1 Bywater Consultancy, Clungunford, UK; 2 Inveresk, Tranent, Scotland, UK; 3 Malaga, Spain; 4 Elanco Animal Health, Greenfield, IN, USA
Received 13 March 2005; returned 14 April 2005; revised 14 June 2005; accepted 7 July 2005
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Abstract |
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Methods: Samples were taken at abattoirs in two successive years (mid-1998mid-1999, year 1; mid-1999mid-2000, year 2) from chickens (France, The Netherlands, Sweden, UK) and pigs (Denmark, The Netherlands, Spain, Sweden). E. faecium was isolated from faecal samples at national laboratories and sent to a central laboratory where MICs of avilamycin, avoparcin, bacitracin, flavophospholipol, spiramycin, tylosin and virginiamycin were determined. Microbiological breakpoints were allocated on the basis of MIC distributions, and comparison was made between host species, country of origin and year of sample.
Results: In total, 2567 isolates were obtained from chickens and 1742 from pigs. In all countries, resistance to avoparcin (banned in 1997) was uncommon, but resistance to bacitracin and flavophospholipol was common (and was probably largely intrinsic). The prevalence of resistance was similar in chicken and pig isolates, with the exception of avilamycin, to which resistance was commoner among chicken isolates. The removal of four compounds as growth promoters (bacitracin, spiramycin, tylosin, virginiamycin) between years 1 and 2 appeared to result in a significant decrease in resistance to three of themspiramycin, tylosin and virginiamycin, with no change in resistance to bacitracin, but an increase in resistance to avilamycin (not discontinued). Associated resistance was shown between some of the compounds.
Conclusions: Resistance prevalence declined rapidly following removal of growth promoters in pigs and chickens, suggesting that in the absence of selective pressure, a susceptible population began to replace phenotypically resistant strains. Associated resistance between different compounds, where seen, could have resulted from either shared resistance mechanisms or from carriage of resistance genes on the same plasmid. Multiresistance to streptogramins, macrolides and glycopeptides was rare.
Keywords: E. faecium , growth promoters , pigs , chickens , animals , surveillance , resistance
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Introduction |
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Data have been published from individual countries such as Denmark,1 Sweden2 and The Netherlands3 showing the susceptibility of E. faecium isolated from farm animals, but an international centralized study comparing the susceptibility patterns in different countries has not hitherto been attempted. This study was set up to establish the susceptibility, over a 2 year period, of E. faecium to seven growth-promoting antimicrobial agents widely used in the early 1990s in Europe. Identical sampling protocols were used in six different EU countries and in two host species, and a single laboratory carried out susceptibility testing, to overcome the technical variations that might occur between laboratories. Seven growth-promoting antibiotics were tested in this study (Table 1). Six of them were available for use in chickens and swine throughout the EU in 1998 (with the exception of certain Scandinavian countries) although one compound, avoparcin (a glycopeptide), had already been withdrawn within the EU. Unexpectedly, after the first year's samples had been collected, but prior to the second year of the study, four others, bacitracin (a polypeptide), spiramycin, tylosin (macrolides) and virginiamycin (a streptogramin), were banned as of July 1999. The removal of the four compounds provided an opportunity to follow the early effects of such removal on the antimicrobial susceptibility of E. faecium in the two host species. The other two compounds tested were avilamycin (an orthosomycin) and flavophospholipol (a glycophospholipid). These latter two compounds were available through both years of the study (but are to be withdrawn in the EU from 1 January 2006).
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Methods |
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Isolates were transported in batches to the laboratories of Inveresk Research, Tranent, Scotland. There, after isolates with yellow colonies had been rejected, the identity of a proportion of the rest was reconfirmed either by the use of commercial identification kits [API 20 Strep or rapid ID 32 Strep, (bioMérieux, Basingstoke, UK)] or by a PCR method7 as later corrected.8 The primers for the PCR, EfeddlF1 (5'+GCAAGGCTTCTTAGAGA 3') and EfeddlF2 (3'-CATCGTGTAAGCTAACTTC 5') were supplied by PE-Applied Biosystems (Beaconsfield, UK) at a concentration of 1 mg/mL. Briefly, the conditions for PCR were as follows. Samples were prepared by the addition of a large loopful of pure overnight culture to 500 µL distilled water in an Eppendorf tube. This was then placed in a thermocycler at 100°C for 10 min and then held at 70°C. Before use, the tube was spun at 9000g for 2 min and the supernatant was added to 50 µL of reaction mix, which consisted of 37.4 µL of dH2O, 10 µL of 10 x PCR buffer (200 mM Tris pH 9.0, 1 M KCl 0.2 mg/mL gelatine), 0.7 µL of Efeddl F1, 0.7 µL of Efeddl F2, 0.8 µL of dNTP mixture (Pharmacia, Milton Keynes, UK) and 0.4 µL of Taq polymerase 250 U (Boehringer Mannheim GmbH, Germany). Tubes were placed in a Hybaid Omnigene Thermocycler and heated as follows: one cycle of 2 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 54°C and 1 min at 72°C, and one cycle of 10 min at 72°C. Samples were stored at 20°C pending analysis. PCR product (10 µL) + 2 µL of 5 x loading dye was added to a 3% agarose gel with X174 HaeIII markers and run at 150 mA for 1 h. The presence of a band of 550 bp in length (subject to the correct performance of negative and positive controls) indicated that the organism was E. faecium.
Minimum inhibitory concentrations of the following antibiotics obtained from their manufacturers were determined by agar dilution as described by the National Committee for Clinical Laboratory Standards (NCCLS, now re-named Clinical and Laboratory Standards Institute CLSI) document M7-A4:9 ampicillin (for quality control purposes), avilamycin (Eli Lilly), avoparcin (Roche), bacitracin (Alpharma), flavophospholipol (Hoechst Roussel Vet), spiramycin (Rhone Poulenc Rorer), tylosin (Eli Lilly) and virginiamycin (Pfizer). The following bacteria were used for quality control: E. faecium ATCC6569, E. faecalis ATCC29212 and Staphylococcus aureus ATCC29213. Although QC values were not available for any of the growth-promoting agents tested, these were generated in the course of the investigation as study-specific quality control ranges. Ampicillin, for which QC values were available, was used to ensure general test validity.
MIC data for each antimicrobial were then tabulated by year, by host animal and by country. Distributions of MICs were determined for each antibiotic and for QC strains, and on this basis the range of susceptibility of the wild-type population was defined for each antibiotic. This wild-type population was labelled susceptible while other populations were defined as resistant, having low-level or high-level microbiological resistance as defined by the European Committee for Antimicrobial Susceptibility Testing (EUCAST, http://www.eucast.org) of the European Society for Clinical Microbiology and Infectious Diseases (ESCMID).10,11 It is emphasized that assigned breakpoints were microbiological, and differed in principle from those used for animal or human therapy, which must also take into account pharmacological parameters and clinical and microbiological results of treatment.9 Associated resistance was identified when bacterial isolates resistant to one antibiotic were significantly more often resistant to a second antibiotic than were susceptible isolates.
The statistical significances of differences in the prevalence of resistance rates were assessed by Fisher's exact test (two-tailed) by the method of summing of small values (http://www.graphpad.com/quickcalcs/CatMenu.cfm).
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Results |
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During year 1 and year 2, respectively, 2353 and 2137 isolates of E. faecium were submitted to the central susceptibility testing laboratory. Of this total, 181 (4.1%) were rejected on the basis of phenotypic or PCR results. Of the remaining 4309 isolates, a further 635 selected at random were confirmed as E. faecium by PCR. The antimicrobial susceptibility of the remaining 4309 isolates was determined.
Antimicrobial susceptibility
Quality control strains. The quality control ranges established during the course of the investigation are shown in Table 2. Although the NCCLS Antimicrobial Susceptibility Testing Subcommittee12 has established procedures to derive multi-laboratory quality control ranges for therapeutic antibiotics, there were no values available to use for these antibiotics during the study. However, study-specific ranges were defined. The tylosin quality control range subsequently established by the NCCLS for ATCC 29212 was 0.54 mg/L,13 with which the range determined by the study-specific calculations conformed, thus supporting the validity of the method.
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The prevalence of resistance for chicken and pig isolates for year 1 and year 2 for each of the countries is shown in Table 4. Results were similar in isolates from pigs and chickens for compounds other than avilamycin, to which isolates from pigs were less often resistant. Comparison of the results by country showed that Swedish isolates had a lower incidence of resistance to the compounds tested except for flavophospholipol and bacitracin, and that avoparcin resistance was uncommon in all countries sampled.
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Discussion |
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By plotting the MIC distributions of each compound and combining the data from isolates obtained in the different countries in the way currently used by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), it was in each case possible to identify apparent wild-type populationsthat is, a group of organisms without acquired resistance mechanisms. Interestingly, the modal MIC for each antibiotic for such populations was around 12 mg/L, although for flavophospholipol it seems possible that such low MICs for a very few isolates may have been aberrant, or that the results were correct but very unusual for the species. In fact, it has been shown that E. faecium is seldom susceptible to the substance, although E. faecalis is susceptible.14 For avoparcin, whose use had been discontinued in all of the countries approximately a year before our study started, the prevalence of resistance was low and about 95% of isolates were in this wild-type group. Indeed, in Sweden, where avoparcin had not been used since 1986, all isolates had wild-type susceptibility, as did all isolates from pigs in Spain. Finally, the very large majority of isolates from pigs were of wild-type susceptibility to avilamycin, perhaps because the compound is not widely used in this species.
For most of the compounds the distribution of MICs was clearly bimodal or, in the case of bacitracin, trimodal. It was therefore relatively easy in each case to allocate microbiological MIC breakpoints in distribution troughs, and where NCCLS human clinical breakpoints were available (glycopeptide, macrolide, streptogramin) they were similar, except for glycopeptides where our breakpoint for avoparcin (>8 mg/L) was lower that the NCCLS equivalent for vancomycin (32 mg/L). The study-specific breakpoints were used to calculate the prevalence of microbiological resistance. In the first year this was of the order of 3050% for the two macrolides and virginiamycin. However, there were considerable differences between countries. In Sweden, where growth-promoting antibiotics had not been used since 1986, more than 85% of isolates were susceptible to all the agents except flavophospholipol (see above) and bacitracin, for which, however, high-level resistance (MICs
128 mg/L) was uncommon. Virginiamycin resistance in Denmark was low at both sample points, perhaps as a result of the unilateral removal of this compound 6 months before the study began. Unfortunately, quantitative antimicrobial consumption data for the host species were neither available for the countries nor for the individual farms from which the isolates originated. Such data would have been helpful in interpreting geographic variation.
The removal of virginiamycin, spiramycin, bacitracin and tylosin as growth promoters (1 July 1999)just 3 months before the start of sampling for year 2apparently resulted in rapid changes in the prevalence of susceptible strains, although the results must be interpreted with caution, given that sampling was in only two time frames. However, the rapid responses to removal were consistent with those reported by others for Denmark,1,15 and suggests instability of the resistant E. faecium population in the absence of selection. Results for year 2 were generally consistent with the removal, in that resistance rates were significantly lower for the newly discontinued compounds virginiamycin, spiramycin and tylosin (despite the latter's continued availability for therapeutic purposes). For bacitracin, despite an apparent shift from high to intermediate MICs, there was no overall change in the prevalence of resistance. Avoparcin resistance rates were already low and remained so with no statistical evidence of change, in keeping with the fact of its earlier removal. Flavophospholipol resistance rates showed no significant change. Finally, resistance rates for avilamycin increased significantly in chickens (but not in pigs). This decrease in susceptibility to avilamycin (not removed) might have resulted from increase usage in the absence of the proscribed compounds.
We were able to detect significant associated resistance between antibiotic classes. For the macrolides and virginiamycin, this could have been cross-resistance based on the shared MLSB mechanism. The only example of multiresistance that might have significance for human medicineassociated resistance to streptogramins, macrolides and glycopeptideswas encountered in only 1.5% of the isolates.
The criteria used for the identification of E. faecium appeared fully supportable, although a very few isolates of E. faecalis may have been included, as suggested by the small number of out-of-range susceptibility test results. These were too few to affect our conclusions. It is notable that whenever surveys of enterococcal antibiotic susceptibility are performed, the problem of misidentification may arise, despite efforts to avoid it.16
Although the procedure used for establishment of breakpoints differed in some respects from the protocol recommended by the NCCLS,12 we believe that we have successfully established provisional parameters which we encourage others to test further. Furthermore, since the MIC results were obtained by the use of the NCCLS method (also now suggested as a reference method by EUCAST),17 comparisons of our quantitative results with others from studies based on these standards are possiblea goal that has been widely supported.18
Transparency declarations
RJB is a pharmaceutical industry consultant. TRS is an employee of Elanco Animal Health, a division of Eli Lilly & Co.
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Acknowledgements |
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