sou · GLB3132d1 · 1998-01-01 00:00:00 — Publications concerning possible effects on gram-positive plasmid-carrying bacteria have not been found. The transfer proteins implicated in the mechanism have not been demonstrated in gram-positives and therefore, such effects would not be expected. and co-workers (1985b) studied antibiotic resistance in the enterococci of commercial broiler flocks fed virginiamycin, bacitracin and avoparcin, and of pigs from commercial farms using in-feed tylosin and virginiamycin. In short, feeding of virginiamycin and/or tylosin resulted in increased resistance to tylosin and variable effects on virginiamycin resistance in both pigs and poultry. Feeding of bacitracin led to an increased resistance to bacitracin. Resistance to avoparcin or other glycopeptides was not tested. Unfortunately, only the pig study included a non-medicated farm as control group, outcome was somewhat variable, and the enterococci were not identified to the species level. The influence of virginiamycin on the prevalence of resistant enterococci within flocks of has recently been reported by Thal and co-workers (1996). Different from the same flock were found to share the same type of streptogramin-resistant % FAECIUM, and the prevalence of resistance to both streptogramins and ampicillin increased over time. All flocks received in-feed virginiamycin, plus other and coccidiostats. A 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 by and co-workers (1988). The results concerning olaquindox resistance in coliforms in farms with and without use of the antibacterial are shown in table 4.III. As is often the case in field studies, there were 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 neighbouring farms not using olaquindox as well, but to a lesser extent. The latter finding is not surprising as the herds were not isolated from the environment. The average percentage of coliforms resistant to olaquindox in each year of the survey (from , 1988) 4ABELL ))) 'ENOMSNITTLIG PROCENTANDEL AV KOLIFORMER RESISTENTA MOT OLAQUINDOX UNDER RESPEKTIVE STUDIE¥R FR¥N ,INTON ET AL 1 Sampled before olaquindox was used in the UK 0ROVTAGNA INNAN OLAQUINDOX ANV¤NDES I 3TORBRITANNIEN Devriese and co-workers (1993) studied the antibacterial resistance patterns for some in # PERFRINGENS isolated from various animal sources and found no notable increase in resistance between 1979 and 1992. Ohmae and co-workers (1981; 1983) reported the incidence of carbadox resistance in % COLI from cattle, pigs and chickens, collected during 1976- 1980. Carbadox resistance was only found in isolates from pigs. All carbadox-resistant isolates originated from 6 farms, where carbadox was used for preventing swine dysentery or promoting piglet growth. All isolates from one farm harboured a conjugative plasmid, carrying carbadox, spectinomycin, streptomycin and ampicillin resistance. An increase in carbadox resistance in salmonellae over time was noted in a study by Mills and (1986). The study included clinical isolates from necropsied swine in during 1980-1983. A steady increase in carbadox resistance, from 1980 to 1983 was noticed. The authors note that in-feed carbadox in is labelled for prevention of swine dysentery and for treatment of salmonella infections. A strong association between the use of avoparcin and prevalence of vancomycin resistant enterococci (VRE) in animals has been reported. and co-workers (1997) investigated poultry and pig farms in a retrospective The relative risk for occurrence of vancomycin resistant %NTEROCOCCUS 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). These findings are supported by observations from countries where avoparcin is not used, no VRE have yet been isolated from animals (Coque ET AL , 1996; Greko and , 1996). Shortly after the introduction of avoparcin, no glycopeptide resistance was 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 4.IV, recent results concerning glycopeptide resistance from the Danish surveillance system are presented. As in the above mentioned study, the isolates investigated were obtained without the use of antibiotic containing media in the course of a monitoring programme. Studies using media favouring resistant isolates indicate that enterococci with high level resistance to glycopeptides (VRE) are widespread among animals including pets and horses (, 1994; , 1995b; Devriese ET AL , 1996). The lack of earlier data on VRE in animals precludes conclusions on whether the resistance trait was present in animal populations at the time of introduction of avoparcin in animal husbandry. As mentioned in 4.3.2, 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 using selective techniques (Coque ET AL , 1996; Greko, 1996). Thus, in the absence of avoparcin, the prevalence of VRE in animals is, at most, very low. Frequency of resistance to glycopeptides (vancomycin) in % FAECIUM from different sources1 4ABELL )6 &REKVENS AV RESISTENS MOT GLYKOPEPTIDER VANCOMYCIN HOS Enterococcus faecium FR¥N OLIKA PROVTYPER 1 Data from DANMAP (1997). In samples from food, only high-level resistance was reported whereas in animals samples, all resistant isolates are included $ATA FR¥N $!.-!0 &¶R PROVER FR¥N LIVSMEDEL HAR ENDAST H¶GGRADIG RESISTENS RAPPORTERATS FR¥N DJURPROVER INKLUDERADES D¤REMOT ALLA RESISTENTA ISOLAT It has often been assumed that resistance observed in animal bacteria is entirely a result of the use of therapeuticals. A comparison between the situation in countries where macrolides are used both for therapy and as , and where they are only used for therapy is therefore of interest. In table 4.V, results from investigations in , and are presented. Spiramycin, being a 16-membered macrolide was chosen in the example as indicator of constitutively expressed ERM-genes, encoding for MLSB type resistance (see annex E). A lower prevalence of resistance is reported from and , using only therapeutical macrolides compared to where they are also used as . The tables above illustrate phenotypic expression of resistance. However, information on phenotype at best only permits inferential conclusions about the genes conferring these resistance traits. All the resistant isolates from and were cross-resistant to erythromycin (a 14-membered macrolide). The enterococci are inherently resistant to moderate concentrations of lincosamides (e.g. clindamycin) but high level resistance can be acquired (, 1990). For all the Swedish isolates that were resistant to 16-membered macrolides, values higher than for the remaining group of isolates were noted. This leads to the assumption that the resistance shown is mediated by constitutively expressed ERM-genes. MICs for clindamycin were not given for the Finnish isolates. Resistance to MLSB antibacterials (indicated by spiramycin resistance) in enterococci from various species and countries (From Greko, 1996; DANMAP, 1997; Greko, 1997; MAF, 1997) 4ABELL 6 2ESISTENS MOT -,3" ANTIBIOTIKA INDIKERAT AV SPIRAMYCINRESISTENS HOS OLIKA DJURSLAG &R¥N '$!.-!0 '-!& Further information on frequency of resistance to can be found in annexes A-F. 4.3.4Some comments on study design Studies performed to date are extremely difficult to compare. Selection criteria, methods used and definition of resistance vary. Further, a serious limitation of most studies on antibacterial resistance is the restricted range of microbes surveyed (Salyers, 1995). This is particularly true if the question asked is how the use of a substance affects the evolution and spread of resistance genes in a certain environment. Inclusion of numerically predominant organisms, such as certain anaerobic genera, would give a more complete picture. For practical reasons, most studies focus on easily cultured bacteria. Until further knowledge about the role of the predominant part of the flora is obtained, we have to accept that much information concerning gene transfer in natural settings is missing (Salyers, 1995). Some species are inherently (naturally) resistant to certain antibacterial substances. For example, # PERFRINGENS and % FAECIUM have been reported to be inherently resistant to flavomycin and enterococci, apart from % FAECIUM are inherently resistant to streptogramin A. Such species will be favoured if a selective pressure is applied. Other species, that are inherently susceptible to the substance in question, may become resistant either by mutation or by the acquisition of already existing resistance genes. When the aim is to assess the influence of antibacterial usage on the prevalence of antibacterial resistance, bacterial populations with a potential for acquiring resistance should be targeted. Enterococci are inherently susceptible to most and are known to easily acquire resistance genes. In view of this, and bearing the above mentioned limitations in mind, the choice of enterococci as one of the indicators of the development of resistance seems acceptable for most . The study time in both experimental and field exposure studies is generally short. Dissemination of antibacterial resistance will be governed by two main factors; presence of the antibacterial in concentrations high enough to inhibit susceptible bacteria and presence of resistance traits (intrinsic or acquired) in bacteria. If either is continually absent, a resistance problem will not emerge (, 1996). Acquisition of the relevant gene and its subsequent adaptation to a new host bacterium may take time (years). As closed experimental herds are often used, resistance may in fact never appear, even if it would with time in a field situation where herds are expected to have numerous direct and indirect contacts with potential gene reservoirs. Given the presence of both a selective pressure and resistance, the resistance trait will be selected for and propagated. The spread of resistant bacteria themselves, as well as cell to cell spread of genes conveying resistance, further adds to the equation. Longer study periods (years) would be expected to give more applicable results, but may be impractical for other reasons. In view of the long standing debate on usage of and resistance, there are surprisingly few studies including data on prevalence of resistance against the substances in question. Even less data is available on development over time. Well-planned screening studies, conducted in several countries and combined with data on consumption of antibacterial feed additives in these countries (amount given to different animal species), as well as therapeutic usage might provide at least some of the information on exposure necessary for an accurate analysis of potential hazards and associated risks. Programmes, such as the Danish monitoring (DANMAP, 1997), as well as the one lately initiated by Commission, are therefore to be commended. Various types of studies show that exposure of bacterial populations to in approved dosages favours resistant clones or subpopulations. 4.4Acquisition of resistance in bacteria Acquired resistance can arise in a bacterium through mutation(s) in existing genes or through the uptake of a pre-existent gene. In the first case, the resistance trait will be confined to the mutant clone. Spread of resistance will depend on the ability of that clone to multiply (vertical transmission) and infect new hosts. In the second case, the resistance trait can also spread to other bacterial clones, to other bacterial species and genera (horizontal transmission). Following uptake, acquired resistance genes may be modified, or optimised, by means of mutation. Once the optimal configuration has been accomplished, change would be expected within the gene, although transfer to a new host species or genus may require some adaptation for maximum expression. Silent resistance genes, i.e. non-expressed genes, may be activated by mutations. 4.4.1Transfer of resistance genes Pre-existent resistance genes are mostly associated with transfer elements such as plasmids or transposons, residing in plasmids or on the chromosome. Detailed studies concerning transfer elements associated with relevant genes have only been published for antibacterials in clinical use. As acquired resistance has been reported for other as well, there is no reason to believe that resistance against these substances would be any different in this respect. In table 4.VI, described genes conveying resistance to macrolides and/or streptogramins and examples of their localisation have been compiled. The plasmids and transposons mentioned are often of a conjugative type, meaning that in addition to the resistance genes, they carry the information necessary to initiate and complete their own transfer to new hosts. Transfer of resistance determinants with relevance for has been shown IN and, for some, IN VIVO. Resistance to the A component of streptogramins is mediated by various genes carried on plasmids. The VATB gene has been shown to transfer between coagulase-negative staphylococci and 3 AUREUS (Allignet ET AL , 1996). Resistance to streptogramin B and 16-membered macrolides (spiramycin, tylosin) is mostly mediated by constitutively expressed ERM-genes of different (Leclercq and Courvalin, 1991b). The product of the gene, a ribosomal methylase, alters the ribosome slightly with the result that macrolides, lincosamides and streptogramin B cannot bind (MLSB resistance). The ERM-genes in staphylococci are often inducible only by 14- membered macrolides (erythromycin), meaning that the methylase will only be produced in presence of erythomycin. The gene can convert to constitutive expression by a single or two point mutation rendering the bacterium phenotypically resistant to all macrolides, to lincosamides and to streptogramin B (see annex E). Poyart-Salmeron and co-workers (1990) showed by way of IN mating experiments that a self-transferable plasmid designated pIP811 carrying an ERMB gene (also carrying resistance determinants for chloramphenicol, tetracycline and streptomycin) could transfer from ,ISTERIA MONOCYTOGENES to, among others, % FAECALIS and vice versa. The donor efficiency3 depended on the combination of donor-recipient tested and varied from 10-3-10-9 with enterococci and streptococci being the most and staphylococci the least efficient. The plasmid, originally identified in ,ISTERIA MONOCYTOGENES, was suggested to have originated from streptococci or enterococci as it is similar to pAMb1, the prototype for broad-host range plasmids in those genera. Acquisition of macrolide resistance through transfer of the chromosomally carried transposon Tn harbouring MLSB (ERMB), kanamycin (APH!3) and tetracycline (TETM) resistance determinants from % FAECALIS to , MONOCYTOGENES has been demonstrated both in by mating experiments and IN VIVO in gnotobiotic mice (Doucet-Populaire ET AL , 1991). and IN VIVO transfer frequencies were around 10-7 and 10-8, respectively. In both cases, subinhibitory concentrations of tetracycline increased the transfer frequency around 10 times. 1The donor efficiency expresses the number of successful transmissions from donors to recipients per total number of recipients. The donor:recipient ratio in experiments is often 1:1 Examples of resistance to macrolides and streptogramins by different mechanisms and genes1 Exempel på resistens mot makrolider och streptograminer, via olika mekanismer och gener1 Examples of bacterial hosts S. aureus, coagulase negative Tn551, pAMb1, S. aureus, S. intermedius, S. Tn917, Tn1545, hyicus, Streptococcus spp, S. pneumoniae, E. faecalis, pE194, plM13, S. aureus, coagulase negative staphylococci, S. hyicus, S. aureus, S. epidermidis 1Data compiled from Leclercq and Courvalin, 1991a; 1991b; Brisson et al, 1988; et al, 1987; Eady et al, 1993; Weisblum, 1995; Mullany et al, 1995; Allignet et al, 1996; Tauch et al, 1995 2 Resistance phenotype; MLSB = macrolide-lincosamide-streptogramin B; S B = streptogramin B; SA = streptogramin A 1Data sammanställda från Leclercq and Courvalin, 1991a;1991b; Brisson et al, 1988; et al, 1987; Eady et al, 1993; Weisblum, 1995; Mullany et al, 1995; Allignet et al, 1996; Tauch et al, 1995 2Resistensfenotyp; MLSB = makrolid-linkosamid-streptogramin B; SB= streptogramin B, SA = streptogramin A In the above cited experiment, germ-free mice were used. McConnell and co-workers (1991) used mice with a complex microflora functionally equivalent to that of conventional mice. They investigated transfer of the plasmid pAMb1, carrying ERMB. Transfer of the plasmid from ,ACTOBACILLUS REUTERI to % FAECALIS was detected within days after birth in the offspring of the mice inoculated with the donor strain. The parent animals were given lincomycin at subtherapeutic concentrations. No transfer was detected in offspring of mice not given lincomycin. The fact that conjugal transfer was easily detectable within a relatively short time period in both IN VIVO experiments cited above confirms that the intestinal environment is highly conductive to conjugal transfer, even when the donor and recipient are of different genera. Further, they show that IN results on transfer and transfer rates can be predictive of IN VIVO results. Transferable glycopeptide resistance is usually mediated by the gene clusters VANA or VANB. These are generally located on plasmids and/or transposons (and Courvalin, 1993). High level resistance to glycopeptides mediated by the VANA-gene cluster has been detected in % FAECIUM, other enterococcal species (and Courvalin, 1993), /ERSKOVIA TURBATA and !RCHANOBACTERIUM HAEMOLYTICUM (ET AL , 1995). cluster is mostly associated with the conjugative transposon Tn and/or self-transferable plasmids (, 1996). Transfer of the VANA-gene cluster has been shown IN from % FAECIUM to ,ISTERIA MONOCYTOGENES, 3TAPHYLOCOCCUS 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 were 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. Resistance to glycopeptides mediated by the VANB gene cluster has, in relation to , attracted less attention. The VANB-gene cluster is transferable either directly from the chromosome by a transposon (Tn ) or through plasmids (Quintiliani and Courvalin, 1994; Woodford ET AL , 1995; Quintiliani and Courvalin, 1996) at a low frequency. The VANB-gene cluster has been found in % FAECALIS, % FAECIUM and, recently, in 3 ( and Courvalin, 1993; Poyart ET AL , 1997). The gene usually confers inducible resistance to glycopeptides by a mechanism similar to that of the VANA gene- cluster. The VANB-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 (and Courvalin, 1993). information, the VANB gene cluster does not seem to be inducible by No information is available on the capacity of ardacin to induce )N as well as during therapy, mutants expressing the VANB gene constitutively have been reported (, 1993; , 1995). Recently, transfer experiments with a strain expressing VANB constitutively were reported (, 1997). The resulting transconjugants were either of constitutive or of inducible type. The use of a non-inducing antibacterial such as avoparcin, and possibly ardacin, could favour strains harbouring VANB-gene clusters with the mutation required for the gene to be constitutively expressed should the gene be present in animal populations or their environments. Further information is needed on this topic. 4.4.2Bacterial interspecies transfer of resistance genes As evident from 4.4.1, similar or identical resistance genes can be found in various bacterial species, indicating that transfer of resistance genes between different bacterial species is not uncommon in nature. Conjugal transfer systems are able to cross genus, phylogenetic, and even kingdom lines (Amabile-Cuevas and Chicurel, 1992). There are broad host range genes that can be expressed in a variety of species as well as broad host range gene transfer elements. As an example, high level resistance to MLSB antibacterials in a clinical isolate of % COLI was characterised by and co-workers (1988). The resistance trait was found to be due to the presence of an ERM-gene highly homologous to ERMB, previously described in % FAECALIS and 3 SANGUIS The occurrence of ERMB-like genes in enterobacteria was confirmed in other % COLI strains and in +LEBSIELLA. The findings indicate that the gene pool of gram-positive cocci has extended to gram-negative bacteria, that such transfer can occur in nature and that the extension is recent. Further inferential evidence of such trans-gram promiscuity exists for other resistance genes (for a review see Courvalin, 1994). 4.4.3Co-transfer of genes and multiresistance A plasmid or transposon may gradually acquire one gene after another. Integrons and transposons appear to be largely responsible for this phenomenon. When they are located on the same transferable element, the transmission of different genes is linked and transfer leads to co-resistance. This means that a bacterium concurrently becomes resistant to two or more different antibacterials, mediated by different resistance mechanisms, governed by different genes. Co-transfer, linked transfer or transfer EN BLOC are different expressions for this phenomenon. The transmission of resistance genes with relevance for can be linked to the transfer of other genes. The % COLI plasmid pNV13 conveys resistance to carbadox, streptomycin, spectinomycin and ampicillin (Ohmae ET AL , 1981; Ohmae ET AL , 1983). Conjugal transfer of resistance to streptogramin A, between 3 AUREUS strains, linked to lincosamide, trimethoprim and penicillin resistance has been reported (Allignet and El Solh, 1995; Allignet ET AL , 1996). Enterococcal plasmids from several host species have been shown to harbour and transfer MLSB, kanamycin, streptomycin and, sometimes, tetracycline resistance (, 1985; LeBlanc ET AL , 1986; , 1987). The transposon Tn has been shown to carry and transfer resistance genes to kanamycin, macrolides and tetracycline (Doucet- Populaire ET AL , 1991). conveying resistance to glycopeptides and macrolides have been found to co-reside on plasmid pIP819 (Leclercq ET AL , 1989). Other examples of co-transfer, have been given in 4.4.1. A similar linkage between different genes has been shown between some resistance and virulence genes, such as haemolysin- or toxin-encoding genes. Co-transfer of virulence and resistance genes has been well studied in enteric pathogens (among others Franklin and Möllby, 1983). Less information is available concerning gram-positive bacteria. and co-workers (1987) demonstrated IN co-transfer of genes encoding for macrolide and tetracycline resistance and haemolysin production from % FAECALIS to "ACILLUS SUBTILIS.