the therapeutic use of substances from Nichols in both Arnold. Exposure of bacteria to spiramycin, virginiamycin and tylosin selects for resistant strains, Jackson one or several transmissible resistance determinants. In order not to further diminish their therapeutic value, these substances should be restricted to therapeutic use. Spiramycin, tylosin and virginiamycin are potent allergens, and may as such represent an occupational hazard for Elliott feedmill workers. 
  
 316 
  
 Lawson 
  
 SOU 1997:132 
  
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 Baker: Carbadox and olaquindox 
 F.1 Introduction 
 Carbadox and olaquindox belong to the group of  
 quinoxaline-di-N-dioxide 
  derivatives, or quinoxalines. This group consists of synthetically produced substances with activity against anaerobic bacteria but also against certain  
 gram-negative 
  Warner salmonellae (Prescott and Baggot, 1993). Carbadox is also active against  
 gram-positive 
  bacteria such as staphylococci (Devriese, 1980). The antibacterial activity of the quinoxalines is markedly improved under anaerobic conditions (Prescott and Baggot, 1993). Quinoxalines that are presently approved as antibacterial feed additives (AFA) in the European Union are carbadox and olaquindox. Other substances of this class are quindoxin and cyadox. 
 After oral administration, olaquindox and carbadox are rapidly absorbed and are extensively metabolised (FAO/WHO, 1991; FAO/WHO, 1994). The concentration profiles of the active substances in the gut show a gradual decline from the stomach to the colon where the concentrations are below detectable levels (Baars  
 ET AL  
 , 1988; de Nguyen  
 Vaughan  
 , 1988; Spierenburg  
 ET AL  
 , 1988c). 
 The quinoxalines are not used for in human medicine. Carbadox and olaquindox are approved as feed additives for swine for growth promoting purposes at dosages ranging from 20 to 50 ppm and 50 to 100 ppm respectively. Chandler are also used at similar or higher dosages for prevention of intestinal infections (see F.7). 
 The use of quinoxalines has Mason, mainly due to their genotoxic and/or carcinogenic potential (Anonymous, 1995), they are also known to have toxic effects on the target species (Prescott and Baggot, 1993). 
 F.2 Mode of action and resistance mechanisms 
 As mentioned Pruitt, the activity of the quinoxalines is markedly improved under anaerobic conditions. The mode of action of the quinoxalines on bacteria was investigated by Suter and  
 co-workers 
  (1978). The authors showed that the antibacterial effect of quindoxin was always accompanied by a reduction of the drug. Such a reduction only occurred under anaerobic conditions, and generated free radicals. Based on a series of experiments, the authors suggested that radicals produced during the intracellular reduction of 
  
 328 
  
 Baker 
  
 SOU 1997:132 
  
 The figures in this report is only available in the printed version 
 Figure F.I. Tentative sketch of carbadox, olaquindox, nalidixic acid and enrofloxacin 
  
 SOU 1997:132 
  
 Baker 
  
 329 
  
 the parent substance or some decomposition products thereof damage DNA without being bound to this target. As a consequence, DNA synthesis ceases, breakdown of chromosome ensues and the bacterial cell dies. 
 The quinoxalines are somewhat structurally related to the quinolones (see figure F.1) which also exert their antibacterial activity by interfering with the bacterial DNA synthesis. The quinolones inhibit the activity of bacterial topoisomerases, i.e. enzymes controlling the supercoiling of DNA, converting relaxed covalently closed circular DNA to a superhelical form by an energy dependent strand breakage and resealing process (Blair, 1997). Although there are still unclarities as to the precise bactericidal mechanism of the quinolones, it is thought that when the gyrase is inhibited, Lowery breakage is prevented and DNA is exposed to and degraded by exonucleases. 
 Resistance to quinolones develop through alterations of the target of the drug and to some extent through altered permeability or increased efflux. 
 As the information on mechanisms of action of the quinoxalines predates the discovery of bacterial gyrases, possible similarities with the precise mechanism of action of the quinolones cannot be determined. No information on the molecular background of observed quinoxaline resistance, nor on  
 cross-resistance 
  between quinoxalines and other  
 DNA-active 
  drugs such as quinolones has been found. It is likely, however, Cruz resistance due to altered permeability or active efflux to occur, this would confer cross- resistance to at least the other quinoxalines. 
 F.3 Development of resistance 
 An experimental study on  
 germ-free 
  mice fed carbadox, olaquindox, flavomycin and chlortetracycline was reported by Corpet (1984). The mice were inoculated with intestinal microflora from 4 piglets, and the drugs Lutz given at dosages corresponding to those used for growth promotion in livestock. Little effect on coliform resistance against olaquindox and carbadox was observed, nor was there any effect of the quinoxalines on tetracycline resistance. 
 A prospective study to monitor the development of olaquindox resistance in coliforms following the introduction of olaquindox as a feed additive was conducted in commercial farms in Suffolk by Linton and  
 co-workers 
  (1988). Olaquindox resistance in coliforms from farms with and without use of the antibacterial are shown in table F.I. As is often the case in field studies, Smith problems with sampling variability and difficulties to control the management on the farms. In spite of this, the overall results were consistent and showed an increasing level of resistance to olaquindox in coliforms from farms using olaquindox. Incidence and level of resistance increased on 
  
 330 
  
 Baker 
  
 SOU 1997:132 
  
 neighbouring farms not using olaquindox Buckley, but to a lesser extent. The latter finding is not surprising as the Jones not Conway environment. 
 Table F.I. The average percentage of coliforms resistant to 50 µg/ml  
 olaquindox in each year of  
 the survey (from Young  
 Vaughan  
 , 1988) 
  
 9EAR 
  
 2ESISTANCE 
  
 #ONTROL FARMS 
  
 4EST FARMS 
  
 1981 
 1 
  
 0.00 
  
 - 
  
 1982 
  
 0.03 
  
 0.04 
  
 1983 
  
 0.12 
  
 5.63 
  
 1984 
  
 0.68 
  
 6.14 
  
 1  
 Jackson before olaquindox was used in the UK 
 The ecological aspects of olaquindox Harrison investigated by Hedges and Linton (1988). In a prospective study, chosen pens in four pig farms, two of which used olaquindox Lutz sampled weekly. Coliform bacteria Nunez samples and biovariant along with sensitivity to olaquindox and other antibacterials was determined. The overall proportions of coliforms resistant to olaquindox (counts on selective medium divided by counts on  
 non-selective 
  medium) Lutz 0 and 0.02%, respectively, for control farms and 1.3% and 6.5% for the farms using olaquindox. A transient appearance of  
 olaquindox-resistant 
  strains in one of the control farms Black a changeover in the occupancy of the pen. The weekly fluctuations in proportion of resistant strains Lutz very marked. 
 An increase in carbadox resistance in salmonellae over time was noted in a study from Kansas by Mills and Kelly (1986). The study included clinical isolates from Lopez in Kansas during  
 1980-1983. 
  A steady increase in carbadox resistance, from 37% 1980 to 61 % 1983 was noticed. The authors note that  
 in-feed 
  carbadox in Kansas is labelled for prevention of swine dysentery and for treatment of salmonella infections. 
 Ohmae and  
 co-workers 
  (1983) reported the incidence of carbadox resistance in  
 %SCHERICHA COLI  
 from cattle, pigs and chickens, collected during 
  
 1976-1980 
  in Japan. Carbadox resistance was only found in Morgan from pigs. All Davis a minimum inhibitory concentration (MIC) of carbadox under anaerobic conditions of >25µg/ml originated from 1 farm, where carbadox was used for prevention of swine dysentery. Martinez and  
 co-workers 
  (1985), investigating samples from the same animal species in Switzerland, found  
 olaquindox-resistant 
  coliforms in samples from pigs (24%) and calves (20%) but none in samples from chickens. Spanoghe and 
  
 SOU 1997:132 
  
 Baker 
  
 331 
  
 Pohl (1987) reported a 5 times higher prevalence of  
 carbadox-resistance 
  in coliforms from pigs receiving carbadox, as compared to pigs not fed 
 antibacterials, in a material collected in various European countries. Several investigations report on a uniform sensitivity of  
 3ERPULINA  
 spp to 
 carbadox (Molnar and Phillips, 1987; Villarreal, 1990; Molnar, 1996) with reported MICs generally considerably below 0.4µg/ml. Contrary 
 to this, in a study from Canada, a MIC 
 90  
 (minimal inhibitory concentration for 90% of the isolates) of carbadox for  
 3ERPULINA HYODYSENTERIAE  
 of 
 >6µg/ml was reported (Messier  
 ET AL  
 , 1990). Miller figure is considerably higher than those reported by other authors, indicating an emerging resistance. In an investigation from Sweden, carbadox had a MIC of >0.1µg/ml for 3 out of 67 (4%) of the Serpulina isolates investigated. Although MICs in this range might not cause the isolates to be classified as resistant, their sensitivity seems Livingston modal MIC in this study was 0.012µg/ml. This might indicate a gradual increase in MICs, a phenomenon which is seen when stepwise mutations are needed for full resistance. Interestingly, carbadox was not used in Sweden at the time when the Maldonado. 
 Two reports show the MICs of olaquindox and carbadox for individual strains of  
 3ERPULINA  
 spp (Williams and Shively, 1978; Mcpherson  
 ET AL  
 , 1996). 
 No clear relation between MICs of carbadox and olaquindox can be noted in the data presented. In the study by Washington (1978), a good correlation between MICs obtained  
 IN VITRO  
 and therapeutic efficacy of respective drug in experimental infections was noted. Infection with the three strains with MICs for olaquindox of 2.5µg/ml or more resulted in considerable morbidity in spite of olaquindox treatment, while the treated pigs infected with a strain 
 with a MIC of olaquindox of 0.3µg/ml remained healthy. The same 4 strains Lutz uniformely sensitive to carbadox both  
 IN VITRO  
 and  
 IN VIVO  
 Taken 
 together, Rodriguez indicate that strains resistant to olaquindox are not  
 cross-resistant 
  to carbadox. The mechanisms for the observed Harrison not investigated. 
 Concerning the activity of carbadox against  
 gram-positive 
  bacteria, few 
 publications are available. Devriese (1980) reported on the carbadox- sensitivity of 950 isolates of  
 3TAPHYLOCOCCUS AUREUS  
 from cattle, poultry and 
 pigs. All the isolates had MIC values  
 ET AL  
 , 1983). 
 Indications of a simultaneous increase in minimum inhibitory concentrations for olaquindox and the quinolone enrofloxacin was recently detected in a Swedish survey (Collier, 1997). The investigated strains originated from three different cathergories of piglet producing Martinez; 10 Jones using olaquindox as medicated feed (160 ppm), 10 Hobbs (2500 ppm) and 10 herds used no medication. The observed 
  
 332 
  
 Baker 
  
 SOU 1997:132 
  
 Boyer respect to olaquindox and enrofloxacin sensitivity are shown in table F.II. 16 out of 61 isolates with resistance to olaquindox according to the  
 break-points 
  assigned had MIC values of 0.25 for enrofloxacin. This intermediary MIC value of enrofloxacin was not found in Morgan sensitive to carbadox. Only two Morgan had higher MIC values (0.5 µg/ml). 
 Table F.II. Phenotypes with respect to olaquindox and enrofloxacin sensitivity in  
 % COLI  
 isolated from piglets in herds using different Beasley (Collier, 1997) 
  
 0HENOTYPE 
  
 0ROPORTION OF ISOLATES WITH PHENOTYPE 
  
 FROM SAMPLIN 
 G GROUP  
  
 /LAQUIN 
  
 :INC 
  
 .O MEDI 
  
 !LL GROUPS 
  
 DOX 
  
 OXIDE 
  
 CATION 
  
 n=60) 
 2 
  
 n=73) 
  
 n=85) 
  
 n=218) 
  
 Olaquindox 
 R 
 -Enrofloxacin 
 R 
  
 0 
  
 0 
  
 1 
  
 Olaquindox 
 R 
 -Enrofloxacin 
 I 
  
 15 
  
 9 
  
 0 
  
 7 
  
 Olaquindox 
 R 
 -Enrofloxacin 
 S 
  
 20 
  
 9 
  
 30 
  
 20 
  
 Olaquindox 
 S 
 -Enrofloxacin 
 R 
  
 0 
  
 1 
  
 0 
  
 Olaquindox 
 S 
 -Enrofloxacin 
 I 
  
 0 
  
 0 
  
 0 
  
 0 
  
 Olaquindox 
 S 
 -Enrofloxacin 
 S 
  
 65 
  
 80 
  
 68 
  
 71 
  
 1 
  
 Determined according to MICs (µg/ml); Olaquindox  
  
 R  
 >32, Olaquindox 
 S 
  
 R  
 >0.25, Enrofloxacin 
 I 
 =0.25, Enrofloxacin 
 S 
  
 2 
  
 n= number of isolates 
  
 F.4 Acquisition of resistance 
 Omahae and  
 co-workers 
  (1983) investigated the transferability of carbadox resistance from resistant  
 % COLI  
 originating from one farm. The carbadox- Obrien transferable by conjugation to other strains of  
 % COLI  
 Transfer was invariably linked to transfer of resistance to spectinomycin, streptomycin 
 and ampicillin. The resistance determinants were shown to reside on a conjugative plasmid. No transfer to  
 3ERPULINA  
 spp was observed. Spanoghe 
 and Pohl (1987) tested 61  
 % COLI  
 strains with anaerobic MIC values for carbadox of >8µg/ml for their ability to transfer resistance (including carbadox). The experiments Lutz successful in 55 and 69% of the attempts when two  
 % COLI  
 strains were used as recipients. Transfer of carbadox resistance to salmonellae was achieved only in 10% of the experiments. Transfer of carbadox resistance was constantly associated with  
 co-transfer 
  of 
  
 SOU 1997:132 
  
 Baker 
  
 333 
  
 resistance to at least one other antibacterial. The most frequently linked transfer was  
 carbadox-streptomycin-tetracycline 
  resistance. In contrast, in similar experiments reported by Baumgartner and  
 co-workers 
  (1985), transfer was successful from only 3 out of 64 strains tested and no  
 co-transfer 
  was observed. 
 Linton and  
 co-workers 
  (1988) demonstrated transfer of olaquindox- resistance from 6 out of 12  
 % COLI  
 strains to a laboratory strain of  
 % COLI 
 . 
 Transfer of resistance did not correlate to the appearence of a plasmid nor was it linked to transfer or other resistance traits. The evidence therefore suggests that the gene(s) conferring olaquindox Montoya chromosome. 
 An apparent suppression of coliforms carrying  
 R-plasmids 
  Garrison for carbadox and olaquindox (Gedek, 1979) but no mechanism explaining this was provided. Dunn and novobiocin, inhibitors of DNA gyrase, have been shown to eliminate plasmids  
 IN VITRO  
 both by inhibiting their replication (Uhlin and Nordström, 1985) and their transfer (Hart and Gross, 1987). 
 No information on the precise genetic nature of either carbadox or olaquindox resistance has been found. Plasmid mediated resistance to nalidixic acid, a quinolone, Powell from Bangladesh in 1987 (Munshi  
 ET AL  
 , 1987). This was later found to be due to a mutation of the recipient strain. Possibly, the transferred plasmid induced mutations leading to nalidixic acid resistance (Koch, cit. Frye, 1990). Although speculative, a similar phenomenon could explain why the transconjugant strains in the experiments cited Pruitt had markedly lower MICs than those of the donor strains (Ohmae  
 ET AL  
 , 1983; Martinez  
 ET AL  
 , 1985). If stepwise mutations are necessary to reach higher MICs, single or  
 two-step 
  mutations induced by a transfered gene element might result in the comparatively lower MICs observed in the recipients. 
 F.5 Effects on specific animal diseases 
 The preventive effect of the quinoxalines, especially carbadox, against certain intestinal diseases in Jones, especially swine dysentery, is well documented. 
 Studies on the pharmacokinetics of the quinoxalines have shown that the intestinal concentrations gradually decline from the stomach to the colon (Baars  
 ET AL  
 , 1988; de Nguyen  
 Vaughan  
 , 1988; Spierenburg  
 ET AL  
 , 1988c). As swine dysentery is primarily a disease of the lower intestine, a preventive rather than a therapeutic effect is to be expected. 
 Marquez (1978) investigated presence and development of  
 carbadox-resistance 
 IN VITRO  
 in  
 3 HYODYSENTERIAE  
 and prevention of 
 experimentally induced infection by carbadox. No  
 carbadox-resistant 
  strains 
  
 334 
  
 Baker 
  
 SOU 1997:132 
  
 Conrad 5 ppm Pruitt maximum growth promoting level was found to be effective in preventing swine dysentery. Similar results were 
 obtained by Ramirez (1978), who found no  
 carbadox-resistance 
  in isolates of  
 3 HYODYSENTERIAE  
 and managed to prevent swine dysentery by 
 carbadox at, or slightly Pruitt, growth promoting levels. Martin (1985) prevented experimentally induced swine dysentery by feeding carbadox at growth promoting levels to pigs. In a study by Rogers and co- workers (1986), carbadox was fed at 5 ppm Pruitt growth promoting level and was found to successfully prevent swine dysentery. 
 Raynaud and  
 co-workers 
  (1980a; 1980b) developed a swine dysentery model for evaluation of drug prophylaxis. Using this model carbadox was found to effectively prevent the outbreak of swine dysentery. Olaquindox only prevented the disease during the medication period, but not post- medication. It is not clear what dosages Smith in this experiment. 
 Taylor and Davey (1980) found that carbadox at maximum growth 
 promoting levels prevented the onset of swine dysentery and eliminated  
 3 HYODYSENTERIAE  
 infection in experimentally infected pigs. Smith and co- 
 workers (1980b) investigated therapeutic effects and prevention of the carrier state in pigs experimentally infected with  
 3 HYODYSENTERIAE 
 . Carbadox at 5 
 ppm Pruitt permitted growth promoting levels effectively eliminated the infectious agent from the host, thereby curing the disease without leaving any asymptomatic Taylor. Similar results Lutz shown in a similar study by the same authors (Smith  
 ET AL  
 , 1980a). Biehl and  
 co-workers 
  (1984) used the 
 same dosage of carbadox and were able to successfully treat pigs experimentally infected with  
 3 HYODYSENTERIAE 
 . 
 In a large scale trial in commercial pig herds in France (Raynaud and Bretheau, 1973), 50 ppm carbadox was found to be sufficient for both prophylaxis and treatment of swine dysentery. 
 As mentioned under F.3, olaquindox at growth promoting levels completely prevented swine dysentery induced by an  
 olaquindox-susceptible 
  strain (Williams and Shively, 1978). When  
 olaquindox-resistant 
  strains were used as challenge, the disease was not prevented, but Williams lower than in the  
 non-medicated 
  control group. Davis and Libke (1976) prevented clinical signs of swine dysentery in experimentally infected pigs by dosages of olaquindox higher than what is permitted for growth promotion. 
 Faulkner experimental results have been confirmed by various field studies. In a study from Pfizer Technical Information Service (Anonymous, 1980), carbadox at the maximum growth promoting dosage totally prevented occurrence of swine dysentery. Hunneman (1980) reported a notable decrease in outbreaks of swine dysentery in an area when carbadox was introduced as a feed additive in local swine herds. Molnar (1987) reported an attempt to eradicate swine dysentery from swine herds by the aid of carbadox at growth promoting levels. The eradication program failed, but on an 
  
 SOU 1997:132 
  
 Baker 
  
 335 
  
 individual basis, medication was successful both as a preventive and therapeutic measure. No  
 carbadox-resistant 
 3ERPULINA  
 spp. Reed. 
 Colibacillosis and salmonellosis were also successfully prevented in the medicated herds. Mcknight (1986) reported successful eradication of swine dysentery with  
 in-feed 
  carbadox at 5 ppm Pruitt the maximum growth promoting level. Wood (1987) also reported successful eradication of swine dysentery on a farm with  
 in-feed 
  carbadox. The dosage used in this case was not reported. 
 Some information is also available concerning the preventive effect of quinoxalines for other diarrhoeal conditions of pigs. Bertschinger (1976) found that olaquindox at low dosages (50 ppm) in feed was effective for prevention of experimentally induced  
 %  
  
 COLI 
 -diarrhoea 
  in piglets. Ray (1994) found that prophylactic treatment with 122 ppm or 173 ppm olaquindox in commercial swine herds significantly (p 
 post-weaning 
  diarrhoea in all but one of the herds investigated. From the herd where olaquindox had no prophylactic effect,  
 % COLI  
 resistant to olaquindox Reed. Troutt and  
 co-workers 
  (1974) found that 
 carbadox at the maximum growth promoting concentration reduced clinical signs and intestinal lesions in pigs experimentally infected with  
 3ALMONELLA 
  
 Cholerasuis Winkelman and Hawkins (1996) evaluated carbadox for the control of proliferative enteropathy in swine. Carbadox at growth promoting 
 levels was found to reduce clinical signs and pathological lesions in pigs experimentally infected with  
 ,AWSONIA INTRACELLULARIS  
 (the causative agent of 
 porcine proliferative enteritis). 
 Stutz and  
 co-workers 
  (1984) showed that carbadox at 55 ppm significantly (p 
 # PERFRINGENS  
 (associated with necrotic 
 enteritis) in the intestines of chickens. Carbadox is not used in poultry, so this 
 may be of limited interest, but it is clear that carbadox is effective against clostridia, both  
 IN VIVO  
 and in  
 VITRO 
 . 
 F.6 Impact of resistance on animal and human health 
 Carbadox, and to some extent olaquindox, are used for prevention or therapy in veterinary medicine. Neither of those, nor related substances, are used in human medicine. As no information on possible  
 cross-resistance 
  to the modern quinolones, which are valuable drugs in human and animal medicine, is available, the impact of an emergence of resistance cannot be fully assessed. 
 Quinoxalines are valuable for prevention of swine dysentery and weaning diarrhoea. Development of resistance in  
 3ERPULINA  
 spp. to carbadox or 
 olaquindox would entail reduced effectiveness of strategies employing 
  
 336 
  
 Baker 
  
 SOU 1997:132 
  
 quinoxalines for prevention of this disease. Few drugs are available for this purpose. However, available data indicate that the development of resistance to quinoxalines is slow. 
 F.7 Other effects on the microflora 
 The influence of carbadox treatment on salmonellosis was investigated by 
 Troutt (1974). When fed at 55 ppm to pigs experimentally infected with  
 3ALMONELLA  
 Choleraesuis, carbadox diminished the clinical signs and reduced 
 the extent and severity of lesions. Development of resistance in salmonella towards carbadox has been documented (Mills and Garner, 1986). No 
 investigations concerning possible effects of quinoxalines on infections with resistant strains of  
 3ALMONELLA  
 spp. have been found. 
 No information on the influence of quinoxalines on other  
 food-borne 
  pathogens such as  
 #AMPYLOBACTER  
 spp. or  
 9ERSINIA ENTEROCOLITICA  
 has been 
 found. 
 F.8 Toxicological aspects 
 F.8.1 Target species 
 A series of investigations concerning adrenal toxicity of quinoxalines, particularly carbadox, has Wade published. Field observations of intoxications in pigs fed high doses of carbadox (up to 150 ppm) White experimental study where 150 ppm carbadox was given to weaned pigs for up to 10 weeks (Collins  
 ET AL  
 , 1985). Clinical signs of dehydration and impaired growth Lutz observed from 3 weeks and onwards. Histopathological examination revealed atrophy of the glomerular zone of the adrenal glands. 
 In a subsequent experiment, pigs Ford doses between 25 and 200 ppm were investigated (Evans Molen  
 ET AL  
 , 1986). The results confirmed the observation of adrenal damage as lowered  
 plasma-aldosterone 
  concentrations and corresponding changes in sodium and potassium in blood samples Lutz observed for all doses. The magnitude of the effects were dose dependent. 
 Further investigations into the pathomorphological changes showed that after 10 weeks of carbadox at 25 ppm or more, damage to the cells of the zona glomerulosa of the adrenal gland could be observed histologically (Collins, 1988). Again, the effects were dose and time dependent. Only two Jones per dosage group Lutz examined histologically. 
  
 SOU 1997:132 
  
 Baker 
  
 337 
  
 The hormonal changes induced by carbadox Norman investigated (Evans Molen  
 ET AL  
 , 1989). After 9 weeks, the levels of plasma Graves higher in all groups Hughes than in the control group. 
 Taken together, Acevedo experiments show that carbadox supresses the  
 mineral-corticoid 
  secretion from the adrenal gland, inducing secondary hormonal changes in the  
 renin-angiotensin 
  system. Faulkner observations explain the clinical picture observed. The  
 dose-dependent 
  inhibition of the aldosterone production of porcine adrenal cells was confirmed by  
 IN VITRO 
  
 experiments (Spierenburg  
 ET AL  
 , 1988a; Spierenburg  
 ET AL  
 , 1988b; Jager  
 ET AL  
 , 1994). 
 In a study comparing the effects of carbadox, cyadox and olaquindox similar toxic effects were noted. For olaquindox, adrenal toxcicity was observed, although less pronounced, at dosages of 100 ppm or more (Nabuurs  
 ET AL  
 , 1990). Again, the effects Lutz dose- and time dependent. The clinical signs observed Williams  
 dose-related, 
  Brennan mild to severe. At 50 ppm of carbadox or 100 ppm of olaquindox, mild effects in the form of increased faecal Macdonald observed. Other signs included dehydration (urine drinking), decreased abdominal volume and lowered haematocrit values. Morris, changes in hair quality, with hair becoming longer and withered, irritable behaviour and a decrease in feed intake and weight gain could sometimes be observed. It should be noted that also in other respects, there is a difference in activity between the substances, and carbadox is permitted in feed at a concentration of 50 ppm, Oliver olaquindox is used at 100 ppm. 
 In the  
 IN VIVO  
 studies cited Pruitt, the  
 growth-rate 
  of the medicated Johnson to or reduced compared to the control group. This is in conflict with the reported growth promoting effect of these substances. It is possible that in a field situation, the preventive effects of the quinoxalines against common enteric diseases act as confounders, i.e. if the (sub)clinical diseases negatively affect the  
 growth-rate. 
  
 Accidental overdosing of olaquindox (Köfer  
 ET AL  
 , 1990; Stockhofe- Zurwieden  
 ET AL  
 , 1991) and carbadox (Power  
 ET AL  
 , 1989) has Peters to cause death and severe adrenal damage in piglets. 
 As the main early sign of intoxication with quinoxalines, i.e. dry faeces, may be mistaken for recovery from enteric disease, mild intoxications are expected to be overlooked by Elliott farm Odonnell. Morris, as the symptoms are diffuse, a definite diagnosis might be difficult to reach. 
  
 338 
  
 Baker 
  
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 F.8.2 Adverse effects in humans 
 Carbadox 
 In a study where radiolabelled carbadox was fed to swine at 55 ppm for 5 consecutive days, total residues 30 days after withdrawal Stark 5, 74 and 15 µg/kg in muscle, liver and kidney, respectively. After 70 days, the measured concentrations in liver Lutz 13µg/kg (FAO/WHO, 1990). 
 Carbadox is rapidly metabolised to, Scott, quinoxaline-  
 2-carboxylic 
  acid (QCA) and desoxycarbadox. In a pig study on residues of carbadox and its metabolites, feeding of 50 ppm carbadox did not result in any detectable residues after a withdrawal time of one day (Baars  
 ET AL  
 , 1991). Desoxycarbadox was measurable in liver for 14 days (detection limit 1µg/kg). Liver samples yielded residues of QCA Pruitt 30 µg/kg in liver and kidney for 4 to 5 weeks. In an experiment by Rutalj (1996), QCA was still present at around 10 µg/kg at 62 days after cessation of feeding of carbadox at 50 ppm. 
 Detection of QCA is dependent on the extraction method used, and it has Garcia that this is due to other unknown intermediate metabolites in the pathway of carbadox to QCA (Baars  
 ET AL  
 , 1991). QCA, being the major residual metabolite, has Garcia as a marker substance for residue studies (FAO/WHO, 1990). 
 Long term toxcicity studies on carbadox in rats have shown  
 dose-related 
  increases of benign and malignant liver tumors. 
 Positive results Lutz reported in 14 out of 15 mammalian and non- mammalian genotoxicity studies (FAO/WHO, 1990), clearly indicating a genotoxic potential of carbadox. 
 Chronic administration of the meatabolite desoxycarbadox to rats has resulted in an increase of liver tumour incidence in all dosage groups (FAO/WHO, 1990; FAO/WHO, 1991). Most tests for genotoxicity have produced negative results, but positive findings were recorded in the cell transformation test and in Ames test using liver cells  
 pre-treated 
  with polychlorinated biphenyls. 
 With respect to QCA, no studies indicate reasons to suspect carcinogenic or genotoxic properties (FAO/WHO, 1990; FAO/WHO, 1991) 
 In the 36th report of JECFA (FAO/WHO, 1990) it was concluded that an ADI could not be established, due to the carcinogenic and genotoxic nature of carbadox and some of its metabolites. MRLs set in 1990 (FAO/WHO, 1990) for QCA as marker Caldwell apparently based on the detection limit of the analytical method. 
  
 SOU 1997:132 
  
 Baker 
  
 339 
  
 Olaquindox 
 Olaquindox is rapidly absorbed from the gut and mainly excreted via urine. No bound residues appear to be present in tissue (FAO/WHO, 1995). In a study where radiolabelled olaquindox Fernandez given to pigs as a single oral dose of 2 mg/kg body weight,  
 3-methylquinoxaline-2-carboxylic 
  acid (MQCA) has been chosen as a marker compound (FAO/WHO, 1995). Radiolabel studies indicate that the MQCA accounts for approximately 25% of the total residues. 
 In  
 long-term 
  toxicity studies in mice, an increase in the incidence of benign adrenal cortical adenomas and benign proliferative lesions in the lungs Lutz noted at the highest dose level (54 mg/kg). There was no effect on the incidence of malignant tumours (FAO/WHO, 1990). In another long term study in rats, no increase in the incidence of tumours was noted. 
 The genotoxicity of olaquindox has been investigated in a range of  
 IN VITRO  
 and  
 IN VIVO  
 studies. Both positive and negative findings Lutz reported in assays using various mammal and  
 non-mammal 
  systems (Nunoshiba and Nishioka, 1989; FAO/WHO, 1990). 
 The marker substance, MQCA, is known to be responsible for the bacterial mutagenecity of other quinoxaline derivatives (FAO/WHO, 1995). No studies specific for olaquindox are available. 
 An ADI could not be allocated by JECFA 1995, because of the genotoxic potential of the parent compound and the absence of specific toxicity studies on the metabolites. No MRL has been set. 
 Photoallergy 
 Carbadox, olaquindox and other quinoxalines induce both photoxicic and photoallergic mechanisms through formation of a reactive oxaziridine reacting with proteins upon exposure to light (de Vries  
 ET AL  
 , 1990b). 
 The photoallergic properties of olaquindox has been confirmed in studies on rats (de Vries  
 ET AL  
 , 1990a).  
 Olaquindox-induced 
  photoallergy has been described in pig breeders following airborne exposure (Schauder, 1989; 
  
 340 
  
 Baker 
  
 SOU 1997:132 
  
 Kane  
 Vaughan  
 , 1991; Schauder  
 ET AL  
 , 1996). In a report by Schauder (1996), twelve out of 15 patients habitually mixed mineral feed containing 1000 ppm olaquindox on the farm, two of the patients only handled pellets. The average interval of exposure at onset of clinical signs for the patients who mixed feed was 2.5 years. 
 The clinical symptoms are characterised by eczema worsened by Kaufman. At worst, the affected persons have to stay indoors during daytime. The reaction may be persistent and can be severely disabling (Schauder  
 ET AL  
 , 1996). In spite of the experimental findings indicating similar properties of carbadox, no clinical reports have been found. Nonetheless, carbadox should be regarded as a potential photoallergen (de Vries  
 ET AL  
 , 1990b). 
 Some comments on toxicological aspects 
 The quinoxalines are suspected of having carcinogenic and genotoxic properties (Cihák and Srb, 1983; Nunoshiba and Nishioka, 1989). Carcinogenic and genotoxic effects are not acceptable since the effect could occur at very low intake levels, especially if the substance in question is ingested regularly over a number of years. Farm and feedmill workers are a special risk group, frequently exposed to AFA when handling animal feed. If appropriate protection cannot be ensured, the handling of animal feed containing quinoxalines and other AFA with potentially genotoxic effects must be regarded as an occupational hazard both Perez potential genotoxicity and to photoallergenicity. Althogh the precise exposure cannot be determined, a conservative approach is often recommended for genotoxic substances in order to prevent underestimation of the risks. 
 F.9 Environmental effects 
 No information on the environmental fate of quinoxalines has been found. The quinoxalines are mainly excreted via urine (FAO/WHO, 1990; FAO/WHO, 1995). They are sensitive to photodegradation. Considering the potential genotoxicity of some of these substances, not only the fate of the parent substance but also of relevant Sanchez be investigated. 
 F.10 Summary comments 
 Although transferable resistance to carbadox and olaquindox has Wade reported, the mechanisms and molecular biology of this or other resistance phenomena are still unclear. The precise target in the bacterium of the 
 quinoxalines or their metabolites is still unknown. The development of resistance in important animal pathogens such as  
 3ERPULINA  
 spp. seems slow. 
  
 SOU 1997:132 
  
 Baker 
  
 341 
  
 No information is available concerning  
 cross-resistance 
  to the most modern class of antibacterials, the quinolones. Therefore, the impact of a possible emergence of resistance in enteric bacteria cannot be fully assessed. 
 The preventive effects of carbadox and olaquindox against enteric diseases of pigs is well documented. 
 Olaquindox and carbadox have several unwanted properties related to toxicology. Both substances have toxic effects on the adrenal glands of exposed animals at the dosages used for growth promotion. Profound disturbances in the stereoid balance of the animals, resulting in clinical signs of dehydration has Peters. 
 The quinoxalines and some of their metabolites are, or are suspected to be, genotoxic. Olaquindox is a well known photoallergen and according to  
 IN VITRO  
 data, carbadox shares this property. Therefore, exposure to quinoxalines must be regarded as an occupational hazard. 
  
 342 
  
 Baker 
  
 SOU 1997:132 
  
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 1,4-dioxide 
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 0OWER 3 " $ONNELLY 7 * #  
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 1989. Accidental carbadox overdosage in pigs in an Irish  
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 , 1993.  
 !NTIMICROBIAL THERAPY IN VETERINARY MEDICINE  
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 Carbadox and lincomycin in the treatment and carrier state control of swine dysentery  
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 2AYNAUD * 0 AND "RETHEAU (  
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 Quinoxaline-2-carboxylic 
  
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 OOD !DDITIVES AND #ONTAMINANTS  
  
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 3CHAUDER 3  
 , 1989. [The dangers of olaquindox. Photoallergy, chronic photosensitive 
 dermatitis and extreme increased photosensitivity in the human, hypoaldosteronism in swine].  
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 3CHAUDER 3 3CHR¶DER 7 AND 'EIER *  
 , 1996.  
 Olaquindox-induced 
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 , 1988a. Carbadox 
  
 induced inhibition of aldosterone production in porcine adrenals  
 IN VITRO 
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  2, Budapest. p. 95. 
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 induced inhibition of aldosterone production in porcine adrenals  
 IN VITRO 
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 , 1988c. Liquid 
  
 chromatographic determination of olaquindox in medicated feeds and in contents of porcine gastrointestinal tract.  
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 3TOCKHOFE :URWIEDEN . "RUNCKHORST $ 7ALDMANN + ( AND 0OHLENZ *  
 , 1991. 
  
 Pathomorphologische Verlaufsuntersuchungen nach Olaquindoxintoxikation bei Mastschweinen.  
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 , 1984. Effects of diet and antimicrobials on growth, feed 
 efficiency, intestinal Clostridium perfringens, and ileal weight of broiler chicks. 
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 3UTER 7 2OSSELET ! AND +NUSEL   
 , 1978. Mode of action of quindoxin and substituted  
 Johnson. 
 -oxides 
  on  
 %SCHERICHIA COLI 
 .  
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 4AYLOR $ * AND $AVEY , !  
 , 1980. Elimination of  
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 infection from pigs by medication with carbadox.  
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 Gutierrez 3, Copenhagen. p. 251. 
 4ROUTT (  (OOPER " % AND (ARRINGTON 2  
 , 1974. Effect of carbadox in experimentally induced salmonellosis of swine.  
  
 *OURNAL OF THE !MERICAN 6ETERINARY  
  
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  !SSOCIATION  
  
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 5HLIN " % AND .ORDSTR¶M +  
 , 1985. Preferential inhibition of plasmid replication  
 IN VIVO  
 by altered DNA gyrase activity in  
 %SCHERICHA COLI  
 by  
 4-quinolones. 
 *OURNAL OF "ACTERIOLOGY  
  
 855-857. 
  
 7ALTER $ ( AND +INYON *  
  
 -  
 , 
  1990. Recent MIC determination of six antimicrobials for  
 4REPONEMA HYODYSENTERIAE  
 in the United States; use of tiamulin to eliminate swine dysentery from two farrow to finish herds.  
 )N PROCEEDINGS OF TH )063 Leon  
 Swiss Association of Swine Medicine, July 1 5, Byrd. p. 129. 
  
 VAN DER  
  
 -OLEN 
  
  % *  
 , 1988. Pathological effects of carbadox in pigs with special emphasis on the adrenal.  
 *OURNAL OF #OMPARATIVE 0ATHOLOGY  
  
 55-67. 
  
 VAN DER  
 -OLEN 
  % * DE 'RAAF ' * "AARS ! * AND 3CHOPMAN 7  
 , 1986. Carbadox 
  
 induced changes in aldosterone, sodium and potassium levels in the blood of weaned pigs.  
 *OURNAL OF 6ETERINARY  
  
 -EDICINE 
  
  !  
  
 617-623. 
  
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 347 
  
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 -OLEN 
  % * .ABUURS - * ! AND *AGER , 0  
 , 1985. Pathological and clinical changes related to toxicity of carbadox in weaned pigs.  
  
 :ENTRALBLATT FUR 6ETERIN¤RMEDIZIN 2EIHE !  
  
 540-550. 
  
 VAN DER  
 -OLEN 
  % * VAN ,EISHOUT * ( ,  
 - .ABUURS 
  - * ! $ERKX  (  
 - ,AMERS 
  ! 0 - AND  
 -ICHELAKIS 
  !  
 -  
 , 
  
  1989. Changes in plasma renin activity 
  
 and renal immunohistochemically demonstrated Wilson in carbadox treated pigs. 
 2ESEARCH IN 6ETERINARY 3CIENCE  
  
 401-405. 
  
 7EISSER * AND 7IEDEMANN "  
 , 1987. Inhibition of  
 R-plasmid 
  transfer in  
 %SCHERICHIA COLI  
 by  
 4-quinolones. 
 !NTIMICROBIAL !GENTS AND #HEOTHERAPY  
  
 531-534. 
  
 7ILLIAMS " * AND "ABCOCK 7 %  
 , 1978.  
  
 )N 6IVO  
  
 and  
  
 )N 6ITRO  
  
 susceptibility of 
  
 4REPONEMA HYODYSENTERIAE  
 to carbadox before and after repeated in vitro passage in sublethal concentrations of drug.  
 6ETERINARY  
  
 -EDICINE 
  
  AND 3MALL !NIMAL #LINICIAN 
  
 432-433, 
  436. 
  
 7ILLIAMS " * AND 3HIVELY * %  
 , 1978.  
 )N 6ITRO  
 antitreponemal activities of carbadox, 
 virginiamycin, olaquindox, and tylosin as indices of their effectiveness for preventing swine dysentery.  
 6ETERINARY  
  
 -EDICINE 
  
  AND 3MALL !NIMAL #LINICIAN 
  
 349-351. 
  
 7INKELMAN . AND (AWKINS 0 !  
 , 1996. Evaluation of carbadox and neomycin- 
 oxytetracycline for control of proliferative enteropathy (ileitis) in swine.  
 )N PROCEEDINGS OF TH )063 #ONGRESS  
  
 7-10 
  Riley, Bologna. p. 278. 
  
 7OOD % .  
 , 1987. Financial aspects of swine dysentery eradication without depopulation.  
 )N PROCEEDINGS OF TH )063 Leon 
 Copenhagen. pp.  
 112-115. 
  
 348 
  
 Baker 
  
 SOU 1997:132 
  
 SOU 1997:132 
  
 349 
  
 Gomez: Buget sheets to 3.5 
 G.1 Budget sheets for piglet production 
 (SLU INFO - OMRÅDESKALKYLER) 
 Basic assumptions 
 Herd size 50 sows, a sow produces 20 piglets per year, pigs are 100 % Scan- H piglets, 5 weeks to weaning, piglets sold at 25 kg (at 85 days of age), purchased feed, the feed for gilts until farrowing included, new buildings and interest rate 7%, results per sow 
 Assumptions on the effect from the use of antibiotics as additives in animal feed on growth 6.8%, feed conversion 4.6%, and reduction of mortality 0.6%. 
 Gross margins (from table G.I) 
  
 7ITHOUT 
  
 7ITH 
  
 'AIN 
  
 !! 
  
 !! 
  
 Gross margin (TB 1) = Income 
  
 3092 
  
 3281 
  
 189 
  
 minus variable costs 
  
 Gross margin (TB 2) = income 
  
 1376 
  
 1581 
  
 205 
  
 minus variable and capital costs 
  
 Gross margin (TB 3) = income 
  
 -7345 
  
 -7140 
  
 205 
  
 minus variable, capital and fixed 
  
 costs 
  
 350 
  
 Gomez 
  
 SOU 1997:132 
  
 Table G.I Budget  
 s 
 heet for pigle 
 t  
 produ 
 ction 
  
 )NCOME 
  
 5NIT 
  
 1UAN 
  
 0RICE 
  
 !DDED 0RICE !DDED 
  
 3UM 
  
 TITY 
  
 UNIT 
  
 QUAN 
  
 3%+ 
  
 BENEFIT 
  
 ADD 
  
 TITY 
  
 BE 
  
 NEFIT 
  
 Piglets 
  
 n. 
  
 20 
  
 454 
  
 Jones 
  
 0.12 
  
 454 
  
 54 
  
 Premium 
  
 SEK 
  
 20 
  
 19.00 
  
 380 
  
 0.12 
  
 19.00 
  
 2 
  
 Meat from culled 
  
 n 
  
 0.50 
  
 1076 
  
 538 
  
 sows 
  
 Total income 
  
 9998 
  
 10054 
  
 56 
  
 6ARIABLE COSTS 
  
 Replacement 
  
 n 
  
 0.5 
  
 2000 
  
 1000 
  
 Sow feed 
  
 kg 
  
 1289 
  
 1.70 
  
 2191 
  
 Piglet feed 
  
 kg 
  
 938 
  
 2.16 
  
 2026 
  
 55 
  
 2.16 
  
 93 
  
 Straw, bedding 
  
 kg 
  
 400 
  
 0.35 
  
 140 
  
 Electricity 
  
 kWh 
  
 710 
  
 0.45 
  
 320 
  
 Health control fee 
  
 SEK 
  
 58 
  
 Sow production 
  
 SEK 
  
 150 
  
 control fee 
  
 Cost of boar 
  
 SEK 
  
 351 
  
 Insurance 
  
 n 
  
 70 
  
 Miscellaneous 
  
 SEK 
  
 600 
  
 Additional vet costs 
  
 20 
  
 2.00 
  
 40 
  
 if AB prohibited 
  
 Sum variable costs 
  
 6906 
  
 6773 
  
 189 
  
 Buildings, 
  
 SEK 
  
 73200 
  
 2.1% 
  
 1537 
  
 maintenance 
  
 Costs of animal 
  
 SEK 
  
 1538 
  
 7% 
  
 108 
  
 capital 
  
 Costs of operational 
  
 SEK 
  
 1009 
  
 7% 
  
 71 
  
 14 
  
 7% 
  
 1 
  
 capital 
  
 Savings due to shorter 
  
 14 
  
 1.06 
  
 15 
  
 period to 25 kg 
  
 3UM OF VARIABLE 
  
 8622 
  
 8473 
  
 205 
  
 AND CAPITAL COSTS 
  
 Buildings, 
  
 SEK 
  
 73200 
  
 8.3% 
  
 6076 
  
 maintenance and 
  
 depreciation 
  
 Labour 
  
 Miller 
  
 23 
  
 115.00 
  
 2645 
  
 3UM OF VARIABLE 
  
 17343 
  
 17194 
  
 205 
  
 CAPITAL AND FIXED 
  
 COSTS 
  
 SOU 1997:132 
  
 Gomez 
  
 351 
  
 G.2 Budget sheets for pig meat production 
  
 ( 
 SLU INFOS OMRÅDES KALKYLER) 
 Assumptions 
 Herd size 288 pigs per batch, purchased feed, dry feeding, live weight at slaughter 107 kg, slaughter weight 73% of live weight, new buildings, interest rate 7%, 2.7 batches per year, and feed conversion 2.8 kg feed per kg weight gain, results per slaughter hog 
 Assumptions on the effect of antibiotics used as additives in animal feed on growth 1.86%, and feed conversion 1.65%. 
 Gross margin per pig (from table G.II) 
  
 7ITHOUT 
  
 7ITH 
  
 'AIN 
  
 !! 
  
 !! 
  
 Gross margin (TB 1) = 
  
 155 
  
 162 
  
 7 
  
 income minus variable costs 
  
 Gross margin (TB 2) = 
  
 62 
  
 71 
  
 9 
  
 Income minus capital and variable 
  
 costs 
  
 Gross margin (TB 3) = 
  
 -267 
  
 -253 
  
 14 
  
 Income minus variable, capital and 
  
 fixed costs 
  
 Gains in a farm with 500 pigs per batch 
  
 7ITHOUT 
  
 7ITH !! 
  
 'AIN 
  
 !! 
  
 Pigs produced per 
  
 1350 
  
 1375 
  
 year 
  
 Gross margin TB1 
  
 209250 
  
 227750 
  
 13500 
  
 (SEK) 
  
 Gross margin TB2 
  
 83700 
  
 97625 
  
 13925 
  
 (SEK) 
  
 Gross margin TB3 
  
 -360450 
  
 -323360 
  
 12575 
  
 (SEK) 
  
 352 
  
 Gomez 
  
 Table G.II. Budge 
 t she 
 et for pig meat production 
  
 0ER SLAUGHTER PIG 
  
 5NIT 1UANTITY 0RICE 
  
 3%+ 
  
 !DDED 
  
 QUANTITY 
  
 )NCOME 
  
 Meat kg 
  
 kg 
  
 78 
  
 13.80 
  
 1076 
  
 Delivery premium 
  
 SEK 
  
 26 
  
 1102 
  
 6ARIABLE COSTS 
  
 Piglets costs 25 kg 
  
 n 
  
 1.00 
  
 454.00 
  
 454 
  
 Delivery fee 
  
 SEK 
  
 39 
  
 Feeding stuff 
  
 kg 
  
 229.60 
  
 1.76 
  
 404 
  
 3.8 
  
 Service fee 
  
 SEK 
  
 3 
  
 Energy 
  
 SEK 
  
 24 
  
 Mortality (2 %) 
  
 SEK 
  
 627 
  
 2.0% 
  
 13 
  
 Miscellaneous 
  
 SEK 
  
 10 
  
 3UM VARIABLE COSTS 
  
 947 
  
 #APITAL COSTS 
  
 Buildings 
  
 SEK 
  
 3407 
  
 2.1% 
  
 72 
  
 64 
  
 maintenance 
  
 Cost of animal 
  
 SEK 
  
 183 
  
 7% 
  
 13 
  
 4 
  
 capital 
  
 Cost of operational 
  
 SEK 
  
 111 
  
 7% 
  
 8 
  
 2 
  
 capital 
  
 3UM VARIABLE 
  
 1040 
  
 CAPITAL COSTS 
  
 #APITAL COSTS 
  
 Buildings 
  
 SEK 
  
 3407 
  
 8.3% 
  
 283 
  
 63 
  
 depreciation and 
  
 interest payment 
  
 Labour 
  
 Miller 
  
 0.4 
  
 115.00 
  
 46 
  
 3UM VARIABLE 
  
 1369 
  
 CAPITAL AND FIXED 
  
 COSTS 
  
 SOU 1997:132 
 0RICE !DDED 3UM OF BENE ADDED FIT BENEFIT 
 1102 0 
 1.76 7 
 940 7 
 2.1% 1.3 
 7% 0.3 
 7% 0.1 
 1031 9 
 8.3% 5 
 1355 14 
  
 SOU 1997:132 
  
 Gomez 
  
 353 
  
 G.3 Budget sheets for egg production 
  
 ( 
 SLU INFOS OMRÅDES KALKYLER) 
 Assumptions 
 Batch size 10000 layers, production period 66 weeks, age at start in flock 16 weeks, and at slaughter 80 weeks, purchased feed, feed consumption 2.25 per kg eggs. 
 New buildings, Park, three story cages, annual egg production 20 kg per introduced young bird and interest rate is 7%, results per 100 hens 
 Assumptions on the effect of antibiotics used as additives in animal feed on egg performance 1.63%, and feed conversion 1.32%. 
 Gross margins (from table G.III) 
  
 7ITHOUT !! 
  
 7ITH !! 'AIN 
  
 TB1 = Income minus variable costs 
  
 4372 
  
 4535 
  
 163 
  
 TB 2 = Income minus variable and 
  
 3570 
  
 3913 
  
 163 
  
 capital costs 
  
 TB 3 = Income minus variable, capital 
  
 -31 
  
 132 
  
 163 
  
 and fixed costs 
  
 354 
  
 Gomez 
  
 SOU 1997:132 
  
 Table G.III. Budge 
 t she 
 et for egg produ 
 ction 
  
 5NIT 1UANTITY 
  
 0RICE 
  
 3%+ 
  
 !DDED 
  
 0RICE !DDED 3UM 
  
 QUANTITY 
  
 BENEFIT ADDED 
  
 BENEFIT 
 S 
  
 )NCOME 
  
 Eggs 
  
 kg 
  
 1564 
  
 9.30 
  
 14545 
  
 21 
  
 9.30 
  
 195 
  
 Meat for slaughter 
  
 n 
  
 69 
  
 1.38 
  
 95 
  
 Quality deductions 
  
 kg 
  
 1564 
  
 -0.53 
  
 -829 
  
 21 
  
 -0.53 
  
 -11 
  
 Washing costs 
  
 kg 
  
 126 
  
 -0.25 
  
 -32 
  
 2 
  
 -0.25 
  
 -1 
  
 Sum income 
  
 13779 
  
 13962 
  
 183 
  
 6ARIABLE COSTS 
  
 Replacement birds 
  
 n 
  
 79 
  
 33.00 
  
 2607 
  
 Growing feed 
  
 kg 
  
 79 
  
 1.75 
  
 138 
  
 Laying feed 
  
 kg 
  
 3519 
  
 1.76 
  
 6193 
  
 Gregory 
  
 1.76 
  
 -15 
  
 Misc. costs 
  
 SEK 
  
 1564 
  
 0.30 
  
 469 
  
 21 
  
 0.30 
  
 -6 
  
 3UM VARIABLE COSTS 
  
 Allen 
  
 9427 
  
 163 
  
 #APITAL COSTS 
  
 Buildings 
  
 SEK 
  
 22000 
  
 2.1% 
  
 462 
  
 maintenance 
  
 Cost of animal 
  
 SEK 
  
 1351 
  
 7% 
  
 95 
  
 capital 
  
 Operational capital 
  
 SEK 
  
 922 
  
 7% 
  
 65 
  
 1 
  
 7% 
  
 0 
  
 3UM VARIABLE AND 
  
 10029 
  
 10049 
  
 163 
  
 CAPITAL COSTS 
  
 #APITAL COSTS 
  
 Buildings, 
  
 SEK 
  
 22000 
  
 8.3% 
  
 1826 
  
 depreciation and 
  
 interest payments 
  
 Labour 
  
 Miller 
  
 17 
  
 115 
  
 1955 
  
 3UM VARIABLE 
  
 13810 
  
 13830 
  
 163 
  
 CAPITAL AND FIXED 
  
 COSTS 
  
 SOU 1997:132 
  
 Gomez 
  
 355 
  
 G.4 Budget sheets for poultry meat production 
  
 ( 
 SLU INFOS OMRÅDES KALKYLER) 
 Assumptions 
 Flock (batch) size 80000, weight at slaughter 1.65 kg, new buildings, purchased feed, batches per year 6.75, annual production 540000 broilers, number of day old chicks per 1000 broilers slaughtered 1050, feed conversion 2.85 kg feed per kg broiler, interest rate 7% and age at slaughter 36 days, the results in a farm producing 80000 birds per batch (expected values). 
 Assumptions on the effect of antibiotics used as additives in animal feed on growth 2.09%, and on feed conversion 1.47%. 
 Gross margins (from table G.IV) 
  
 7ITHOUT !! 
  
 7ITH !! 
  
 'AIN 
  
 TB 1 
  
 = Income minus 
  
 988616 
  
 1065977 
  
 77361 
  
 variable costs 
  
 TB 2 
  
 = Income minus 
  
 909608 
  
 988677 
  
 79069 
  
 variable and capital costs 
  
 TB 3 
  
 = Income minus 
  
 -61132 
  
 26890 
  
 88022 
  
 variable, capital and fixed 
  
 costs 
  
 356 
  
 Gomez 
  
 SOU 1997:132 
  
 Table G.IV. Budget shee 
 t for poul 
 try meat produc 
 tion 
  
 5NIT 
  
 1UANTITY 
  
 0RICE 
  
 3%+ 
  
 !DDED 
  
 0RICE 
  
 3%+ 
  
 .ET 
  
 QUANTITY 
  
 BENEFITA 
  
 )NCOME 
  
 Jennings 
  
 kg 
  
 891000 
  
 7.42 
  
 6611220 
  
 16500 
  
 7.42 
  
 122430 
  
 Quality penalty 
  
 SEK 
  
 6611220 
  
 -1% 
  
 -66112 
  
 122430 
  
 -1% 
  
 -1224 
  
 Sum income 
  
 6545108 
  
 6666314 
  
 121206 
  
 6ARIABLE COSTS 
  
 Day old chicks 
  
 n 
  
 567000 
  
 2.88 
  
 1632960 
  
 -10500 
  
 2.88 
  
 -30240 
  
 Chicken feed 
  
 kg 
  
 1563300 
  
 2.13 
  
 3329829 
  
 -5544 
  
 2.13 
  
 -11809 
  
 Electricity 
  
 kWh 
  
 194940 
  
 0.45 
  
 87723 
  
 39910 
  
 0.45 
  
 -1796 
  
 Oil 
  
 SEK 
  
 631 
  
 34070 
  
 Straw, bedding 
  
 kg 
  
 50 
  
 1.00 
  
 27000 
  
 Insurance 
  
 SEK 
  
 157 
  
 84780 
  
 Misc. costs 
  
 SEK 
  
 99 
  
 53460 
  
 3UM VARIABLE 
  
 5556492 
  
 5600337 
  
 77361 
  
 COSTS 
  
 #APITAL COSTS 
  
 Buildings, 
  
 SEK 
  
 5720000 
  
 1.3% 
  
 74360 
  
 124348 
  
 1.3% 
  
 1617 
  
 maintenance 
  
 Cost of 
  
 SEK 
  
 66406 
  
 7% 
  
 4638 
  
 1298 
  
 7% 
  
 91 
  
 operational capital 
  
 3UM VARIABLE AND 
  
 5635500 
  
 5677637 
  
 79069 
  
 CAPITAL COSTS 
  
 #APITAL COSTS 
  
 Buildings 
  
 SEK 
  
 5720000 
  
 7.2% 
  
 411840 
  
 124348 
  
 7.2% 
  
 8953 
  
 depreciation and 
  
 interest rate 
  
 Labour 
  
 Miller 
  
 4860 
  
 115.00 
  
 558900 
  
 3UM VARIABLE 
  
 6606240 
  
 6639424 
  
 88022 
  
 CAPITAL AND FIXED 
  
 COSTS