Combined Antimicrobial Resistance in Enterococcus faecium Isolated from Chickens

Abstract

Nineteen E. faecium strains isolated from chicken caecum samples, collected in slaughterhouses and highly resistant to vancomycin or gentamicin, were coresistant to erythromycin, and/or tetracyclines, and/or streptogramins, and/or avilamycin. Multiple antibiotic resistance was related to the presence in various combinations of aac(6′)-aph(2"), erm(B), emtA, mef(A), tet(L), tet(M), and vanA genes.

Enterococcus faecalis and Enterococcus faecium belong to the normal digestive floras of humans and animals but are also known as occasional human pathogens responsible for nosocomial and community-acquired infections (26). In addition to the presence of intrinsic resistance to several classes of antimicrobials, enterococci can acquire high-level resistance to a variety of antimicrobials by horizontal transfer of mobile genetic determinants. In particular, strains belonging to the species E. faecium have been reported as multiply resistant to antimicrobials, including penicillins, aminoglycosides (at a high level), and vancomycin, therefore limiting the therapeutics available for the treatment of severe enterococcal infections.

Since, in Europe, vancomycin-resistant enterococci have been isolated not only from humans but also from farm animals and environmental samples, the role of the food chain in the spread of resistant enterococci has been questioned (7, 24, 29, 31, 34). This question has been the subject of controversy and finally led to the ban by the EU of avoparcin in 1997 and of most other antimicrobials used as growth promoters in 1999 (28).

Although strains of vancomycin-resistant enterococci isolated in animals have been extensively studied, less is known of combined resistance in these microorganisms. The aim of this study was to characterize the genes responsible for multiple antimicrobial resistance in vancomycin-resistant E. faecium isolated from chicken caeca.

Bacterial isolates were collected in the course of a national monitoring program on antimicrobial resistance in zoonotic enteropathogens (Salmonella spp., Campylobacter spp.) and commensal bacteria (Escherichia coli, enterococci) of animal origin, first implemented for poultry in 1999 in France and funded by the Ministry of Agriculture (P. Sanders, F. Humbert, M. Gicquel, and A. Perrin-Guyomard, Abstr. Int. Conf. Antimicrob. Agents Vet. Med., abstr. P.122, 2002). In 1999, independent samples of chicken caeca were collected in 10 slaughterhouses representative of French poultry farming and screened on m-Enterococcus agar free of antibiotic (Difco Laboratories, Sparks MD). A total of 620 samples from 620 flocks (1 sample per flock) were studied. From these samples, 295 isolates (1 per sample) were identified as Enterococcus faecium using API Strept galleries (bioMérieux, La-Balme-les-Grottes, France). Of these isolates, 16 and 3 were resistant to vancomycin and to high levels of gentamicin, respectively, and were studied further. Identification of these isolates was confirmed by amplification of a fragment internal to the ddl_E. _faecium gene encoding the d-alanyl-d-alanine ligase of E. faecium using specific oligonucleotide primers as described previously (14).

MICs of antibiotics were determined by a broth microdilution technique according to the NCCLS procedure (27). The antibiotic breakpoints used were those of the NCCLS and those of the Comité de l'Antibiogramme de la Société Française de Microbiologie for virginiamycin (12).

Isolates resistant to antimicrobials were tested for the presence of resistance genes by PCR. Specific oligonucleotide primers previously described were used to amplify the major erm genes [erm(A), erm(B), and erm(C) genes encoding ribosomal methylases], the mef(A) and msr(A) genes encoding macrolide efflux proteins, the vat(D) and vat(E) genes encoding streptogramins A acetyltransferases, the vgb gene encoding a streptogramin B lyase (6, 10), the emtA gene conferring resistance to avilamycin by methylation (22), the aac(6′)-aph(2") gene conferring high-level resistance to gentamicin (16), the vanA, vanB, vanC1, and vanC2 genes involved in vancomycin resistance (14), the tetracycline resistance genes encoding ribosomal protection proteins (5), and the tet(L) and tet(K) genes conferring resistance to tetracycline by efflux (32). Strains of Staphylococcus aureus HM1080 [erm(A)], S. aureus HM1054/R [erm(C)], Streptococcus pneumoniae HM30 [erm(B), tet(M)], E. faecium HM1032 [vat(D) and vgb] from our collection, S. pneumoniae [mef(A)], E. faecium UW 1965 [vat (E)], and E. coli with plasmids carrying cloned resistance genes, and pAT392 [aac(6′)-aph(2")] and pPV72 [tet(L)] were used as PCR positive controls.

Since resistance to avilamycin may be due to mutations in genes encoding the domain V of rRNA 23S (19, 36) and the ribosomal protein L16 (1, 2), two overlapping fragments of 386 and 372 bp of ribosomal DNA encoding domain V of 23S rRNA and a fragment of 414 bp of the gene encoding the L16 protein were amplified from DNA of all enterococci, whether resistant or susceptible to avilamycin, by using primers described previously (4, 11) (Table 1). The amplicons were analyzed for the presence of mutations by single-strand conformational polymorphism, as described previously (11), and PCR products were sequenced.

TABLE 1.

PFGE type, MICs of selected antimicrobials, and resistance gene content for Enterococcus faecium strains

PFGE type	Slaughter- house	Strain	MIC (μg/ml) of indicated antibiotic [resistance gene]a
			ERY	VIR	GEN	VAN	TET	AVI
A	8	1	—b	8 [vat(E)]	—	>64 [vanA]	8 [tet(L)]	>32 [unknown]
B	10	2	>64 [erm(B)]	—	—	>64 [vanA]	>64 [tet(L)]	>32 [unknown]
	10	3	>64 [erm(B)]	—	—	>64 [vanA]	>64 [tet(L) + tet(M)]	>32 [unknown]
	10	4	>64 [erm(B)]	—	—	>64 [vanA]	>64 [tet(L) + tet(M)]	>32 [unknown]
	10	5	>64 [erm(B)]	—	—	>64 [vanA]	>64 [tet(L) + tet(M)]	>32 [emtA]
	2	6	>64 [erm(B)]	—	—	>64 [vanA]	>64 [tet(L) + tet(M)]	>32 [unknown]
C	10	7	>64 [erm(B)]	—	—	>64 [vanA]	64 [tet(M)]	—
	10	8	—	—	—	>64 [vanA]	64 [tet(M)]	—
D	10	9	—	—	—	>64 [vanA]	64 [tet(M)]	>32 [emtA]
E	10	10	>64 [erm(B)]	—	—	>64 [vanA]	>64 [tet(L) + tet(M)]	—
F	5	11	>64 [erm(B)]	>16 [unknown]	>512 [aac(6′)-aph(2′′)]	—	>64 [tet(L) + tet(M)]	>32 [emtA]
G	6	12	>64 [erm(B)]	—	—	>64 [vanA]	>64 [tet(L) + tet(M)]	>32 [emtA]
H	4	13	—	8 [vat(E)]	—	>64 [vanA]	4 [tet(L)]	>32 [unknown]
I	9	14	>64 [erm(B)]	—	>512 [aac(6′)-aph(2′′)]	—	>64 [tet(L) + tet(M)]	>32 [unknown]
J	1	15	>64 [erm(B)]	—	—	>64 [vanA]	>64 [tet(L) + tet(M)]	>32 [unknown]
	7	16	>64 [erm(B)]	—	>512 [aac(6′)-aph(2′′)]	—	>64 [tet(L) + tet(M)]	—
K	9	17	—	—	—	>64 [vanA]	>64 [tet(L) + tet(M)]	—
L	2	18	4 [mef(A)]	—	—	>64 [vanA]	>64 [tet(L) + tet(M)]	>32 [emtA]
M	3	19	—	—	—	>64 [vanA]	64 [tet(M)]	—

^aAVI, avilamycin; ERY, erythromycin; GEN, gentamicin; TET, tetracycline; VAN, vancomycin; VIR, virginiamycin.

^b—, not shown (only MICs for resistant strains are shown).

Genomic DNA was analyzed by pulsed-field gel electrophoresis (PFGE) after digestion with SmaI (BioLabs), as described previously (25). Interpretation of gels was performed using the criteria of Tenover et al. (30).

PFGE typing allowed the identification of 13 different genotypes (Fig. 1, Table 1). Three genotypes, B, C, and J, included five, two, and two isolates, respectively. The genotypes E and M were closely related to type B with one or two bands difference. Eight isolates (four of type B, two of type C, one of type D, and one of type E) came from the same slaughterhouse. The type J was unrelated with the other types, and the two isolates included in this genotype came from two different slaughterhouses.

FIG. 1.

PFGE patterns of SmaI-restricted genomic DNA of some of Enterococcus faecium isolates from chicken caecum samples. M, lambda ladder DNA standard. Lanes 3 through 19, strain number (genotype).

The MICs of antibiotics for the resistant strains are shown in Table 1. Of the 19 isolates, 10 had a MIC of ampicillin greater than 4 μg/ml (including three strains with a MIC greater than 16 μg/ml). Among the 16 isolates that displayed high-level resistance to vancomycin (MIC > 64 μg/ml), 14 had from two to four additional resistances to other antibiotics, in particular, avilamycin, erythromycin, and tetracyclines. The three isolates highly resistant to gentamicin were also resistant to several other antimicrobials.

All isolates highly resistant to vancomycin (n = 16) or to erythromycin (MIC > 64 μg/ml) (n = 12) contained vanA- or erm(B)-like genes, respectively (Table 1). Only one strain with an MIC of erythromycin equal to 4 μg/ml (intermediate resistance) contained a mef(A)-like gene. To the best of our knowledge, resistance to erythromycin by active efflux has never been reported for enterococci from animals. Resistance to streptogramin was associated with the presence of a vat(E)-like gene responsible for streptogramin A inactivation in two strains, whereas resistance could not be explained in one strain. Resistance to tetracyclines present in all strains but one was due to ribosomal protection [tet(M) gene] or to drug efflux [tet(L) gene]. In 12 isolates, both genes were combined. Resistance to avilamycin was present in 13 isolates but could be related to an emtA-like gene responsible for ribosomal methylation in only 5 isolates. PCR products corresponding to the two overlapping fragments of rrl gene (domain V of 23S rRNA) and to the gene for ribosomal protein L16 were screened for mutations by PCR-single-strand conformational polymorphism. No difference in migration patterns was observed for the avilamycin-susceptible or -resistant strains. In addition, the PCR products of three strains resistant to avilamycin were sequenced. Sequence comparison with the genomic sequence of E. faecium (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) did not reveal any difference.

In conclusion, although only 16 of the 295 E. faecium isolates obtained from 620 samples were resistant to vancomycin (5.4%), these isolates often combined multiple antibiotic resistance, confirming the propensity of this species to acquire antibiotic resistance (26). Isolation of vancomycin-resistant enterococci from animals 2 years after the ban of avoparcin could be surprising. Other studies reported similar findings (3, 9), showing that reversion to susceptibility after antibiotic discontinuation does not occur rapidly. Alternatively, the fact that the strains were multiply resistant to antibiotics might have favored coselection of glycopeptide resistance by nonglycopeptide antimicrobials still used until 1999 as growth promoters.

The resistance genes detected by PCR are also found in enterococci causing infections in humans (16, 18, 20, 21, 33). The detection in this study of a mef(A) gene in an animal enterococcus further confirms that enterococci from humans and animals share the same pool of resistance genes. Most strains displayed high MICs of avilamycin (>32 μg/ml), an oligosaccharide that has been used in Europe in animals for several years (2). Similar findings have been reported for vancomycin-resistant enterococci isolated in New Zealand from poultry fecal samples with 63% of strains displaying MICs of avilamycin > 128 μg/ml and in Japan with 12.4% of E. faecium isolated from chicken fecal samples resistant to this antibiotic (23, 35). Avilamycin has the same binding site on the 50S ribosomal subunit as another oligosaccharide, evernimicin, developed initially for human therapeutics but withdrawn in 2000 for toxicity reasons (8). Evernimicin showed excellent activity against enterococci, including vancomycin-resistant isolates from human infections (17), and emergence of resistance to avilamycin in enterococci raised concerns about possible spread of resistant strains to humans (13). Only five strains carried the emt(A) gene. The lack of detection of known resistance genes in the eight remaining isolates resistant to avilamycin and in one isolate resistant to streptogramins suggested the presence of new resistance genes in enterococci from chickens.

Further study with recent strains should be carried out to assess the effects of the withdrawal of growth promoters on multiple antimicrobial resistance. Recently, Emborg et al. (15) observed a decline in the frequency of resistance to avilamycin, erythromycin, vancomycin, and virginiamycin among E. faecium from broilers and broiler meat in relation with the withdrawal of these antibiotics.