Epidemiological Study of the Incidence of Antimicrobial Resistant Bacteria in Pigs at Slaughter

Final report summary:
This project was linked to a portfolio of Defra-funded abattoir surveillance studies involving pigs, sheep and cattle that were performed in 1999 and 2000. The main objectives of these abattoir studies were to determine the prevalence of certain zoonotic organisms (eg Salmonella spp., Campylobacter spp., Yersinia enterocolitica and E. coli O157) and also to determine the prevalence of resistance in these organisms to certain antimicrobials of relevance to public or animal health. Project VM02110 complemented the other work that had been performed, by providing details of antimicrobial resistance in Campylobacter spp. and Enterococcus faecium recovered from pigs. The aims of this project were as follows:

<UL> <LI> To determine the antimicrobial resistance of Campylobacter species (representing a zoonotic organism of public health importance) and the indicator organism Enterooccus faecium isolated from pigs at slaughter, against a panel of antimicrobials of veterinary and medical importance.
<LI> To ensure that the methodology used to determine resistance allows direct comparison of veterinary results with results collected in the medical field.
<LI> To provide data on the extent and patterns of antimicrobial resistance in these organisms that will assist in formulating policy designed to limit the development of antimicrobial resistance in bacteria of animal origin and to control the spread of antimicrobial resistance genes both within animal populations and to humans.
<LI> To include therapeutic antimicrobials and also, in the case of E. faecium, growth promoting antimicrobials, in the panel of antimicrobials tested.
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Campylobacter spp. and E. faecium isolates were collected from pigs after slaughter as part of Defra-funded surveillance studies in abattoirs. Sampling procedures were designed to ensure that a representative number of animals were examined.
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The identity of E. faecium isolates was confirmed using a published PCR method, which amplifies internal fragments of genes encoding D-alanine:D-alanine ligases and related glycopeptide resistance genes (Dutka-Malen et al. 1995).
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E.faecium isolates were stored at -70oC and later their antimicrobial susceptibility determined using a semi-automated microbroth minimum inhibitory concentration (MIC) method (Sensititre, Trek Diagnostic Systems). Each sample was tested against a panel of twelve antimicrobials prepared as a customised ¡®Sensititre¡¯ plate.
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A breakpoint method was used to determine the antimicrobial susceptibility of Campylobacter strains. Antimicrobials were incorporated into Isosensitest agar (Oxoid) containing 5% laked horse blood at the concentrations described in full in the later part of this report. <P>

Enterococcus faecium. <BR>

A total number of 158 isolates, provisionally identified using phenotypic tests as E. faecium, were recovered from culture of the 2,509 pig caecal samples [work performed as part of a previous Defra-funded study]. Examination of these isolates by a published PCR method, which amplifies internal fragments of genes encoding D-alanine:D-alanine ligases and related glycopeptide resistance genes (Dutka-Malen et al. 1995) confirmed that 148 of these isolates were E. faecium. Ten isolates were not therefore E. faecium and these isolates are excluded from the susceptibility test results.
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The results of susceptibility tests performed on the 148 porcine isolates of E. faecium are shown in table 1. A single isolate was found out of this total of 148 which was resistant to vancomycin and teicoplanin. 98.6% of isolates were resistant to tetracyclines, whilst 89.9% of isolates were resistant to tylosin, with many isolates resistant to both tetracyclines and tylosin. 1.4% of isolates were resistant to ampicillin and no high-level aminoglycoside resistance was detected to gentamicin. 47.3% of isolates were resistant to dalfopristin/quinupristin and the distribution of MIC values for this antimicrobial combination was similar to that obtained for virginiamycin. For salinomycin a unimodal distribution of MICs was observed with the modal MIC at 1¦Ìg/ml and accounting for 70% of isolates. 8.1% of isolates had an MIC of 8¦Ìg/ml to salinomycin.
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Campylobacter coli and C. jejuni. <BR>

Considering the overall results which are shown in table 2, resistance was more frequent in the C. coli isolates (n= 702) than in the C.jejuni isolates (n=27) tested. The majority of both C.coli and C.jejuni isolates were resistant to tetracyclines (79 % and 70 % respectively). Resistance to erythromycin was also high in the C. coli strains (85%). Interestingly, none of the C.jejuni isolates were resistant to nalidixic acid or ciprofloxacin. In contrast some of the C. coli strains did show resistance to these antibiotics (17% and 10% respectively). 16% of C.coli isolates and 4% of C.jejuni isolates were resistant to ampicillin.
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The principal findings in the abattoir surveys were the high level of resistance in bacterial isolates from pigs to certain antimicrobials, including tetracyclines and macrolides. In general, a higher level of resistance was detected in bacterial organisms of porcine origin when compared to the other domestic species that had been surveyed previously (cattle and sheep). However, the porcine data from Great Britain compared on the whole favourably with published data from other parts of Europe. Speciation of Enterococcus spp. bacteria is important from the antimicrobial resistance perspective because different enterococcal species are intrinsically resistant to different antimicrobial agents. Van A enterococci are typically resistant to high levels of vancomycin (MIC ¡Ý 128¦Ìg/ml) and teicoplanin (MIC ¡Ý 16¦Ìg/ml). A single E.faecium isolate was recovered with this phenotype. No high-level aminoglycoside resistance (HLAR) was detected in any of the isolates tested (i.e. MIC ¡Ý 512¦Ìg/ml gentamicin).

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There is no internationally-agreed reference method for susceptibility testing of campylobacter organisms. The technique used in this study to determine antimicrobial resistance was that in use in the Campylobacter Reference Laboratory of the HPA. Thus, it is anticipated that comparison between the results of this veterinary study and the previously-published medical studies relating to England and Wales (and future human studies) will be facilitated. In general the resistance patterns for these porcine strains differed from the patterns seen in human isolates in GB, suggesting that pigs are not a major reservoir for resistant campylobacter in humans. A large proportion (85%) of porcine C.coli isolates were resistant to erythromycin, with lower numbers (41%) of C.jejuni showing resistance. These figures are significantly higher than those reported for humans from England and Wales in 1997 (12.8% for C.coli and 1% for C.jejuni) (Thwaites and Frost 1999). Most human campylobacter infections are untreated. However, erythromycin is one of the antimicrobials of choice when treatment of campylobacter infections is necessary. High levels of resistance in porcine campylobacter strains are undesirable even if the contribution of these strains to human disease is relatively small. No resistance to the quinolone nalidixic acid or the fluoroquinolone ciprofloxacin was detected in any of the C.jejuni isolates recovered in the survey. 10.5% of C.jejuni isolates from man in 1997 showed resistance to both nalidixic acid and ciprofloxacin, with 21.8% of C.coli showing resistance (Thwaites and Frost 1999). The C.coli recovered from pigs in this study showed 10% resistance to ciprofloxacin when using a breakpoint of 1mg/L to discriminate between sensitive and resistant strains and 17% of C.coli were resistant to nalidixic acid (breakpoint 16mg/L nalidixic acid).
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