Vancomycin-Resistance Phenotypes,Vancomycin-Resistance Genes,and Resistance to Antibiotics of Enterococci Isolated from Food of Animal Origin

Abstract

In the present study, 500 raw beef, pork, and chicken meat samples and 100 pooled egg samples were analyzed for the presence of vancomycin-resistant enterococci, vancomycin-resistance phenotypes, and resistance genes. Of 141 isolates of enterococci, 88 strains of Enterococcus faecium and 53 strains of E. faecalis were identified. The most prevalent species was E. faecium. Resistance to ampicillin (n=93, 66%), ciprofloxacin (n=74, 52.5%), erythromycin (n=73, 51.8%), penicillin (n=59, 41.8%) and tetracycline (n=52, 36.9%) was observed, while 53.2% (n=75) of the isolates were multiresistant and 15.6% (n=22) were susceptible to all antibiotics. Resistance to vancomycin was exhibited in 34.1% (n=30) of the E. faecium isolates (n=88) and 1.9% (n=1) of the E. faecalis isolates (n=53) using the disc-diffusion test and the E-test. All isolates were tested for vanA and vanB using real-time polymerase chain reaction (PCR) and multiplex PCR, and for vanC, vanD, vanE, vanG genes using multiplex PCR only. Among E. faecalis isolates, no resistance genes were identified. Among the E. faecium isolates, 28 carried the vanA gene when tested by multiplex PCR and 29 when tested with real-time PCR. No isolate carrying the vanC, vanD, vanE, or vanG genes was identified. Melting-curve analysis of the positive real-time PCR E. faecium isolates showed that 22 isolates carried the vanA gene only, 2 isolates the vanB2,3 genes only, and seven isolates carried both the vanA and vanB2,3 genes. Enterococci should be considered a significant zoonotic pathogen and a possible reservoir of genes encoding resistance potentially transferred to other bacterial species.

Introduction

During the recent two decades, enterococci have emerged as an important cause of nosocomial and community-acquired infections, which are difficult to treat due to the emergence of antibiotic resistance (Tacconelli and Cataldo, 2008; Staley et al., 2014). Also, a rapid increase of vancomycin-resistant enterococci (VRE), isolated from livestock and food, has been observed (Hayes et al., 2003; Chan et al., 2008). Since the first isolation of VRE in late 1980s, isolations from humans, livestock, and food have been reported worldwide (Aarestrup et al., 2000; Robredo et al., 2000; Ramos et al., 2012; Pesavento et al., 2014).

Vancomycin and teicoplanin, which are glycopeptide antimicrobials, are used for the treatment of human infections in case of resistance or allergic reactions to β-lactams; however, the therapeutic action of vancomycin has been limited due to the emergence of VRE (Mayhall, 1996; Harada et al., 2012). Enterococci of foodborne origin are not identified as a direct cause of resistant enterococci in humans, but they could pose a risk in transfer of resistance determinants to human-adapted strains (Hayes et al., 2003; Werner et al., 2013). Glycopeptide resistance may be due to the transfer of acquired genes such as vanA, vanB, vanD, vanE, vanL, and vanN; vanC1 and vanC2/3 are responsible for intrinsic resistance in Enterococcus gallinarum and Enterococcus casseliflavus, respectively (Hegstad et al., 2010; Bourdon et al., 2011). The vanA and vanB clusters encoding vancomycin resistance are localized in mobile elements and could be transferred when there is appropriate genetic background. On the basis of sequence differences, vanB is classified in three different subtypes (vanB1, vanB2, and vanB3) and encodes the D-Ala-D-Lac ligase (Courvalin, 2006; Hegstad et al., 2010). Formerly, the vanB1 was referred as vanB gene. The vanB3 DNA heterogeneity differs from vanB1 by 5% and from vanB2 by 3.6% (Lu et al., 2001). In Greece, the first human colonization by VRE was detected in 1999, and within 2 years a polyclonal nosocomial dissemination of multiresistant vanA E. faecium was reported (Papaparaskevas et al., 1999; Maniatis et al., 2001). However, reports on VRE from livestock and food are limited (Gousia et al., 2011; Tzavaras et al., 2012). The present investigation aimed to assess the prevalence of vancomycin resistance in enterococci isolated from food samples of animal origin, to explore the genetic background of the VRE isolates by real-time polymerase chain reaction (PCR) and multiplex PCR, and to assess the antimicrobial susceptibility of Enterococcus spp. from food of animal origin.

Materials and Methods

Samples

From September 2010 to January 2012, 500 raw meat samples and 100 pooled egg samples were collected from supermarkets, butcheries, and poultry farms in Northwestern Greece. The meat samples consisted of 300 chicken, 100 porcine, and 100 bovine samples. Although eggs are an unusual source of enterococci they were included in this study due to their potential to contaminate humans when consumed raw or undercooked. The samples were collected in sterile plastic jars and were transferred to the laboratory within 4 h in insulated thermoboxes.

Enterococcus isolation and identification

From each meat sample, 25 g was weighed, placed in a stomacher bag with 225 mL of maximum-recovery diluent (NaCl 8.5 g/L, peptone 1.0 g/L, CM0733, Oxoid), and homogenized for 2 min. The homogenate was inoculated onto Slanetz-Bartley agar plates (CM0377, Oxoid) and Slanetz-Bartley agar supplemented with 6 mg/L vancomycin (Sigma-Aldrich). Each egg was flamed for 5–10 s after dipping in 70% ethanol and a hole was made by a sterile scalpel on the shell through which the contents were emptied into a stomacher bag. Pooled sets of five contents were used. Subsequently, the egg contents were diluted with an appropriate volume of maximum recovery diluent to a final dilution of 1:10, blended for 30 s in a stomacher, and 0.1 mL of the homogenate was plated onto Slanetz-Bartley agar and Slanetz-Bartley agar supplemented with vancomycin (6 mg/L). The plates were incubated at 37°C for 24–48 h. One or two characteristic colonies on Slanetz-Bartley agar plates were pure-cultured and identified to the genus level by Gram staining, catalase test, and API STREP (Biomerieux).

Phenotypic antimicrobial susceptibility

The phenotype of the antimicrobial susceptibility of the isolates was examined by both the disc-diffusion and minimum inhibitory concentration (MIC) methods. The disc-diffusion method was performed according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI, 2013). Concerning the antibiotics tested, the harmonized set of antimicrobials included in the European Food Safety Authority (EFSA) recommendations has been used, with the exception of streptomycin and gentamicin (EFSA, 2008). Teicoplanin has been included to complement vancomycin in characterizing the vancomycin-resistance phenotype. Also, ciprofloxacin and penicillin have been included, as performed by other researchers (Hayes et al., 2003; Busani et al., 2004; Eisner et al., 2005; López et al., 2009). Antibiotic discs containing ampicillin (10 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), erythromycin (15 μg), penicillin (10 IU), tetracycline (30 μg), linezolid (30 μg), quinupristin-dalfopristin (30 μg), vancomycin (30 μg), and teicoplanin (30 μg) were used. All antibiotic susceptibility discs were supplied by Oxoid (UK).

The MIC was performed with the use of the E-test against vancomycin and teicoplanin (Biomerieux, France). The CLSI-recommended breakpoint for vancomycin and teicoplanin resistance was used (32 μg/mL) (CLSI, 2013). Strains were considered as VRE when MIC was ≥32 μg/mL for vancomycin. For quality-control purposes the E. faecalis ATCC 29212 and E. faecalis ATCC 51299 strains were used.

DNA extraction

The High Pure PCR Template Preparation Kit (Roche, USA) was used for the extraction of total DNA according to the manufacturer's instructions. In brief, 200 μL from an overnight broth culture were centrifuged at 3000×g for 5 min, washed with phosphate-buffered saline twice and, after lysis with 5 μL of lysozyme (10 μg in 10 μM Tris-HCl; Roche), the binding buffer and proteinase K were added. The reaction was stopped by addition of 100 μL of isopropanol, filtered through a High Pure filter tube and washed 3×with appropriate wash buffer. A total of 200 μL of eluted DNA solution was obtained, containing 1–3 μg of nucleic acid.

Multiplex PCR

A previously published multiplex PCR method (Depardieu et al., 2004) with primers targeting the genes vanA, vanB, vanC, vanD, vanE, vanG, and ddl of E. faecium and E. faecalis was used. The ddl used permit the specific identification of E. faecium and E. faecalis (Depardieu et al., 2004). The primers used were from Lab Supplies Scientific (Athens, Greece). According to the multiplex PCR we used, the vanB primers derived from the sequences of the vanB1, vanB2, and vanB3 subtypes (Patel et al., 1998; Depardieu et al., 2004) in order to identify all three alleles.

The final volume of the PCR reaction was 50 μL containing 2–3 μL total DNA, 1×Gene Amp PCR buffer (10 mM Tris [pH 8.3], 50 mM KCl, 1.5 mM MgCl₂, 0.001% [wt/vol] gelatin), 200 μM dNTP (Life Technologies, USA), 40 pmol of each specific primer, and 1.25 U of AmpliTaq Gold DNA polymerase (Life Technologies, USA). The amplification program included an initial denaturation at 95°C for 10 min followed by 35 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min with a final extension at 72°C for 7 min. PCR products were analyzed by electrophoresis on a 2% agarose gel in 1×TBE buffer stained with ethidium bromide (10 μM) and visualized under ultraviolet light (UVP PhotoDoc-It Imaging System, USA).

Real time PCR

For the real-time PCR, the LightCycler^® VRE Research Use Only Detection Kit (Roche, USA) was used according to the manufacturer's instructions. The amplification and detection was performed on a LightCycler^®1.5 instrument (Roche, USA). The LightCycler^® VRE Research Use Only Detection Kit is sequence-specific for vancomycin-resistance genotypes vanA, vanB, and vanB2,3 (Roche, USA). All solutions were included in the detection kit with no additional information regarding their composition. The following conditions were applied: denaturation at 95°C for 10 min, 45 cycles of 95°C for 10 s, 55°C for 10 s, and 72°C for 12 s with a final cooling step at 40°C for 30 s. The amplification procedure was followed by melting-curve analysis by continuous measurement of fluorescence in a gradient transition from 45°C to 95°C in 40 s. In every PCR run, a negative control (sample without template) and a positive control (a stabilized solution of plasmid DNA—approximately 200 copies/μL—encoding a 232-bp fragment of the vanA gene and a 187 bp fragment of the vanB gene) were included. The melting curve analysis was used so as to verify the amplification of van genes and for the differentiation of vanA, vanB, and vanB2,3 genes targets.

Statistical analysis

The statistical analysis of the data was performed using the statistical package STATISTICA 8 (StatSoft, USA). The chi-square test was employed to investigate the agreement between vancomycin resistance phenotype and genotype and the detection of vanA and vanB gene with multiplex PCR and real-time PCR. Confidence intervals (95%) were set to compare the prevalence of enterococci in the samples containing the vanA and vanB genes. The analysis of phenotypic data was performed using the Spearman's rank correlation to measure the relationships between phenotypic susceptibility data and the genotypic data presented.

Results

Prevalence of Enterococcus spp. in the food samples

The prevalence of Enterococcus spp. in the analyzed food samples is presented in Table 1. Twenty percent (20/100) of the bovine, 32% (32/100) of the porcine, 21.7% (65/300) of the chicken meat samples, and 24% (24/100) of the egg samples were found to be contaminated with enterococci, with E. faecium the prevailing species. Of 141 isolates of enterococci, the amplification of specific ddl genes by PCR identified 88 strains of E. faecium and 53 strains of E. faecalis.

Table 1.

Prevalence of Enterococcus Species in Various Foods

		E. faecium		E. faecalis
	N	n	%	n	%
Bovine meat	100	10	10.0	10	10.0
Porcine meat	100	26	26.0	6	6.0
Poultry meat	300	37	12.3	28	9.3
Eggs	100	15	15.0	9	9.0
Total	600	88	14.7	53	8.8

N, number of samples; n, number of isolates.

Antimicrobial susceptibility of Enterococcus spp. isolates

The results of the susceptibility tests are presented in Table 2. Resistance to ampicillin (n=93, 66%) was observed followed by resistance to ciprofloxacin (n=74, 52.5%), erythromycin (n=73, 51.8%), penicillin (n=59, 41.8%), and tetracycline (n=52, 36.9%), while 53.2% (n=75) of the isolates were multiresistant (resistant to 3 or more antibiotic classes) and 15.6% (n=22) were susceptible to all antibiotics. Resistance to vancomycin was exhibited by 34.1% (n=30) of the E. faecium and 1.9% (n=1) of the E. faecalis strains. From the 53 E. faecalis strains, 26 (49.1%) were multiresistant, 20 (37.7%) were resistant up to 2 classes of antibiotics, and 7 (13.2%) were susceptible to all antibiotics, whereas for the 88 E. faecium strains, 49 (55.7%) were multiresistant, 24 (27.3%) were resistant to up to 2 classes of antibiotics, and 15 (17%) were susceptible.

Table 2.

Occurrence of Antimicrobial Resistance to 10 Antibiotics (Number of Isolates in Bold, Percentage Against Total Isolates in Italics)

	Enterococcus faecalis					Enterococcus faecium
Origin	Bovine (n=10)	Egg (n=9)	Porcine (n=6)	Poultry (n=28)	Total (n=53)	Bovine (n=10)	Egg (n=15)	Porcine (n=26)	Poultry (n=37)	Total (n=88)
AMP	6 (60%)	6 (66.7%)	3 (50%)	18 (64.3%)	33 (62.3%)	10 (100%)	12 (80%)	15 (57.7%)	23 (62.2%)	60 (68.2%)
C	0	3 (33.3%)	0	9 (32.1%)	12 (22.6%)	2 (20%)	2 (13.3%)	4 (15.4%)	6 (16.2%)	14 (15.9%)
CIP	1 (10%)	2 (22.2%)	1 (16.7%)	14 (50%)	18 (34%)	10 (100%)	7 (46.7%)	16 (61.5%)	23 (62.2%)	56 (63.6%)
E	0	2 (22.2%)	3 (50%)	17 (60.7%)	22 (41.5%)	8 (80%)	6 (40%)	19 (73.1%)	18 (48.6%)	51 (58%)
LZD	0	0	0	0	0	0	2 (13.3%)	0	1 (2.7%)	3 (3.4%)
P	0	5 (55.6%)	0	3 (10.7%)	8 (15.1%)	8 (80%)	8 (53.3%)	16 (61.5%)	19 (51.4%)	51 (58%)
QD	0	1 (11.1%)	0	0	1 (1.9%)	0	2 (13.3%)	1 (3.8%)	0	3 (3.4%)
TE	2 (20%)	6 (66.7%)	1 (16.7%)	20 (71.4%)	29 (54.7%)	0	4 (26.7%)	4 (15.4%)	15 (40.5%)	23 (26.1%)
TEC	0	0	0	0	0	6 (60%)	4 (26.7%)	11 (42.3%)	7 (18.9%)	28 (31.8%)
VA	0	0	0	1 (3.6%)	1 (1.9%)	8 (80%)	4 (26.7%)	11 (42.3%)	7 (18.9%)	30 (34.1%)

AMP, ampicillin; C, chloramphenicol; CIP, ciprofloxacin; E, erythromycin; P, penicillin; TE, tetracycline; LZD, linezolid; QD, quinupristin-dalfopristin; VA, vancomycin; TEC, teicoplanin.

Molecular detection of vancomycin-resistance genes

The real-time PCR detected the vanA gene in 22 E. faecium strains, the vanB2,3 genes in two E. faecium strains, and both the vanA and vanB2,3 genes in 7 E. faecium strains. The prevalence among VRE strains (n=31) carrying the vanA gene was higher (n=29, 93.5%) than that of the vanB2,3 genes (n=9, 29%). No E. faecalis carrying vanA, vanB, or vanB2,3 genes was identified. Both the phenotypic and molecular tests showed that almost all vanA carriers expressed resistance (p<0.05). The E-test revealed that with the exception of 2 strains, the remaining 27 vanA strains presented high MICs to vancomycin and teicoplanin (Table 3).

Table 3.

Resistant Phenotypes, Resistance Gene Carriage and Minimum Inhibitory Concentration (MIC) of Vancomycin Resistant Enterococcus faecium (Genotype or Phenotype) Strains Isolated from Various Foods, According to Real-Time Polymerase Chain Reaction

	MIC (μg/mL)
Source	VA	TEC	Vancomycin resistance genes	Resistance phenotype for other antibiotics
Poultry	>256	>256	van A, van B2,3	AMP, CIP, E, P, TE, TEC, VA
Bovine	>256	>256	van A, van B2,3	AMP, CIP, E, P, TEC, VA
Bovine	>256	64	van A, van B2,3	AMP, CIP, E, P, TEC, VA
Bovine	>256	>256	van A, van B2,3	AMP, CIP, E, P, TEC, VA
Bovine	>256	64	van A, van B2,3	AMP, CIP, E, P, TEC, VA
Egg^a	2	0.125	van A, van B2,3	AMP, P
Egg^b	2	0.125	van A, van B2,3	AMP, P
Porcine	>256	>256	van A	AMP, CIP, E, P, TE, TEC, VA
Porcine	>256	>256	van A	AMP, CIP, E, P, TE, TEC, VA
Porcine	>256	>256	van A	AMP, CIP, E, P, TE, TEC, VA
Poultry	>256	>256	van A	AMP, CIP, E, P, TE, TEC, VA
Porcine	>256	>256	van A	AMP, CIP, E, P, TE, TEC, VA
Egg	>256	64	van A	AMP, CIP, E, LZD, P, QD, TE, TEC, VA
Egg	>256	64	van A	AMP, CIP, E, LZD, P, QD, TE, TEC, VA
Egg	>256	64	van A	AMP, CIP, E, P, TEC, VA
Porcine	>256	>256	van A	AMP, CIP, E, P, TEC, VA
Porcine	>256	>256	van A	AMP, CIP, E, P, TEC, VA
Porcine	>256	>256	van A	AMP, CIP, E, P, TEC, VA
Bovine	>256	64	van A	AMP, CIP, E, P, TEC, VA
Poultry	>256	64	van A	AMP, CIP, E, P, TEC, VA
Egg	>256	64	van A	AMP, CIP, E, P, TEC, VA
Porcine	>256	>256	van A	AMP, CIP, E, P, TEC, VA
Porcine	>256	>256	van A	AMP, CIP, E, P, TEC, VA
Porcine	>256	>256	van A	AMP, CIP, E, P, TEC, VA
Bovine	>256	64	van A	AMP, CIP, E, P, TEC, VA
Poultry	>256	64	van A	AMP, CIP, E, P, TEC, VA
Poultry	>256	>256	van A	AMP, CIP, E, TE, TEC, VA
Poultry	>256	>256	van A	AMP, CIP, E, TE, TEC, VA
Porcine	>256	>256	van A	AMP, QD, TEC, VA
Bovine	64	4	van B2,3	AMP, C, CIP, E, P, VA
Bovine	64	4	van B2,3	AMP, C, CIP, E, P, VA
Poultry^c	>256	48	No gene	AMP, CIP, E, LZD, P, TE, TEC, VA

a,b

E. faecium. Strains with non-VanA or non-VanB phenotype, but vanA and vanB2,3 genotype.

E. faecium. Strain with VanA phenotype, but not vanA genotype.

AMP, ampicillin; C, chloramphenicol; CIP, ciprofloxacin; E, erythromycin; P, penicillin; TE, tetracycline; LZD, linezolid; QD, quinupristin-dalfopristin; VA, vancomycin; TEC, teicoplanin.

The multiplex PCR detected the vanA gene in 28 E. faecium isolates, vanB in 2 E. faecium isolates, and both vanA and vanB in 6 E. faecium isolates. No isolate carrying the vanC, vanD, vanE, or vanG genes was identified. All the strains gave only the expected band corresponding to the conserved region of 16S rRNA.

The choice of two methodologies was based on the complementarities of the methods, since the LightCycler^® VRE Research Use Only Detection kit is detecting only vanA, vanB and vanB2,3 genes, while the multiplex PCR method of Depardieu et al. (2004) detects the vanA, vanB, vanC, vanD, vanE, and vanG genes.

Discussion

The consumption of food carrying resistant enterococci strains is considered a possible route of transfer from animals to humans (Lester et al., 2006; Vignaroli et al., 2011; Sparo et al., 2012; Klibi et al., 2013). In this study, 20% (n=20) of the bovine, 32% (n=32) of the porcine, 21.7% (n=65) of the chicken, and 24% (n=24) of the egg samples were contaminated with enterococci (Table 1). Higher prevalences have been reported from researchers elsewhere (McGowan et al., 2006; Pesavento et al., 2014). Enterococci occurrence in egg content has been scarcely reported before, yet their potential of foodborne transmission exists. In a previous study carried out in Northwestern Greece, E. faecalis was isolated from only 0.4% (n=1) of eggs (Papadopoulou et al., 1997), probably due to use of nonselective culture media. Schwaiger et al. (2010) investigated the presence of Enterococcus spp. in eggs and reported 21% (n=19) to be contaminated.

The most prevalent species were E. faecium (n=88, 62.4%) and E. faecalis (n=53, 37.6%). E. faecium comprised 50% (n=10), 81.3% (n=26), 56.9% (n=37) and 62.5% (n=15) of the total enterococci isolated from beef (n=20), pork (n=32), chicken (n=65) and eggs (n=24) respectively. Analogous results have been reported from Turkey (Kasimoglu-Dogru et al., 2010), Italy (Busani et al., 2004), Spain (Martin et al., 2005), and United States (Hayes et al., 2003). E. faecalis was more predominant in Denmark (Aarestrup et al., 2000), Greece (Gousia et al., 2011), and Italy (Pesavento et al., 2014). In Germany (Lemcke and Bülte, 2000) and Tunisia (Klibi et al., 2013), the higher prevalence was shared by E. faecalis and E. faecium. The discrepancies in the reported results are attributed to different study periods, sources, and geographical regions, and diverse methodologies utilized.

Resistance to ampicillin (n=93, 66%), ciprofloxacin (n=74, 52.5%), erythromycin (n=73, 51.8%), penicillin (n=59, 41.8%) and tetracycline (n=52, 36.9%) along with a considerable number of multiresistant isolates (53.2%, n=75) was observed. To our knowledge, there are no officially available data about the veterinary use of antimicrobials in Greece. However, the antibiotics commonly used in poultry farms are penicillins (amoxicillin), quinolones (enrofloxacin), tetracyclines (doxycycline, oxytetracycline), macrolides (erythromycin, tylosine), aminoglycosides, the sulfonamide and trimethoprim combination, polymyxins (colistin), and other antimicrobials (tiamulin). Comparing the susceptibility results relating to eggs with those of a previous study in this region (Papadopoulou et al., 1997), a notable resistance increase was observed.

VanA and VanB are considered the most frequent phenotypes, encountered in >95% of VRE strains (Lambert, 2013). The VanA phenotype is responsible for high resistance to both vancomycin and teicoplanin, while the VanB phenotype is characterized by various levels of resistance to vancomycin (MIC, 4–1024 μg/mL) and susceptibility to teicoplanin (Courvalin, 2006). In this study, resistance to vancomycin was exhibited mostly by E. faecium (n=30, 34.1% of E. faecium strains), with only 1 resistant E. faecalis (1.9%) isolated. Comparable resistance to vancomycin (n=12, 21.1%) in isolates from porcine meat was reported by Gousia et al., 2011). Messi et al. (2006) reported VRE prevalence of 35.1% in meat samples (n=59). Novais et al. (2005) reported higher VRE prevalence (48%) in poultry samples, but the breakpoints for the classification of VRE were higher in our study (VA≥32 μg/mL versus VA≥8 μg/mL).

In this study, 22 strains contained the vanA gene, 2 strains contained the vanB2,3 genes, and 7 strains contained both vanA and vanB2,3. This is the first report of acquired resistance genes from Enterococcus spp. isolated from food samples in Greece. The overall prevalence of VRE in food samples was 5.1% (31 VRE-van genotypes in 600 food samples). López et al. (2009), using a similar methodological approach, reported an analogous percentage (n=9, 3.9%) of acquired resistance genes in enterococci from food in Spain.

In the analyzed food samples, the prevalence of VREs carrying the vanA gene (n=29, 4.8%) is significantly higher (p<0.05) than the vanB2,3 genes (n=9, 1.5%). This is probably due to the greater mobility of the vanA gene cluster than that of the vanB gene cluster (Corso et al., 2007). The vanB cluster is similar in its organization and regulation to that of vanA, but differs in that vancomycin, but not teicoplanin, is an inducer of the vanB cluster (Courvalin, 2006). Furthermore, the good fit between the phenotypic and molecular tests indicates that almost all vanA carriers expressed resistance. The E-test revealed that 27 vanA strains presented high MICs to vancomycin and teicoplanin. Among the VRE strains, no E. faecalis carrying vanA, vanB, or vanB2,3 genes was identified. It has been reported that though E. faecalis strains are most frequently isolated, they have a lower incidence of vanA or vanB genes (Freitas et al., 2009, 2011). In our study, no strain was carrying the vanC, vanD, vanE, or vanG genes.

The statistical analysis of the phenotypic susceptibility data revealed correlations between vancomycin-MIC and vanA (r_s=0.485), vanB (r_s=−0.602) genes and between teicoplanin-MIC and vanA (r_s=0.480), vanB (r_s=−0.378). Weak correlation was found between food source and vanB (r_s=0.353). Strong correlations were observed between E. faecium phenotypic susceptibility data and food sources. For example, the E. faecium susceptibility pattern from bovine samples correlated strongly with the corresponding pattern from eggs (r_s=0.76), porcine (r_s=0.84), and poultry samples (r_s=0.84). All correlations are significant at p<0.05. However, for E. faecalis the susceptibility pattern from porcine correlated only with the poultry samples (r_s=0.90).

Concerning detection of vanA and vanB genes among the E. faecium isolates, no statistically significant difference (p>0.05) was observed. The majority were resistant to vancomycin and teicoplanin by the disc diffusion and MIC tests, with the exception of two egg isolates, expressing unusually low resistance to vancomycin and teicoplanin. Although these isolates displayed a non-VanA phenotype (MIC for vancomycin=2 μg/mL, MIC for teicoplanin=0.125 μg/mL), vanA and vanB2,3 were detected by both PCRs. VanA genotype with non-VanA phenotype has been reported by other researchers (Huh et al., 2004; Naas et al., 2005; Chan et al., 2008). In a study on clinical and dairy enterococci, strains containing the vanA gene exhibited MIC values <6 μg/mL (Ribeiro et al., 2007). Also, vanB genotype with non-VanB phenotype has been reported by Klare et al. (2012).

Regarding the vanB genes, eight E. faecium isolates carried the vanB gene by the multiplex PCR, instead of nine E. faecium strains that carried the vanB2,3 genes when tested by the real-time PCR. Among them, seven E. faecium strains carried both vanA and vanB2,3 genes according to both real-time and multiplex PCR. The performance of the employed molecular assays was comparable for both vanA and vanB identification (p<0.05). This observation is based on the evaluation of the detection of the vanA or vanB genes in E. faecium isolates and not on E. faecalis isolates because of the low incidence of vancomycin-resistant E. faecalis. Similar observations have been reported before (Aarestrup et al., 2002; Freitas et al., 2009; Freitas et al., 2011; Werner, 2012).

Interestingly, one E. faecium strain of poultry origin, negative for the van genes, exhibited a VanA phenotype (MIC for vancomycin=256 μg/mL, MIC for teicoplanin=48 μg/mL). Although a mutation to the tested van genes could not be ruled out, this phenotype is compatible with the carriage of vanM gene, which is reported to express MIC ≥256 μg/mL for vancomycin and MIC for teicoplanin between 0.75 and 48 μg/mL (Werner, 2012; Johnson and Cashara, 2013).

The results indicate that a variety of potentially transferable antibiotic resistance determinants, particularly vanA, vanB2, and vanB3, occur among E. faecium strains from food. VanA is similarly disseminated in enterococci from eggs, bovine and porcine meat samples, and its presence corresponds to a resistant behavior. However, the results concerning vanB suggest that the porcine isolates are not in contact with vanB genes. Generally, food isolates are not often in contact with vanB genes (Biavasco et al., 2007; Werner et al., 2013). Conclusively, the combination of antibiotic resistance and virulence determinants may be a health risk for the dissemination of VRE through the food chain; therefore, surveillance and vigilance of their emergence in food is imperative in order to safeguard public health.

Footnotes

Disclosure Statement

No competing financial interests exist.

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