The characterization of Listeria spp. isolated from food products and the food‐processing environment

Aim: To enhance the information pertaining to the epidemiology of a collection of 378 Listeria spp. isolates obtained from several food‐processing plants in Ireland over a 3‐ year period (2004–2007). Methods and results: The collection was characterized by pulsed‐field gel electrophoresis (PFGE). The most prevalent pulse‐type was PFGE profile I (n = 14·5%) that consisted mainly of environmental Listeria spp. samples. Serotyping of 145 Listeria monocytogenes isolates was performed. The most common serovar was 1/2a and comprised 57·4% (n = 77) of the L. monocytogenes collection. The other serovars were as follows: 4b (14·1%, n = 19), 1/2b (9·7%, n = 13), 4c (4·4%, n = 6) and 1/2c (6·7%, n = 9), respectively. Eleven isolates were identified as non‐Listeria spp., the remaining ten L. monocytogenes isolates were nontypeable. The antimicrobial susceptibility testing revealed the antibiotic that isolates displayed the most resistance to was gentamicin (5%) followed by sulfamethoxazole‐trimethoprim (2%), tetracycline and ciprofloxacin (1·5%). Conclusions: The subtyping has indicated the diversity of the Listeria spp. The presence of serotype 1/2a, 1/2b and 4b in both raw and cooked ready‐to‐eat food products is a public health concern, as these serotypes are frequently associated with foodborne outbreaks and sporadic cases of human listeriosis. In addition, the emergence of antimicrobial‐resistant L. monocytogenes isolates could have serious therapeutic consequences. Significance and Impact of Study: The molecular subtyping and the further characterization of these isolates may be valuable particularly in the context of a suspected common source outbreak in the future.     

Isolation and Confirmation of <Emphasis Type="Italic">Listeria</Emphasis> Species from Seafood off Goa Region by Polymerase Chain Reaction

Several food borne outbreaks have highlighted the importance of Listeria monocytogenes to the public health and have been recognized as an emerging, important food borne pathogen, and a causative agent of listerioses. A number of genes are involved in the manifestation of Listeria virulence, hlyA is one among them. In the present study, 111 marine fish samples including prawns, finfishes and bivalves were screened for the presence of Listeria species. The isolates were characterized biochemically and further L. monocytogenes were confirmed by polymerase chain reaction (PCR) technique using the hlyA gene as a tool to differentiate between L. monocytogenes and other non-pathogenic Listeria species. Out of 111 samples 5 (4.5%) samples were positive for L. monocytogenes. Among the three different types of samples bivalves were found to have maximum percent (12.5) of L. monocytogenes followed by prawns (3.84) and finfishes (2.9). Among all the 111 samples, 15 (13.51%) samples were positive for other Listeria species. It was observed that Listeria occurrence is more in shellfishes than in fin fishes. All the isolates were sensitive towards five different antibiotics in sequence ciprofloxacin > sulphafurazole > norfloxacin > ampicillin and gentamicin.     

Identification of tetracycline‐ and erythromycin‐resistant Gram‐positive cocci within the fermenting microflora of an Italian dairy food product

Aims: Microbiological and molecular analysis of antibiotic resistance in Gram‐positive cocci derived from the Italian PDO (Protected Designation of Origin) dairy food product Mozzarella di Bufala Campana. Methods and Results: One hundred and seven coccal colonies were assigned to Enterococcus faecalis, Lactococcus lactis and Streptococcus bovis genera by ARDRA analysis (amplified ribosomal DNA restriction analysis). Among them, 16 Ent. faecalis, 26 L. lactis and 39 Strep. bovis displayed high minimum inhibitory concentration (MIC) values for tetracycline, while 17 L. lactis showed high MIC values for both tetracycline and erythromycin. Strain typing and molecular analysis of the phenotypically resistant isolates demonstrated the presence of the tet(M) gene in the tetracycline‐resistant strains and of tet(S) and erm(B) in the double‐resistant strains. Southern blot analysis revealed plasmid localization of L. lactis tet(M), as well as of the erm(B) and tet(S) genes. Genetic linkage of erm(B) and tet(S) was also demonstrated by PCR amplification. Conjugation experiments demonstrated horizontal transfer to Ent. faecalis strain JH2‐2 only for the plasmid‐borne L. lactis tet(M) gene. Conclusions: We characterized tetracycline‐and erythromycin‐resistance genes in coccal species, representing the fermenting microflora of a typical Italian dairy product. Significance and Impact of the Study: These results are of particular relevance from the food safety viewpoint, especially in the light of the potential risk of horizontal transfer of antibiotic‐resistance genes among foodborne commensal bacteria.     

Mechanisms of antimicrobial resistance and genetic relatedness among enterococci isolated from dogs and cats in the United States

Aims: In this study, mechanisms of antimicrobial resistance and genetic relatedness among resistant enterococci from dogs and cats in the United States were determined. Methods and Results: Enterococci resistant to chloramphenicol, ciprofloxacin, erythromycin, gentamicin, kanamycin, streptomycin, lincomycin, quinupristin/dalfopristin and tetracycline were screened for the presence of 15 antimicrobial resistance genes. Five tetracycline resistance genes [tet(M), tet(O), tet(L), tet(S) and tet(U)] were detected with tet(M) accounting for approx. 60% (130/216) of tetracycline resistance; erm(B) was also widely distributed among 96% (43/45) of the erythromycin‐resistant enterococci. Five aminoglycoside resistance genes were also detected among the kanamycin‐resistant isolates with the majority of isolates (25/36; 69%) containing aph(3′)‐IIIa. The bifunctional aminoglycoside resistance gene, aac(6′)‐Ie‐aph(2″)‐Ia, was detected in gentamicin‐resistant isolates and ant(6)‐Ia in streptomycin‐resistant isolates. The most common gene combination among enterococci from dogs (n = 11) was erm(B), aac(6′)‐Ie‐aph(2″)‐Ia, aph(3′)‐IIIa, tet(M), while tet(O), tet(L) were most common among cats (n = 18). Using pulsed‐field gel electrophoresis (PFGE), isolates clustered according to enterococcal species, source and antimicrobial gene content and indistinguishable patterns were observed for some isolates from dogs and cats. Conclusion: Enterococci from dogs and cats may be a source of antimicrobial resistance genes. Significance and Impact of the Study: Dogs and cats may act as reservoirs of antimicrobial resistance genes that can be transferred from pets to people. Although host‐specific ecovars of enterococcal species have been described, identical PFGE patterns suggest that enterococcal strains may be exchanged between these two animal species.     

Human heat-shock protein 60 receptor-coated paramagnetic beads show improved capture of Listeria monocytogenes in the presence of other Listeria in food

Aims: To investigate the suitability of human Hsp60, a receptor for Listeria adhesion protein (LAP), on paramagnetic beads (PMB) to capture Listeria monocytogenes from food in the presence of other Listeria to facilitate rapid and specific detection of this pathogen. Methods and Results: Commercially available streptavidin‐coated PMBs were linked with biotinylated Hsp60 (PMB‐Hsp60), and the bacterial capture efficiency from pure culture and meat samples was determined. Capture rate was also compared with the monoclonal antibody (MAb)‐C11E9‐coated beads (PMB‐C11E9) and the commercial Dynabeads anti‐Listeria. Captured cells were detected and quantified by plating on selective medium, quantitative real‐time PCR (qPCR) and a light‐scattering sensor. Overall, all ligand‐coated beads had similar capture efficiency (varied from 1·8 to 9·2%) for L. monocytogenes under the conditions employed, and the minimum cell number required to achieve such capture was 10³ CFU ml^?1. PMB‐Hsp60 had significantly greater capture efficiency for pathogenic Listeria (P < 0·0001) than the nonpathogenic Listeria. In contrast, PMB‐C11E9 and Dynabeads anti‐Listeria had similar capture efficiency for both. The efficacy of all PMBs to capture L. monocytogenes in the presence of Listeria innocua from food matrices was compared. Although Dynabeads anti‐Listeria had the overall best capture efficiency, PMB‐Hsp60 was able to selectively capture L. monocytogenes even in the presence of 10–100‐fold more L. innocua cells from enriched meat samples. Conclusions: Data show that the human cell receptor, Hsp60, is suitable for the capture of pathogenic Listeria on PMB in the presence of other Listeria in food. Significance and Impact of the Study: As pathogen interaction with host cells is highly specific, host cell receptors could be used as alternate capture molecules on PMB to aid in specific detection of pathogens.     

Antimicrobial resistance in faecal enterococci and Escherichia coli isolates recovered from Iberian wolf

The aim of this study was to report the antimicrobial resistance, the molecular mechanisms associated and the detection of virulence determinants within faecal Enterococcus spp. and Escherichia coli isolates of Iberian wolf. Enterococci (n = 227) and E. coli (n = 195) isolates were obtained from faecal samples of Iberian wolf (Canis lupus signatus). High rates of resistance were detected for tetracycline and erythromycin among the enterococci isolates, and most of resistant isolates harboured the tet(M) and/or tet(L) and erm(B) genes, respectively. The bla_TEM, tet(A) and/or tet(B), and aadA or strA‐strB genes were detected among most ampicillin‐, tetracycline‐ or streptomycin‐resistant E. coli isolates, respectively. E. coli isolates were ascribed to phylogroups A (n = 56), B1 (91), B2 (13) and D (35). The occurrence of resistant enterococci and E. coli isolates in the faecal flora of Iberian wolf, including the presence of resistant genes in integrons, and virulence determinants was showed in this study. Iberian wolf might act as reservoir of certain resistance genes that could be spread throughout the environment.

Significance and Impact of the Study

This study shows antimicrobial resistance in commensal bacteria from the free‐range, Portuguese, Iberian wolf population. The results indicate that the Iberian wolf could contribute to the spread of resistant bacteria throughout the environment. Additionally, in case of infection, an increased risk of therapeutic failure due to the presence of multiresistant bacteria may represent a health problem for this endangered species. Future studies must be performed to analyse the possible contamination of these animals through the environment and/or the food chain.     

Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua

Aims: Detectability of Listeria monocytogenes at 10⁰ CFU per food sample in the presence of Listeria innocua using standard microbiological detection was evaluated and compared with the real‐time PCR‐based method. Methods and Results: Enrichment in half‐Fraser broth followed by subculture in Fraser broth according to EN ISO 11290‐1 was used. False‐negative detection of 10⁰ CFU L. monocytogenes was obtained in the presence of 10¹ CFU L. innocua per sample using the standard detection method in contrast to more than 10⁵ CFU L. innocua per sample using real‐time PCR. Identification of L. monocytogenes on the chromogenic medium by the standard procedure was impossible if L. innocua was able to overgrow L. monocytogenes by more than three orders of magnitude after the enrichment in model samples. These results were confirmed using naturally contaminated food samples. Conclusions: Standard microbiological method was insufficient for the reliable detection of 10⁰ CFU L. monocytogenes in the presence of more than 10⁰ CFU of L. innocua per sample. On the other hand, if the growth of L. monocytogenes was sufficient to reach the concentration equal to the detection limit of PCR, the amount of the other microflora present in the food sample including L. innocua was not relevant for success of the PCR detection of L. monocytogenes. Significance and Impact of the Study: After the enrichment, the PCR detection is more convenient than the standard one as PCR detection is not compromised by other present microflora.     

Antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food

Aim: To develop antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food products. Methods and Results: Aptamer, a single‐stranded oligonucleotide ligand that displays affinity for the target molecule, was used in the assay to provide sensor specificity. Aptamer‐A8, specific for internalin A, an invasin protein of L. monocytogenes, was used in the fibre‐optic sensor together with antibody in a sandwich format for detection of L. monocytogenes from food. Biotinylated polyclonal anti‐Listeria antibody, P66, was immobilized on streptavidin‐coated optical waveguide surface for capturing bacteria, and Alexa Fluor 647‐conjugated A8 was used as a reporter. The biosensor was able to selectively detect pathogenic Listeria in pure culture and in mixture with other bacteria at a concentration of approx. 10³ CFU ml^?1. This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 10² CFU 25 g^?1) ready‐to‐eat meat products such as sliced beef, chicken and turkey after 18 h of enrichment. Conclusion: Based on the data presented in this study, the antibody–aptamer functionalized fibre‐optic biosensor could be used as a detection tool for sensitive and specific detection of L. monocytogenes from foods. Significance and Impact of the Study: The study demonstrates feasibility and novel application of aptamer on fibre‐optic biosensor platform for the sensitive detection of L. monocytogenes from food products.     

Combined effect of synthetic enterocin CRL35 with cell wall,membrane‐acting antibiotics and muranolytic enzymes against Listeria cells

Aims: To evaluate the inhibition effectiveness of enterocin CRL35 in combination with cell wall, membrane‐acting antibiotics and muranolytic enzymes against the foodborne pathogen Listeria. Methods and Results: Synthetic enterocin CRL35 alone and in combination with monensin, bacitracin, gramicidin, mutanolysin and lysozyme were used in this study. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) index assays were performed using Listeria innocua 7 and Listeria monocytogenes FBUNT as sensitive strains. Antibiotics showed positive interactions with the bacteriocin in both strains tested. On the other hand, when mutanolysin and enterocin CRL35 were added to resting cells in a buffer system, the lytic effect of mutanolysin was enhanced. However, the addition of mutanolysin showed no effect on the growth of L. innocua 7 cells in a culture medium. Moreover, mutanolysin allowed the overgrowth of L. innocua 7 cells to an OD similar to control cells in the presence of inhibitory concentration of enterocin CRL35. In contrast, the combination of lysozyme and enterocin CRL35 resulted in a 50% inhibition of the L. innocua 7 growth. Conclusions: Based on our results, we conclude that the combination of synthetic enterocin CRL35 with some antibiotics is effective against L. innocua 7 and L. monocytogenes FBUNT cells, and more importantly the amount of these agents to be used was considerably reduced. The effectiveness of the combination of synthetic enterocin CRL35 with muramidases seems to depend on complex environments, and more detailed studies need to be performed to elucidate this issue. Significance and Impact of the Study: Enterocin CRL35 represents a promising agent that not only can ensure the quality and safety of food but it can also be combined with several antimicrobial agents important in the medical field.     

Detection and Differentiation of Listeria spp. by a Single Reaction Based on Multiplex PCR

The iap gene encodes the protein p60, which is common to all Listeria species. A previous comparison of the DNA sequences indicated conserved and species-specific gene portions. Based on these comparisons, a combination consisting of only five different primers that allows the specific detection and differentiation of Listeria species with a single multiplex PCR and subsequent gel analysis was selected. One primer was derived from the conserved 3′ end and is specific for all Listeria species; the other four primers are specific for Listeria monocytogenes, L. innocua, L. grayi, or the three grouped species L. ivanovii, L. seeligeri, and L. welshimeri, respectively. The PCR method, which also enables the simultaneous detection of L. monocytogenes and L. innocua, was evaluated against conventional biotyping with 200 food hygiene-relevant Listeria strains. The results indicated the superiority of this technique. Thus, this novel type of multiplex PCR may be useful for rapid Listeria species confirmation and for identification of Listeria species for strains isolated from different sources.     

Prevalence and characteristics of Listeria monocytogenes in meat,meat products,food handlers and the environment of the meat processing and the retail facilities of a company in Northern Greece

In this study, we investigated the incidence of Listeria monocytogenes in the receiving meat, the meat products, the personnel and the environment of a vertically integrated company in Northern Greece owing a processing plant and three trading facilities. A total of 303 samples were examined from the receiving raw meat, raw meat preparations, ready-to-eat meat products, processing surfaces and the environment of these facilities as well as the food handlers’ hands and nasal cavities. MALDI-TOF MS was used for Listeria identification; from the 22 (7·26%) positive to Listeria spp. isolates, 12 (3·96%) identified as L. monocytogenes, eight (2·64%) as Listeria innocua and two (0·66%) as Listeria welshimeri. Molecular serotyping of L. monocytogenes isolates by multiplex PCR revealed 11 strains belonging to serogroup IIa (1/2a and 3a) and one to IIc (1/2c and 3c). The assay for the detection of the virulence-associated genes revealed eight isolates carrying all the examined genes (inlA, inlB, inlC, plcA, prfA, actA, hlyA and iap) and four carrying all except the actA gene. Eleven (91·7%) of the isolates showed a strong ability to form biofilm. All isolates were multidrug resistant. The MALDI-TOF Main Spectrum Profile (MSPs), revealed three clusters: one with five isolates (four from environmental samples and one from a food handler), one with five isolates (all from environmental samples) and one with two isolates (both from raw meat products). MALDI-TOF MS seems to be a reliable tool for the identification of niches and contamination routes in processing plants, contributing also to the evaluation and improvement of the applied preventive measures to control L. monocytogenes.     

Prevalence of Antimicrobial Resistance and Transfer of Tetracycline Resistance Genes in Escherichia coli Isolates from Beef Cattle

The aim of this study was to investigate the prevalence and transferability of resistance in tetracycline-resistant Escherichia coli isolates recovered from beef cattle in South Korea. A total of 155 E. coli isolates were collected from feces in South Korea, and 146 were confirmed to be resistant to tetracycline. The tetracycline resistance gene tet(A) (46.5%) was the most prevalent, followed by tet(B) (45.1%) and tet(C) (5.8%). Strains carrying tet(A) plus tet(B) and tet(B) plus tet(C) were detected in two isolates each. In terms of phylogenetic grouping, 101 (65.2%) isolates were classified as phylogenetic group B1, followed in decreasing order by D (17.4%), A (14.2%), and B2 (3.2%). Ninety-one (62.3%) isolates were determined to be multidrug resistant by the disk diffusion method. MIC testing using the principal tetracyclines, namely, tetracycline, chlortetracycline, oxytetracycline, doxycycline, and minocycline, revealed that isolates carrying tet(B) had higher MIC values than isolates carrying tet(A). Conjugation assays showed that 121 (82.9%) isolates could transfer a tetracycline resistance gene to a recipient via the IncFIB replicon (65.1%). This study suggests that the high prevalence of tetracycline-resistant E. coli isolates in beef cattle is due to the transferability of tetracycline resistance genes between E. coli populations which have survived the selective pressure caused by the use of antimicrobial agents.     

MLST and a genetic study of antibiotic resistance and virulence factors in vanA‐containing Enterococcus from buzzards (Buteo buteo)

Aims: To analyse the occurrence of faecal carriage of vancomycin‐resistant enterococci (VRE) in Buteo buteo and to study the associated resistance and virulence genes. Methods and Results: The presence of VRE was investigated in 33 faecal samples of B. buteo. Samples were seeded in Slanetz–Bartley agar plates supplemented with vancomycin for VRE recovery. Genes encoding antimicrobial resistance and virulence were studied by polymerase chain reaction. Vancomycin‐resistant Enterococcus faecium isolates were characterized by multilocus sequence typing. VRE with an acquired mechanism of resistance (vanA genotype) were detected in 9% of samples analysed (Ent. faecium and Enterococcus durans). In addition, 27% of samples contained VRE with an intrinsic mechanism of resistance (Enterococcus gallinarum, vanC1). All vanA‐containing isolates showed resistance to tetracycline and erythromycin and harboured the tet(M) and/or tet(L) genes, in addition to the ermB gene. The vat(E) and/or vat(D), cat(A) and aph(3′)‐IIIa genes were identified in quinupristin–dalfopristin‐, chloramphenicol‐, and kanamycin‐resistant vanA‐containing strains, respectively. The sequence types ST273 and ST5 were identified in two vanA‐positive Ent. faecium isolates, and the presence of hyl, gelE, cylA, cylL and cylM virulence genes and gelatinase activity were identified in Ent. faecium ST5 strain. Conclusions: The intestinal tract of B. buteo could be a reservoir of vanA‐positive enterococci. Significance and Impact of the Study: First study focused to define the occurrence of vanA‐containing Enterococcus strains in B. buteo.     

Molecular diversity and transferability of the tetracycline resistance gene <Emphasis Type="Italic">tet</Emphasis>(M), carried on Tn<Emphasis Type="Italic">916-1545</Emphasis> family transposons,in enterococci from a total food chain

In the present study, 20 enterococci belonging to the species Enterococcus faecalis (12 strains), Enterococcus faecium (4), Enterococcus durans (2), Enterococcus hirae (1) and Enterococcus mundtii (1) and originating from a total production chain of swine meat commodities were analysed to investigate the diversity of their tetracycline resistance gene tet(M). PCR–RFLP and sequence analysis showed that the tet(M) gene of most strains can be correlated with the Tn916 transposon. Conversely, tet(M) of six E. faecalis and the E. hirae strain, all isolated from pig faecal samples, may be associated with previously undescribed members of the Tn916-1545 transposon family. In vitro filter conjugation trials showed the ability of 50% of the enterococcal strains, including E. mundtii, to transfer the tet(M) gene (and the associated Tn916 and new transposons) to E. faecalis or Listeria innocua recipient strains. tet(M) gene transfer to L. innocua recipient was also directly observed in meat food products. Collectively, these sequence and conjugation data indicate that various transposons can be responsible of the spread of tetracycline resistance in enterococci and validate the opinion that Enterococcus species are important sources of antibiotic resistance genes for potentially pathogenic bacteria occurring in the food chain. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of the prfA Virulence Gene Cluster Insertion Site in Non-Hemolytic Listeria spp.: Probing the Evolution of the Listeria Virulence Gene Island

The prfA virulence gene cluster is present between prs and ldh in the pathogenic L. monocytogenes and L. ivanovii, but absent from the non-pathogenic L. innocua and L. welshimeri. To probe the evolution of this virulence gene cluster, we sequenced the prs-ldh intergenic region in L. welshimeri and L. innocua. Two ORFs (ORFA and ORFB) were found in both species as well as in L. monocytogenes. Another ORF of unknown function (ORFZ) was found in L. monocytogenes and L. innocua, while two unique ORFs were present in L. welshimeri. ORFA and ORFB showed significant functional constraint, suggesting that further investigations in the functions of these genes, including possible roles in horizontal gene transfer or sequence deletion, are warranted. DNA sequences homologous to Tn1545 integration consensus sequences were found downstream of prs and ORFB, thus defining the likely junctions of the virulence gene island and indicating that the prs-ldh intergenic region may represent a Tn insertion hot spot. Our results are consistent with the hypothesis that a combination of horizontal gene transfer and deletion events may have been involved in the evolution of the prfA virulence gene cluster in Listeria. Received: 27 November 2000 / Accepted: 20 February 2001     

Typing of Listeria monocytogenes strains isolated in Italy by inlA gene characterization and evaluation of a new cost‐effective approach to antisera selection for serotyping

Aims: In this study, 105 Listeria monocytogenes strains isolated from humans, foods and environmental samples were characterized using several typing methods. Moreover, serotyping procedure was evaluated, and a cost‐effective methodological approach based on preliminary PCRs screening was proposed. Methods and Results: The isolates were analysed by conventional serotyping, multiplex‐PCRs for serogroup and lineage identification and PCR–RFLP of inlA gene to identify potentially noninvasive L. monocytogenes. Among the strains, only the serotypes 1/2a, 1/2c, 1/2b, 4b and 3a were identified. The isolates were classified into serogroups I (58·10%), II (22·85%), III (12·38%) and IV (6·67%). Among clinical strains, lineage I was more represented (68·75%) than lineage II; whereas, lineage II was more associated with food (90·24%) and environmental (85·72%) isolates. Most of food (89·02%) and environmental (85·71%) isolates were classified into truncated InlA profiles, whereas the 93·75% of clinical strains were associated with a complete form of the protein. Conclusion: Molecular techniques were sensitive and specific for classifying strains into serogroup and lineage and in agreement with the serotyping. Moreover, a preliminary PCRs‐based screening was proposed to select only the necessary antisera by a flow chart; this methodological approach allows cost saving up to 42%. Our results further suggest the role of InlA protein in human listeriosis, particularly in immunocompetent individuals, and a correlation between truncated protein and serotype. Significance and Impact of the Study: This study further validates molecular methods for L. monocytogenes analysis and proposed a new cost‐effective approach for serotyping. It could help to improve a national surveillance network for L. monocytogenes infections in Italy.     

Detection of tetracycline and macrolide resistance determinants in enterococci of animal and environmental origin using multiplex PCR

An occurrence of resistance to tetracycline (TET) and erythromycin (ERY) was ascertained in 82 isolates of Enterococcus spp. of animal and environmental origin. Using E test, 33 isolates were resistant to TET and three isolates to ERY. Using polymerase chain reaction (PCR; single and multiplex), the TET determinants tet(M) and tet(L) were detected in 35 and 13 isolates, respectively. Twelve isolates carried both tet(M) and tet(L) genes. Eight isolates possessed ermB gene associated with ERY resistance. Multiplex PCR was shown to be a suitable method for simultaneous determination of all three resistance determinants that occurred most frequently in bacteria isolated from poultry. This study also demonstrates that gastrointestinal tract of broilers may be a reservoir of enterococci with acquired resistance to both TET and ERY that can be transferred to humans via food chain.