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. 2015 Apr 10;59(5):2926–2929. doi: 10.1128/AAC.05072-14

Persistence of Vancomycin Resistance in Multiple Clones of Enterococcus faecium Isolated from Danish Broilers 15 Years after the Ban of Avoparcin

Valeria Bortolaia a,✉, Manuela Mander a, Lars B Jensen b, John E Olsen a, Luca Guardabassi a
PMCID: PMC4394765  PMID: 25712360

Abstract

The occurrence and diversity of vancomycin-resistant Enterococcus faecium (VREF) were investigated in 100 Danish broiler flocks 15 years after the avoparcin ban. VREF occurred in 47 flocks at low fecal concentrations detectable only by selective enrichment. Vancomycin resistance was prevalently associated with a transferable nontypeable plasmid lineage occurring in multiple E. faecium clones. Coselection of sequence type 842 by tetracycline use only partly explained the persistence of vancomycin resistance in the absence of detectable plasmid coresistance and toxin-antitoxin systems.

TEXT

The public health impact of the transfer of vanA plasmids from animal to human enterococci is unclear (1, 2, 3). As a result of the ban on the use of the vancomycin analogue avoparcin as a growth promoter in livestock in Denmark in 1995, the prevalence of vancomycin-resistant Enterococcus faecium (VREF) in Danish broilers gradually decreased from 80% in 1995 (4) to undetectable levels in 2010 (5). However, these data were generated by testing one random E. faecium isolate per flock (5), which is not optimal for detecting antimicrobial resistance occurring in fecal samples at low bacterial concentrations (6, 7). The objective of this study was to investigate the occurrence and diversity of VREF in Danish broilers 15 years after the avoparcin ban using selective and nonselective isolation methods.

Cloacal swabs were collected at slaughter from 500 broilers between April and September 2010 within a stratified random-sampling scheme representative of the Danish broiler population (5). These samples represented 100 broiler flocks from farms geographically distributed across the regions where most Danish broiler holdings are located and accounted for approximately 25% of the broiler samples collected for the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) 2010 (5). Fecal material from five pooled cloacal swabs per broiler flock was suspended in 2 ml of sodium chloride (0.9%). A drop of each suspension was plated on Slanetz-Bartley agar by the nonselective procedure employed in the DANMAP, followed by testing one randomly selected isolate per plate for vancomycin susceptibility by broth microdilution (5). In parallel, 1 ml of each suspension was added to 5 ml of Enterococcus selective broth (Conda, Denmark) containing 16 μg/ml of vancomycin (Sigma-Aldrich, Denmark). After overnight incubation at 42°C, 100 μl of enrichment culture was spread onto vancomycin-containing (16 μg/ml) Slanetz-Bartley agar (Oxoid, Denmark) plates. On each sampling occasion, positive (E. faecium BM4147) and negative (E. faecium BM4105) controls were used. Following 48 h of incubation at 42°C, a presumptive E. faecium colony was subcultured on blood agar, tested for vanA (8), and identified to the genus and species levels (9, 10).

VREF harboring vanA was detected in 47 (47%) samples by selective isolation, whereas no VREF isolates were detected by nonselective isolation. As determined by broth microdilution (11, 12) using the Sensititre Gram-positive (GPALL1F) plate (Thermo Scientific, United Kingdom), VREF displayed resistance to tetracyclines (40%), ciprofloxacin (30%), erythromycin (19%), and penicillin (8%). In addition, 36 of the 47 isolates (77%) had reduced susceptibility to daptomycin (MIC > 4 μg/ml) (Table 1). No resistance to ampicillin, chloramphenicol, gentamicin, levofloxacin, linezolid, quinupristin-dalfopristin, streptomycin, or tigecycline was observed. Multilocus sequence typing (MLST) (http://efaecium.mlst.net/) revealed the presence of 18 sequence types (STs) clustering in three groups according to analysis with the BAPS (Bayesian analysis of population structure) software (http://www.helsinki.fi/bsg/software/BAPS/) (13, 14). By filter mating conjugation experiments (15) using fusidic acid-resistant and rifampin-resistant E. faecium R4 (BM4105) as the recipient, vanA-positive transconjugants were obtained from 31 (66%) isolates. Four (13%) transconjugants displayed additional resistance to erythromycin (Table 1), while the cotransfer of resistance to chloramphenicol, gentamicin, linezolid, and tetracycline was not detected, as determined by disk diffusion (11, 12). Pulsed-field gel electrophoresis (PFGE) of S1 nuclease-digested genomic DNA (16) of transconjugants showed that the transferred plasmids ranged in size from approximately 33 to 244 kb, with plasmids of approximately 54 kb detected in all isolates. At least 17 transconjugants harbored multiple plasmids (Table 1). PCRs to identify rep families (17) revealed rep1 (within the theta-replicating Inc18 plasmid group), rep14 (within the Rep_trans plasmid group with rolling circle replication), and rep18 (within the theta-replicating Rep_3 plasmid group) in 8 (26%), 10 (32%), and 5 (16%) transconjugants, respectively (Table 1). Five (16%) transconjugants were simultaneously positive for rep1 and rep14, and 13 (42%) transconjugants yielded plasmids nontypeable by this method. Of note, the pLG1 rep family recently identified in VREF in hospitalized humans and pigs (18) is not targeted by the method used. No axe-txe or ω-ε-ζ toxin-antitoxin (TA) systems (19) were detected. DNA from an S1-PFGE gel of 12 transconjugants displaying different rep types and obtained from donors with distinct STs was used for Southern hybridization using digoxigenin-labeled probes (Roche Applied Science, Denmark), revealing that vanA was located on nontypeable plasmids of ∼54 kb, with the exception of one rep14 plasmid. In five transconjugants, the vanA probe additionally hybridized to nontypeable plasmids between 216 kb and 244 kb in size (Table 1).

TABLE 1.

Phenotypic and genotypic traits of vancomycin-resistant Enterococcus faecium isolated from Danish broiler flocks in 2010

Isolatea Sequence type (BAPS group)b Antimicrobial resistance profile(s)c Transconjugant's plasmid(s)
 Farm/flock identifierf Isolation date (day-mo)
Plasmid size (kb)d Replicon family(ies)e Cotransfer of resistance
VAN307 10 (2.1b) VAN NA NA NA 6100-B 17-Jun
VAN333 10 (2.1b) VAN NA NA NA 9690-B 24-Jun
VAN331 12 (2.1b) VAN, DAP NA NA NA 6740-A-1 24-Jun
VAN342 22 (3.1) VAN, DAP, ERY, TET 54, 244 18, NT VAN 6100-A-2 24-Jun
VAN330 26 (2.1b) VAN, PEN NA NA NA 9240 24-Jun
VAN336 26 (2.1b) VAN, CIP, DAP,PEN 54, 138 1, 14, NT VAN 9670-C 24-Jun
VAN486 26 (2.1b) VAN, TET NA NA NA 6000 16-Sep
VAN474 26 (2.1b) VAN, DAP NA NA NA 6715 16-Sep
VAN332 38 (7.1) VAN, CIP, DAP 54, 138 1, 14 VAN 7300 24-Jun
VAN345 38 (7.1) VAN, CIP, DAP 54, 138 1, 14 VAN 7400 24-Jun
VAN458 38 (7.1) VAN, DAP 54, 140 1, 14 VAN 7120 9-Sep
VAN382 157 (2.1b) VAN, DAP, PEN NA NA NA 7323 12-Aug
VAN335 417 (2.3a) VAN, DAP, ERY 54, 138 1, NT VAN, ERY 5500-A-1 24-Jun
VAN337 417 (2.3a) VAN, DAP, ERY 54, 140 1 VAN, ERY 5500-A-2 24-Jun
VAN327 417 (2.3a) VAN, DAP 54, 138 1, 14, NT VAN 6400-A-1 24-Jun
VAN340 417 (2.3a) VAN, DAP, ERY 54, 140 1 VAN, ERY 6980 24-Jun
VAN220 520 (3.3b) VAN, DAP NA NA NA 7870 29-Apr
VAN490 520 (3.3b) VAN, DAP NA NA NA 9460-A-3 16-Sep
VAN488 587 (2.1b) VAN, CIP, DAP, PEN NA NA NA 9500 16-Sep
VAN219 784* (3.3b) VAN, CIP, DAP 54 14 VAN 6622-A-1 29-Apr
VAN222 784* (3.3b) VAN, CIP, DAP, ERY 54 NT VAN 7441 29-Apr
VAN489 784* (3.3b) VAN, CIP, DAP, ERY 54, 50 14 VAN 6622-A-2 16-Sep
VAN476 785* (3.3b) VAN, CIP, ERY ND 14 VAN, ERY 8450 16-Sep
VAN482 785* (3.3b) VAN, CIP, ERY 54, 216 14, NT VAN 9460-A-6 16-Sep
VAN301 839* (7.1) VAN, DAP 54, 244 NT VAN 9670-B 17-June
VAN221 840* (2.1b) VAN, CIP, DAP 33–54, 54, 216–244 NT VAN 5853 29-Apr
VAN296 841* (3.3b) VAN, CIP, DAP 54, 216–244 NT VAN 9670-A 17-Jun
VAN240 842* (2.3a) VAN, TET 54 NT VAN 6100-A-1 6-May
VAN234 842* (2.3a) VAN, DAP, TET NA NA NA 9690-A-1 6-May
VAN308 842* (2.3a) VAN, TET 54 NT VAN 7752 17-Jun
VAN305 842* (2.3a) VAN, DAP, TET 54 18 VAN 9460-A-2 17-Jun
VAN299 842* (2.3a) VAN, DAP, TET NA NA NA 9690-A-2 17-Jun
VAN328 842* (2.3a) VAN, DAP, TET 54 NT VAN 7600 24-Jun
VAN338 842* (2.3a) VAN, TET NA NA NA 8983 24-Jun
VAN461 842* (2.3a) VAN, CIP, DAP, TET 54 NT VAN 6100-A-3 9-Sep
VAN463 842* (2.3a) VAN, CIP, DAP, TET 33–54, 54, 138 18, NT VAN 9460-A-5 9-Sep
VAN471 842* (2.3a) VAN, DAP, TET NA NA NA 6330-A-1 16-Sep
VAN492 842* (2.3a) VAN, TET 54 NT VAN 6400-A-2 16-Sep
VAN484 842* (2.3a) VAN, DAP, TET 33–54, 54 18 VAN 9460-A-7 16-Sep
VAN479 842* (2.3a) VAN, DAP, TET 54 NT VAN 9460_B 16-Sep
VAN470 842* (2.3a) VAN, DAP, TET 54 NT VAN 9690-C-1 16-Sep
VAN477 842* (2.3a) VAN, DAP, TET 54 18 VAN 9690-C-2 16-Sep
VAN481 842* (2.3a) VAN, TET 54, 33 NT VAN Missing 16-Sep
VAN510 842* (2.3a) VAN, DAP, TET ND 14 VAN 6330-A-2 23-Sep
VAN343 843* (3.3b) VAN, DAP ND NT VAN 6200 24-Jun
VAN483 905* (3.1) VAN, CIP, DAP, ERY NA NA NA 5772 16-Sep
VAN472 906* (3.3a) VAN, DAP NA NA NA 6740-A-2 16-Sep
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a

Isolates in bold type were selected for Southern blot hybridization.

b

Sequence type was determined by multilocus sequence typing (MLST). Bayesian analysis of population structure (using BAPS software) was done on the entire MLST database including 966 sequence types on 4 February 2015. An asterisk indicates new STs identified in this study.

c

CIP, ciprofloxacin; DAP, daptomycin; ERY, erythromycin; PEN, penicillin; TET, tetracyclines; VAN, vancomycin.

d

Plasmid size was deduced by comparing the migration of the transconjugant's bands with the closest corresponding marker's bands. Plasmids in bold type were proven to harbor vanA by Southern blotting hybridization. NA, not applicable as no transconjugants were obtained; ND, not determined due to presence of smears in the S1-PFGE gels.

e

rep types are indicated with the same font type (bold and underlined) as that for the corresponding plasmid as determined by Southern blotting hybridization. Not all rep types identified by PCR were assigned to a band on the S1-PFGE likely because the corresponding plasmids could not be visualized by such techniques. NT, nontypeable by rep typing.

f

Farms are identified by the zip code and a capital letter if more than one farm was located in the same area. Flock numbers are reported if more than one flock was sampled in the same farm.

Our study shows that VREF isolates are still frequent 15 years after the discontinuation of avoparcin use in Danish broiler production. VREF isolates were detected only by selective enrichment, indicating that they occurred at low proportions in the total E. faecium population in broiler feces. Accordingly, the risk of carcass contamination at slaughter and foodborne transmission to humans should be lower than that in the past, when VREF isolates were detected at high frequencies by nonselective isolation (4). Persistence of VREF isolates in poultry after the avoparcin ban has been reported in several European countries (6, 20, 21, 22, 23, 24, 25). In Norway, VREF strains were found to occur at low percentages (0.8% to 4.6%) among total enterococci in broiler feces collected up to 8 years after the avoparcin ban (20).

The VREF population in Danish broilers was genetically diverse, as evidenced by identification of 18 STs among 47 isolates. These STs clustered in three different BAPS populations. ST842 was the most prevalent clone isolated from 17 (36%) flocks and 8 farms broadly distributed geographically (Table 1 and Fig. 1). All ST842 isolates were tetracycline resistant, which may have contributed to the epidemiological success of this clone. However, strain coselection by antimicrobial use may only partly explain the presence of VREF after the avoparcin ban since 16 (34%) VREF isolates did not display resistance to penicillins, macrolides, or tetracyclines, which are the antimicrobials mostly used in Danish broiler production that may affect enterococci (5). Vertical transmission may be an alternative port of entry of VREF in the Danish broiler production, as previously shown with ampicillin- and fluoroquinolone-resistant Escherichia coli (26, 27). Nearly all broiler flocks raised in Denmark derive from parent birds imported from Sweden, and the high occurrence of VREF was also observed in Swedish broiler flocks until 2011 (28).

FIG 1.

FIG 1

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Geographical distribution of Danish broiler flocks positive for vancomycin-resistant Enterococcus faecium. Numbers represent sequence types (STs), and identical STs are indicated by the same color.

Vancomycin resistance was mainly mediated by transferable nontypeable plasmids of ∼54 kb, suggesting horizontal transfer of a specific plasmid lineage across multiple E. faecium clones. This plasmid lineage lacked TA systems previously identified on vanA plasmids and did not carry genes conferring resistance to antimicrobials used in Danish broiler production (5). Thus, its maintenance in multiple E. faecium clones is not attributable to plasmid coselection by antimicrobial use. The reasons for the persistence of VREF in the intestinal microbiota of Danish broilers 15 years after the avoparcin ban remain largely unknown and go beyond the possible coselection of ST842 by tetracycline use.

ACKNOWLEDGMENTS

This work was supported by grant HEALTH-F3-2011-282004 (EvoTAR) from the European Union.

We thank AnneMette Seyfarth for valuable assistance in collecting samples and retrieving sample information.

We declare no conflicts of interest.

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