Patient-derived Enterococcus faecium with inflammatory genotypes promote colitis.

BACKGROUND: Dysbiosis of gut microbiota promotes colitis in ulcerative colitis (UC). Enterococcus faecium is an important constituent of dysbiotic microbiota. However, the mechanisms underlying E. faecium-induced colitis remain unclear. METHODS: Overall, 23 E. faecium strains isolated from human feces and 3 commercial strains were inoculated into Il10-/- mice. Mouse colons were histologically evaluated and analyzed using real-time PCR analysis of cytokines. Genes in 26 E. faecium strains were identified by whole-genome shotgun sequencing of genomic DNA. The production of reactive oxygen species (ROS) from each strain was measured. An antioxidant, lipoic acid, was orally administered to the colitis mouse model. RESULTS: Inoculation of E. faecium derived from patients with UC resulted in colitis in Il10-/- mice. The genotypes of 26 strains were characterized by identifying 1893 known genes; clustering all the strains based on the genotypes showed two major groups-inflammatory and probiotic clusters. Additionally, linear discriminant analysis clarified that lipoic acid metabolism was a significantly abundant pathway in the probiotic cluster compared to the inflammatory cluster. Further, the production of ROS was greater in inflammatory than in probiotic strains. Administration of lipoic acid in E. faecium-inoculated mice ameliorated colitis. CONCLUSIONS: Enterococcus faecium strains in the inflammatory cluster promoted colitis with higher production of ROS than the strains in the probiotic cluster.

Characterisation of the manganese superoxide dismutase of Enterococcus faecium.

The manganese superoxide dismutase (SodA) of E. faecium strain AUS0004 has been characterised. It is most closely related to Enterococcus hirae, Enterococcus durans, Enterococcus villorium, and Enterococcus mundtii with 100%, 91,55%, 90,85%, and 90,58% homology, respectively, but more distant from SodA of E. faecalis (81.68%). A sodA deletion mutant has been constructed. Compared to the parental strain, the ΔsodA mutant was affected in aerobic growth and more sensitive to hydrogen peroxide (H2O2), cumene hydroperoxide (CuOOH), and the superoxide anion (O2•-) generator menadione. The E. faecium strain AUS0004 is part of those bacteria accumulating H2O2 to high concentrations (around 5 mM) starting from late exponential growth phase. Accumulation of the peroxide was around 25% less in the mutant suggesting that this part of H2O2 is due to the dismutation of O2•- by SodA. The sodA gene of E. faecium AUS0004 was induced by oxygen, peroxides and menadione but the corresponding regulator remains hitherto unknown. Finally, we showed that SodA activity is important for virulence in the Galleria mellonella model.

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.

Liquid Extraction Surface Analysis Mass Spectrometry of ESKAPE Pathogens.

The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae) represent clinically important bacterial species that are responsible for most hospital-acquired drug-resistant infections; hence, the need for rapid identification is of high importance. Previous work has demonstrated the suitability of liquid extraction surface analysis mass spectrometry (LESA MS) for the direct analysis of colonies of two of the ESKAPE pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) growing on agar. Here, we apply LESA MS to the remaining four ESKAPE species (E. faecium E745, K. pneumoniae KP257, A. baumannii AYE, and E. cloacae S11) as well as E. faecalis V583 (a close relative of E. faecium) and a clinical isolate of A. baumannii AC02 using an optimized solvent sampling system. In each case, top-down LESA MS/MS was employed for protein identification. In total, 24 proteins were identified from 37 MS/MS spectra by searching against protein databases for the individual species. The MS/MS spectra for the identified proteins were subsequently searched against multiple databases from multiple species in an automated data analysis workflow with a view to determining the accuracy of identification of unknowns. Out of 24 proteins, 19 were correctly assigned at the protein and species level, corresponding to an identification success rate of 79%.

Purification and characterization of bacteriocins-like inhibitory substances from food isolated Enterococcus faecalis OS13 with activity against nosocomial enterococci.

Nosocomial infections caused by enterococci are an ongoing global threat. Thus, finding therapeutic agents for the treatment of such infections are crucial. Some Enterococcus faecalis strains are able to produce antimicrobial peptides called bacteriocins. We analyzed 65 E. faecalis isolates from 43 food samples and 22 clinical samples in Egypt for 17 common bacteriocin-encoding genes of Enterococcus spp. These genes were absent in 11 isolates that showed antimicrobial activity putatively due to bacteriocins (three from food, including isolate OS13, and eight from clinical isolates). The food-isolated E. faecalis OS13 produced bacteriocin-like inhibitory substances (BLIS) named enterocin OS13, which comprised two peptides (enterocin OS13α OS13ß) that inhibited the growth of antibiotic-resistant nosocomial E. faecalis and E. faecium isolates. The molecular weights of enterocin OS13α and OS13ß were determined as 8079 Da and 7859 Da, respectively, and both were heat-labile. Enterocin OS13α was sensitive to proteinase K, while enterocin OS13ß was resistant. Characterization of E. faecalis OS13 isolate revealed that it belonged to sequence type 116. It was non-hemolytic, bile salt hydrolase-negative, gelatinase-positive, and sensitive to ampicillin, penicillin, vancomycin, erythromycin, kanamycin, and gentamicin. In conclusion, BLIS as enterocin OS13α and OS13ß represent antimicrobial agents with activities against antibiotic-resistant enterococcal isolates.

Gut Microbiota Predict Enterococcus Expansion but Not Vancomycin-Resistant Enterococcus Acquisition.

Vancomycin-resistant Enterococcus (VRE) is a leading cause of hospital-acquired infections and continues to spread despite widespread implementation of pathogen-targeted control guidelines. Commensal gut microbiota provide colonization resistance to VRE, but the role of gut microbiota in VRE acquisition in at-risk patients is unknown. To address this gap in our understanding, we performed a case-control study of gut microbiota in hospitalized patients who did (cases) and did not (controls) acquire VRE. We matched case subjects to control subjects by known risk factors and "time at risk," defined as the time elapsed between admission until positive VRE screen. We characterized gut bacterial communities using 16S rRNA gene amplicon sequencing of rectal swab specimens. We analyzed 236 samples from 59 matched case-control pairs. At baseline, case and control subjects did not differ in gut microbiota when measured by community diversity (P = 0.33) or composition (P = 0.30). After hospitalization, gut communities of cases and controls differed only in the abundance of the Enterococcus-containing operational taxonomic unit (OTU), with the gut microbiota of case subjects having more of this OTU than time-matched control subjects (P = 0.01). Otherwise, case and control communities after the time at risk did not differ in diversity (P = 0.33) or community structure (P = 0.12). Among patients who became VRE colonized, those having the Blautia-containing OTU on admission had lower Enterococcus relative abundance once colonized (P = 0.004). Our results demonstrate that the 16S profile of the gut microbiome does not predict VRE acquisition in hospitalized patients, likely due to rapid and profound microbiota change. The gut microbiome does not predict VRE acquisition, but it may be associated with Enterococcus expansion, suggesting that these should be considered two distinct processes.IMPORTANCE The Centers for Disease Control and Prevention estimates that VRE causes an estimated 54,000 infections and 539 million dollars in attributable health care costs annually. Despite improvements in hand washing, environmental cleaning, and antibiotic use, VRE is still prevalent in many hospitals. There is a pressing need to better understand the processes by which patients acquire VRE. Multiple lines of evidence suggest that intestinal microbiota may help some patients resist VRE acquisition. In this large case-control study, we compared the 16S profile of intestinal microbiota on admission in patients that did and did not subsequently acquire VRE. The 16S profile did not predict subsequent VRE acquisition, in part due to rapid and dramatic change in the gut microbiome following hospitalization. However, Blautia spp. present on admission predicted decreased Enterococcus abundance after VRE acquisition, and Lactobacillus spp. present on admission predicted Enterococcus dominance after VRE acquisition. Thus, VRE acquisition and domination may be distinct processes.

Clonal distribution of vancomycin-resistant Enterococcus faecium in Turkey and the new singleton ST733.

BACKGROUND: The aim of this study was to provide information about the spread and characteristics of the vancomycin-resistant Enterococcus faecium isolates (VREfm) in Turkey. METHODS: Seventy-one nonduplicate consecutive isolates of VREfm were obtained from various clinical specimens of inpatients treated at university or training hospitals in seven regions of Turkey. Further characteristics included antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA, and multilocus sequence typing (MLST) of selected isolates. The presence of vancomycin resistance and virulence genes (esp and hyl) was investigated by polymerase chain reaction (PCR). RESULTS: All VREfm isolates had MICs to vancomycin of ≥32 mg/L and contained the vanA gene. The presence of esp gene was identified in 64 and hyl in eight VREfm isolates. All VREfm showed the multiresistance phenotype, including ampicillin (99%), penicillin (99%), imipenem (99%), ciprofloxacin (87%), moxifloxacin (87%), erythromycin (97%), streptomycin (86%), gentamicin (82%), tetracycline (70%), and teicoplanin (99%). All were susceptible to tigecycline while quinupristin-dalfopristin (97%) and linezolid (93%) were the most active other agents. Analysis of the PFGE profiles showed that 53 (74.6%) VREfm isolates shared a similar electrophoretic profile, designed as type 1, and were closely related (>85%). The sequence type was identified by MLST in 44 VRE isolates with unrelated or closely related PFGE patterns. MLST revealed that nosocomial spread of VREfm resulted from dissemination of lineage C1 E faecium clones. Sequence types ST78, ST203, and ST117 were the most frequently isolated. This is the first report of ST733 around the world. CONCLUSIONS: Lineage C1 clones are disseminated among clinical VREfm isolates in seven different regions in Turkey. Regarding VREfm isolates, the worldwide epidemic strains are in circulation in Turkey.

Biofilm synthesis and other virulence factors in multidrug-resistant uropathogenic enterococci isolated in Northern India.

Purpose: Enterococci express high degree of resistance towards wide range of antibiotics. Production of biofilm and many virulence factors along with drug resistance makes it difficult to eradicate the infection from urinary tract. The present study detected the expression of such factors including biofilm production by multidrug-resistant (MDR) enterococci. Materials and Methods: Drug susceptibility of 103 uropathogenic enterococci was performed followed by estimation of minimum inhibitory concentration of high-level gentamicin and vancomycin by microbroth dilution method. Vancomycin-resistant genes were detected by multiplex polymerase chain reaction. Production of virulence factors such as haemagglutination, caseinase, lipase, gelatinase, haemolysin and ß-lactamase was detected by phenotypic methods in MDR strains. Biofilm production was detected by calcofluor-white fluorescence staining and semi-quantitative adherence assay. Results: 45% and 18.4% of the isolates were high-level gentamicin-resistant and vancomycin-resistant enterococci (VRE), respectively. vanA gene was detected in 14 and vanB gene in 5 strains. Biofilm, caseinase and gelatinase were the most expressed virulence factor. Expression of caseinase, gelatinase and lipase was significantly higher in Enterococcus faecalis (P < 0.05). Expression of haemagglutination, gelatinase and haemolysin among the vancomycin-resistant isolates was significantly higher (P < 0.05). Conclusion: VanA and vanB are the prevalent genotypes responsible for vancomycin resistance. The high prevalence of MDR enterococcal strains producing biofilm and virulence determinants raises concern. asa1, hyl, esp, gelE, cyl and other genes are known to express these factors and contribute to biofilm formation. Most uropathogenic enterococci expressed biofilm at moderate level and can be detected effectively by calcofluor-white staining. No correlation was noted between vancomycin resistance and biofilm production.

What Differentiates Probiotic from Pathogenic Bacteria? The Genetic Mobility of Enterococcus faecium Offers New Molecular Insights.

Enterococcus faecium is a lactic acid bacterium with applications in food engineering and nutrigenomics, including as starter cultures in fermented foods. To differentiate the E. faecium probiotic from pathogenic bacteria, physiological analyses are often used but they do not guarantee that a bacterial strain is not pathogenic. We report here new findings and an approach based on comparison of the genetic mobility of (1) probiotic, (2) pathogenic, and (3) nonpathogenic and non-probiotic strains, so as to differentiate probiotics, and inform their safe use. The region of the 16S ribosomal DNA (rDNA) genes of different E. faecium strains native to Pernambuco-Brazil was used with the GenBank query sequence. Complete genomes were selected and divided into three groups as noted above to identify the mobile genetic elements (MGEs) (transposase, integrase, conjugative transposon protein and phage) and antibiotic resistance genes (ARGs), and to undertake pan-genome analysis and multiple genome alignment. Differences in the number of MGEs were found in ARGs, in the presence and absence of the genes that differentiate E. faecium probiotics and pathogenic bacteria genetically. Our data suggest that genetic mobility appears to be informative in differentiating between probiotic and pathogenic strains. While the present findings are not necessarily applicable to all probiotics, they offer novel molecular insights to guide future research in nutrigenomics, clinical medicine, and food engineering on new ways to differentiate pathogenic from probiotic bacteria.

Metagenomic Profiling of Ocular Surface Microbiome Changes in Meibomian Gland Dysfunction.

Purpose: Ocular surface microbiome changes can affect meibomian gland dysfunction (MGD) development. This study aimed to delineate differences among the microbiome of eyelid skin, conjunctiva, and meibum in healthy controls (HCs) and patients afflicted with MGD. Methods: Shotgun metagenomic analysis was used to determine if there are differences between the microbial communities in ocular sites surrounding the meibomian gland in healthy individuals and patients afflicted with MGD. Results: The meibum bacterial content of these microbiomes was dissimilar in these two different types of individuals. Almost all of the most significant taxonomic changes in the meibum microbiome of individuals with MGD were also present in their eyelid skin, but not in the conjunctiva. Such site-specific microbe pattern changes accompany increases in the gene expression levels controlling carbohydrate and lipid metabolism. Most of the microbiomes in patients with MGD possess a microbe population capable of metabolizing benzoate. Pathogens known to underlie ocular infection were evident in these individuals. MGD meibum contained an abundance of Campylobacter coli, Campylobacter jejuni, and Enterococcus faecium pathogens, which were almost absent from HCs. Functional annotation indicated that in the microbiomes of MGD meibum their capability to undergo chemotaxis, display immune evasive virulence, and mediate type IV secretion was different than that in the microbiomes of meibum isolated from HCs. Conclusions: MGD meibum contains distinct microbiota whose immune evasive virulence is much stronger than that in the HCs. Profiling differences between the meibum microbiome makeup in HCs and patients with MGD characterizes changes of microbial communities associated with the disease status.

Evolution of vancomycin-resistant Enterococcus faecium during colonization and infection in immunocompromised pediatric patients.

Patients with hematological malignancies or undergoing hematopoietic stem cell transplantation are vulnerable to colonization and infection with multidrug-resistant organisms, including vancomycin-resistant Enterococcus faecium (VREfm). Over a 10-y period, we collected and sequenced the genomes of 110 VREfm isolates from gastrointestinal and blood cultures of 24 pediatric patients undergoing chemotherapy or hematopoietic stem cell transplantation for hematological malignancy at St. Jude Children's Research Hospital. We used patient-specific reference genomes to identify variants that arose over time in subsequent gastrointestinal and blood isolates from each patient and analyzed these variants for insight into how VREfm adapted during colonization and bloodstream infection within each patient. Variants were enriched in genes involved in carbohydrate metabolism, and phenotypic analysis identified associated differences in carbohydrate utilization among isolates. In particular, a Y585C mutation in the sorbitol operon transcriptional regulator gutR was associated with increased bacterial growth in the presence of sorbitol. We also found differences in biofilm-formation capability between isolates and observed that increased biofilm formation correlated with mutations in the putative E. faecium capsular polysaccharide (cps) biosynthetic locus, with different mutations arising independently in distinct genetic backgrounds. Isolates with cps mutations showed improved survival following exposure to lysozyme, suggesting a possible reason for the selection of capsule-lacking bacteria. Finally, we observed mutations conferring increased tolerance of linezolid and daptomycin in patients who were treated with these antibiotics. Overall, this study documents known and previously undescribed ways that VREfm evolve during intestinal colonization and subsequent bloodstream infection in immunocompromised pediatric patients.

Hydrophobic and adhesive patterns of lactic acid bacteria and their antagonism against foodborne pathogens on tomato surface (Solanum lycopersicum L.).

AIMS: To evaluate tomato epiphyte lactic acid bacteria (LAB) hydrophobicity and auto-aggregation as an indicator of bacteria adhesion to tomato. Likewise, use LAB adhesion and co-aggregation as mechanisms to antagonize pathogen attachment. METHODS AND RESULTS: Fifty-four LAB were screened to evaluate their hydrophobic, auto- and co-aggregative properties against Salmonella Typhimurium, Saintpaul, Montevideo and Escherichia coli O157:H7. Subsequently, tomato adhesion of Enterococcus faecium Col1-1C, Weisella cibaria 11-E-2 and W. confusa Col 1-13 with high, medium and low hydrophobicity and high co-aggregation was investigated as well as their pathogen antagonism. Results indicate that bacteria hydrophobicity and auto-aggregation correspond to LAB adhesion to tomato. Enterococcus faecium Col1-1C interfered in most of the pathogen adhesion and micrographs revealed that such effect could be related to the inhibition of structures-type biofilm on E. coli O157:H7 and the aggregate formation on Salmonella. CONCLUSIONS: Lactic acid bacteria hydrophobicity and auto-aggregation can estimate bacteria adhesion to tomato and adhesive and co-aggregative properties could serve as a tool to antagonize foodborne pathogens under specific conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evidence the interference of Ent. faecium Col1-1C in E. coli O157:H7 biofilm formation and Salmonella colonization.

Improved detection of esp, hyl, asa1, gelE, cylA virulence genes among clinical isolates of Enterococci.

OBJECTIVE: Virulence factors (VFs) among the clinical strains of enterococci play a vital role in pathogenesis. This study was aimed to screen for cylA, asa1, gelE, esp and hyl among Enterococcus faecalis (n = 89) and E. faecium (n = 51) by multiplex PCR. The previously reported multiplex PCR was modified to 2 duplex (asa1 and gelE, cylA and esp) PCRs and 1 simplex (hyl) PCR. The idea of the modification of the multiplex PCR proposed here emerged in the course of the research study when majority of the isolates which phenotypically exhibited virulence traits were found to be negative for the respective gene. RESULTS: cylA, gelE and asa1 were significantly predominant in E. faecalis (59.55%, 85.39%, 86.51%) than E. faecium (1.96%, 60.78%, 9.80%) (p < 0.0001, p = 0.001967, p < 0.0001). hyl was detected in E. faecium (5.9%) only. The number of VFs detected in each isolate was recorded as the VF score. E. faecalis isolates had a VF score pattern of score 4 (34.83%), score 3 (26.96%), score 2 (28.08%) and score 1 (8.98%) while E. faecium had score 4 (1.96%), score 3 (7.84%), score 2 (25.49%) and score 1 (41.18%). This modification of the PCR protocol could resolve the problem of decreased detection of virulence determinants in enterococci.

Epidemiology, treatment and outcomes of bloodstream infection due to vancomycin-resistant enterococci in cancer patients in a vanB endemic setting.

BACKGROUND: Vancomycin-resistant enterococcus (VRE) is an important cause of infection in immunocompromised populations. Few studies have described the characteristics of vanB VRE infection. We sought to describe the epidemiology, treatment and outcomes of VRE bloodstream infections (BSI) in a vanB predominant setting in malignant hematology and oncology patients. METHODS: A retrospective review was performed at two large Australian centres and spanning a 6-year period (2008-2014). Evaluable outcomes were intensive care admission (ICU) within 48 h of BSI, all-cause mortality (7 and 30 days) and length of admission. RESULTS: Overall, 106 BSI episodes were observed in 96 patients, predominantly Enterococcus faecium vanB (105/106, 99%). Antibiotics were administered for a median of 17 days prior to BSI, and 76/96 (79%) were neutropenic at BSI onset. Of patients screened before BSI onset, 49/72 (68%) were found to be colonised. Treatment included teicoplanin (59), linezolid (6), daptomycin (2) and sequential/multiple agents (21). Mortality at 30-days was 31%. On multivariable analysis, teicoplanin was not associated with mortality at 30 days. CONCLUSIONS: VRE BSI in a vanB endemic setting occurred in the context of substantive prior antibiotic use and was associated with high 30-day mortality. Targeted screening identified 68% to be colonised prior to BSI. Teicoplanin therapy was not associated with poorer outcomes and warrants further study for vanB VRE BSI in cancer populations.

Surveillance of Enterococcus spp. reveals distinct species and antimicrobial resistance diversity across a One-Health continuum.

For a One-Health investigation of antimicrobial resistance (AMR) in Enterococcus spp., isolates from humans and beef cattle along with abattoirs, manured fields, natural streams, and wastewater from both urban and cattle feedlot sources were collected over two years. Species identification of Enterococcus revealed distinct associations across the continuum. Of the 8430 isolates collected, Enterococcus faecium and Enterococcus faecalis were the main species in urban wastewater (90%) and clinical human isolates (99%); Enterococcus hirae predominated in cattle (92%) and feedlot catch-basins (60%), whereas natural streams harbored environmental Enterococcus spp. Whole-genome sequencing of E. faecalis (n = 366 isolates) and E. faecium (n = 342 isolates), revealed source clustering of isolates, indicative of distinct adaptation to their respective environments. Phenotypic resistance to tetracyclines and macrolides encoded by tet(M) and erm(B) respectively, was prevalent among Enterococcus spp. regardless of source. For E. faecium from cattle, resistance to ß-lactams and quinolones was observed among 3% and 8% of isolates respectively, compared to 76% and 70% of human clinical isolates. Clinical vancomycin-resistant E. faecium exhibited high rates of multi-drug resistance, with resistance to all ß-lactam, macrolides, and quinolones tested. Differences in the AMR profiles among isolates reflected antimicrobial use practices in each sector of the One-Health continuum.

Plasmids Shaped the Recent Emergence of the Major Nosocomial Pathogen Enterococcus faecium.

Enterococcus faecium is a gut commensal of humans and animals but is also listed on the WHO global priority list of multidrug-resistant pathogens. Many of its antibiotic resistance traits reside on plasmids and have the potential to be disseminated by horizontal gene transfer. Here, we present the first comprehensive population-wide analysis of the pan-plasmidome of a clinically important bacterium, by whole-genome sequence analysis of 1,644 isolates from hospital, commensal, and animal sources of E. faecium Long-read sequencing on a selection of isolates resulted in the completion of 305 plasmids that exhibited high levels of sequence modularity. We further investigated the entirety of all plasmids of each isolate (plasmidome) using a combination of short-read sequencing and machine-learning classifiers. Clustering of the plasmid sequences unraveled different E. faecium populations with a clear association with hospitalized patient isolates, suggesting different optimal configurations of plasmids in the hospital environment. The characterization of these populations allowed us to identify common mechanisms of plasmid stabilization such as toxin-antitoxin systems and genes exclusively present in particular plasmidome populations exemplified by copper resistance, phosphotransferase systems, or bacteriocin genes potentially involved in niche adaptation. Based on the distribution of k-mer distances between isolates, we concluded that plasmidomes rather than chromosomes are most informative for source specificity of E. faeciumIMPORTANCEEnterococcus faecium is one of the most frequent nosocomial pathogens of hospital-acquired infections. E. faecium has gained resistance against most commonly available antibiotics, most notably, against ampicillin, gentamicin, and vancomycin, which renders infections difficult to treat. Many antibiotic resistance traits, in particular, vancomycin resistance, can be encoded in autonomous and extrachromosomal elements called plasmids. These sequences can be disseminated to other isolates by horizontal gene transfer and confer novel mechanisms to source specificity. In our study, we elucidated the total plasmid content, referred to as the plasmidome, of 1,644 E. faecium isolates by using short- and long-read whole-genome technologies with the combination of a machine-learning classifier. This was fundamental to investigate the full collection of plasmid sequences present in our collection (pan-plasmidome) and to observe the potential transfer of plasmid sequences between E. faecium hosts. We observed that E. faecium isolates from hospitalized patients carried a larger number of plasmid sequences compared to that from other sources, and they elucidated different configurations of plasmidome populations in the hospital environment. We assessed the contribution of different genomic components and observed that plasmid sequences have the highest contribution to source specificity. Our study suggests that E. faecium plasmids are regulated by complex ecological constraints rather than physical interaction between hosts.

[Investigation of Antibiotic Resistance and Virulence Factors of Enterococcus faecium and Enterococcus faecalis Strains Isolated from Clinical Samples]. / Klinik Örneklerden Izole Edilen Enterococcus faecium ve Enterococcus faecalis Izolatlarinin Antibiyotik Direnci ve Virülans Faktörlerinin Arastirilmasi.

Enterococci, which are commonly found in the environment, cause serious infections despite the absence of well-defined virulence factors and toxins. Knowing the virulence properties of enterococci is important to understand the complex pathogenic structures. In this study, we aimed to investigate the virulence factors (asa1, hyl, cylA, efa, ebp, ace, esp, gelE, sprE, fsrA, fsrB, fsrC genes, gelatinase activity, hemolysin, hydrogen peroxide and biofilm production) and antibiotic resistance of Enterococcus faecium and Enterococcus faecalis strains isolated from clinical specimens. A total of 110 enterococcus isolates which were accepted as infectious agents were included in the study. The polymerase chain reaction method was used to identify the isolates and to detect virulence genes. Characteristics of hemolysis, biofilm formation, hydrogen peroxide production and gelatinase activity were investigated by phenotypic methods. The antibiotic susceptibility test was performed with VITEK 2 automated system. E.faecalis ATCC 29212 standard strain was used as a quality control in all tests. Of the 110 enterococci isolates included in the study, 61 were identified as E.faecium and 49 as E.faecalis. The efa gene was the most frequently detected virulence gene (92.7%), followed by ace (83.6%), esp (66.4%), ebp (60.0%), cylA (50.9%), hyl (46.4%), asa1 (45.5%), gelE, sprE, fsrC (33.6%), fsrA (12.7%) and fsrB (11.8%). All genes except hyl were higher in E.faecalis isolates and the difference was statistically significant (p<0.05). Twenty-five (51%) E.faecalis and 1 (1.6%) E.faecium isolates had beta-hemolysis and the difference was statistically significant (p= 0.000). Seven (11.5%) E.faecium and 4 (8.2%) E.faecalis isolates formed biofilm, but the difference was not statistically significant (p> 0.05). Two (3.3%) E.faecium and 14 (28.6%) E.faecalis isolates exhibited gelatinase activity and the difference between the two species was statistically significant (p= 0.000). Hydrogen peroxide production was not detected in any of the isolates. The highest resistance rate was determined against ciprofloxacin (70.9%). The resistance to ampicillin was 69.1%, high level streptomycin 65.1%, high level gentamicin 39.4%, vancomycin and teicoplanin 4.5%, and linezolid 1.8%. In conclusion, our data indicated that virulence factors except hyl gene and biofilm production were higher in E.faecalis isolates but E.faecium isolates were more resistant to antibiotics. In order to prevent infection of such virulent or resistant isolates in the hospital setting, infection control measures must be followed. In vivo studies are needed for the better understanding of the virulence of enterococci.

Immunization with the basic membrane protein (BMP) family ABC transporter elicits protection against Enterococcus faecium in a murine infection model.

Enterococcus faecium is evolving as a multi-resistant pathogen causing infections with high morbidity and mortality. A protective vaccine against E. faecium is lacking up till now. ATP-binding cassette (ABC) transporter proteins have important functions in bacteria to maintain survival and homeostasis. In the present study, we evaluated the basic membrane protein (BMP) family ABC transporter substrate-binding protein, designated herein as BMP, as a potential vaccine candidate against E. faecium. Recombinant BMP of E. faecium was expressed in Escherichia coli, and purified by metal affinity chromatography. Swiss albino mice were immunized with the recombinant BMP combined with Bacillus Calmette-Guérin (BCG) and/or alum as adjuvants. Mice immunized with BMP combined with alternating BCG and alum developed BMP-specific IgG and were protected against E. faecium challenge as evidenced from organ bioburden and histopathological examination. Furthermore, serum from immunized mice showed enhanced opsonophagocytic activity and protected mice against E. faecium challenge by passive immunization. Bioinformatic analysis revealed appreciable degrees of homology between E. faecium BMP and proteins from other pathogens which suggests BMP could be a useful vaccine against multiple pathogens. To our knowledge, this is the first report of in-vivo evaluation of BMP as a potential vaccine candidate against E. faecium.

Comparative analysis of Enterococcus faecalis and Enterococcus faecium strains isolated from clinical samples and traditional cheese types in the Northwest of Iran: antimicrobial susceptibility and virulence traits.

This research was conducted using 50 samples of popular traditional cheeses and 160 enterococcal clinical isolates. Phenotypic and genotypic methods used for identification of enterococci. Then, the incidences of antibacterial resistance and virulence traits were investigated. In total, 165 E. faecalis and 43 E. faecium obtained from traditional cheeses and different clinical isolates were analyzed in the study. Antibiotic susceptibility testing revealed 175(84.1%) isolates with multi-drug resistance (MDR) patterns, which was more common among clinical sources. The predominant virulence profile, including gelE, asa1 and cpd was detected within 47 (22.6%) of the MDR isolates. Our results showed that traditional cheeses and clinical E. faecalis isolates have distinct patterns of virulence traits. The identified enterococci with antibiotic resistance and associated virulence factors, could provide a potential risk to the public health.