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Listeria ivanovii is one of the two pathogenic species within the genus Listeria, the other being L. monocytogenes. In this study, we generated a stable pediocin resistant mutant Liv-r1 of a L. ivanovii strain, compared phenotypic differences between the wild-type and the mutant, localised the pediocin-induced mutations in the chromosome, and analysed the mechanisms behind the bacteriocin resistance. In addition to pediocin resistance, Liv-r1 was also less sensitive to nisin. The growth of Liv-r1 was significantly reduced with glucose and mannose, but less with cellobiose. The cells of Liv-r1 adsorbed less pediocin than the wild-type cells. Consequently, with less pediocin on the cell surface, the mutant was also less leaky, as shown as the release of intracellular lactate dehydrogenase to the supernatant. The surface of the mutant cells was more hydrophobic than that of the wild-type. Whole genome sequencing revealed numerous changes in the Liv-r1 chromosome. The mutations were found e.g., in genes encoding sigma-54-dependent transcription regulator and internalin B, as well as in genes involved in metabolism of carbohydrates such as glucose and cellobiose. Genetic differences observed in the mutant may be responsible for resistance to pediocin but no direct evidence is provided.

Objectives. To evaluate the long-term effect of chlorhexidine (CHX) and dimethyl sulfoxide (DMSO) on the sealing ability and biomineralization of two different calcium silicate cements (CSC) in root canal. Methods. Sixty human third molar root canals were obturated with ProRoot MTA or Biodentine. Before obturation the canals were irrigated with saline (control), 2% CHX or 5% DMSO. Microleakage was tested after three days and after six months. After additional six months (12 months after root filling) the roots were cut into 2 mm thick dentine discs. The discs were stored in artificial saliva for one year. The bond strength was measured with the push-out method, and the failure mode was evaluated with a stereomicroscope. The most apical disc of each tooth was used for Vickers hardness test. Results. No significant differences between the groups was found in initial microleakage. The leakage increased significantly during the 6-month storage in all groups except in Biodentine-CHX group and Biodentine-DMSO group. CHX and DMSO irrigation significantly increased the leakage with ProRoot MTA with time, but there was no statistically significant difference compared to the ProRoot MTA-control group at six months' time point. CHX significantly reduced the push-out bond strength of ProRoot MTA. With Biodentine irrigation with CHX or DMSO resulted with significantly higher push-out strength compared to the Biodentine control group. Fracture analysis showed statistically significant difference in the distribution of the fractures between the groups, but neither CHX nor DMSO change the fracture pattern statistically significantly. With Vickers hardness test ProRoot MTA with and without DMSO as the final irrigant showed significantly higher dentin hardness than any Biodentine-group. Significance. Considering that aging increased the leakage in all groups except with Biodentine-DMSO and the differences in the push-out strength and surface microhardness data, it appears that the time-related biomineralizing effect of MTA and Biodentine does not improve sealing to dentin. CHX significantly reduced ProRoot MTA bond strength and increased pure adhesive failures with both cements. (C) 2020 Published by Elsevier Inc. on behalf of The Academy of Dental Materials.

Historical antimicrobial use in NZ agriculture has driven the evolution of ST108, a VRE lineage carrying a range of clinically relevant antimicrobial resistances. The persistence of this lineage in NZ for over a decade indicates that coselection may be an important stabilizing mechanism for its persistence., Enterococcus faecalis and Enterococcus faecium are human and animal gut commensals. Vancomycin-resistant enterococci (VRE) are important opportunistic pathogens with limited treatment options. Historically, the glycopeptide antibiotics vancomycin and avoparcin selected for the emergence of vancomycin resistance in human and animal isolates, respectively, resulting in global cessation of avoparcin use between 1997 and 2000. To better understand human- and animal-associated VRE strains in the postavoparcin era, we sequenced the genomes of 231 VRE isolates from New Zealand (NZ; 75 human clinical, 156 poultry) cultured between 1998 and 2009. E. faecium lineages and their antibiotic resistance carriage patterns strictly delineated between agricultural and human reservoirs, with bacitracin resistance ubiquitous in poultry but absent in clinical E. faecium strains. In contrast, one E. faecalis lineage (ST108) predominated in both poultry and human isolates in the 3 years following avoparcin discontinuation. Both phylogenetic and antimicrobial susceptibility (i.e., ubiquitous bacitracin resistance in both poultry and clinical ST108 isolates) analyses suggest an agricultural origin for the ST108 lineage. VRE isolate resistomes were carried on multiple, heterogeneous plasmids. In some isolate genomes, bacitracin, erythromycin, and vancomycin resistance elements were colocalized, indicating multiple potentially linked selection mechanisms. IMPORTANCE Historical antimicrobial use in NZ agriculture has driven the evolution of ST108, a VRE lineage carrying a range of clinically relevant antimicrobial resistances. The persistence of this lineage in NZ for over a decade indicates that coselection may be an important stabilizing mechanism for its persistence.

Summary Vancomycin-resistant Enterococcus (VRE) are highly antibiotic-resistant and readily transmissible pathogens that cause severe infections in hospitalized patients. We discovered that lithocholic acid (LCA), a secondary bile acid prevalent in the cecum and colon of mice and humans, impairs separation of growing VRE diplococci, causing the formation of long chains and increased biofilm formation. Divalent cations reversed this LCA-induced switch to chaining and biofilm formation. Experimental evolution in the presence of LCA yielded mutations in the essential two-component kinase yycG/walK and three-component response regulator liaR that locked VRE in diplococcal mode, impaired biofilm formation, and increased susceptibility to the antibiotic daptomycin. These mutant VRE strains were deficient in host colonization because of their inability to compete with intestinal microbiota. This morphotype switch presents a potential non-bactericidal therapeutic target that may help clear VRE from the intestines of dominated patients, as occurs frequently during hematopoietic stem cell transplantation.

Vancomycin is a glycopeptide antibiotic that inhibits transpeptidation during cell wall synthesis by binding to the D-Ala-D-Ala termini of lipid II. For long, it has been used as a last resort antibiotic. However, since the emergence of the first vancomycin-resistant enterococci in 1987, vancomycin resistance has become widespread, especially in hospitals. We have synthesized and evaluated 110 vancomycin analogs modified at the C-terminal carboxyl group of the heptapeptide moiety with R2NHR1NH2 substituents. Through iterative optimizations of the substituents, we identified vancomycin analogs that fully restore (or even exceed) the original inhibitory activity against vancomycin-resistant enterococci (VRE), vancomycin-intermediate (VISA) and vancomycin-resistant Staphylococcus aureus (VRSA) strains. The best analogs have improved growth inhibitory activity and in vitro therapeutic indices against a broad set of VRE and methicillin-resistant S. aureus (MRSA) isolates. They also exceed the activity of vancomycin against Clostridium difficile ribotypes. Vanc-39 and Vanc-42 have a low probability to provoke antibiotic resistance, and overcome different vancomycin resistance mechanisms (VanA, VanB, and VanC1). ispartof: Frontiers in Microbiology vol:9 issue:JUN ispartof: location:Switzerland status: published

Genomic comparison of the first six Dutch vanD -type vancomycin-resistant Enterococcus faecium (VRE) isolates with four vanD gene clusters from other enterococcal species and anaerobic gut commensals revealed that the vanD gene cluster was located on a genomic island of variable size. Phylogenetic inferences revealed that the Dutch VRE isolates were genetically not closely related and that genetic variation of the vanD -containing genomic island was not species specific, suggesting that this island is transferred horizontally between enterococci and anaerobic gut commensals.

In this study, the presence of genes responsible for the pathogenicity and antibiotic resistance profile of enterococci isolated from various foodstuffs of animal origin was investigated. The percentage prevalence of enterococci was 54.1% (203/375) and the average count was found to be 3.81 log cfu/ml-g. Species-specific primers revealed Enterococcus faecalis as the predominant species carrying one or more virulence-associated traits of efa, gelE, ace, esp and agg genetic markers. Only one E. faecium isolate (from milk) was positive for the esp gene. Regarding antibiotic resistance, the highest frequency of resistance was observed for tetracycline (21.7%), followed by quinupristin/dalfopristin (13.3%), ciprofloxacin (2.0%), penicillin (2.0%), linezolid (1.0%), ampicillin (1.0%), streptomycin (1.0%), and gentamicin (0.5%). Enterococcus faecalis showed a higher prevalence of antibiotic resistance than other enterococci. The percentage of multidrug resistance among the isolates was 3.4%. Twenty-nine E. faecalis isolates (26.6%) carrying one of the virulence-associated traits were at the same time resistant to at least one antibiotic. Our results show that foods of animal origin, including ready-to-eat products, may be reservoirs of antibiotic-resistant and potentially virulent enterococci.

The need for novel antibiotics in an era where antimicrobial resistance is on the rise, and the number of new approved antimicrobial drugs reaching the market is declining, is evident. The underused potential of post-translationally modified peptides for clinical use makes this class of peptides interesting candidates. In this study, we made use of the vast amounts of available genomic data and screened all publicly available prokaryotic genomes (~3000) to identify 394 novel head-to-tail cyclized antimicrobial peptides. To verify these in silico results, we isolated and characterized a novel antimicrobial peptide from Bacillus pumilus that we named pumilarin. Pumilarin was demonstrated to have a circular structure and showed antimicrobial activity against several indicator strains, including pathogens., MML was supported by ALW-NWO project SynMod (project number 855.01.162) and the FP7 project Synpeptide. AJvH was supported by NWO-STW programme GenBiotics (project number 10466).

The aim of this study was to determine the association between Clostridium difficile (C. difficile) and vancomycin-resistant Enterococcus (VRE) and efficacy of screening stools submitted for C. difficile toxin assay for prevalence of VRE. Between April 2012 and February 2014, 158 stool samples submitted for C. difficile toxin to the Marmara University Microbiology Laboratory, were included in the study. Stool samples were analyzed by enzyme immuno assay test; VIDAS (bioMerieux, France) for Toxin A&B. Samples were inoculated on chromID VRE (bioMerieux, France) and incubated 24 h at 37 degrees C. Manuel tests and API20 STREP (bioMerieux, France) test were used to identify the Enterococci species. After the species identification, vancomycin and teicoplanin MIC's were performed by E test and molecular resistance genes for vanA vs vanB were detected by polymerase chain reaction (PCR). Of the 158 stool samples, 88 were toxin positive. The prevalence of VRE was 17%(n:19) in toxin positives however, 11.4% in toxin negatives(n:70). All VRE isolates were identified as Enterococcus faecium. These results were evaluated according to Fischer's exact chi-square test and p value between VRE colonization and C. difficile toxin positivity was detected 0.047 (p < 0.05). PPV and NPV were 79% and 47% respectively. In our study, the presence of VRE in C. difficile toxin positives is statistically significant compared with toxin negatives (p < 0.05). Screening for VRE is both additional cost and work load for the laboratories. Therefore VRE screening among C. difficile toxin positive samples, will be cost effective for determination of high risk patients in the hospitals especially for developing countries (C) 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda.

WOS: 000382446000075, Enterococci, which have useful biotechnological applications, produce bacteriocins, including those that exert anti-Listerial activity. The present study aimed to determine the antibiotic susceptibility patterns and antimicrobial activity of Enterococcus faecium strains isolated from human breast milk. The strains were identified using carbohydrate fermentation tests and ribotyping. Subsequently, the antibacterial activity of the isolates was investigated, and the quantities of lactic acid and hydrogen peroxide produced, and the proteolytic activity of E. faecium, were determined. In addition, biofilm formation by E. faecium strains was assessed. E. faecium strains exhibited antimicrobial activity against food-borne and clinical bacterial isolates. Furthermore, following 24 h incubation, the tested strains exhibited resistance to a pH range of 2.0-9.5 and tolerance of bile acid, lysozyme activity and phenol. Supernatants of the E. faecium TM13, TM15, TM17 and TM18 strains were shown to be effective against Listeria monocytogenes, and were also resistant to heat. Further studies are required in order to determine whether certain strains of E. faecium may be used for the development of novel antibacterial agents., Anadolu University Council of Research Project Fund [41020], The present study was supported by the Anadolu University Council of Research Project Fund (grant no. 41020).

Enterococcus faecium belonging to the polyclonal subcluster CC17, with a typical ampicillin-resistant E. faecium (AREfm) phenotype, have become prevalent among nosocomial infections around the world. High-density intestinal AREfm colonization could be one of the factors contributing to the successful spread of these pathogens. We aimed to quantify the enterococcal intestinal colonization densities in stool samples from AREfm-colonized and non-colonized patients using fluorescent in situ hybridization (FISH). Stool samples were collected from AREfm-colonized (n = 8) and non-colonized (n = 8) patients. The relative number of Enterococcus faecalis and E. faecium was determined by FISH using specific 16S rRNA probes, while the total amount of bacterial cells was counted by staining the sample with 4',6-diamidino-2-phenylindole (DAPI). The median bacterial cell numbers in fecal samples, counted by DAPI staining, were 7.7 x 10(9) and 4.8 x 10(9) cells/g for AREfm-colonized and non-colonized patients, respectively (p = 0.34). The E. faecium densities in AREfm-colonized patients, accounting for 0.5-7% of all fecal bacterial cells, exceeded E. faecalis levels by over ten-fold. E. faecium was not detected in non-colonized patients. This study demonstrated high E. faecium cell densities in stool samples from patients colonized with AREfm. Increased cell densities may contribute to host-to-host transmission and environmental contamination, facilitating the spread of AREfm in the hospital setting.

A collection of 147 isolates obtained from 23 samples of traditional Azerbaijani dairy products was screened for the presence of proteolytic enzymes. Six Enterococcus faecalis strains obtained from three cheese samples have been identified as proteinase-producing strains, according to their ability to hydrolyze caseins. RAPD-PCR profiles of their total DNA showed different patterns for strains isolated from different cheese samples. The proteolytic activities of these strains were studied during their growth in milk and in non-proliferative cells system. Isolated strains were able to hydrolyze alpha(S1)-, alpha(S2)-, beta-caseins and BLG albeit to different extents, at optimal pH in the range 6.0-7.2 and optimal temperature in the range 37-45 A degrees C, depending on the strain. Proteolysis was strongly inhibited in the presence of EDTA-specific inhibitor of metalloproteases-but the presence of other types of proteases cannot be excluded. The potential pathogenicity of the strains was evaluated investigating the presence of the genes coding different virulence factors and their resistance to antibiotics. The obtained results yield new information about technological characteristics and safety of studied Enterococci strains from Azerbaijani artisanal dairy products. Many from the isolated strains contribute certainly to the differences in flavor, texture, and taste of Azerbaijani traditional cheeses and could represent new adjunct cultures for the dairy industry.

BACKGROUND: Enterococcus hirae-associated endocarditis, characterized by a peak in mortality during the second week of the grow-out, and occasionally lameness, was diagnosed at Dutch broiler farms. OBJECTIVES: Field cases were studied to increase knowledge on clinical and pathological characteristics, pathogenesis and epidemiology of these infections. ANIMALS AND METHODS: In total, 1266 birds of 25 flocks from 12 farms were examined. Post-mortem examinations, bacteriology, histopathology, PCR and DNA fingerprinting was carried out. Six flocks were followed longitudinally (n = 1017 birds). RESULTS: Average mortality was 4.1% for the entire grow-out, of which 36% was attributed to endocarditis. Fibrinous thromboendocarditis of the right atrioventricular (AV) valve was found in 24% of hearts, compared to 7% and 4% with lesions of left and both AV valves, respectively. Thrombotic lesions were found in 24% (n = 432) of lungs, but only in larger branches of the Arteria pulmonalis. Occasionally, thrombi were found in the Arteria ischiadica externa and in liver and brain vessels. Enterococcus was cultured from 54% (n = 176) of heart and in 75% (n = 28), 62% (n = 106) and 31% (n = 16) of liver, bone marrow and lung samples, respectively. Further identification, using the Rapid ID Strep 32 API system and a PCR targeting mur-2 and mur-2(ed) genes was carried out on a subset of Enterococcus positive isolates (n = 65): both techniques identified the isolates as Enterococcus hirae. Pulsed-field gel electrophoresis did not indicate evidence of clonality between farms and flocks. CONCLUSIONS: The relevance of these findings for pathogenesis and epidemiology of E. hirae infections is discussed. CLINICAL IMPORTANCE. This study may facilitate diagnosis of field cases and may contribute to the design of further research and development of control measures.

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.

29 p.- 4 tab.-2 fig., Fluorescent reporter genes are valuable tools for real-time monitoring of gene expression in living cells. In this study we describe the construction of novel promoter-probe vectors containing a synthetic mCherry fluorescent protein gene, codon-optimized for lactic acid bacteria, divergently linked, or not, to a gene encoding the S65T and F64L variant of the green fluorescent protein. The utility of the transcriptional fusion vectors was demonstrated by the cloning of a single or two divergent promoter regions and by the quantitative evaluation of fluorescence during growth of Lactococcus lactis, Enterococcus faecalis, and Escherichia coli., This work was supported by the Spanish Ministry of Science and Innovation (grants AGL2009-12998-C03-01, AGL2009- 13361-C02-02, and CSD2007-00063 Consolider Ingenio 2010 FUN-CFOOD), the Comunidad de Madrid (grant ALIBIRD P2009/AGR-1469), and the European Union VII framework program (Initial Training Network, grant no. 238490). We thank Dr. Stephen Elson for the critical reading of the manuscript. We are grateful to M. Angeles Corrales for her technical assistance.