1 US Food and Drug Administration, Center for Veterinary Medicine, 8401 Muirkirk Road, Laurel, MD 20708; 2 3304 Marie Mount Hall, College Park, MD 20742; 3 Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA
Received 2 April 2002; returned 19 June 2002; revised 29 August 2002; accepted 9 September 2002
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Abstract |
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Keywords: Enterococcus faecium, quinupristindalfopristin, resistance
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Introduction |
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Resistance to streptogramins was first reported in staphylococci in 1980.4 Only resistance to the A component is required for streptogramin resistance; however, resistance to both A and B components may result in higher MICs.4 A number of genes have been reported that confer streptogramin A resistance in both staphylococci and enterococci.47
In both Europe and the United States, only small numbers of quinupristindalfopristin-resistant E. faecium have been recovered from human sources.8,9 In contrast, the frequency of quinupristindalfopristin-resistant E. faecium of animal origin on both these continents has been reported to be much higher 6,10,11 especially from poultry.7,10,12,13 With the banning of virginiamycin in Europe there has been a steady decrease in the number of quinupristindalfopristin-resistant E. faecium being isolated from animals.3 For example, data from Denmark have shown that in 1997 there was a 66.2% prevalence of virginiamycin-resistant E. faecium being recovered from broilers.3 In 1998 the use of virginiamycin as a growth promotor was banned. By 2000, the prevalence of quinupristindalfopristin-resistant E. faecium being recovered from broilers in Denmark had been reduced to 33.9%.3
In this study we investigated the mechanisms of quinupristindalfopristin resistance in non-Enterococcus faecalis enterococci recovered from pre-packaged retail poultry purchased in the Greater Washington DC area in 2000.
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Material and methods |
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Chicken carcasses (n = 43) and turkey breasts (n = 32) were collected randomly from retail stores of four supermarket chains in the Greater Washington DC area. Stores of the four supermarket chains in the area were identified by using phone books, store web sites and store maps. Each store was assigned an identification number in order to form a store database. Sampling visits were made between June 1999 and July 2000. On each sampling day, four stores were randomly chosen from the store database by using a statistical program (SAS Institute Inc., Cary, NC, USA). Prepackaged raw meat products were randomly selected and transported on ice to the laboratory. On the day of collection, retail meat samples were brought to the laboratory and immediately transferred from commercial packaging into sterile plastic bags and rinsed in buffered peptone water. One millilitre of rinse from meat samples was placed in 10 mL of Enterococcosel (BBL Microbiology Systems, Cockeysville, MD, USA) broth and incubated at 45°C for up to 48 h. Aesculin-positive cultures were streaked on Enterococcosel agar and incubated at 35°C for 24 h. Colonies characteristic of Enterococcus spp. were streaked on to trypticasesoy agar with 5% sheep blood to assure purity and to check for haemolysis. Where presumptive enterococcal isolates with different morphotypes were observed, one colony of each morphotype was selected. Single colonies were picked from the blood agar and streaked on brainheart infusion agar and tested for catalase and pyruvate (PYR) reactions and by Grams stain. Catalase-negative, Gram-positive, PYR-positive isolates were confirmed as Enterococcus spp. using the AccuProbe Enterococcus identification test (Gen-Probe, Inc., San Diego, CA, USA). AccuProbe-positive isolates were identified to species using the Vitek Automated Microbial Identification System (bioMerieux Vitek, Inc., St Louis, MO, USA).
Antimicrobial susceptibility determination of enterococci
Antimicrobial MICs for enterococci were determined via the Sensititre Automated Antimicrobial Susceptibility System (Trek Diagnostic Systems, Westlake, OH, USA) and interpreted according to the NCCLS guidelines for broth microdilution methods.14 Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and E. faecalis ATCC 29212 were used as quality control microorganisms.
DNA extraction, PCR studies and DNA sequencing
Total bacterial DNA was extracted using the guanidium thiocyanate method as described previously to act as template for the PCR.15 Known streptogramin resistance genes, as well as the macrolide resistance gene, ermB, were amplified by PCR using oligonucleotide primers and PCR conditions described previously.6 The PCRs were done using the AmpliTaq Gold PCR system (Perkin Elmer, San Diego, CA, USA). All PCR-positive samples were sequenced commercially (SeqWright, Houston, TX, USA).
Pulsed-field gel electrophoresis
Pulsed-field gel electrophoresis (PFGE) was performed after DNA digestion with SmaI as described previously.16 To analyse the PFGE results for strain relatedness of the E. faecium isolates, we used the interpretive criteria of Tenover et al.17 Comparisons of the PFGE fingerprinting were made using computer-assisted analysis (BioNumerics; Applied Maths, Austin, TX, USA).
Conjugation of streptogramin resistance determinants
The in vitro transfer frequency of the streptogramin resistance determinants was examined using the filter mating method as described previously.15 The recipient strain used for conjugation studies was a plasmid-free E. faecium, GE-1, which displays resistance to rifampicin and fusidic acid. Transconjugants were selected on trypticasesoy agar base supplemented with 5% defibrinated sheep blood, rifampicin (50 mg/L), fusidic acid (25 mg/L) and quinupristindalfopristin (4 mg/L) following incubation at 37°C for 48 h. From each mating experiment 10 transconjugants were selected for further analysis.
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Results |
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A total of 70 enterococcal isolates were recovered from the retail meat samples analysed, comprising 32 E. faecalis, 33 E. faecium, four Enterococcus gallinarum and one Enterococcus hirae (Table 1). Antimicrobial susceptibility studies identified 31 non-E. faecalis isolates with MICs of quinupristindalfopristin of 4 mg/L (resistance breakpoint is 4 mg/L), E. faecium (chicken n = 23, turkey n = 4), E. gallinarum (chicken n = 1, turkey n = 2) and E. hirae (chicken n = 1); however, no isolate had quinupristindalfopristin MICs > 16 mg/L. Seventy-five per cent of the turkey E. faecium isolates showed resistance to erythromycin (breakpoint 8 mg/L) and penicillin (breakpoint 16 mg/L), and all were resistant to tetracycline (breakpoint 16 mg/L). In contrast, among the E. faecium recovered from chickens, 13% were resistant to erythromycin, 75% were resistant to tetracycline and 69% were resistant to penicillin. The two turkey E. gallinarum isolates showed resistance to erythromycin and tetracycline; however, both were susceptible to penicillin. The single E. hirae isolate recovered from chicken meat was susceptible to erythromycin and penicillin but showed resistance to tetracycline. The MIC profiles of the non-E. faecalis isolates are shown in Table 1.
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PCR amplification identified the presence of vat(E) in 43% (n = 10) of the chicken E. faecium and two of the four turkey E. faecium. The vat(E) gene was not detected by PCR in either E. hirae or E. gallinarum. The vat(E) genes identified in the 12 E. faecium isolates were sequenced in their entirety, in both directions. Five allelic variations were obtained and have been described elsewhere.18 We were unable to detect vat(D) or any of the other streptogramin resistance genes by PCR. In addition, we did PCR analysis for the macrolide resistance gene erm(B). This gene was detected in 30% (n = 7) of chicken E. faecium and all of four turkey E. faecium and two E. gallinarum from turkeys. We could not detect erm(B) in E. hirae by PCR. DNA sequencing confirmed the identity of the amplified PCR products. The PCR results are summarized in Table 1.
PFGE
PFGE was conducted on all the E. faecium isolates recovered in this study. A total of 19 different PFGE patterns were observed among the 23 chicken E. faecium isolates, whilst three PFGE patterns were observed in the four turkey E. faecium isolates. The different PFGE patterns were arbitrarily assigned to groups AU (Table 1). Interestingly, the two turkey vat(E) isolates did not belong to the same PFGE group. However, of the 10 chicken vat(E)-positive isolates, two isolates belonged to PFGE group F and two belonged to PFGE group L. The remaining six isolates were distinguishable by PFGE.
Conjugation of streptogramin resistance determinants
We were able to transfer vat(E) by conjugation from only two E. faecium isolates, one recovered from chicken (CVM3475; conjugation frequency of 1.2 x 102/recipient) and the other from turkey (CVM3980; conjugation frequency of 1.4 x 102/recipient). We confirmed the presence of vat(E) in the transconjugants by PCR amplification. No other quinupristindalfopristin-resistant enterococcal isolate was able to transfer vat(E) by conjugation. In addition to quinupristindalfopristin resistance, all the transconjugants had erythromycin susceptibility profiles of the respective donor strains. Resistance to gentamicin, tetracycline and penicillins varied between the transconjugants examined.
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Discussion |
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In the present study we were able to isolate 70 enterococci from retail poultry meat samples purchased in the Greater Washington DC area. Thirty-one of 38 (81%) non-E. faecalis enterococci that were recovered displayed resistance to quinupristindalfopristin and virginiamycin (MIC 4 mg/L). These figures parallel those that have been reported in Europe and in the USA.10,12,13,19 In addition, the E. faecium from turkey and chicken meat samples displayed differences in the prevalence of susceptibility to other antimicrobial agents. There was a higher prevalence of resistance to erythromycin in turkey E. faecium compared with chicken E. faecium (75% versus 13%). There was a prevalence of 75% resistance to tetracycline in E. faecium recovered from chicken but those E. faecium recovered from turkey were all susceptible to tetracycline.
When PCR studies were carried out to characterize the mechanism(s) of resistance to quinupristindalfopristin, only 38% (n = 12) of the quinupristindalfopristin-resistant isolates showed the presence of vat(E). We were able to transfer the vat(E) gene by conjugation from only two of the 12 (16%) vat(E)-positive E. faecium isolates and none of the PCR-negative isolates. The failure of most strains to donate the vat(E) gene by conjugation suggests that the gene is frequently present on non-conjugative or non-mobile elements.
None of the isolates examined in this study carried the vat(A), vat(B), vat(C), vat(D), vga or vga(B) genes, conferring resistance to streptogramin A, or vgb or vgb(B), conferring resistance to streptogramin B. Five allelic variations of vat(E) were identified and have been described elsewhere.18 These variations lead us to believe that the vat(E) in these isolates may have originated from independent sources and substantiates the reports of Soltani et al.20 that it is not always possible to trace the epidemiological spread of the vat(E) gene based on PCR results alone, and that DNA sequencing information is necessary to obtain a more complete picture of vat(E) gene dissemination in veterinary and human environments.
The PFGE data indicate that in both chicken and turkey E. faecium, the vat(E) gene is not limited to a single clone but is distributed in a range of different E. faecium clones. This is not surprising since vat(E) is known to be disseminated via plasmids.6
The prevalence of vat(E) in low-level quinupristindalfopristin-resistant isolates of E. faecium reported in this study is in contrast to that reported in Europe. In the European studies, in those E. faecium isolates showing low-level quinupristindalfopristin resistance (MICs < 32 mg/L) none of the known streptogramin resistance genes could be detected by PCR.6 However, in E. faecium isolates with high-level quinupristindalfopristin resistance (MIC > 32 mg/L) either vat(D) or vat(E) could be detected by PCR and both were shown to be associated with a plasmid as determined by Southern hybridization studies.1,2,6 In addition, a single study from Europe reported the presence of vgb in a quinupristindalfopristin-resistant E. faecium isolated from a human.2 This gene was absent from our isolates. We did, however, note the presence of erm(B) in 30% of the chicken E. faecium and in 100% of the turkey E. faecium and E. gallinarum. The prevalence of erm(B) is comparable to that reported in other countries.6,10,11
It has been reported that msrC is prevalent, but not intrinsic, in E. faecium isolates and encodes an efflux pump, MsrC.21,22 Expression of msrC can result in a two- to eight-fold increase in the MICs of quinupristin (streptogramin B) and erythromycin.21 Although we could hypothesize that erm(B) may be acting in concert with msrC in some isolates to confer the low levels of resistance observed, both of these resistance mechanisms act on the streptogramin B subunit only. The streptogramin A subunit would still remain an active component of the quinupristindalfopristin combination. Therefore, this hypothesis would not account for the low levels of resistance observed. Active efflux of streptogramin A, due to ATP-binding cassette transporters, is another mechanism associated with streptogramin resistance, but has only been documented in staphylococci.23,24 It is possible that a staphylococcal homologue is present in the enterococcal isolates described in both this study and those from Europe.
In conclusion, we have presented data documenting a high prevalence of vat(E) in enterococci recovered from retail poultry meat. It seems that vat(D), which is prevalent in European countries, has yet to appear in US quinupristindalfopristin-resistant E. faecium isolates. The data presented in this study, along with other studies, indicate that in the majority of low-level quinupristindalfopristin-resistant isolates, an undefined mechanism(s) confers quinupristindalfopristin resistance. It will be necessary to identify the full complement of transmissible genes responsible for quinupristindalfopristin resistance before the impact of virginiamycin use on the outcome of human quinupristindalfopristin therapy can be understood.
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Acknowledgements |
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Footnotes |
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References |
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