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This study investigated the safety characteristics and potential probiotic properties of Enterococcus faecium by using whole genome analysis, and then explored the effect of this strain on the virulence of Listeria monocytogenes in vitro and during the storage of fermented sausages. Results showed that E. faecium B1 presented enterocin A, B, and P, enterolysin A, and UviB, and the exotoxin related genes and exoenzyme related genes were not detected in the genome of E. faecium B1. However, the adherence genes including acm and scm were present in this strain, which also positively correlated with characteristics related to probiotic potential. In addition, E. faecium could adapt to the condition of fermented sausages, and decrease the survival of L. monocytogenes in vitro and in vivo. The expression of the virulence genes (prfA , hly , inlA , and inlB) and sigB -related genes (prli42 , rsbT , rsbU , rsbV , rsbW , and sigB) were all inhibited by E. faecium B1 to different extents during the storage of fermented sausages at 4 °C. Moreover, compared with the E. faecium B1 group, the expression level of entA , entB , and entP genes of E. faecium B1 in the co-culture of fermented sausages was increased during the storage, which may be the inhibition mechanism of E. faecium B1 on L. monocytogenes. These results demonstrated that E. faecium B1 could potentially be used as bio-protection to control L. monocytogenes in meat products. • E. faecium B1 exhibited better safety characteristics by whole genome analysis. • E. faecium B1 inhibited the survival of L. monocytogenes in fermented sausages. • The expression of entA / B / P in E. faecium B1 was increased during co-culture. • E. faecium plays an important regulatory role in the virulence genes of pathogens. [ABSTRACT FROM AUTHOR]

The continuous detection of multi-drug-resistant enterococci in food source environments has aroused widespread concern. In this study, 198 samples from chicken products, animal feces, raw milk, and vegetables were collected in Japan and Egypt to investigate the prevalence of e nterococci and virulence characterization. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed for species identification and taxonomic analysis of the isolates. The results showed that the rates of most virulence genes (efaA , gelE , asa1 , ace, and hyl) in the Japanese isolates were slightly higher than those in the Egyptian isolates. The rate of efaA was the highest (94.9 %) among seven virulence genes detected, but the cylA gene was not detected in all isolates, which was in accordance with γ-type hemolysis phenotype. In Enterococcus faecalis , the rate of kanamycin-resistant strains was the highest (84.75 %) among the antibiotics tested. Moreover, 78 % of E. faecalis strains exhibited multi-drug resistance. Four moderately vancomycin-resistant strains were found in Egyptian isolates, but none were found in Japanese isolates. MALDI-TOF MS analysis correctly identified 98.5 % (68/69) of the Enterococcus isolates. In the principal component analysis dendrogram, strains isolated from the same region with the same virulence characteristics and similar biofilm-forming abilities were characterized by clustered distribution in different clusters. This finding highlights the potential of MALDI-TOF MS for classifying E. faecalis strains from food sources. • 77.1 % of raw chicken, raw milk and compost were positive for Enterococcus in Japan. • 20 % of food source derived samples were positive for Enterococcus in Egypt. • 78 % of the E. faecalis isolates showed multi-drug resistance. • efaA was positive for 94.9 % of the isolates among 7 virulence genes tested. • PCA classified strains by same region, virulence trait and biofilm-forming ability [ABSTRACT FROM AUTHOR]

Enterococcus faecalis is a phylogenetically and industrially relevant microorganism associated with Lactic Acid Bacteria. Some strains of this bacterium are employed as probiotics in commercial applications, while others serve as the principal component in starter cultures for artisanal regional cheese production. However, over the last decade, this species has emerged as an opportunistic multiresistant pathogen, raising concerns about its impact on human health. Recently, we identified multiple potassium transporter systems in E. faecalis , including the Ktr systems (KtrAB and KtrAD), Kup, KimA and Kdp complex (KdpFABC). Nevertheless, the physiological significance of these proteins remains not fully understood. In this study, we observed that the kup gene promoter region in the JH2–2 strain was modified due to the insertion of a complete copy of the IS6770 insertion sequence. Consequently, we investigated the influence of IS6770 on the expression of the kup gene. To achieve this, we conducted a mapping of the promoter region of this gene in the E. faecalis JH2–2 strain, employing fluorescence gene reporters. In addition, a transcriptional analysis of the kup gene was executed in a strain derived from E. faecalis V583 that lacks the IS30-related insertion element, facilitating the identification of the transcriptional start site. Next, the expression of the kup gene was evaluated via RT-qPCR under different pH stressful conditions. A strong upregulation of the kup gene was observed at an initial pH of 5.0 in the strain derived from E. faecalis V583. However, the activation of transcription was not observed in the E. faecalis JH2–2 strain due to the hindrance caused by the presence of IS6770. Besides that, our computational analysis of E. faecalis genomes elucidates a plausible association between transposition and the regulation of the kup gene. Remarkably, the ubiquitous presence of IS6770 throughout the phylogenetic tree implies its ancient existence within E. faecalis. Moreover, the recurrent co-occurrence of IS6770 with the kup gene, observed in 30 % of IS6770-positive strains, alludes to the potential involvement of this genomic arrangement in the adaptive strategies of E. faecalis across diverse niches. • The study sheds light on bacterial adaptation and the role of the IS6770 in evolutionary variation in different environments. • IS6770 insertion specifically disrupts the kup gene promoter region, abrogating its induction under acidic conditions. • kup gene expression in Enterococcus faecalis isolated from various niches is impacted by the IS6770 insertion. • This study emphasizes the significance of IS6770 in modifying the kup gene promoter in E. faecalis. [ABSTRACT FROM AUTHOR]

Enterococci are emerging nosocomial pathogens. Their widespread distribution causes them to be food contaminants. Furthermore, Enterococci can colonize various ecological niches and diffuse into the food chain via contaminated animals and foods because of their remarkable tolerance to unfavorable environmental circumstances. Due to their potential dissemination to humans, antimicrobial-resistant Enterococci in fish are a worldwide health issue. This study characterized AMR, ARGs, VAGs, gelatinase activity, and biofilm formation in Enterococcus spp. recovered from fish and seafood and evaluated potential correlations. 54 Enterococcus spp. strains(32.73 %)were isolated from 165 samples (75 Oreochromis niloticus , 30 Argyrosomus regius , and 60 Shrimp), comprising 30 Enterococcus faecalis (55.6 %) and 24 Enterococcus faecium (44.4 %) with total 32.73 % (54/165), The maximum prevalence rate of Enterococcus spp. was observed in Nile tilapia (34/54; 63 %), followed by shrimp (14/54; 25.9 %) and Argyrosomus regius (6/54; 11.1 %). The maximum prevalence rate of E. faecalis was observed in Nile tilapia (22/30; 73.3 %), followed by shrimp (8/30; 26.7 %) with significant differences. The prevalence rate of E. faecium was observed in Nile tilapia (12/24; 50 %), followed by shrimp (6/24,25 %). E. faecium is only isolated from Argyrosomus regius (6/24,25 %). Isolates exhibited high resistance against both tetracycline (90.7 %) and erythromycin(88.9 %), followed by gentamycin (77.8 %), ciprofloxacin (74.1 %), levofloxacin (72.2 %), penicillin (44.4 %), vancomycin (37 %), and linezolid (20.4 %). 50 strains (92.6 %) exhibited resistance to more than two antibiotics, 5 strains (10 %) were XDR, and the remaining 45 strains (90 %) were classified as MDR. 92.6 % of the isolates had MARindices >0.2, indicating they originated in settings with a high risk of contamination. Additionally, ten ARGs were identified, with tet (M) 92.6 %, followed by erm (B) (88.9 %), aac (6 ′)- Ie - aph (2 ″)- Ia (77.8 %), tet (K) (75.9 %), gyr A (74.1 %), bla Z (48.1 %), van A (37 %), van B (31.5 %), optr A (20.4 %), and cat A(3.7 %). Biofilm formation and gelatinase activity were observed in 85.2 %, and 61.1 % of the isolates, respectively. A total of 11 VAGs were detected, with gel E as the most prevalent (83.3 %) followed by agg (79.6 %), pil (74.1 %), both spr E and asa 1 (72.2 %), hyl (70.4 %), eps (68.5 %), EF3314 (57.4 %), ace (50 %), and cyl A (35.2 %) with no detection of cyl B. In conclusion, the emergence of linezolid-resistant -vancomycin-resistant enterococci recovered from Egyptian fish and shrimp, suggests that fish and seafood might participate a fundamental part in the emergence of antimicrobial resistance among humans. • Enterococcus spp. particularly E. faecalis and E. faecium is an emerging pathogen commonly isolated from fish and seafood. • Enterococcus spp. isolates exhibited multidrug and extensive resistance to various antibiotics • Veterinary and human health risks can be posed by infections in fish and shrimp, which can be a source of VRE and LRE. • A significant association was detected between AMR, ARGs, VAGs, gelatinase, and biofilm is demonstrated. • Routine antibiotic susceptibility testing and detect its genes are necessary to hinder the MDR and XDR Enterococcus spread. [ABSTRACT FROM AUTHOR]

Raw almonds have been associated with Salmonella outbreaks and multiple recalls related to Listeria monocytogenes contamination. While steam treatment has been approved for pasteurizing both conventional and organic whole almonds, there is limited understanding of how water activity (a w) influences the effectiveness of steam treatments in decontaminating almonds. Hence, this study aimed to assess and compare the efficacy of steam treatments against Listeria innocua and Enterococcus faecium NRRL B-2354, the known non-pathogenic surrogates, on almonds. It also sought to investigate the impact of almond's a w on bacterial resistance during steam treatments. Almond kernels were inoculated with ~8 log 10 CFU/g of either E. faecium or L. innocua and equilibrated to a w 0.25 or 0.45 before being subjected to steam treatments at temperatures of 100–135 °C. Our results revealed that L. innocua exhibited lower resistance to steam compared to E. faecium , with 1.2–2.6 log 10 CFU/g reductions for L. innocua and 1.0–2.0 log 10 CFU/g reductions for E. faecium when the surface temperature of almonds reached 100–130 °C, depending on the a w of the almonds. The obtained D L. innocua , 100–130°C -values were 2.0–16.6 s, and D E. faecium , 100–130°C -values were 4.0–21.8 s, depending on the a w of almonds. In general, elevating steam temperatures and almond a w decreased the tolerance of L. innocua and E. faecium during steam inactivation. In addition, the z -values indicated that E. faecium on almonds was less sensitive to change in steam temperature compared to L. innocua , especially at lower a w. The z L. innocua -values were 36.6 °C and 35.7 °C, while z E. faecium -values were 48.9 °C and 42.7 °C in almonds with a w 0.25 and 0.45, respectively. Results from this study suggest that steam treatments serve as effective interventions for controlling pathogen contaminations in raw almonds. • The impact of almond a w on the efficacy of steam treatments was assessed. • L. innocua (Li) on almonds had lower resistance to steam than E. faecium (Ef). • Increasing almond a w from 0.25 to 0.45 enhanced steam efficacy against Li and Ef. • D 100–130°C -values were 2–17 s for Li and 4–22 s for Ef in almonds a w 0.25–0.45. [ABSTRACT FROM AUTHOR]

Grep-chhurpi, peha, peron namsing and peruñyaan are lesser-known home-made fermented soybean foods prepared by the native people of Arunachal Pradesh in India. Present work aims to study the microbiome, their functional annotations, metabolites and recovery of metagenome-assembled genomes (MAGs) in these four fermented soybean foods. Metagenomes revealed the dominance of bacteria (97.80 %) with minor traces of viruses, eukaryotes and archaea. Bacillota is the most abundant phylum with Bacillus subtilis as the abundant species. Metagenome also revealed the abundance of lactic acid bacteria such as Enterococcus casseliflavus, Enterococcus faecium, Mammaliicoccus sciuri and Staphylococcus saprophyticus in all samples. B. subtilis was the major species found in all products. Predictive metabolic pathways showed the abundance of genes associated with metabolisms. Metabolomics analysis revealed both targeted and untargeted metabolites, which suggested their role in flavour development and therapeutic properties. High-quality MAGs, identified as B. subtilis, Enterococcus faecalis, Pediococcus acidilactici and B. velezensis, showed the presence of several biomarkers corresponding to various bio-functional properties. Gene clusters of secondary metabolites (antimicrobial peptides) and CRISPR-Cas systems were detected in all MAGs. This present work also provides key elements related to the cultivability of identified species of MAGs for future use as starter cultures in fermented soybean food product development. Additionally, comparison of microbiome and metabolites of grep-chhurpi, peron namsing and peruñyaan with that of other fermented soybean foods of Asia revealed a distinct difference. • Some spontaneously fermented soybean foods of India were studied using metagenomics and metabolomics approaches. • Bacillus subtilis as the abundant species, few lactic acid bacteria, and coagulase-negative staphylococci. • Untargeted metabolites including flavour compounds, isoflavones, immunomodulators and bio-peptides detected in samples. • High-quality MAGs B. subtilis, B. velezensis Enterococcus faecalis, Pediococcus acidilactici recovered with biomarkers. [ABSTRACT FROM AUTHOR]

Bacterial biofilm is a major concern of dairy industry due to its association with milk contamination and its derived products. Algerian pasteurized milk shelf-life does not exceed one day, which may reflect the high level of contamination of this product and presence of extracellular enzymes such as lipases and proteases. This work aimed to investigate the microbial biodiversity in milk-processing surfaces of a dairy plant in Algeria. Therefore, stainless steel cylinders were placed in piping system of the dairy system before and after pasteurization of the milk, being removed after 7 days, for biofilm maturation and microorganism isolation and identification by mass spectrometry. Fifty-nine Gram-positive isolates were identified, namely Bacillus altitudinis , Bacillus cereus , Bacillus pumilus , Bacillus subtilis , Bacillus weithenstephanensis , Enterococcus casseliflavus , Enterococcus faecium , and Staphylococcus epidermidis. In addition, twenty-four Gram-negative isolates were identified, namely Acinetobacter schindleri Enterobacter cloacae , Enterobacter xiangfangensis , Leclercia adecarboxylata , and Raoultella ornithinolytica. Bacterial isolates showed ability for production of extracellular enzymes, being 49 % capable of both proteolytic and lipolytic activities. Milk isolates were tested for the ability to form biofilms on stainless steel. The cell numbers recovered on plate count agar plates from stainless steel biofilms ranged from 3.52 to 6.92 log10 CFU/cm2, being the maximum number detected for Enterococcus casseliflavus. Bacterial isolates showed intermediate and/or resistant profiles to multiple antibiotics. Resistance to amoxicillin, cefoxitin and/or erythromycin was commonly found among the bacterial isolates. • Standard cleaning and sanitation procedures related to milk pasteurization processes need improvements in Algeria. • Multiple bacteria were found adhered to the stainless steel pipelines used in the dairy industry. • Bacillus cereus and Enterococcus faecium were found before and after pasteurization processes in both cold and hot seasons. • Bacterial isolates were capable to form biofilms on stainless steel and produce extracellular proteases and lipases. • Antibiotic resistant isolates were found, being resistance to amoxicillin, cefoxitin and/or erythromycin the most common. • No resistance to chloramphenicol was observed. [ABSTRACT FROM AUTHOR]

Wheat, the raw material for flour milling, can be contaminated with enteric pathogens, leading to outbreaks linked to flour. In previous lab-scale studies, vacuum steam treatment was able to reduce Salmonella Enteritidis PT30 and Shiga-toxin producing E. coli (STEC) O121 levels on soft wheat kernels while maintaining flour quality and gluten functionality. This study used a newly designed lab-scale vacuum steam pasteurizer (VSP) to evaluate its efficacy to inactivate multiple strains of Salmonella and STEC on soft wheat by modeling the non-isothermal time-temperature history during treatment and reduction of the microbial populations. The results demonstrated that vacuum steam treatment could effectively disinfect wheat grains inoculated with enteric pathogens. In this study, Salmonella strains were less thermally resistant than STEC strains. The D 75°C of Salmonella strains were 2.8 and 3.2 min, and the D 75°C of STEC ranged from 3.1 to 4.6 min. E. faecium had a D 75°C of 3.3 min, which indicates that it could be used as surrogate for larger scale evaluation of vacuum steam pasteurization in the future but was not conservative compared to some of the STEC strains. • VSP treatment at 75 °C for 8 min led to >5-log pathogen reduction on wheat grain. • Enterococcus faecium could be used as a surrogate for Salmonella for VSP treatment. • Salmonella strains used were more sensitive to VSP treatment than STEC strains used. • D 75°C of Salmonella ranges from 2.8 to 3.2 min, and of STEC from 3.1 to 4.5 min. [ABSTRACT FROM AUTHOR]

Abstract The feasibility of using gaseous ozone, alone or in combination with other treatments, to decontaminate in-shell almonds and pistachios, prepared under different pathogen-inoculation conditions, was explored. Nuts were inoculated with either Salmonella enterica serovar Enteritidis or a potential Salmonella surrogate, Enterococcus faecium OSY 31284. The effect of inoculation method (with or without vacuum application), and of drying inoculated nuts (up to three days) on treatment efficacy was investigated. Inoculated nuts were subjected to gaseous ozone alone (almonds, pistachios) or ozone in combination with heating in brine solution (pistachios). Ozone treatment included application of vacuum (10 in Hg), followed by vessel pressurization to 12.5 psig with ozone‑oxygen mixture (160 g ozone/m3 gas mixture) and holding for 30 min. Heating was conducted in a brine solution (5% NaCl) at 70 °C, for 10 min. Ozone-based treatments were significantly more effective (P < 0.05) on almonds than pistachios, with maximum S. Enteritidis reduction of 2.9 vs. 0.8 log CFU/g, regardless of inoculation method or the drying time. Treatment of inoculated pistachios with heated brine and gaseous ozone reduced S. Enteritidis population by 5.0 to 7.0 log CFU/g and was not significantly more effective than treatment with heated brine alone (reduction of 4.8 to 7.1 log CFU/g). Application of vacuum during inoculation increased bacterial population on nut kernels by approximately 1.2 log CFU/g, but the increase in inoculum population had no effect on inactivation of either species of inoculated bacteria. Decontamination treatments were less effective against both bacteria by up to 2 log CFU/g when drying time of inoculated nuts increased. E. faecium was significantly more resistant to heat and ozone treatment (P < 0.05) than was S. Enteritidis on pistachios, but not on almonds. Results of this study show that laboratory methodology affects observed treatment effectiveness. Considering its high resistance to the heat-ozone combination, E. faecium may not be a suitable surrogate for S. Enteritidis during processing of pistachios by this treatment. Efficacy of ozone gas to decontaminate S. Enteritidis - inoculated nuts depends heavily on the type of nut. Although reductions of S. Enteritidis populations on in-shell pistachios are low, treatment of in-shell almonds resulted in greater reductions, indicating the promise of this technology to enhance the safety of specific nut products. Highlights • Gaseous ozone is more effective against Salmonella on almonds than on pistachios. • E. faecium OSY 31284 is not a valid Salmonella surrogate for ozone-based processes. • Inoculation method and drying time alter lethality of ozone, heat-based treatments. [ABSTRACT FROM AUTHOR]

Abstract The heavy use or abuse of antimicrobials in food animals has caused an increase in antimicrobial resistance in enterococci of animal origin, which could get transmitted to those of human origin via the food chain. Since duck meat consumption has been on the rise in Korea, we conducted this study to provide information about the antimicrobial resistance of the enterococci obtained from healthy ducks and their carcasses. A total of 82 Enterococcus faecium and 174 E. faecalis isolated from duck fecal and carcass samples were investigated for antimicrobial resistance to 16 agents, using broth dilution method, and were further characterized using molecular methods. Most of E. faecium (84.1%) and E. faecalis (87.9%) isolates were resistant to one or more antimicrobials. Multi-drug resistant (MDR) isolates were observed in both E. faecium (40.2%) and E. faecalis (33.9%) with high frequencies. High rate of resistance was observed for tetracycline, ciprofloxacin, chloramphenicol, and erythromycin in both E. faecium and E. faecalis. Resistance to gentamicin, vancomycin, and daptomycin, in both E. faecium and E. faecalis , was, if at all, very rare. However, linezolid resistance was observed in nine E. faecium (11.0%) and one E. faecalis (0.6%). All, but one, Linezolid resistant (LR) isolates were also resistant to chloramphenicol and florfenicol. The novel transferable oxazolidinone and phenicol resistant gene, optrA , was found in six E. faecium isolates. All of them co-carried phenicol exporter gene fexA. None of the LR isolates had mutation in the 23S ribosomal RNA and in the ribosomal protein L3. Six LR E. faecium isolates had Asn130Lys mutation in the ribosomal protein L4, of which five also carried optrA gene. None of the isolates carried the multi-resistance gene cfr. Transfer of oxazolidinone and phenicol resistance was observed in five among the 10 LR isolates; two of them had optrA and fexA genes. Multi-drug resistant Enterococcus that also carried the resistance gene to a last-resort antimicrobial is a major concern for public health. Thus, to prevent the introduction of last-resort antimicrobial resistance into food chain, continuous surveillance of antimicrobial resistance in duck is imperative. Highlights • E. faecium and E. faecalis , resistant to antibiotics used by humans, were reported. • Overall, nine E. faecium and one E. faecalis were resistant to linezolid. • Among them, six optrA -positive isolates co-carried phenicol export gene fexA. • First report of linezolid resistance and optrA gene in isolates from duck [ABSTRACT FROM AUTHOR]

The members of the Enterococcus genus are widely distributed in nature. Its strains have been extensively reported to be present in plant surfaces, soil, water and food. In an attempt to assess their potential application in food industry, four Enterococcus faecium group-strains recently isolated from Argentinean regional cheese products were evaluated using a combination of whole genome analyses and in vivo assays. In order to identify these microorganisms at species level, in silico analyses using their newly reported sequences were conducted. The average nucleotide identity (ANI), in silico DNA-DNA hybridization, and phylogenomic trees constructed using core genome data allowed IQ110, GM70 and GM75 strains to be classified as E. faecium while IQ23 strain was identified as E. durans . Besides their common origin, the strains showed differences in their genetic structure and mobile genetic element content. Furthermore, it was possible to determine the absence or presence of specific features related to growth in milk, cheese ripening, probiotic capability and gut adaptation including sugar, amino acid, and peptides utilization, flavor compound production, bile salt tolerance as well as biogenic amine production. Remarkably, all strains encoded for peptide permeases, maltose utilization, bile salt tolerance, diacetyl and tyramine production genes. On the other hand, some variability was observed regarding citrate and lactose utilization, esterase, and cell wall-associated proteinase. In addition, while strains were predicted to be non-human pathogens by the in silico inspection of pathogenicity and virulence factors, only the GM70 strain proved to be non-virulent in Galleria mellonella model. In conclusion, we propose that, in order to improve the rational selection of strains for industrial applications, a holistic approach involving a comparative genomic analysis of positive and negative features as well as in vivo evaluation of virulence behavior should be performed. [ABSTRACT FROM AUTHOR]

Caciofiore della Sibilla is an Italian specialty soft cheese manufactured with Sopravissana raw ewes' milk and thistle rennet prepared with young fresh leaves and stems of Carlina acanthifolia All. subsp. acanthifolia , according to an ancient tradition deeply rooted in the territory of origin (mountainous hinterland of the Marche region, Central Italy). In this study, the impact of thistle rennet on the bacterial dynamics and diversity of Caciofiore della Sibilla cheese was investigated by applying a polyphasic approach based on culture and DNA-based techniques (Illumina sequencing and PCR-DGGE). A control cheese manufactured with the same batch of ewes' raw milk and commercial animal rennet was analyzed in parallel. Overall, a large number of bacterial taxa were identified, including spoilage, environmental and pro-technological bacteria, primarily ascribed to Lactobacillales . Thistle rennet was observed clearly to affect the early bacterial dynamics of Caciofiore della Sibilla cheese with Lactobacillus alimentarius / paralimentarius and Lactobacillus plantarum / paraplantarum / pentosus being detected in the phyllosphere of C . acanthifolia All., thistle rennet and curd obtained with thistle rennet. Other bacterial taxa, hypothetically originating from the vegetable coagulant ( Enterococcus faecium, Lactobacillus brevis, Lactobacillus delbrueckii, Leuconostoc mesenteroides/pseudomesenteroides ), were exclusively found in Caciofiore della Sibilla cheese by PCR-DGGE. At the end of the maturation period, Illumina sequencing demonstrated that both cheeses were dominated by Lactobacillales ; however curd and cheese produced with thistle rennet were co-dominated by Lactobacillus and Leuconostoc, whereas Lactoccous prevailed in curd and cheese produced with commercial animal rennet followed by Lactobacillus . Differences in the bacterial composition between the two cheeses at the end of their maturation period were confirmed by PCR-DGGE analysis. [ABSTRACT FROM AUTHOR]

Microbiological limits are widely used in food processing as an aid to reduce the exposure to hazardousmicroorganisms for the consumers. However, in pork, the prevalence and concentrations of Salmonella are generally low and microbiological limits are not considered an efficient tool to support hygiene interventions. The objective of the present studywas to develop an approachwhich couldmake it possible to define potential risk-basedmicrobiological limits for an indicator, enterococci, in order to evaluate the risk frompotential growth of Salmonella. A positive correlation between the concentration of enterococci and the prevalence and concentration of Salmonellawas shown for 6640 pork samples taken at Danish cutting plants and retail butchers. The sampleswere collected in five different studies in 2001, 2002, 2010, 2011 and 2013. The observations that both Salmonella and enterococci are carried in the intestinal tract, contaminate pork by the same mechanisms and share similar growth characteristics (lag phase and maximum specific growth rate) at temperatures around 5-10 ?C, suggest a potential of enterococci to be used as an indicator of potential growth of Salmonella in pork. Elevated temperatures during processingwill lead to growth of both enterococci and, if present, also Salmonella. By combining the correlation between enterococci and Salmonellawith risk modelling, it is possible to predict the risk of salmonellosis based on the level of enterococci. The riskmodel used for this purpose includes the dose-response relationship for Salmonella and a reduction factor to account for preparation of the fresh pork. By use of the riskmodel, it was estimated that themajority of salmonellosis cases, caused by the consumption of pork in Denmark, is caused by the small fraction of pork products that has enterococci concentrations above 5 log CFU/g. This illustrates that our approach can be used to evaluate the potential effect of differentmicrobiological limits and therefore, the perspective of this novel approach is that it can be used for definition of a risk-basedmicrobiological limit for enterococci. The limit for enterococci can then be used for development of a process hygiene criterion in cutting plants and retail butcher shops, with the purpose of reducing the risk of Salmonella for the consumer. [ABSTRACT FROM AUTHOR]

Enterococcus has been considered one of the most important nosocomial pathogens for human infections, and the hospital environment is an important reservoir for vancomycin-resistant enterococci (VRE) that leads to antimicrobial therapeutic failure. However, infant foods and their production environments could pose risks for the immature population, while this question remains unaddressed. This study conducted an extensive and thorough Enterococcus isolation, VRE risk assessment of the Chinese infant food production chains and additional online-marketing infant foods, including powdered infant formula (PIF) and infant complementary food (ICF). To investigate the prevalence of Enterococcus along infant food chains and commodities, a total of 482 strains of Enterococcus , including E. faecium (n = 363), E. faecalis (n = 84), E. casseliflavus (n = 13), E. mundtii (n = 12), E. gallinarum (n = 4), E. hirae (n = 4), and E. durans (n = 2) were recovered from 459 samples collected from infant food production chains (71/254) and food commodities (67/205). A decreasing trend for Enterococcus detection rate was found in the PIF production chain (PIF-PC), particularly during the preparation of the PIF base powder (From 100 % in raw milk to 8.70 % in end products), while an increasing trend was observed in the ICF production chain (ICF-PC) mainly during the initial processing of farm crops and the further processing of the product (20 % at farm crops increasing to 76.92 % at end products). The result indicated that the PIF-PC process effectively reduced Enterococcus contamination, while the ICF-PC showed the opposite trend. Importantly, eleven VRE isolates were recovered from the infant food production chain, including seven E. casseliflavus isolates carrying vanC2/C3 and four E. gallinarum isolates carrying vanC1. Ten VRE isolates were from food production environments. Collectively, our study demonstrated that infant food production environments represent potential reservoirs for VRE non-nosocomial infections in vulnerable populations. [Display omitted] • Powdered infant formula production chain has higher Enterococcus contamination, particularly in production/food-processing environment. • The Enterococcus contamination rate of domestic PIF (45.37%) is relatively higher than that of imported PIF (25.37%). • Among 11 vancomycin-resistant isolates, 10 were from infant food production environments. • Antimicrobial and virulent genes were detected in vancomycin-resistant enterococci. [ABSTRACT FROM AUTHOR]

In this study, PDO Provola dei Nebrodi cheese was deeply characterized for its bacterial community and chemical composition. Four dairy factories (A-D) were monitored from milk to ripened cheese. Wooden vat biofilms were dominated by thermophilic rod LAB (4.6-6.5 log CFU/cm 2 ). Bulk milk showed consistent levels of total mesophilic microorganisms (TMM) (5.0-6.0 log CFU/mL) and, after curdling, a general increase was recorded. The identification of the dominant LAB in wooden vat biofilms and ripened cheeses showed that the majority of wooden vat LAB were lactococci and Streptococcus thermophilus, while cheese LAB mainly belonged to Lacticaseibacillus paracasei and Enterococcus. Illumina sequencing identified 22 taxonomic groups; streptococci, lactococci, lactobacilli and other LAB constituted the majority of the total relative abundance % of the wooden vat (69.01-97.58 %) and cheese (81.57-99.87 %) bacterial communities. Regarding chemical composition, the effect of dairy factories was significant only for protein content. Inside cheese color was lighter and yellower than surface. Differences in fatty acids regarded only myristic acid and total amount of monounsaturated fatty acids. The sensory evaluation indicated some differences among cheeses produced in the four dairies regarding color, homogeneity of structure, overall intensity, salty, spicy, and hardness. The integrated approach applied in this study showed that PDO Provola dei Nebrodi cheese characteristics are quite stable among the dairy factories analyzed and this has to be unavoidably imputed to the application of the same cheese making protocol among different dairies., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest for this research., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Our study investigated the potential of Annona squamosa (L.) fruit as a reservoir of yeasts and lactic acid bacteria having biotechnological implications, and phenolics capable of modifying the ecology of microbial consortia. Only a single species of lactic acid bacteria (Enterococcus faecalis) was identified, while Annona fruit seemed to be a preferred niche for yeasts (Saccharomyces cerevisiae, Hanseniaspora uvarum), which were differentially distributed in the fruit. In order to identify ecological implications for inherent phenolics, the antimicrobial potential of water- and methanol/water-soluble extracts from peel and pulp was studied. Pulp extracts did not show any antimicrobial activity against the microbial indicators, while some Gram-positive bacteria (Staphylococcus aureus , Staphylococcus saprophyticus , Listeria monocytogenes , Bacillus megaterium) were susceptible to peel extracts. Among lactic acid bacteria used as indicators, only Lactococcus lactis and Weissella cibaria were inhibited. The chemical profiling of methanol/water-soluble phenolics from Annona peel reported a full panel of 41 phenolics, mainly procyanidins and catechin derivatives. The antimicrobial activity was associated to specific compounds (procyanidin dimer type B [isomer 1], rutin [isomer 2], catechin diglucopyranoside), in addition to unidentified catechin derivatives. E. faecalis , which was detected in the epiphytic microbiota, was well adapted to the phenolics from the peel. Peel phenolics had a growth-promoting effect toward the autochthonous yeasts S. cerevisiae and H. uvarum. • A. squamosa fruits had a specific spatial distribution of microbes and phenolics. • Peel phenolics had antimicrobial activity against several Gram-positive bacteria. • Antimicrobial activity of peel phenolics was associated to specific compounds. • E. faecalis was well adapted to the phenolics from the peel. • Peel phenolics had a growth-promoting effect toward autochthonous yeasts. [ABSTRACT FROM AUTHOR]

Poultry meat has been a vehicle of antibiotic resistant bacteria and genes. Yet, the diversity of selective pressures associated with their maintenance in the poultry-production chain remains poorly explored. We evaluated the susceptibility of Enterococcus spp. from chicken meat collected 20 years apart to antibiotics, metals, acidic pH and peracetic acid-PAA. Contemporary chicken-meat samples (n = 53 batches, each including a pool of neck skin from 10 single carcasses) were collected in a slaughterhouse facility using PAA as disinfectant (March–August 2018, North of Portugal). Broilers were raised in intensive farms (n = 29) using CuSO 4 and organic acids as feed additives. Data were compared with that of 67 samples recovered in the same region during 1999–2001. All 2018 samples had multidrug resistant-MDR isolates, with >45 % carrying Enterococcus faecalis, Enterococcus faecium or Enterococcus gallinarum resistant to tetracycline, erythromycin, ampicillin, quinupristin-dalfopristin, ciprofloxacin, chloramphenicol or aminoglycosides. Resistance rates were similar (P > 0.05) to those of 1999–2001 samples for all but five antibiotics. The decrease of samples carrying vancomycin-resistant isolates from 46 % to 0 % between 1999-2001 and 2018 was the most striking difference. Isolates from both periods were similarly susceptible to acid pH [minimum-growth pH (4.5-5.0), minimum-survival pH (3.0-4.0)] and to PAA (MIC 90 = 100–120 mg/L/MBC 90 = 140–160 mg/L; below concentrations used in slaughterhouse). Copper tolerance genes (tcrB and/or cueO) were respectively detected in 21 % and 4 % of 2018 and 1999-2001 samples. The tcrB gene was only detected in E. faecalis (MIC CuSO4 > 12 mM), and their genomes were compared with other international ones of chicken origin (PATRIC database), revealing a polyclonal population and a plasmid or chromosomal location for tcrB. The tcrB plasmids shared diverse genetic modules, including multiple antimicrobial resistance genes (e.g. to tetracyclines, chloramphenicol, macrolide-lincosamide-streptogramin B-MLS B , aminoglycosides, bacitracin, coccidiostats). When in chromosome, the tcrB gene was co-located closely to merA (mercury) genes. Chicken meat remains an important vehicle of MDR Enterococcus spp. able to survive under diverse stresses (e.g. copper, acid) potentially contributing to these bacteria maintenance and flux among animal-environment-humans. • Chicken-meat samples collected 20 years apart have multidrug-resistant Enterococcus. • Significant decrease of vancomycin-resistant Enterococcus in recent years. • Plasmids with copper/antibiotic resistance genes and other adaptive features. • Enterococcus survive to low pH found in poultry production-chain. • Peracetic acid remains with good activity against multidrug resistant Enterococcus. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a relevant foodborne pathogen causing invasive listeriosis in humans, a disease with high mortality rates. Its ubiquity and growth characteristics enable this pathogen to survive harsh food processing environments. The addition of bacteriocins, antimicrobial peptides ribosomally synthesized by certain bacteria, appears as a natural alternative to control this pathogen in food. However, the emergence of L. monocytogenes strains resistant to the inhibitory action of bacteriocins has been detected. In order to analyse the development of this resistance, different properties of L. monocytogenes strains susceptible to bacteriocins (strains 01/155, 99/287 and 99/267) and their respective resistant isolates (strains 01/155B6R, 99/287B6R, 99/286C1R, 99/287 Mo1R, 99/287 M1bR, 99/287 M2dR, 99/267B6R), were compared in this work. Differences were analysed in: a) growth of the pathogen strains in direct contact with bacteriocin solution, in co-cultures with the producing strain, or with different sugars; b) response to antibiotics typically used against listeriosis; c) changes in cell morphology, observed by transmission or scanning electron microscopy; d) expression of mobility and haemolysin activity, two of L. monocytogenes main virulence factors; and e) biofilm formation ability. For all the isolates, the acquired resistance was permanent and crossed between the different bacteriocins under study. An inhibitory effect was observed for resistant strains only when they were grown in mixed culture with any of the bacteriocin-producing strains, with an acidified medium as additional growth stress. In all cases, the decrease in viability was lower for resistant strains and followed a particular profile for each strain. The variation of sugar substrate influenced resistant variants growth ability, with a more pronounced difference in the medium supplemented with glucose. Susceptibility to antibiotics was similar or higher for resistant variants, while neither the mobility nor the haemolytic activity presented differences among resistant or susceptible strains. Finally, the resistant variants showed a greater capacity to form biofilms, although this effect was reversed when grown in the presence of bacteriocins. Each resistant isolate had a particular behaviour pattern, and the acquisition of resistance appeared to be strain and bacteriocin dependent. These results contribute to the knowledge of L. monocytogenes bacteriocin-resistance development, which is essential to favour the use of these peptides as biopreservatives. • Listeria monocytogenes isolates resistant to bacteriocin activity were detected. • Acquired resistance was permanent and crossed between the bacteriocins under study. • Each resistant isolate had a particular behaviour pattern. • The acquisition of resistance appeared to be strain and bacteriocin dependent. [ABSTRACT FROM AUTHOR]

Forty enterococci isolated along the production chains of three traditional cheeses (PDO Pecorino Siciliano, PDO Vastedda della Valle del Belìce, and Caciocavallo Palermitano) made in Sicily (southern Italy) were studied for the assessment of their antibiotic resistance and virulence by a combined phenotypic/genotypic approach. A total of 31 Enterococcus displayed resistance to at least one or more of the antimicrobials tested. The strains exhibited high percentages of resistance to erythromycin (52.5%), ciprofloxacin (35.0%), quinupristin–dalfopristin (20.0%), tetracycline (17.5%), and high-level streptomycin (5.0%). The presence of tet (M), cat (pC221), and aad E genes for resistance to tetracycline, chloramphenicol, and streptomycin, respectively, was registered in all strains with resistance phenotype. The erm (B) gene was not detected in any erythromycin-resistant strain. The Enterococcus strains were further tested by PCR for the presence of virulence genes, namely, gel E, asa1 , efaA , ace , and esp . Twenty strains were positive for all virulence genes tested. Among the enterococci isolated from final cheeses, three strains (representing 33.3% of total cheese strains) were sensible to all antimicrobials tested and did not carry any virulence factor. Although this study confirmed that the majority of dairy enterococci are vectors for the dissemination of antimicrobial resistance and virulence genes, only two strains showed a high resistance to aminoglycosides, commonly administered to combat enterococci responsible for human infections. Furthermore, the presence of the strains E. casseliflavus FMAC163, E. durans FMAC134B, and E. faecium PON94 without risk determinants, found at dominating levels over the Enterococcus populations in the processed products, stimulates further investigations for their future applications in cheese making. All strains devoid of the undesired traits were isolated from stretched cheeses. Thus, this cheese typology represents an interesting environment to deepen the studies on the risk/benefit role of enterococci in fermented foods for their qualified presumption of safety (QPS) assessment. [ABSTRACT FROM AUTHOR]

This work presents the first study on the bacterial communities in Pico cheese, a traditional cheese of the Azores (Portugal), made from raw cow's milk. Pyrosequencing of tagged amplicons of the V3–V4 regions of the 16S rDNA and Operational Taxonomic Unit-based (OTU-based) analysis were applied to obtain an overall idea of the microbiota in Pico cheese and to elucidate possible differences between cheese-makers (A, B and C) and maturation times. Pyrosequencing revealed a high bacterial diversity in Pico cheese. Four phyla (Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes) and 54 genera were identified. The predominant genus was Lactococcus (77% of the sequences). Sequences belonging to major cheese-borne pathogens were not found. Staphylococcus accounted for 0.5% of the sequences. Significant differences in bacterial community composition were observed between cheese-maker B and the other two units that participated in the study. However, OTU analysis identified a set of taxa ( Lactococcus , Streptococcus , Acinetobacter , Enterococcus , Lactobacillus , Staphylococcus , Rothia , Pantoea and unclassified genera belonging to the Enterobacteriaceae family) that would represent the core components of artisanal Pico cheese microbiota. A diverse bacterial community was present at early maturation, with an increase in the number of phylotypes up to 2 weeks, followed by a decrease at the end of ripening. The most remarkable trend in abundance patterns throughout ripening was an increase in the number of sequences belonging to the Lactobacillus genus, with a concomitant decrease in Acinetobacter , and Stenotrophomonas . Microbial rank abundance curves showed that Pico cheese's bacterial communities are characterized by a few dominant taxa and many low-abundance, highly diverse taxa that integrate the so-called “rare biosphere”. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the virulence, antibiotic resistance and biogenic amine production in bacteriocinogenic lactococci and enterococci isolated from goat milk in order to evaluate their safety. Twenty-nine bacteriocinogenic lactic acid bacteria (LAB: 11 Lactococcus spp., and 18 Enterococcus spp.) isolated from raw goat milk were selected and subjected to PCR to identify gelE, cylA, hyl, asa1, esp, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc genes. The expression of virulence factors (gelatinase, hemolysis, lipase, DNAse, tyramine, histamine, putrescine) in different incubation temperatures was assessed by phenotypic methods, as well as the resistance to vancomycin, gentamicin, chloramphenicol, ampicillin and rifampicin (using Etest(r)). The tested isolates presented distinct combinations of virulence related genes, but not necessarily the expression of such factors. The relevance of identifying virulence-related genes in bacteriocinogenic LAB was highlighted, demanding for care in their usage as starter cultures or biopreservatives due to the possibility of horizontal gene transfer to other bacteria in food systems. [ABSTRACT FROM AUTHOR]

Bacterial biocide resistance is becoming a matter of concern. In the present study, a collection of biocide-resistant, Gram-positive bacteria from organic foods (including 11 isolates from genus Bacillus, 25 from Enterococcus and 10 from Staphylococcus) were analyzed for genes associated to biocide resistance efflux pumps and antibiotic resistance. The only qac-genes detected were qacA/B (one Bacillus cereus isolate) and smr (one B. cereus and two Staphylococcus saprophyticus isolates). Efflux pump genes efrA and efrB genes were detected in Staphylococcus (60% of isolates), Bacillus (54.54%) and Enterococcus (24%); sugE was detected in Enterococcus (20%) and in one Bacillus licheniformis; mepA was detected in Staphylococcus (60%) and in one Enterococcus isolate (which also carried mdeA), and norE gene was detected only in one Enterococcus faecium and one S. saprophyticus isolate. An amplicon for acrB efflux pump was detected in all but one isolate. When minimal inhibitory concentrations (MICs) were determined, it was found that the addition of reserpine reduced the MICs by eight fold for most of the biocides and isolates, corroborating the role of efflux pumps in biocide resistance. Erythromycin resistance gene ermB was detected in 90% of Bacillus isolates, and in one Staphylococcus, while ereA was detected only in one Bacillus and one Staphyloccus, and ereB only in one Staphylococcus. The ATP-dependent msrA gene (which confers resistance to macrolides, lincosamides and type B streptogramins) was detected in 60% of Bacillus isolates and in all staphylococci, which in addition carried msrB. The lincosamide and streptogramin A resistance gene lsa was detected in Staphylococcus (40%), Bacillus (27.27%) and Enterococcus (8%) isolates. The aminoglycoside resistance determinant aph (3_)-IIIa was detected in Staphylococcus (40%) and Bacillus (one isolate), aph(2_)-1d in Bacillus (27.27%) and Enterococcus (8%), aph(2_)-Ib in Bacillus (one isolate), and the bifunctional aac(6_)1e-aph(2_)-Ia in Staphylococcus (20%), Enterococcus (8%) and Bacillus (one isolate). Chloramphenicol resistance cat gene was detected in Enterococcus (8%) and Staphylococcus (20%), and blaZ only in Staphylococcus (20%). All other antibiotic or biocide resistance genes investigated were not detected in any isolate. Isolates carrying multiple biocide and antibiotic determinants were frequent among Bacillus (36.36%) and Staphylococcus (50%), but not Enterococcus. These results suggest that biocide and antibiotic determinants may be co-selected. [ABSTRACT FROM AUTHOR]

Abstract: Twenty-nine Enterococcus strains from raw and fermented meat products were screened for the presence of virulence genes, including those for aggregation substances (asa1 and asa373), cytolysin activator (cylA), collagen binding protein (ace), endocarditis antigen (efaA), enterococcal surface protein (esp) and gelatinase (gelE). Virulence gene occurrence, expression of gelatinase and pheromone aggregation was greater in Enterococcus faecalis than in Enterococcus faecium strains. All E. faecalis and 54% of E. faecium were positive for at least one or more virulence gene. The only strain of Enterococcus gallinarum tested also contained virulence genes. The effect of different growth temperatures (25 and 37°C) on biofilm formation using polystyrene plates was also assessed. Strong biofilm formation occurred at lower than optimum temperature in all three species of enterococci. Neither esp nor gelE was necessary for biofilm formation and this relationship was species rather than strain specific. This study emphasizes the importance of enterococci as a reservoir of virulence genes and the potential for their genetic transfer to human strains following consumption of uncooked or undercooked contaminated meat. [Copyright &y& Elsevier]

Adequate surrogate identification is critical for validating in-plant thermal process controls for Salmonella inactivation in different food matrices. This study compared the thermal inactivation parameters (D- and z-values) and evaluated the heat resistance of Enterococcus faecium (8459) as a surrogate for a 5-serovar Salmonella cocktail in cornmeal. The cornmeal was spray inoculated with the respective bacteria to achieve ~9 log CFU/g population and set to the desired moisture contents (16, 22, and 28% w.b.). The inoculated cornmeal was then heat-treated at pre-determined temperatures (60, 64, and 68 °C) in sealed aluminum thermal-death-time disks in hot water baths for pre-determined time intervals. Injury-recovery media [brain heart infusion (BHI) agar overlaid with xylose lysine deoxycholate (XLD) agar for Salmonella or BHI agar overlaid m-enterococcus agar for E. faecium ] were used for microbial enumeration to account for thermally injured bacterial cells. The D-values of Salmonella in cornmeal at 16, 22, and 28% moisture content were 37.5, 8.4, and 2.4 min at 60 °C, 19.9, 3.5, and 1.1 min at 64 °C, and 10.1, 1.4, and 0.5 min at 68 °C, respectively. The D-values of E. faecium in cornmeal at 16, 22, and 28% moisture content were 140.4, 18.9, and 3.3 min at 60 °C, 78.4, 7.1, and 1.6 min at 64 °C, and 37.3, 2.8, and 0.8 min at 68 °C, respectively. The z-values of E. faecium and Salmonella in cornmeal at 16, 22, and 28% moisture content were 13.9, 9.7, and 12.5 °C, and 14.0, 10.4, and 11.7 °C, respectively. These results indicated similar or higher thermal resistance (D-values) and equivalent thermal sensitivity (z-values) of E. faecium compared to Salmonella at different moisture contents and respective temperatures (P ≤ 0.05). Therefore, E. faecium could be used as a surrogate for Salmonella during thermal process validation of cornmeal processing. • E. faecium showed higher thermal resistance (D- values) in cornmeal compared to Salmonella. • Thermal sensitivity (z-values) of E. faecium and Salmonella were similar at different moisture contents (16, 22, and 28%, wb). • E. faecium can be considered as fail-safe Salmonella surrogate for the thermal process validation of cornmeal. [ABSTRACT FROM AUTHOR]

Abstract: The increase demand for fresh vegetables is causing an expansion of the market for minimally processed vegetables along with new recognized food safety problems. To gain further insight on this topic we analyzed the microbiological quality of Portuguese ready-to-eat salads (RTS) and their role in the spread of bacteria carrying acquired antibiotic resistance genes, food products scarcely considered in surveillance studies. A total of 50 RTS (7 brands; split or mixed leaves, carrot, corn) were collected in 5 national supermarket chains in Porto region (2010). They were tested for aerobic mesophilic counts, coliforms and Escherichia coli counts as well as for the presence of Salmonella and Listeria monocytogenes. Samples were also plated in different selective media with/without antibiotics before and after enrichment. The E. coli, other coliforms and Enterococcus recovered were characterized for antibiotic resistance profiles and clonality with phenotypic and genetic approaches. A high number of RTS presented poor microbiological quality (86%—aerobic mesophilic counts, 74%—coliforms, 4%—E. coli), despite the absence of screened pathogens. In addition, a high diversity of bacteria (species and clones) and antibiotic resistance backgrounds (phenotypes and genotypes) were observed, mostly with enrichment and antibiotic selective media. E. coli was detected in 13 samples (n=78; all types and 4 brands; phylogenetic groups A, B1 and D; none STEC) with resistance to tetracycline [72%; tet(A) and/or tet(B)], streptomycin (58%; aadA and/or strA-strB), sulfamethoxazole (50%; sul1 and/or sul2), trimethoprim (50%; dfrA1 or dfrA12), ampicillin (49%; bla TEM ), nalidixic acid (36%), ciprofloxacin (5%) or chloramphenicol (3%; catA). E. coli clones, including the widespread group D/ST69, were detected in different samples from the same brand or different brands pointing out to a potential cross-contamination. Other clinically relevant resistance genes were detected in 2 Raoultella terrigena carrying a bla SHV-2 and 1 Citrobacter freundii isolate with a qnrB9 gene. Among Enterococcus (n=108; 35 samples; Enterococcus casseliflavus—40, Enterococcus faecalis—20, Enterococcus faecium—18, Enterococcus hirae—9, Enterococcus gallinarum—5, and Enterococcus spp.—16) resistance was detected for tetracyclines [6%; tet(M) and/or tet(L)], erythromycin [3%; erm(B)], nitrofurantoin (1%) or ciprofloxacin (1%). The present study places ready-to-eat salads within the spectrum of ecological niches that may be vehicles for antibiotic resistance bacteria/genes with clinical interest (e.g. E. coli-D-ST69; bla SHV-2 ) and these findings are worthy of attention as their spread to humans by ingestion cannot be dismissed. [Copyright &y& Elsevier]

Abstract: Enterococci are predominantly found in the gastrointestinal tract of humans and animals, but species commonly resident on vegetation are known. Their presence in large numbers in foods may indicate a lapse in sanitation and their ability to serve as a genetic reservoir of transferable antibiotic resistance is of concern. Conventional culture methods for identification of enterococci are slow and sometimes give false results because of the biochemical diversity of the organisms in this genus. This work reports the development of a PCR-based assay to detect enterococci at the genus level by targeting a 16S rRNA sequence. Published 16S rRNA sequences were aligned and used to design genus specific primers (EntF and EntR). The primers were able to amplify a 678bp target region from Enterococcus faecalis ATCC 7080 and 20 other strains of enterococci from 11 different species, but there was no amplification by 32 species from closely related genera (Pediococcus, Lactobacillus, Streptococcus and Listeria) or species of Escherichia coli and Salmonella. The PCR positive samples were plated, screened by a colony patch technique and their identities were confirmed by API 20 Strep panels and sequencing. When dry fermented sausage and ham as well as fresh meat batter for dry cured sausage manufacture were tested for enterococci by the method, 29 Enterococcus strains (15 E. faecalis, 13 E. faecium, and one E. gallinarum) were identified. When susceptibility of these enterococci to 12 antibiotics was tested, the highest incidence of resistance was to clindamycin (89.6%), followed by tetracycline hydrochloride (65.5%), tylosin (62%), erythromycin (45%), streptomycin and neomycin (17%), chloramphenicol (10.3%), penicillin (10.3%), ciprofloxacin (10.3%) and gentamicin (3.4%). None was resistant to the clinically important drugs vancomycin or ampicillin. Most strains (27/29) were resistant to more than one antibiotic while 17 of 29 strains were resistant to three to 8 antibiotics. The molecular method developed was validated for speciation of enterococci and was useful in assessing uncooked processed meat products as a reservoir for multi-drug resistant Enterococcus species. [Copyright &y& Elsevier]

Abstract: Increasing consumption and year-round consumer demand for fresh, minimally processed green vegetables have been observed in Canada and other developed countries. However, in the past two decades, produce has been increasingly implicated in outbreaks and correspondingly recognized as a vector for the transmission of pathogenic microorganisms. To this end, we examined the microbiological quality of imported produce available at retail across Canada during a period of limited domestic availability. In total, 106 samples obtained from five Canadian cities were purchased from retail outlets and subjected to microbiological analyses, including aerobic plate (APC) and coliform counts, and enrichments for enterococci, indicator Escherichia coli, E. coli O157:H7 and Salmonella spp. Also, recovered Enterococcus faecalis and Enterococcus faecium were screened for antimicrobial resistance (AMR). Overall, samples included herbs (n=61), leafy greens (n=25), and spinach (n=20) deriving from five countries (Columbia, Dominican Republic, Guatemala, Mexico, and the United States [US]). APCs were consistent across commodities regardless of country, ranging from mean log10 CFU/g of 6.1 to 7.4, with no significant differences observed. Excluding a single leafy green sample from Guatemala, the lowest prevalence of coliforms was for Mexican herbs (22.2%), with a high of 66.7% on US leafy greens. With the exception of spinach, concentrations of coliforms varied widely, ranging from undetectable to too numerous to count (>8.5log10 CFU/g). Of the commodities assessed, Mexican and US spinach had the lowest coliform concentrations (undetectable to 4.0log10 CFU/g). Organic herbs and conventional leafy greens possessed significantly lower (p <0.05) prevalence of coliforms compared to conventional herbs and organic leafy greens, respectively. The most frequent recovery of indicator E. coli was observed for herbs, with 11.1, 8.3, and 3.7% prevalence observed in samples from Columbia, US, and Mexico, respectively. For spinach, 0 and 6.7% of Mexican and US samples tested positive, while no leafy green samples from either country were positive. No E. coli O157:H7 or Salmonella spp. were detected. E. faecium and E. faecalis were recovered from 15.1 and 5.7% of samples, respectively. Although no glycopeptide resistance was observed, resistance to other clinically relevant antibiotics was noteworthy in both species. Overall, though microbiological quality indicators were frequently high, E. coli O157:H7 and Salmonella were not detected. However, the presence of resistance and reduced susceptibility to clinically relevant antimicrobials in recovered enterococci demonstrate imported fresh produce may serve as a vehicle for the transmission of antimicrobial resistance across national borders. [Copyright &y& Elsevier]

Abstract: The objective of this study was to characterize antimicrobial resistance (AMR) and virulence genotypes of Enterococcus spp. particularly Enterococcus faecalis isolated from retail meats purchased (2007–2008) in Alberta, Canada. Unconditional statistical associations between AMR pheno- and genotypes and virulence genotypes were determined. A total of 532 enterococci comprising one isolate from each positive sample were analyzed for antimicrobial susceptibility. A customized enterococcal microarray was used for species identification and the detection of AMR and virulence genes. E. faecalis was found in >94% of poultry samples and in about 73% of beef and 86% of pork samples. Enterococcus faecium was not found in turkey meat and its prevalence was 2% in beef and pork and 4% in chicken samples. None of the enterococci isolates were resistant to the clinically important drugs ciprofloxacin, daptomycin, linezolid and vancomycin. Multiresistance (≥3 antimicrobials) was more common in E. faecalis (91%) isolated from chicken and turkey (91%) than those isolated from beef (14%) or pork (45%). Resistance to aminoglycosides was also noted at varying degrees. The most common resistance genes found in E. faecalis were aminoglycosides (aac, aphA3, aadE, sat4, aadA), macrolides (ermB, ermA), tetracyclines (tetM, tetL, tetO), streptogramin (vatE), bacitracin (bcrR) and lincosamide (linB). Virulence genes expressing aggregation substances (agg) and cytolysin (cylA, cylB, cylL, cylM) were found more frequently in poultry E. faecalis and were unconditionally associated with tetM, linB and bcrR resistance genes. Other virulence genes coding for adhesion (ace, efaAfs), gelatinase (gelE) were also found in the majority of E. faecalis. Significant statistical associations were found between resistance and virulence genotypes, suggesting their possible physical link on a common genetic element. This study underscores the importance of E. faecalis as a reservoir of resistance and virulence genes and their potential transfer to humans through consumption of contaminated undercooked meat. [Copyright &y& Elsevier]

Abstract: Enterococci are reported to survive pasteurisation but the extent of their survival is unclear. Sixty-one thermoduric enterococci isolates were selected from laboratory pasteurised milk obtained from silos in six dairy factories. The isolates were screened to determine log10 reductions incurred after pasteurisation (63°C/30min) and ranked from highest to lowest log10 reduction. Two isolates each of Enterococcus faecalis, Enterococcus faecium, Enterococcus durans and Enterococcus hirae, exhibiting the median and the greatest heat resistance, as well as E. faecalis ATCC 19433, were selected for further heat resistance determinations using an immersed coil apparatus. D values were calculated from survival curves plotted from viable counts obtained after heating isolates in Brain Heart Infusion Broth at 63, 69, 72, 75 and 78°C followed by rapid cooling. At 72°C, the temperature employed for High Temperature Short Time (HTST) pasteurisation (72°C/15s), the D values extended from 0.3min to 5.1min, depending on the isolate and species. These data were used to calculate z values, which ranged from 5.0 to 9.8°C. The most heat sensitive isolates were E. faecalis (z values 5.0, 5.7 and 7.5°C), while the most heat resistant isolates were E. durans (z values 8.7 and 8.8°C), E. faecium (z value 9.0°C) and E. hirae (z values 8.5 and 9.8°C). The data show that heat resistance in enterococci is highly variable. [Copyright &y& Elsevier]

Abstract: The isolation rate of high-level vancomycin-resistant enterococci (VRE) from poultry samples in Japan has increased in recent years. As this raises concerns for the potential spread of genes encoding vancomycin resistance, poultry is routinely screened for VRE. Here, we report the isolation and characterization of a vanA genotype vancomycin-resistant Enterococcus cecorum strain (E. cecorum IPHa84) from retail domestic poultry in September 2009. The species identification was performed by biochemical testing and sequencing of the 16S rRNA and manganese-dependent superoxide dismutase genes. The vancomycin and teicoplanin susceptibility tests showed that E. cecorum IPHa84 was resistant to vancomycin and susceptible to teicoplanin, demonstrating that this isolate was VanB phenotype-vanA genotype VRE. Moreover, a vanA gene cluster was found in a chromosomally encoded Tn1546-related element, which exhibited the characteristic structure of the prototype Tn1546 element, but contained eight point mutations. The vanS sequence of E. cecorum IPHa84 contained three point mutations and was 100% identical to those of VRE isolated from different broiler droppings in Japan prior to the banning of avoparcin, indicating that the Tn1546-related element may be stable in poultry production environments, even in the absence of selective pressure. The isolation of a novel enterococcal species harboring the vanA gene reconfirms that poultry can serve as a reservoir of VanA-type VRE or vancomycin resistance genes, and suggests that the transmission of these risk factors from poultry to humans through the food chain remains a potential threat in Japan. [Copyright &y& Elsevier]

Abstract: The technological and safety properties of 35 indigenous strains of Enterococcus faecium isolated from two Egyptian cheeses were characterised in order to determine their ability for rational manufacture of diversified and typical dairy products in the Middle East. A great diversity was observed within the 35 strains on the basis of their technological properties. A statistical analysis made it possible to distribute the 35 strains of E. faecium into different groups. Three groups were identified in terms of their acidification activity, measured by the Cinac system: a group of strains that quickly acidified milk, a second that moderately acidified milk, and a last cluster that revealed weak acidification activity. On the basis of texturing properties that were evaluated using a texturometer and a viscometer, a cluster of strains produced viscous and firm gels, a second cluster included fairly viscous, firm and cohesive gels, and a last group generated slightly viscous, but firm and very cohesive gels. By considering the aroma profiles that were determined by gas chromatography coupled with mass spectrometry, four clusters were identified. One cluster displayed a high dimethyl disulfide level, a second group of strains was highly aromatic, a third cluster led to typical “lactic” products, and the last cluster made it possible to obtain low aromatic products. None of the 35 strains proved to be β-haemolytic on the basis of the characterisation of their safety properties. The resistance to 20 antibiotics was assessed by the disc diffusion method. The 35 isolates were sensitive to 12 antibiotics, and among them, one isolate was resistant to only two antibiotics (nalixidic acid and streptomycin). The resistance to eight antibiotics was strain-dependant. Finally, this study demonstrates that some indigenous strains of E. faecium displayed interesting technological properties for cheese manufacture, together with good safety characteristics. They could be useful for the manufacture of typical products in Egypt. [Copyright &y& Elsevier]

Abstract: Enterococci belong to the lactic acid bacteria (LAB) and they are of importance in foods due to their involvement in food spoilage and fermentations, as well as their utilisation as probiotics in humans and slaughter animals. However, they are also important nosocomial pathogens that cause bacteraemia, endocarditis and other infections. Some strains are resistant to many antibiotics and possess virulence factors such as adhesins, invasins, pili and haemolysin. The role of enterococci in disease has raised questions on their safety for use in foods or as probiotics. Studies on the incidence of virulence traits among enterococcal strains isolated from food showed that some can harbour virulence traits, but it is also thought that virulence is not the result of the presence of specific virulence determinants alone, but is rather a more intricate process. Specific genetic lineages of hospital-adapted strains have emerged, such as E. faecium clonal complex (CC) 17 and E. faecalis CC2, CC9, CC28 and CC40, which are high risk enterococcal clonal complexes. These are characterised by the presence of antibiotic resistance determinants and/or virulence factors, often located on pathogenicity islands or plasmids. Mobile genetic elements thus are considered to play a major role in the establishment of problematic lineages. Although enterococci occur in high numbers in certain types of fermented cheeses and sausages, they are not deliberately added as starter cultures. Some E. faecium and E. faecalis strains are used as probiotics and are ingested in high numbers, generally in the form of pharmaceutical preparations. Such probiotics are administered to treat diarrhoea, antibiotic-associated diarrhoea or irritable bowel syndrome, to lower cholesterol levels or to improve host immunity. In animals, enterococcal probiotics are mainly used to treat or prevent diarrhoea, for immune stimulation or to improve growth. From a food microbiological point of view, the safety of the bacteria used as probiotics must be assured, and data on the major strains in use so far indicate that they are safe. The advantage of use of probiotics in slaughter animals, from a food microbiological point of view, lies in the reduction of zoonotic pathogens in the gastrointestinal tract of animals which prevents the transmission of these pathogens via food. The use of enterococcal probiotics should, in view of the development of problematic lineages and the potential for gene transfer in the gastrointestinal tract of both humans and animals, be carefully monitored, and the advantages of using these and new strains should be considered in a well contemplated risk/benefit analysis. [Copyright &y& Elsevier]

Abstract: The aim of this study was to elucidate whether and to what extent fresh produce from Germany plays a role as a carrier and reservoir of antibiotic resistant bacteria. For this purpose, 1001 vegetables (fruit, root, bulbous vegetables, salads and cereals) were collected from 13 farms and 11 supermarkets in Germany and examined bacteriologically. Phenotypic resistance of Enterobacter cloacae (n=172); Enterobacter gergoviae (n=92); Pantoea agglomerans (n=96); Pseudomonas aeruginosa (n=295); Pseudomonas putida (n=106) and Enterococcus faecalis (n=100) against up to 30 antibiotics was determined by using the microdilution method. Resistance to ß-lactams was most frequently expressed by P. agglomerans and E. gergoviae against cefaclor (41% and 29%). Relatively high resistance rates were also observed for doxycycline (23%), erythromycin (21%) and rifampicin (65%) in E. faecalis, for spectinomycin (28%) and mezlocillin (12%) in E. cloacae, as well as for streptomycin (19%) in P. putida. In P. aeruginosa, relatively low resistance rates were observed for the aminoglycosides amikacin, apramicin, gentamicin, neomycin, netilmicin and tobramycin (<4%); 11% was resistant to streptomycin. No glycopeptide-resistant enterococci were observed. Resistance rates of bacteria isolated from farm samples were higher than those of the retail markets whenever significant differences were observed. This suggests that expressing resistance is at the expense of bacterial viability, since vegetables purchased directly at the farm are probably fresher than at the supermarket, and they have not been exposed to stress factors. However, this should not keep the customer from buying directly at the farm, since the overall resistance rates were not higher than observed in bacteria from human or animal origin. Instead, peeling or washing vegetables before eating them raw is highly recommended, since it reduces not only the risk of contact with pathogens, but also that of ingesting and spreading antibiotic resistant bacteria. [Copyright &y& Elsevier]

Abstract: In this work, biogenic amine production (histamine, tyramine and putrescine) by a collection of 74 lactic acid bacteria of aquatic origin has been investigated by means of amino acid decarboxylation by growth on decarboxylase differential medium, biogenic amine detection by thin-layer chromatography (TLC) and decarboxylase gene detection by PCR. None of the evaluated strains showed neither production of histamine and putrescine, nor presence of the genetic determinants encoding the corresponding decarboxylase activities. However, the tyrosine decarboxylase gene (tdc) was present in all the enterococcal strains, and tyramine production was detected by TLC in all of them but Enterococcus faecium BCS59 and MV5. Analysis of the tyrosine decarboxylase operon of these strains revealed the presence of an insertion sequence upstream tdc that could be responsible for their lack of tyrosine decarboxylase activity. [Copyright &y& Elsevier]

Abstract: A complex microbial consortium derived from raw milk and composed of populations classified in 4 groups (lactic acid bacteria (A), Gram positive catalase positive bacteria (B), Gram negative bacteria (C) and yeasts (D)) can contribute to the inhibition of Listeria monocytogenes in the core of an uncooked pressed cheese. To identify what groups may be involved in the inhibition, the consortium was simplified by successively omitting one group at a time. Pasteurized milk was inoculated with these more or less complex consortia and their effects on L. monocytogenes count, pH, acids and volatile compounds in the core of uncooked pressed cheese were evaluated. The growth of L. monocytogenes was the highest in cheeses prepared with pasteurized milk and only St. thermophilus. Inhibition in other cheeses was expressed by comparison with growth in these ones. All the consortia containing both lactic acid bacteria (group A) and Gram positive catalase positive bacteria (group B) — ABCD, ABD, ABC, AB — were more inhibitory than those containing lactic acid bacteria on its own (A) or associated only with yeasts (AD) or/and Gram negative (ADC). Consortia without lactic acid bacteria were weakly inhibitory or had no effect. Gram positive catalase positive bacteria alone were not inhibitory although most of the species became established in the cheeses. The Lactobacillus population (Lb. casei, Lb. plantarum, Lb. curvatus and Lb. farciminis) was predominant in cheeses (9logCFU/g) with a higher count than Leuconostoc (7logCFU/g) and Enterococcus (7logCFU/g). Lactobacillus counts were negatively correlated with those of L. monocytogenes (r=−0.84 at 18days) and with the level of d-lactic acid. There was no correlation between L. monocytogenes and Leuconostoc or Enterococcus counts. Complex consortium ABCD and AB not only had a stronger inhibitory power in cheeses than consortium AD, they were also associated with the highest levels of l-lactic and acetic acids. All cheeses inoculated with lactic acid bacteria differed from those without by higher levels of ethyl formiate, pentane and alcohols (2-butanol, 2-pentanol), and lower levels of ketones (2-hexanone, 2,3-butanedione) and aldehydes (2-methyl-butanal). Levels of 2-methyl-butanal, 2-butanol and 2-pentanol were higher in ABCD and AB cheeses than in AD cheeses. Beside their contribution to the inhibition, their effect on cheese flavour must be evaluated. [Copyright &y& Elsevier]

Abstract: Untreated drinking water is frequently overlooked as a source of antibiotic resistance in developed countries. To gain further insight on this topic, we isolated the indicator bacteria Enterococcus spp. from water samples collected in wells, fountains and natural springs supplying different communities across Portugal, and characterized their antibiotic resistance profile with both phenotypic and genetic approaches. We found various rates of resistance to seven antibiotic families. Over 50% of the isolates were resistant to at least ciprofloxacin, tetracyclines or quinupristin–dalfopristin and 57% were multidrug resistant to ≥3 antibiotics from different families. Multiple enterococcal species (E. faecalis, E. faecium, E. hirae, E. casseliflavus and other Enterococcus spp) from different water samples harbored genes encoding resistance to tetracyclines, erythromycin or gentamicin [tet(M)-46%, tet(L)-14%, tet(S)-5%, erm(B)-22%, aac(6´)-Ie-aph(2″)-12%] and putative virulence factors [gel-28%, asa1-16%]. The present study positions untreated drinking water within the spectrum of ecological niches that may be reservoirs of or vehicles for antibiotic resistant enterococci/genes. These findings are worthy of attention as spread of antibiotic resistant enterococci to humans and animals through water ingestion cannot be dismissed. [Copyright &y& Elsevier]

Abstract: Enterococcus isolates (1500) obtained from the feces of 48 humans, 209 domesticated food animals, and 155 wild geese in South Korea were characterized with respect to species status by PCR analyses and resistance to antibiotics. Of the 1500 strains examined, the majority (n=577) were Enterococcus faecalis from 224 (54.4%) of the samples feces, while 299 were of E. faecium from 125 of the samples (30.3%), 224 were E. hirae from 101 (24.5%) of the samples, 94 were E. casseliflavus from 43 (10.4%) of the samples, and one was E. gallinarum. While 305 isolated from 125 (30.3%) of the samples were unidentified species. Approximately 15, 60, 50, 55, 3, and 40% of samples obtained from beef cattle, chickens, ducks, swine, wild geese, and humans, respectively, yielded Enterococcus isolates that were resistant to high-levels of aminoglycosides (i.e., of gentamicin, kanamycin, and streptomycin, minimum inhibitory concentrations were >1000mg/l). The 180 Enterococcus isolates that showed high levels of resistance to aminoglycoside antibiotics (HLAR) were screened for virulence genes encoding for aggregation substance (agg), cytolysin activator (cylA), gelatinase (gelE) and surface protein (esp). Of those, the gelE gene was found most frequently in chickens and ducks of the HLAR isolates, while 56 E. faecalis and 13 E. faecium HLAR were gelatinase positive and showed hemolysin activity. Multiple antibiotic resistant Enterococcus isolates carrying virulence genes were most frequently isolated from poultry and swine, and were mostly E. faecalis or E. faecium. These findings suggest that restriction of the use of antibiotics in food animal operations in South Korea, especially those involved in poultry and swine production would be desirable. [ABSTRACT FROM AUTHOR]

Abstract: The goal of this research was to determine the effect of different doses of galactooligosaccharide (GOS) on the fecal microbiota of healthy adults, with a focus on bifidobacteria. The study was designed as a single-blinded study, with eighteen subjects consuming GOS-containing chocolate chews at four increasing dosage levels; 0, 2.5, 5.0, and 10.0g. Subjects consumed each dose for 3weeks, with a two-week baseline period preceding the study and a two-week washout period at the end. Fecal samples were collected weekly and analyzed by cultural and molecular methods. Cultural methods were used for bifidobacteria, Bacteroides, enterobacteria, enterococci, lactobacilli, and total anaerobes; culture-independent methods included denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qRT-PCR) using Bifidobacterium-specific primers. All three methods revealed an increase in bifidobacteria populations, as the GOS dosage increased to 5 or 10g. Enumeration of bifidobacteria by qRT-PCR showed a high inter-subject variation in bifidogenic effect and indicated a subset of 9 GOS responders among the eighteen subjects. There were no differences, however, in the initial levels of bifidobacteria between the responding individuals and the non-responding individuals. Collectively, this study showed that a high purity GOS, administered in a confection product at doses of 5g or higher, was bifidogenic, while a dose of 2.5g showed no significant effect. However, the results also showed that even when GOS was administered for many weeks and at high doses, there were still some individuals for which a bifidogenic response did not occur. [ABSTRACT FROM AUTHOR]

Abstract: In this work we report on the main bacterial microflora typical for fermentation and ripening of traditional Istrian cheese. Samples from milk as well as Istrian cheese were analyzed during the ripening process by using culture independent molecular fingerprinting methods as well as culture based approaches. Our results indicate changes in bacterial diversity pattern during the ripening process. Differences in bacterial diversity at the same ripening stage among different farms investigated were comparably low. Sequence analysis of the most prominent bands of denaturing gradient gel electrophoresis fingerprints revealed dominance of Lactococcus lactis subs. lactis in all samples and a strong presence of Enterococcus spp. which was also confirmed by plate count analysis. [ABSTRACT FROM AUTHOR]

Abstract: The Enterococcus genus, a member of the Lactic Acid Bacteria (LAB) is found in various environments, but more particularly in the intestines of humans and other animals. Although sometimes associated with pathogenicity these bacteria have many benefits. They have been found in traditional artisanal fermented products, are used as probiotic cultures and nowadays extensively studied for the production of bacteriocins — the enterocins. Many of these enterocins have been found to be active against Listeria monocytogenes, and a few have also been reported to be active even against Gram negative bacteria, an unusual property for the bacteriocins produced by LAB. These properties have resulted in many studies describing the use of enterocins as preservatives in foods of animal and vegetable origin. This review covers the most recent information on the use of enterocins as food preservatives, either produced in - situ by the addition of enterocin producing strains or as external preservatives in the form of purified or semi-purified extracts, to prevent the growth of spoilage and pathogenic microorganisms. [Copyright &y& Elsevier]

Abstract: Two-hundred-twenty-nine food samples of animal origin were tested to know the prevalence of vancomycin-resistant enterococci (VRE) after a decade of avoparcin ban as animal growth promoter in Spain. VRE with acquired mechanism of resistance were detected in 9 of these 229 samples (3.9%, obtained from chicken, veal and rabbit), and one VRE per food sample was further characterized. The vanA gene was identified in seven isolates (2 E. faecium, 3 E. durans, and 2 E. hirae), and the vanB2 gene in the remaining 2 isolates (identified as E. faecium). The two vanB2 isolates showed a phenotype of multiresistance that included, in addition to vancomycin, also ampicillin, erythromycin, tetracycline, streptomycin, kanamycin, ciprofloxacin, chloramphenicol and trimethoprim-sulfamethoxazole and contained, among others, erm(B), tet(M), ant(6), and aph(3′)-III genes. Most of vanA enterococci showed erythromycin and tetracycline resistance and contained the erm(B) and tet(M) genes. One vanA- and both vanB2-positive E. faecium isolates were classified by MLST analysis into the CC17 clonal complex (ST17 and ST78), and one additional vanA isolate was included in a new sequence type named ST425 (singleton). Co-transference by conjugation of erm(B) and vanA genes was demonstrated in one vanA-positive E. faecium isolate. The inclusion of vanB2 cluster into Tn5382 structure was demonstrated in the two vanB2 isolates, as well as the linkage pbp5-Tn5382, and beta-haemolysis and gelatinase production was identified in one of them. Food sample of animal origin could be a vehicle of transference of VRE of vanA and vanB2 type that could be transferred to humans. [Copyright &y& Elsevier]