Species prevalence, virulence genes, and antibiotic resistance of enterococci from food-producing animals at a slaughterhouse in Turkey.

Healthy cattle, sheep, and goats can be reservoirs for gastrointestinal pathogenic fecal enterococci, some of which could be multidrug-resistant to antimicrobials. The objective of this study was to determine the prevalence and diversity of Enterococcus species in healthy sheep, goat, and cattle carcasses, as well as to analyze the antimicrobial resistance phenotype/genotype and the virulence gene content. During 2019-2020, carcass surface samples were collected from 150 ruminants in a slaughterhouse. A total of 90 enterococci, comprising five species, were obtained. The overall prevalence of enterococci was found to be 60%, out of which 37.7% were identified as Enterococcus (E.) hirae, 33.3% as E. casseliflavus, 15.5% as E. faecium, 12.2% as E. faecalis, and 1.1% as E. gallinarum. Virulence-associated genes of efaA (12.2%) were commonly observed in the Enterococcus isolates, followed by gelE (3.3%), asaI (3.3%), and ace (2.2%). High resistance to quinupristin-dalfopristin (28.8%), tetracycline (21.1%), ampicillin (20%), and rifampin (15.5%) was found in two, four, four, and five of the Enterococcus species group, respectively. The resistance of Enterococcus isolates to 11 antibiotic groups was determined and multidrug resistant (MDR) strains were found in 18.8% of Enterococcus isolates. Characteristic resistance genes were identified by PCR with an incidence of 6.6%, 2.2%, 1.1%, 1.1%, 1.1%, and 1.1% for the tetM, ermB, ermA, aac(6')Ie-aph(2")-la, VanC1, and VanC2 genes in Enterococcus isolates, respectively. Efflux pump genes causing multidrug resistance were detected in Enterococcus isolates (34.4%). The results showed that there were enterococci in the slaughterhouse with a number of genes linked to virulence that could be harmful to human health.

Identification of Enterococcus spp. from food sources by matrix-assisted laser desorption ionization-time of flight mass spectrometry and characterization of virulence factors, antibiotic resistance, and biofilm formation.

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 enterococci 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.

Both biofilm cytotoxicity and monocytes' adhesion may be used as estimators of enterococcal virulence.

Our study aimed to identify markers of enterococci's virulence potential by evaluating the properties of strains of different sites of isolation. Enterococcal strains were isolated as commensals from faeces and as invasive strains from the urine and blood of patients from the University Clinical Centre, Gdansk, Poland. Changes in monocytes' susceptibility to the cytotoxic activity of isolates of different origins and their adherence to biofilm were evaluated using a flow cytometer. The bacterial protein profile was estimated by matrix assisted laser desorption ionization-time of flight mass spectrometer. The cytotoxicity of biofilm and monocytes' adherence to it were the most accurate factors in predicting the prevalence of the strain in the specific niche. Additionally, a bacterial protein with mass-to-charge ratio (m/z) 5000 was found to be responsible for the increased bacterial cytotoxicity, while monocytes' decreased adherence to biofilm was linked with the presence of proteins either with m/z 3330 or 2435. The results illustrate that monocytes' reaction when exposed to the bacterial biofilm can be used as an estimator of pathogens' virulence potential. The observed differences in monocytes' response are explainable by the bacterial proteins' profile. Additionally, the results indicate that the features of both bacteria and monocytes impact the outcome of the infection.

Non-faecium non-faecalis enterococci: a review of clinical manifestations, virulence factors, and antimicrobial resistance.

SUMMARYEnterococci are a diverse group of Gram-positive bacteria that are typically found as commensals in humans, animals, and the environment. Occasionally, they may cause clinically relevant diseases such as endocarditis, septicemia, urinary tract infections, and wound infections. The majority of clinical infections in humans are caused by two species: Enterococcus faecium and Enterococcus faecalis. However, there is an increasing number of clinical infections caused by non-faecium non-faecalis (NFF) enterococci. Although NFF enterococcal species are often overlooked, studies have shown that they may harbor antimicrobial resistance (AMR) genes and virulence factors that are found in E. faecium and E. faecalis. In this review, we present an overview of the NFF enterococci with a particular focus on human clinical manifestations, epidemiology, virulence genes, and AMR genes.

Patient-derived Enterococcus mundtii and its capsular polysaccharides cause depression through the downregulation of NF-κB-involved serotonin and BDNF expression.

The genus Enterococcus is commonly overpopulated in patients with depression compared to healthy control in the feces. Therefore, we isolated Enterococcus faecalis, Enterococcus durans, Enterococcus gallinarum, Enterococcus faecium, and Enterococcus mundtii from the feces of patients with comorbid inflammatory bowel disease with depression and examined their roles in depression in vivo and in vitro. Of these Enterococci, E. mundtii NK1516 most potently induced NF-κB-activated TNF-α and IL-6 expression in BV2 microglia cells. NK1516 also caused the most potent depression-like behaviors in the absence of sickness behaviors, neuroinflammation, downregulated brain-derived neurotrophic factor (BDNF), and serotonin (5-HT) levels in the hippocampus of mice. Furthermore, E. mundtii NK1516 reduced the mRNA expression of Htr1a in the hippocampus. Its capsular polysaccharide (CP), but not cytoplasmic components, also caused depression-like behaviors and reduced BDNF and serotonin levels in the hippocampus. Conversely, this was not observed with E. mundtii ATCC882, a well-known probiotic, or its CP. Orally gavaged fluorescence isothiocyanate (FITC)-conjugated NK1516 CP was detected in the hippocampus of mice. The NK1516 genome exhibited unique CP biosynthesis-related genes (capD, wbjC, WecB, vioB), unlike that of ATCC882. These findings suggest that E. mundtii may be a risk factor for depression.

Enterococci enhance Clostridioides difficile pathogenesis.

Enteric pathogens are exposed to a dynamic polymicrobial environment in the gastrointestinal tract1. This microbial community has been shown to be important during infection, but there are few examples illustrating how microbial interactions can influence the virulence of invading pathogens2. Here we show that expansion of a group of antibiotic-resistant, opportunistic pathogens in the gut-the enterococci-enhances the fitness and pathogenesis of Clostridioides difficile. Through a parallel process of nutrient restriction and cross-feeding, enterococci shape the metabolic environment in the gut and reprogramme C. difficile metabolism. Enterococci provide fermentable amino acids, including leucine and ornithine, which increase C. difficile fitness in the antibiotic-perturbed gut. Parallel depletion of arginine by enterococci through arginine catabolism provides a metabolic cue for C. difficile that facilitates increased virulence. We find evidence of microbial interaction between these two pathogenic organisms in multiple mouse models of infection and patients infected with C. difficile. These findings provide mechanistic insights into the role of pathogenic microbiota in the susceptibility to and the severity of C. difficile infection.

Emerging enterococcus pore-forming toxins with MHC/HLA-I as receptors.

Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.

Tertiary hospital sewage as reservoir of bacteria expressing MDR phenotype in Brazil / Esgoto de hospital terciário como reservatório de bactérias expressando o fenótipo MDR no Brasil

High doses of antibiotics used in hospitals can affect the microbial composition of sewers, selecting resistant bacteria. In this sense, we evaluated the antibiotic resistance profile and the multiresistant phenotype of bacteria isolated in sewage from a tertiary hospital in the interior São Paulo state, Brazil. For bacteria isolation, 10 µL of sewage samples were sown in selective culture media and the isolates were identified using VITEK-2 automatized system. The antibiotic sensitivity test was performed by disk diffusion. High percentages of resistance were found for amoxicillin, ampicillin, ceftazidime, clindamycin, vancomycin and the multidrug-resistant phenotype (MDR) was attributed to 60.7% of the isolates. Our results show bacteria classified as critical/high priority by WHO List of Priority Pathogens (Enterococcus and Staphylococcus aureus resistant to vancomycin and Enterobacteriaceae resistant to carbapenems) in hospital sewage. Therefore, the implementation of disinfection technologies for hospital sewage would reduce the bacterial load in the sewage that will reach urban wastewater treatment plants, minimizing superficial water contamination and bacterial resistance spread in the environment.

Altas doses de antibióticos utilizados em hospitais podem afetar a composição microbiana dos esgotos, selecionando bactérias resistentes. Nesse sentido, avaliamos o perfil de resistência a antibióticos e o fenótipo multirresistente de bactérias isoladas em esgoto de um hospital terciário no interior do estado de São Paulo, Brasil. Para o isolamento de bactérias, foram semeados 10 µL das amostras de esgoto em meios de cultura seletivos e os isolados foram identificados usando o sistema automatizado VITEK-2. O teste de sensibilidade aos antibióticos foi realizado por disco-difusão em ágar. Elevadas porcentagens de resistência foram encontradas para amoxicilina, ampicilina, ceftazidima, clindamicina, vancomicina e o fenótipo multirresistente (MDR) foi atribuído a 60,7% dos isolados. Nossos resultados mostram bactérias classificadas como prioridade crítica/alta pela Lista de Patógenos Prioritários da OMS (Enterococcus e Staphylococcus aureus resistentes à vancomicina e Enterobacteriaceae resistentes aos carbapenêmicos) no esgoto hospitalar. Sendo assim, implementação de tecnologias de desinfecção do esgoto hospitalar reduziriam a carga bacteriana no esgoto que chegará às estações de tratamento de esgoto urbanas, minimizando a contaminação dos ecossistemas hídricos receptores e a disseminação da resistência bacteriana no ambiente.

Oxandrastins: Antibacterial Meroterpenes from an Australian Mud Dauber Wasp Nest-Associated Fungus, Penicillium sp. CMB-MD14.

The ethyl acetate extract of an ISP-2 agar cultivation of the wasp nest-associated fungus Penicillium sp. CMB-MD14 exhibited promising antibacterial activity against vancomycin-resistant enterococci (VRE), with a bioassay guided chemical investigation yielding the new meroterpene, oxandrastin A (1), the first andrastin-like metabolite with an extra oxygenation at C-2. A culture media optimisation strategy informed a scaled-up rice cultivation that yielded 1, together with three new oxandrastins B-D (2-4), two known andrastins C (5) and F (6), and a new meroterpene of the austalide family, isoaustalide F (7). Structures of 1-7 were assigned based on detailed spectroscopic analysis and chemical interconversion. A GNPS molecular networking analysis of the rice cultivation extract detected the known austalides B (8), H (9), and H acid (10), tentatively identified based on molecular formulae and co-clustering with 7. That the anti-VRE properties of the CMB-MD14 extract were exclusively attributed to 1 (IC50 6.0 µM, MIC99 13.9 µM), highlights the importance of the 2-OAc and 3-OAc moieties to the oxandrastin anti-VRE pharmacophore.

Identification and characterization of a novel Enterococcus bacteriophage with potential to ameliorate murine colitis.

Increase of the enteric bacteriophages (phage), components of the enteric virome, has been associated with the development of inflammatory bowel diseases. However, little is known about how a given phage contributes to the regulation of intestinal inflammation. In this study, we isolated a new phage associated with Enterococcus gallinarum, named phiEG37k, the level of which was increased in C57BL/6 mice with colitis development. We found that, irrespective of the state of inflammation, over 95% of the E. gallinarum population in the mice contained phiEG37k prophage within their genome and the phiEG37k titers were proportional to that of E. gallinarum in the gut. To explore whether phiEG37k impacts intestinal homeostasis and/or inflammation, we generated mice colonized either with E. gallinarum with or without the prophage phiEG37k. We found that the mice colonized with the bacteria with phiEG37k produced more Mucin 2 (MUC2) that serves to protect the intestinal epithelium, as compared to those colonized with the phage-free bacteria. Consistently, the former mice were less sensitive to experimental colitis than the latter mice. These results suggest that the newly isolated phage has the potential to protect the host by strengthening mucosal integrity. Our study may have clinical implication in further understanding of how bacteriophages contribute to the gut homeostasis and pathogenesis.

Experimental infection of Asian house geckos with Enterococcus lacertideformus demonstrates multiple disease transmission routes and the in-vivo efficacy of antibiotics.

The disease caused by Enterococcus lacertideformus is multisystemic and ultimately fatal. Since its emergence, the bacterium has significantly impacted the captive breeding programs of the extinct in the wild Christmas Island Lister's gecko (Lepidodactylus listeri) and blue-tailed skink (Cryptoblepharus egeriae). The bacterium's pathogenicity, inability to grow in-vitro, and occurrence beyond the confines of Christmas Island necessitated the development of an experimental infection and treatment model. Asian house geckos (Hemidactylus frenatus) were challenged with a single dose of E. lacertideformus inoculum either by mouth, application to mucosal abrasion or skin laceration, subcutaneous injection, coelomic injection, or via co-housing with an infected gecko. Five healthy geckos acted as controls. Each transmission route resulted in disease in at least 40% (n = 2) geckos, expanding to 100% (n = 5) when E. lacertideformus was applied to skin laceration and mucosal abrasion groups. Incubation periods post-infection ranged between 54 and 102 days. To determine the efficacy of antibiotic treatment, infected geckos were divided into six groups (enrofloxacin 10 mg/kg, per os (PO), every 24 h (q24), amoxicillin-clavulanic acid 10 mg/kg, PO, q24, enrofloxacin 10 mg/kg combined with amoxicillin-clavulanic acid 10 mg/kg, PO, q24, rifampicin 15 mg/kg, PO, q24, clarithromycin 15 mg/kg, PO, q24, and untreated controls) for 21 days. Response to treatment was assessed by the change in lesion size, bacterial dissemination, and histological evidence of a host immune response. Irrespective of the antibiotic given, histology revealed that geckos inoculated by skin laceration were observed to have more extensive disease spread throughout the animal's body compared to other inoculation routes. The reduction in the average surface area of gross lesions was 83.6% for geckos treated with enrofloxacin, followed by the combination therapy amoxicillin-clavulanic acid and enrofloxacin (62.4%), amoxicillin-clavulanic acid (58.2%), rifampicin (45.5%), and clarithromycin (26.5%). Lesions in geckos untreated with antibiotics increased in size between 100 and 300%. In summary, enrofloxacin and amoxicillin-clavulanic acid show promising properties for the treatment of E. lacertideformus infection in geckos. The Asian house gecko E. lacertideformus infection model therefore provides foundational findings for the development of effective therapeutic treatment protocols aimed at conserving the health of infected and at-risk reptiles.

Genomic-based characterization of Enterococcus spp.: an emerging pathogen isolated from human gut.

BACKGROUND: Enterococci are ubiquitous microorganisms having diverse ecological niches but most prominently in gastrointestinal tract of humans and animals. Production of enterocins makes them a good probiotic candidate. However, their role as probiotics has become ambiguous in the last few years because of the presence of virulence factors and antibiotic resistance genes. These virulence traits are known to be transferred genetically, which makes them opportunistic pathogens in the gastrointestinal tract leading to serious concerns about their being used as probiotics. In the present study, Enterococcusspp. isolated from the human gut were subjected to Whole-Genome Sequencing (WGS) to determine the presence of resistance and virulence genes. METHODS AND RESULTS: Four human origins Enterococcus spp. including Enterococcus faecalis, Enterococcus casseliflavus, and two Enterococcus gallinarum were isolated from human fecal samples and further cultured on blood agar. Sanger sequencing was done using Applied Biosystems 3730xl DNA Analyzer. These strains were further subjected to WGS using oxford nanopore technology MinION. Raw data were analyzed using the free online tool epi2me. The Comprehensive Antibiotic Resistance Database (CARD) and RAST (Rapid Annotation using Subsystem Technology) software were used to look for the presence of antibiotic resistance genes in these strains. Resistance determinants for clinically important antibiotics (vancomycin) and functional virulence factor genes were detected. G-view server was used for comparative genomics of all strains. CONCLUSION: The genomic sequencing of Enterococcus suggested that E. faecalis, E. casseliflavus, and E. gallinarum strains are opportunistic pathogens, having antibiotic resistance genes. All isolates had vancomycin resistance genes, which were expressed phenotypically. Genes related to bacteriocin resistance were also present in E. casseliflavus and E. gallinarum.

Novel genomic islands and a new vanD-subtype in the first sporadic VanD-type vancomycin resistant enterococci in Norway.

BACKGROUND: Vancomycin-resistant enterococci (VRE) represent several types of transferable vancomycin resistance gene clusters. The vanD type, associated with moderate to high level vancomycin resistance, has only sporadically been described in clinical isolates. The aim of this study was to perform a genetic characterization of the first VanD-type VRE strains detected in Norway. METHODS: The VanD-type VRE-strains (n = 6) from two patient cases were examined by antimicrobial susceptibility testing and whole genome sequencing (WGS) to uncover Van-phenotype, strain phylogeny, the vanD gene clusters, and their genetic surroundings. The putative transferability of vanD was examined by circularization PCR and filter mating. RESULTS: The VanD-type Enterococcus faecium (n = 4) and Enterococcus casseliflavus (n = 2) strains recovered from two cases (A and B), expressed moderate to high level vancomycin resistance (MIC 64->256 mg/L) and various levels of teicoplanin susceptibility (MIC 2->256 mg/L). WGS analyses revealed phylogenetically different E. faecium strains (A1, A2, and A3 of case A and B1 from case B) as well as vanD gene clusters located on different novel genomic islands (GIs). The E. casseliflavus strains (B2 and B3 of case B) were not clonally related, but harbored nearly identical novel GIs. The vanD cluster of case B strains represents a novel vanD-subtype. All the vanD-GIs were integrated at the same chromosomal site and contained genes consistent with a Clostridiales origin. Circular forms of the vanD-GIs were detected in all strains except B1. Transfer of vanD to an E. faecium recipient was unsuccessful. CONCLUSIONS: We describe the first VanD-type E. casseliflavus strains, a novel vanD-subtype, and three novel vanD-GIs with a genetic content consistent with a Clostridiales order origin. Despite temporal occurrence, case A and B E. faecium strains were phylogenetically diverse and harbored different vanD subtypes and vanD-GIs.

Identification and safety assessment of Enterococcus thailandicus TC1 isolated from healthy pigs.

Enterococci have the dual characteristics of being opportunistic pathogens and promising probiotics. The isolation from patients of CDC PNS-E2, a newly described Enterococcus species Enterococcus sanguinicola, may pose potential hazards. Enterococcus thailandicus from fermented sausage is a senior subjective synonym of E. sanguinicola. In this study, Enterococcus thailandicus TC1 was first isolated in healthy pigs in Tongcheng, China and identified by phenotypic analysis and 16S rRNA-based techniques. To evaluate the strain safety, an approach including virulence factors, antibiotic resistance, and animal experiments was adopted. The results show that cylA, gelE, esp, agg, ace, efaAfm, efaAfs, ptsD genes were undetected, and that the strain was sensitive or poorly resistant to some clinically relevant antibiotics. However, the isolated strain demonstrated ß-hemolytic activity in rabbit blood agar plates. Analysis of animal experiments revealed that the isolated strain had no adverse effect on translocation and the internal organ indices, though significant differences in histology (villi height, crypts height) of ileum were observed. The data acquired suggest that E. thailandicus TC1 may be associated with a potential health risk.

Time to positivity as a prognostic factor in bloodstream infections with Enterococcus spp.

Time to positivity (TTP) is the delay of time from incubation to blood culture positivity. Short TTP can predict mortality and source of infection. The aim of this study was to investigate the value of TTP of patients with bloodstream infections with enterococci (E-BSI).In a single centre retrospective cohort study in Germany, the data of 244 patients with monomicrobial E-BSI were analyzed with hospital mortality as the primary outcome of interest from January 1 2014 to December 31 2016. Mortality rate of patients with bloodstream infections (BSI) with E. faecalis was 16.7%, Vancomycin sensitive E. faecium (VSEfm) 26.7% and Vancomycin resistant E. faecium (VREfm) 38.2%. Cut-offs showed a significantly higher mortality rate when compared to longer TTP (E. faecalis: P=0.047; VSEfm: P=0.02), but were not risk factors in survival analysis (E.faecalis: HR (hazard ratio): 2.73; P=0.17; VSEfm: HR: 1.63; P=0.15; VREfm: HR: 1.24; P=0.63). TTP≤10.5 hours with E. faecalis BSI was a discriminator for cardiovascular source of infection (AUC: 0.75). A short TTP could predict mortality rates and source of infection but was not an independent parameter for risk of death in survival analysis.

Endocarditis with spondylodiscitis: clinical characteristics and prognosis.

BACKGROUND: The association of infective endocarditis (IE) with spondylodiscitis (SD) was first reported in 1965, but few data are available about this issue. This study aimed to evaluate the prevalence of SD in patients with IE, and to determine the clinical features and the prognostic impact of this association. METHODS: We retrospectively analysed 363 consecutive patients admitted to our Department with non-device-related IE. Radiologically confirmed SD was revealed in 29 patients (8%). Long-term follow-up (average: 3 years) was obtained by structured telephone interviews; in 95 cases (13 of whom had been affected by SD), follow-up echocardiographic evaluation was also available. RESULTS: At univariable analysis, the combination of IE with SD was associated with male gender (p = 0.017), diabetes (p = 0.028), drug abuse (p = 0.009), Streptococcus Viridans (p = 0.009) and Enterococcus (p = 0.015) infections. At multivariable analysis, all these factors independently correlated with presence of SD in patients with IE. Mortality was similar in patients with and without SD. IE relapses at 3 years were associated with the presence of SD (p = 0.003), Staphylococcus aureus infection (p < 0.001), and drug abuse (p < 0.001) but, at multivariable analysis, only drug abuse was an independent predictor of IE relapses (p < 0.001; HR 6.8, 95% CI 1.6-29). At echocardiographic follow-up, SD was not associated with worsening left ventricular systolic function or valvular dysfunction. CONCLUSIONS: The association of IE with SD is not rare. Hence, patients with IE should be screened for metastatic infection of the vertebral column, especially if they have risk factors for it. However, SD does not appear to worsen the prognosis of patients with IE, either in-hospital or long-term.

Risk factors for enterococcal urinary tract infections: a multinational, retrospective cohort study.

Complicated urinary tract infection (cUTI) is a frequent cause of morbidity. In this multinational retrospective cohort study, we aimed to demonstrate risk factors for enterococcal UTI. Univariate and multivariate analyses of risk factors for enterococcal infection were performed. Among 791 hospitalized patients with cUTI, enterococci accounted for approximately 10% of cases (78/791). Risk factors for enterococcal UTI in multivariable analysis were male gender, age range of 55-75 years, catheter-associated UTI, and urinary retention. This information may assist treating physicians in their decision-making on prescribing empiric anti-enterococcus treatment to hospitalized patients presenting with cUTI and thus improve clinical outcomes.

Phenotypic and genotypic characterization of Enterococcus spp. from yolk sac infections in broiler chicks with a focus on virulence factors.

Bacterial infections of yolk sacs contribute to increased mortality of chicks, chronic infections during their rearing, or increased selection in the flock, which in turn leads to high economic losses in poultry production worldwide. The aim of this study was a phenotypic and genotypic characterization of enterococci isolated from yolk sac infections (YSI) of broiler chickens from Poland and the Netherlands. Biochemical, matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) MS, and rpoA gene sequencing identification was performed. Moreover, phenotypic and genotypic characterization of virulence factors and analysis of the clonal relationship of isolates by MALDI-TOF MS and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) were performed. The biochemical test identified 70 isolates as Enterococcus faecalis and 6 as Enterococcus mundtii. The results of MALDI-TOF MS were 100% concordant with those obtained by rpoA gene sequencing, and all 76 isolates were identified as E. faecalis. Differences were noted in the ß-glucuronidase, ß-glucosidase, α-galactosidase, phosphatase, melibiose, lactose, and raffinose tests that is going about the results of biochemical identification. None of the isolates were beta-hemolytic on blood agar in aerobic conditions, but all but one were gelatinase positive. Among biofilm-forming isolates (30/76; 39.5%), as many as 66.7% (20/30) were Polish E. faecalis strains. Most of the isolates carried virulence genes, that is gelE, ace, asa1, efaAfs, fsrA, fsrB, fsrC, cob, cpd, and ccf, but none had the hyl gene. Some isolates harbored cyl operon genes. One Polish strain (ST16) had all of the tested cyl genes and the esp gene, considered clinically important, and showed the highest biofilm-forming ability. Nearly 50% of the isolates showed close genetic relatedness in ERIC typing. In contrast with MALDI-TOF MS cluster analysis, ERIC-PCR results did not show a relationship with the origin of the strains. Using MALDI-TOF MS, 7 peaks were found in Polish and Dutch isolates, which may type them as species-specific biomarkers in E. faecalis from YSI.