sou · GLB3132d3 · 1998-01-01 00:00:00 — 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 been found in staphylococci, but without presenting any data or information on how these results had been obtained. Flavomycin is reportedly mainly against gram-positive bacteria. However, several publications indicate that the compound is sometimes against gram-negative organisms, such as 3ALMONELLA spp. and %SCHERICHIA COLI (and Moeller, 1976; and Moeller, 1977b; and Moeller, 1977a; , 1996). Unfortunatley, pure flavomycin for laboratory use is not commercially available. Thus, no experiments could be performed to clarify this issue. The figures in this report is only available in the printed version Tentative sketch of flavomycin C.3 Development of resistance 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 and Moeller (1977a) shows a significant (p<0.001) increase in the percentage of flavomycin-resistant % COLI isolated from calves fed flavomycin at growth promoting levels, as compared to non-medicated calves. Since flavomycin is mainly against gram-positive bacteria it would be interesting to see similar data on, for instance, enterococci. Prevalence of resistance to flavomycin in various bacterial species * total number of strains: C.perfringens = 142, C. sporogenes = 6, C. botulinum = 3 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 %NTEROCOCCUS FAECIUM (Dutta and Devriese, 1984). However, susceptible strains of % FAECIUM have been reported (DANMAP, 1997). discrepancies could 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 and (1984) investigated the effect of flavomycin on plasmid-mediated antimicrobial resistance 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 wall causing 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 were 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 conclusions was conducted by Sepulchre (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 (1975), also showed decreased transfer frequency of some, but not all, R plasmids between % COLI strains in the presence of flavomycin. 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 isolates was lower in the flavomycin-fed group than in the control group. It is doubtful, though, whether this mouse 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 (Huber, 1979). If the presence of R-plasmids in some gram-negative organisms causes alteration of the cell wall surface, this may facilitate the uptake of flavomycin into the cell and thereby render these cells susceptible to flavomycin. C.5 Effects on specific animal diseases The fact that all # PERFRINGENS seem to be flavomycin-resistant may cause some concern regarding necrotic enteritis in poultry. (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. Thus, at present no adverse effects would be expected from an increased resistance to flavomycin among human and animal bacteria. Several other phosphorus-containing glycolipids with similar chemical properties and antibacterial have been described (Huber and Nesemann, 1968; Meyers ET AL , 1968; Huber, 1979). Some of these are prasinomycin, diumycin (macarbomycin), 11837 R.P, quebemycin, 19402 R.P., ensanchomycin, prenomycin and pholipomycin. As these substances are all highly against 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, with increasing 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 A few studies on the effects of in-feed flavomycin on 3ALMONELLA colonisation have been published (and , 1975; and Moeller, 1976; and Moeller, 1977b; , 1982; , 1991). 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 The results indicate that the use of flavomycin reduced the duration and prevalence of 3ALMONELLA shedding in both pigs and calves. experimental groups are rather small and, 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 whether this is unusual or not. If the infecting strain is sensitive to flavomycin, feeding this drug would be expected to reduce shedding. This might be regarded as therapy and not growth promotion, though. In the study by and 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 were 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 given as to the design of this study, regarding culturing methods, presentation of the results and length of study period (see annex B). and 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. and (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 and co-workers, where selective 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. The toxicity of flavomycin is very low, even after intravenous administration (Huber, 1979), and is only absorbed from the gut in small quantities (Sambeth ET AL , 1974).Therefore, no toxic effects would be expected in either animals or humans due to the usage of flavomycin in animal feed. 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. Like other , 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 (Jagnow, 1978). According to Jagnow (1978), it takes 17 weeks for complete aerobic degradation of 10 ppm flavomycin in fresh manure, and 2 weeks in a soil mixture. Galvachin and (1994) found that at or more, flavomycin, in a soil-faeces mixture, was degraded within 25 days. At 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, areas with a temperature below . 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 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. Brenes, A., Trevino, J., Centeno, C. and Yuste, P., 1989. Influence of peas (Pisum 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. 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. DANMAP, 1997. Consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in . , J. and Moeller, M.W., 1976. Influence of bambermycins on Salmonella infection and antibiotic resistance in swine. , J. and Moeller, M.W., 1977a. Effect of bambermycins on Escherichia coli and antibiotic resistance in calves. , J. and Moeller, M.W., 1977b. Influence of bambermycins on Salmonella infection and antibiotic resistance in calves. Devriese, L.A., 1980. Sensitivity of staphylococci from farm animals to antibacterial agents used for growth promotion and therapy - a ten year study. Annales de Recherches Veterinaires. Devriese, L.A., Daube, G., Hommez, J. and Haesebrouck, F., 1993. In vitro susceptibility of Clostridium perfringens isolated from farm animals to growth- enhancing antibiotics. Devriese, L.A. and Haesebrouck, F., 1996. Susceptibility of enterococci and intestinal streptococci from pigs to the growth enhancing antibiotics flavomycin and avoparcin. In proceedings of: 14th 7-10 , . Dutta, G.N. and Devriese, L.A., 1982. Susceptibility of fecal streptococci of poultry origin to nine growth-promoting agents. Applied and Environmental Microbiology. Dutta, G.N. and Devriese, L.A., 1984. Observations on the in vitro sensitivity and resistance of positive intestinal bacteria of farm animals to growth promoting antimicrobial agents. , E. and Blobel, H., 1973. Testing the antigenicity of Flavomycin. Zentrablatt fur Veterinarmedizin. Gavalchin, J. and , S.E., 1994. The persistence of fecal-borne antibiotics in soil. and , D.J., 1984. Effect of bambermycins, in vitro, on plasmid- mediated antimicrobial resistance. , , , D.J., Quarles, C.L., , J.M. and McKinley, G.A., 1982. Effect of bambermycins on quantity, prevalence, duration, and antimicrobial resistance of Salmonella typhimurium in experimentally infected broiler chickens. Huber, G., 1979. Moenomycin and related phosphorus-containing antibiotics. In: F.E. ), Antibiotics; Vol. 5, pt 1. Huber, G. and Nesemann, G., 1968. Moenomycin, an inhibitor of cell wall synthesis. Hudd, D.L., 1983. The addition of antibiotics to feedingstuffs. In : Pharmacological basis of large animal medicine. Eds Lees, J.A.B.P., Yoxall, A.T. Balckwell Scientific Publications, , UK. , F., Lalande, F., lHospitalier, R., Salvat, G. and Bennejean, G., 1991. Effect of four antibiotic additives on the Salmonella contamination of chicks protected by an adult caecal flora. Jagnow, G., 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. Meyers, E., Miraglia, G.J., , D.A., Basch, H.I., , F.E., Trejo, W.H. and Donovick, R., 1968. Biological characterization of prasinomycin, a phosphorus- containing antibiotic. , P., Laub Kupersztejn, R., , J. and van Robaeys, G., 1975. Effets de la flavomycine et de quelques agents antiparasitaires sur une souche colibacillaire hébergeant divers facteurs-R.Annales de Médecine Vétérinaire119:. Sambeth, W., , , , G. and Nesemann, G., 1974. Evaluation of the saferty of flavomycin in poultry. In proceedings of: XV World's 11- 16, . Sepulchre, M., 1979. Flavomycine: inhibition de lantibioresistance chez les enterobacteries. Bulletin Mensuel de la Societé Vétérinaire Pratique de . and , J.F., 1975. The effect of feeding diets containing permitted antibiotics on the faecal excretion of Salmonella typhimurium by experimentally infected chickens. Stutz, M.W. and , G.C., 1984. Effects of diet and antimicrobials on growth, feed efficiency, intestinal Clostridium perfringens, and ileal weight of broiler chicks. 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. van Heijenoort, Y., Leduc, M., , H. and van Heijenoort, J., 1987. Effects of moenomycin on Escherichia coli. , W., 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. Whiley, Chichester (Symposium 207), 16-18, . : Ardacin and avoparcin Ardacin and avoparcin are glycopeptide antibiotics. 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 (, 1990). Glycopeptides are against gram-positive bacteria such as staphylococci, streptococci, enterococci, corynebacteria, clostridia and ,ISTERIA spp. Presently known glycopeptides are not against gram- negative bacteria because the antibiotic molecules are unable to pass through the outer membrane and hence cannot reach their target (, 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 accordance with Directive 97/6/EC, presently not approved in the EU. Similarly, ardacin has been approved 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 well 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 . The assembly of precursors by cross linking takes place on the outer surface of the cell membrane. At this step, glycopeptides inhibit cell wall formation by binding to peptide stems of the precursor ending with D-alanine-D-alanine (D-Ala-D-Ala). The figures in this report is only available in the printed version Tentative sketch of ardacin, avoparcin, vancomycin and teicoplanin The figures in this report is only available in the printed version 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 (, 1996). Bacteria producing peptidoglycan precursors not ending in D-Ala-D-Ala will be partially or completely resistant to glycopeptides. 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 %NTEROCOCCUS GALLINARIUM group are resistant to low levels of vancomycin but sensitive to teicoplanin (VanC phenotype). These enterococci produce peptidoglycan precursors where D-Ala is substituted by D-serine (D-Ser). Vancomycin, but not teicoplanin, have a lower affinity for precursors ending in D-Ser (, 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 In a study by (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 were isolated from both avoparcin-fed birds and from the control group. Similarly, vancomycin-resistant strains were detected in all groups. The author stated that there was no evidence of cross-resistance between avoparcin and other antibiotics tested (vancomycin, among others). The results are not presented in a form that makes it possible to substantiate this claim. No species identification of resistant isolates was 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 non-supplemented feed. The incidence of resistance to therapeutic antibiotics, expressed as the Antibiotic Resistance Index (), was higher (p=0.003) in all groups receiving antibacterials, as compared to the control This increase could be associated with an increase in the proportion of % FAECIUM in the enterococcal population of the treated birds. 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 (in birds given different antibacterial feed additives and in control birds). An association between the use of avoparcin and prevalence of vancomycin resistant enterococci (VRE) in animals has been reported (Aarestrup, 1995; , 1995a; , 1995; van den Bogaard ET AL , 1996). In studies from where avoparcin has never been used, and from where avoparcin has not been used for 10 years, no VRE were found in samples from animals when selective techniques were used (Coque ET AL , 1996; Greko, 1996). Thus, in the absence of avoparcin, the prevalence of VRE in animals is, at most, very low. and co-workers (1997) investigated poultry and pig farms in in a retrospective cohort study.