sou · GLB3132d3 · 1998-01-01 00:00:00 — 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. 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 D.I, results concerning prevalence of glycopeptide resistance from and are presented. The results in these studies were 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 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. Table D.I Prevalence of glycopeptide resistance D.4 Resistance 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 VANA-gene cluster (and Courvalin, 1993; , 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 VANB-gene cluster ( AL , 1996). The recently described VanD phenotype is encoded by a gene cluster designated VAND (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 vanSB-vanRB (, 1996). encoded for by Tn (the VANA-gene cluster) and Tn (the VANB-gene cluster), their products and main functions (based on information from , 1996) &UNCTION OF RESPECTIVE transcription of vanH, vanX and vanA/B Open reading frame, Open reading frame, Production of VanH, VanA and VanX (VanHAX), encoded for by the genes in the operon VANHAX, is normally inducible. This means that they are 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 VANR). Recent evidence indicate that VanS, encoded for by the VANS 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 VANHAX operon (, 1997). VanA- type resistance is induced by vancomycin, teicoplanin and apparently also by avoparcin (, 1995b). The ability of ardacin to induce VanA-type resistance has 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 antibiotics such as moenomycin (flavomycin), bacitracin, daptomycin, penicillin, cephalotin have been presented (and , 1995). Recent evidence indicate that apart from glycopeptides, only flavophospholipol (moenomycin/flavomycin) has the capacity to act as an inducer (, 1996). The regulatory system for production of VanHB, VanB and VanXB in strains with VanB-type resistance is similar to the above described. However, the regulatory system VanRB- VanSB is only activated by vancomycin, explaining the phenotypic susceptibility of VanB strains to teicoplanin (and Courvalin, 1996). Teicoplanin resistant derivatives of VanB type strains have been reported (, 1993; , 1995) These isolates are likely to be the result of spontaneous mutations altering the specificity of the VanSB sensor domain. Both the VANA and VANB gene clusters 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 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 VANB-gene cluster has, with respect to avoparcin and ardacin, attracted less attention. The VANB-gene cluster is transferable either directly from the chromosome by a transposon (Tn1547) or through plasmids (Quintiliani and Courvalin, 1994; Woodford ET AL , 1995b; Quintiliani and Courvalin, 1996) at a low frequency. gene cluster has been found in % FAECALIS, % FAECIUM and, recently, in 3 (and Courvalin, 1993; Poyart ET AL , 1997). As mentioned, 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). According to available information, the VANB- gene cluster does not seem to be inducible by avoparcin. No information is available on the capacity of ardacin to induce VANB. Selection of mutants expressing the VANB-gene constitutively have been reported both from IN studies as well as in clinical isolates ( AL , 1993; , 1995). Recently, transfer experiments with strains 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. VRE harbouring the VANA gene cluster, have been isolated from humans, both in hospitals and community, from swine, rabbits, dogs, cats, horses chickens, , pheasants, ducks, foods of animal origin and sewage (, 1994; Torres ET AL , 1994; , 1995b; Chadwick ET AL , 1996; Devriese ET AL , 1996; DANMAP, 1997). A polyclonal nature of the VRE strains has been demonstrated (, 1995b). As shown in table D.II, the VANA 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 been used to colonise mice experimentally (Whitman 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 Bogaard ET AL , 1997). The prevalence of VRE in , farmers, slaughterers and urban residents was found to be , , and respectively. Further investigations showed that VRE isolated from one of the farmers and his could not be differentiated by phenotypic or genotypic (pulsed- field gel electrophoresis) methods. Investigations of the VANA-gene cluster by polymerase chain 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 VANX and VANY 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. gene cluster contains 9 genes (7 and two transposition genes). Between those genes are intergenic, non-coding regions. The coding regions would be expected to be highly conserved once their 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 polymerase chain reaction (PCR) and sequencing of the genes and/or their intergenic regions (, 1996; Haaheim ET AL , 1997; , 1997). In the Norwegian study (Haaheim ET AL , 1997), PCR for the VANA and VANB genes combined with restriction fragment analysis of a long PCR covering the entire gene cluster and sequencing of the intergenic VANS-VANH region were used to analyse the vanA gene cluster from VRE of Norwegian poultry and humans of various nationality (Swedish, Norwegian and American). In 9/12 human isolates and 7/10 poultry the results were identical, indicating horizontal transfer of the gene cluster. and co-workers (1997), investigated 37 VRE isolates from one UK hospital and 36 VRE isolates from poultry meat bought in national By PCR, three intergenic regions of the VANA-cluster were investigated (VANS-VANH, VANX-VANY and VANY-VANZ). VANX, VANY and VANZ was also determined. In the chicken isolates, all three investigated genes were amplified as well as the three intergenic regions. Evidence of a not previously described insertion sequence in various locations of the intergenic region between VANX and VANY 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 (, 1993; Handwerger ET AL , 1995), as the VANY region as well as the intergenic regions VANX-VANY and VANY-VANZ failed to amplify with the primers used. Only the regions VANS-VANH, and the genes VANX and VANZ were amplified, indicating the presence of the VANX and VANZ genes but not the VANY gene. These results may be caused by an insertion or a deletion in the primer binding site for the VANY gene. In a recent publication by and co-workers (1997), a novel insertion sequence element designated IS located within the VANY gene, was reported in one out of 124 clinical isolates, indicating the occasional finding of such variations. The fact that all the gene clusters from human strains in the study by and co- workers (1997) apparently had a deletion and/or insertion in the VANY 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 multiple origin, this study does not seem suitable for any general conclusions about the relation between human and animal strains. Another report, cited above, of comparisons between the VANA-clusters of different strains indicates the transmission between animal and man (van den Bogaard ET AL , 1997). VRE isolated from a farmer and his could not be differentiated by phenotypic or genotypic methods. The VANA gene clusters also appeared to be identical, having an insertion between the VANX and VANY gene, and a deletion in the right end of the cluster. The focus of a Danish study, reported by (1996), was slightly different. In order to investigate the degree of variation within the VANA gene cluster, isolates from different animals and humans from a wider geographic range were investigated. Similar to and co-workers, found evidence of an insertion sequence in the VANX-VANY region in 7 of 12 British human isolates. Based on sequencing of coding and non-coding regions, the remaining isolates could be divided into 3 groups, each containing isolates 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 VANX and an insertion sequence (IS 6) in a specific position in the transposon (Tn ). The group contained isolates from humans (and ) and pigs (and UK). Mutations within the coding regions appear to be rare. Insertion sequences are highly mobile, 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 those isolates must be very closely related and their presence the result of horizontal transfer. As mentioned, transfer of the VANA-gene cluster has been shown IN 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 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. The localisation of, for instance, MLS resistance determinants on mobile gene elements together with glycopeptide resistance means 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 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 (, 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 been reported with increasing frequency 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 (, 1996). A further threat is the possible spread of vancomycin resistance genes from their enterococcal hosts to Although transfer of the VANA gene cluster from enterococci to 3TAPHYLOCOCCUS AUREUS on the skin of mice in an IN VIVO model has been reported (Noble ET AL , 1992), no such resistance has yet been reported in clinical isolates of staphylococci. Multiple factors predispose a person to infection with VRE, but colonisation precedes most infections (, 1995). In Europe, a community reservoir has been demonstrated. The reported rates of VRE carriage in non-hospitalised humans range from 2 to (Jordens ET AL , 1994; Gordts ET AL , 1995; van den Bogaard ET AL , 1996). Whether this colonisation is transient or persistent is not known. According to the findings of Whitman (1996), administration of glycopeptides seems to be an important factor in establishing a persistent infection. (1996) studied the effects of administration of oral glycopeptides to healthy volunteers. Before exposure, no VRE were recovered from the faecal samples whereas after exposure, of the volunteers gave VRE positive samples. It is not clear whether these findings were due to a pre-exposure 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 been demonstrated (, 1994; Aarestrup, 1995; , 1995a; , 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 VANA-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 for 10 minutes. Thus, food-borne transmission is possible even without recontamination if food is not heated to higher temperatures for longer times. Transmission of VRE from animals 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 Of all , 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 animals. Already in 1978, and (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. were either directly infected with of a nalidixic acid-resistant strain of 3 Typhimurium at a concentration of 109 CFU/ml, or through contact with experimentally infected chickens. Faecal samples were cultured on selective media containing nalidixic acid. However, this method also 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 (and , 1980), the earlier results were confirmed. In this study, 5 different serovars were used (3. Typhimurium, 3. Oranienburg, 3. Infantis and 3. Three different poultry breeds and 3 different commercial diets were also 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 were also 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, Leuchtenberger (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 (103 or 104 organisms of 3 Typhimurium) were tried in various combinations. Most experiments were performed in duplicate, with groups of 20-30 animals. 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 were sampled on all occasions, though. The author concluded that feeding avoparcin, virginiamycin or tylosin, at both levels 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 of infection, as well as the frequency of dosing with infectious organisms, and on the housing system. It is rather surprising that so 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 were 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 were 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 and co-workers published a study where chickens fed avoparcin, virginiamycin or no antimicrobial were compared after receiving 3Typhimurium via the drinking water (GustafsonET AL, 1981). 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. This proportion of positive birds then decreased to become lower than that in the control group at about 4 weeks 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 animals in each group (all animals were 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.