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Enterococcal bloodstream infections (E-BSI) constitute the second cause of Gram-positive bacterial BSI in Europe with a high rate of in-hospital mortality. Furthermore, E-BSI treatment is still challenging because of intrinsic and acquired antibiotic resistances. We conducted a retrospective, 2-year, observational, single-centre study to evaluate clinical outcome and risk factors for E-BSI mortality in internal medicine wards. 201patients with E-BSI were included in the analysis. Infection rate was 2.4/1000 days of hospital admission. Most E-BSI were hospital acquired (78.1%). The median age was 68 years. Charlson Comorbidity Index, adjusted for age, was 5 (range 4–6). Patients with E-BSI frequently had at least one invasive device, predominantly a central venous (73%) or a bladder catheter (61.7%). Enterococcus faecium accounted for 47.94% of E-BSI (resistance rate to ampicillin or vancomycin was 22.2 and 23.3%, respectively) and Enterococcus faecalis for 52.08% (resistance rate to ampicillin or vancomycin was 3.1 and 2.2%, respectively). Among all E-BSI, 25% of patients received appropriate therapy. In total, 59% of E-BSI underwent echocardiography. At the multivariate analysis, resistance to vancomycin (OR 2.09, p = 0.025), sepsis (OR 2.57, p = 0.003) and septic shock (OR 3.82, p = 0.004) was a predictor of mortality. No difference in 28-day survival was observed between appropriate or inappropriate treatment, except for endocarditis. However, E-BSI sources in clinical practices are not always properly investigated, including the rule-out of intracardiac vegetations. We did not demonstrate a difference in mortality for inappropriate therapy in the absence of endocarditis in comorbid patients with a long history of medicalization.

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.

We present the case of a 73-year-old immunosuppressed male with a history of multiple benign, colonic adenomas who was admitted to our hospital with Enterococcus faecalis (E. faecalis) bacteremia. The patient also had a prior history of dual-chamber pacemaker placement for sick sinus syndrome. Two days before the admission, the patient had undergone radiofrequency ablation of the atrioventricular (AV) node for refractory atrial flutter without receiving any peri-procedural antibiotic prophylaxis. Despite high-grade bacteremia and a high NOVA (Number of positive blood cultures, Origin of the bacteremia, previous Valve disease, Auscultation of heart murmur) score, there was no evidence of infective endocarditis on transesophageal echocardiogram (TEE). The patient was treated successfully with appropriate intravenous antibiotics, and he recovered well. To the best of our knowledge, this is the first reported case of post-AV node ablation E. faecalis bacteremia. We conclude that the presence of colonic lesions and immunosuppression can increase the risk of peri-procedural E. faecalis bacteremia, and clinicians should consider antibiotic prophylaxis in this high-risk patient group.

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.

Enterococcus 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., 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. faecium.

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.

OBJECTIVES: In enterococci, resistance to linezolid is often mediated by mutations in the V domain of the 23S rRNA gene (G2576T or G2505A). Furthermore, four genes [optrA, cfr, cfr(B) and poxtA] encode linezolid resistance in enterococci. We aimed to develop a Web tool for detection of the two mutations and the four genes encoding linezolid resistance in enterococci from whole-genome sequence data.METHODS: LRE-Finder (where LRE stands for linezolid-resistant enterococci) detected the fraction of Ts in position 2576 and the fraction of As in position 2505 of the 23S rRNA and the cfr, cfr(B), optrA and poxtA genes by aligning raw sequencing reads (fastq format) with k-mer alignment. For evaluation, fastq files from 21 LRE isolates were submitted to LRE-Finder. As negative controls, fastq files from 1473 non-LRE isolates were submitted to LRE-Finder. The MICs of linezolid were determined for the 21 LRE isolates. As LRE-negative controls, 26 VRE isolates were additionally selected for linezolid MIC determination.RESULTS: LRE-Finder was validated and showed 100% concordance with phenotypic susceptibility testing. A cut-off of 10% mutations in position 2576 and/or position 2505 was set in LRE-Finder for predicting a linezolid resistance phenotype. This cut-off allows for detection of a single mutated 23S allele in both Enterococcus faecalis and Enterococcus faecium, while ignoring low-level sequencing noise.CONCLUSIONS: A Web tool for detection of the 23S rRNA mutations (G2576T and G2505A) and the optrA, cfr, cfr(B) and poxtA genes from whole-genome sequences from enterococci is now available online.

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).

Enterococci can produce enterocins which have antimicrobial activity against Gram-positive and also Gram-negative pathogenic, toxigenic and food-spoilage bacteria. The aim of this study was to determine the antimicrobial activity of Enterococcus spp. isolated from traditional Turkish cheeses such as Kashar, Manyas, Sepet, Kelle, Mihalic, Tulum. The isolates were tested against Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Staphylococcus aureus and Enterococcus faecalis and also detected the presence of entA and entB genes of these isolates. Total 66 of enterococcal isolates were obtained from 34 of cheese samples and 25 of these isolates showed antimicrobial activity against tested reference bacteria by using agar spotting method. Also it was determined most of Enterococcus spp. carried enterocin encoding entA and entB genes. We concluded that these isolates or their enterocins may have a potential for food preservation, however they should be evaluated in terms of food safety. Enterokoklar, patojenik, toksijenik ve gıdalarda bozulma yapan Gram-pozitif ve hatta Gram-negatif bakterilere karşı antimikrobiyal etkiye sahip enterosinler üretebilmektedir. Bu çalışmanın amacı, Kaşar, Manyas, Sepet, Kelle, Mihaliç, Tulum gibi geleneksel Türk peynirlerinden elde edilen Enterococcus spp. izolatlarının antimikrobiyal aktivitelerinin belirlenmesidir. İzolatların, Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Staphylococcus aureus ve Enterococcus faecalis’e karşı aktiviteleri test edilmiş ve ayrıca izolatlarda entA ve entB gen varlığı araştırılmıştır. Çalışmada 34 peynir örneğinden toplam 66 adet enterokok izole edilmiş ve yapılan testte bunların 25 tanesinin test edilen bakterilere karşı antimikrobiyal aktivite gösterdiği belirlenmiştir. Ayrıca bu izolatların çoğunun enterosin kodlayan entA ve entB genlerini taşıdıkları görülmüştür. Bu izolatların ya da bu izolatlardan elde edilecek enterosinlerin gıda koruyucusu olarak kullanım potansiyeli olduğu ancak izolatların gıda güvenliği yönüyle değerlendirilmesinin gerektiği düşünülmektedir.

As alterações locomotoras representam um desafio na produção avícola moderna em todo o mundo e podem ter origem não infecciosa e infecciosa. A osteomielite vertebral é uma doença bacteriana descrita em surtos em diversos países caracterizada por infecção da vértebra torácica móvel (T4) que resulta em compressão da medula espinhal, dificuldade de locomoção e morte das aves acometidas. O objetivo deste trabalho foi determinar a frequência da doença em frangos de corte do estado de Minas Gerais, além de conhecer e caracterizar molecularmente os agentes etiológicos envolvidos na doença. Para isso, foram analisados 608 frangos de corte com problemas locomotores que tiveram os sinais clínicos registrados e foram submetidos à necropsia. Amostras de corpo vertebral e articulações com alterações macroscópicas foram coletadas para isolamento bacteriano, histopatologia e análise molecular. Osteomielite vertebral foi encontrada em 5,1% (31/608) das aves, as quais apresentaram diferentes graus de dificuldade locomotora relacionados ao nível de compressão da medula espinal. As bactérias mais frequentemente isoladas das lesões foram: Enterococcus spp. (53,6%), E. faecalis (32,1%) e E. hirae (7,1%); Escherichia coli (42,8%) em co-infecção E. faecalis em dois casos; Staphylococcus aureus (14,3%) em dois casos em co-infecção com Enterococcus spp. ou E. hirae. Os isolados de E. coli apresentaram diferentes padrões genéticos por PFGE, independentemente do lote estudado e tipo de lesão (osteomielite vertebral ou artrite). Os isolados pertenciam a sete sequence types (STs) descritos anteriormente (ST117, ST101, ST131, ST371 e ST3107) ou descritos pela primeira vez neste estudo (ST5766 e ST5856). A maioria dos isolados pertenciam ao grupo filogenético D (66,7%) e diversos sorogrupos (O88, O25, O12 e O45), alguns de importância mundial. O perfil de susceptibilidade antimicrobiana também refletiu a diversidade dos isolados e revelou alta frequência de cepas multirresistentes (73%), principalmente às quinolonas e beta-lactâmicos. A análise do Multilocus sequence typing (MLST) revelou que os isolados de E. faecalis pertenciam a oito STs distintos. Desses, seis (ST49, ST100, ST116, ST202, ST249 e ST300) foram previamente descritos, enquanto ST708 e ST709 foram descritos pela primeira vez nesse estudo. E. faecalis ST49 foi o mais frequentemente isolado das lesões vertebrais. Os isolados da bactéria apresentaram maior percentual de resistência antimicrobiana aos aminoglicosídeos, principalmente à gentamicina (40.0%), e baixa resistência à vancomicina (10%). Os resultados desse estudo demonstram que, no Brasil, osteomielite vertebral em frangos de corte pode não ser causada por um único agente infeccioso e sugere diferenças geográficas relativas à frequência e etiologia da doença entre esta região do Brasil e outros países. Além disso, nossos resultados demonstraram a diversidade de STs de E. faecalis envolvidos na doença com alta frequência de isolados resistentes a aminoglicosídeos e baixa frequência de E. faecalis resistentes à vancomicina. Nossos resultados demonstram, ainda, que osteomielite vertebral e artrite podem estar associadas à E. coli altamente diversas, as quais são frequentementeresistentes a múltiplas drogas antimicrobianas. Alguns isolados de E. coli pertencem a STs descritos também em seres humanos, o que representa uma preocupação para a saúde pública e animal. Locomotor disorders represent a major challenge in modern poultry production worldwide and they may be related to non-infectious and infectious etiologies. Vertebral osteomyelitis is a bacterial disease described in outbreaks in many countries, characterized by infection of the mobile thoracic vertebra (T4), which results in the compression of the spine, reduced mobility and death of affected broilers. The objective of this study was to determine the frequency of vertebral osteomyelitis in broilers in the state of Minas Gerais, and to determine the bacterial etiologies involved in disease and their molecular characteristics. For this, we analyzed 608 broilers with locomotor disorders, which had their clinical signs recorded and then necropsied. Vertebral column samples and joints with gross changes were collected for bacterial isolation, molecular and histopathological analysis. Vertebral osteomyelitis was found in 5.1% (31/608) of the birds, which had different degrees of limited mobility, related to the level of spinal cord compression. The bacteria most frequently isolated from lesions were: Enterococcus spp.(53.6%), E. faecalis (32.1%) and E. hirae (7.1%); Escherichia coli (42.8%) in co-infection with E. faecalis in two cases; Staphylococcus aureus (14.3%) in co-infection with Enterococcus spp. or E. hirae in two cases. E. coli strains harbored different genetic pattern as assessed by PFGE, regardless of flock origin and lesion site (vertebral osteomyelitis or arthritis). The E. coli strains belonged to seven sequence types (STs) described previously (ST117, ST101, ST131, ST371 and ST3107) or newly described in this study (ST5766 and ST5856). Most strains belonged to ECOR phylogenetic group D (66.7%) and diverse serogroups (O88, O25, O12 and O45), some of worldwide importance. The antimicrobial susceptibility profile also showed the diversity of the strains and revealed a high proportion of multidrug-resistant strains (73%), mainly to quinolones and beta-lactams. Multilocus sequence typing (MLST) analysis of E. faecalis revealed that the strains belonged to eight different STs, being six (ST49, ST100, ST116, ST202, ST249, and ST300) previously described and ST708 and ST709 first identified in this study. ST49 was the most frequently isolated from vertebral osteomyelitis lesions. E. faecalis strains showed the highest resistance to aminoglycoside antibiotics, mainly to gentamicin (40.0%), and low resistance to vancomycin (10%). The results indicated that, in Brazil, vertebral osteomyelitis in broilers may not be caused by a single infectious agent and suggested geographical differences concerning the frequency and etiology of the disease, as comparing our region in Brazil with reports in other countries. Furthermore, our results showed the diversity of E. faecalis STs involved with this disease and high frequency of aminoglycoside resistance and low frequency of vancomycin-resistance. Also, vertebral osteomyelitis and arthritis could be associated with highly diverse E. coli, which were often multidrug-resistant. Some E. coli strains belonged to STs described also in humans, which may represent a concern to public and animal health.

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.

Enterococci are ubiquitous microorganisms, found as part of the normal intestinal microbiota of many animals. They can be present in food products, for example, the Portuguese dry fermented sausage chourico. Twenty enterococci were isolated from chourico in two processing units; after identification and typification by conventional-molecular methods, the isolates were screened for virulence factors and antibiotic resistance. Identification allocated all enterococci to the species Enterococcus faecalis, and PCR fingerprinting demonstrated that each isolate was specific to the processing unit and chourico from which it was recovered. Regarding the screening for virulence factors, I strain produced cytolysin and 4 were gelatinase positive, but none produced lipase. The ace gene was detected in I enterococci, ebpABC and efaA(fs). in 16 isolates each, esp in 3, fsrB in 5, gelE in 7, and cylA in I. A multiresistant phenotype was observed in 8 isolates, 6 belonging to factory A. The antibiotic resistance gene ere(B) was detected in 9 enterococci, whereas the genes tet(M), aac(6')-le-aph(2 ''), and vanA were detected in 8 isolates each. As some of the E. faecalis chourico isolates present a multiresistant profile and harbor virulence and/or resistance genes, to assess further the safety of Portuguese dry sausages, a larger number of products and processing units must by analyzed.

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 an opportunistic pathogen responsible for numerous outbreaks worldwide. The basis for the colonization capacities, host persistence and environmental stress response of the hospital-adapted clones emerging from E. faecium are poorly understood. In this study, we propose the use of Galleriamellonella as a simple nonmammalian model to assess E. faecium host persistence. Various strains (n = 10), including hospital-adapted, commensal or animal isolates and a SodA-deficient strain were used to assess the relevance of this model. Compared to Enterococcus faecalis, E. faecium strains do not appear very lethal in a Galleria killing assay. The ability of E. faecium strains to overcome host-immune responses and multiply within the host system was evaluated by monitoring bacterial loads following Galleria infection. Among the E. faecium strains, two hospital-adapted isolates displayed increased colonization ability. In contrast, inactivation of sodA, encoding a putative manganese-dependent superoxide dismutase, significantly reduced survival of E. faecium to Galleria defenses. Galleria appears to be a suitable and convenient surrogate model to study E. faecium survival to host defenses and the role of suspected virulence factors in the colonization process.

AIM: To study the influence of 15 microbial isolates on the prevalence of charge-heterogeneous and charge-homogeneous Enterococcus faecalis strains, all isolated from biliary stents, in mixed-species biofilms.METHODS AND RESULTS: Six Enterococcus faecalis strains were paired with 15 other microbial isolates to form mixed-species biofilms in a microtitre plate assay. Charge-heterogeneous Enterococcus faecalis strains display two subpopulations with different surface charges, expressed as a biomodal zeta potential distribution. It was found that, in general, the prevalence of the charge-heterogeneous, biofilm forming Enterococcus faecalis was reduced in mixed-species biofilms. The prevalence of charge-homogeneous, nonbiofilm-forming Enterococcus faecalis strains was increased only in the presence of Citrobacter freundii BS5126, Stenotrophomonas malthophilia BS937, and Candida lusitaniae BS8256, all of which introduced sizeable charge heterogeneity in the mixed microbial population.CONCLUSIONS: Charge-homogeneous Enterococcus faecalis strains are stimulated to form biofilm only by the presence of another microbial species with a considerably less negative zeta potential, thereby creating a charge-heterogeneous microbial population.SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococcus faecalis is one of the predominant species isolated from mixed-species biofilms in clogged biliary stents. The current study shows how charge-homogeneous Enterococcus faecalis strains form more biofilm in the presence of other microbial species.

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.

The aim of the study was to determine the biofilm production, genotypes, antibiotics resistance patterns and antibiotypes of 82 Enterococcus faecium strains isolated from dog, cat and human. Of examined strains biofilm production detected totally 72 (87.8%) in 35 (97.2%) dog, 22 (78.6%) cat and 15 (83.3%) human isolates. Genotyping of isolates was performed by RAPD-PCR and 16, 3 and 4 different profiles were detected in dog, cat and human isolates, respectively. In total of 98.8% with a maximum resistance to nalidixic acid and 4.9% with the lowest resistance to vancomycin was found. None of vancomycin resistance 4 isolates, vancomycin resistance genes (vanA, vanB, vanC1/C2 or vanD) has been detected. Antibiotyping of isolates was performed with UPGMA and 5 groups of dog, 10 groups of cat and 7 groups of human isolates were determined. The results from this study indicate that healthy dogs and cats are a source of Antibiotic resistant enterococci and may act as a reservoir of resistance that can be transferred from pets to people. Also our results demonstrated that the phenotype and genotype patterns found among enterococci strains from dogs, cats and humans were heterogeneous.

Los resultados del presente trabajo permiten afirmar que la especie Rodophyta y Heterocontophyta inhiben el crecimiento del Enterococcus faecalis ATCC: 7080 con halos inhibitorios de 46.56mm y66.47mm respectivamente, en la prueba de pocillos

Conventional root canal therapy consists of chemo-mechanical treatment of the root canal to eliminate the microbial infection. The root canal is obturated with a root filling, which normally consists of a core and an endodontic sealer. The purpose of the sealer is to obturate the root canal, and should in addition have an antibacterial activity against bacteria that are left in the root canal after instrumentation and cleaning. The aim of this study was to investigate the antibacterial activity of three endodontic sealers with different chemical composition, AH Plus, RealSeal SE and TotalFill against Enterococcus faecalis in vitro. Methods: Agar diffusion test (ADT), Direct contact test (DCT) and Modified direct contact test (MDCT) were used to evaluate the antibacterial activity of the three endodontic sealers. Results: For the ADT a small clear zone of inhibition could be identified around AH Plus, which were absent for Real Seal SE and TotalFill. For the DCT, no bacterial growth was evident for all three sealers AH Plus, RealSeal SE and TotalFill compared to the control inoculum. This was in accordance with the MDCT, which showed that all three sealers eliminated all bacterial after 1 hour contact time with the sealers. Conclusions: Freshly prepared samples of AH Plus, Real Seal SE and TotalFill showed bactericidal activity against E. faecalis.

Enterococcus faecalis is an opportunistic pathogen responsible for a wide range of life-threatening nosocomial infections, such as septicemia, peritonitis, and endocarditis. E. faecalis infections are associated with a high mortality and substantial health care costs and cause therapeutic problems due to the intrinsic resistance of this bacterium to antibiotics. Several factors contributing to E. faecalis virulence have been identified. Due to the variety of infections caused by this organism, numerous animal models have been used to mimic E. faecalis infections, but none of them is considered ideal for monitoring pathogenesis. Here, we studied for the first time E. faecalis pathogenesis in zebrafish larvae. Using model strains, chosen isogenic mutants, and fluorescent derivatives expressing green fluorescent protein (GFP), we analyzed both lethality and bacterial dissemination in infected larvae. Genetically engineered immunocompromised zebrafish allowed the identification of two critical steps for successful establishment of disease: (i) host phagocytosis evasion mediated by the Epa rhamnopolysaccharide and (ii) tissue damage mediated by the quorum-sensing Fsr regulon. Our results reveal that the zebrafish is a novel, powerful model for studying E. faecalis pathogenesis, enabling us to dissect the mechanism of enterococcal virulence.

Synthetic methods to glycerol teichoic acids

1. La evidencia acumulada en los últimos 15 o 20 años pone de manifiesto la efectividad de las medidas de prevención y control aplicadas en la asistencia sanitaria en la reducción de transmisión de las enfermedades transmisibles, los avances en las formas de transmisión de estas enfermedades, así como el estudio de los riesgos ocupacionales han constituido el elemento fundamental en los programas de control de las enfermedades infecciosas en la consulta odontológica. (1) 2. Algunas especies de bacterias productoras de pigmento negro, los peptoestreptococos, los peptococos y las especies fusobacterium, eubacterium y actinomices han sido consideradas las causantes de ciertos síntomas y signos clínicos. Sin embargo, no se ha establecido ninguna relación absoluta entre cualquier especie de bacterias y la gravedad de las infecciones endodonticas. Es probable que tal hecho guarde relación con la naturaleza poli microbiana de las infecciones endodonticas, y con la relación sinérgica entre bacterias o factores de virulencia, que aumentan el efecto patogénico global. (2) 3. Los microbios poseen numerosos factores de virulencia, entre los que se incluyen cápsulas bacterianas, fimbrias (Pili), lipopolisacaridos (LPS), enzimas, vesículas extracelulares, ácidos grasos, poliaminas, amoniaco y sulfuros de hidrógeno. Las bacterias tanto Gram positivas como Gram negativas tienen capsulas que pueden protegerlas de la fagocitosis.

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.

The strictly aerobic, polyphosphate-accumulating Acinetobacter johnsonii strain 210A degrades its polyphosphate when oxidative phosphorylation is impaired. The endproducts of this degradation, divalent metal ions and inorganic phosphate, are excreted as a neutral metal-phosphate (MeHPO(4)) chelate via the electrogenic MeHPO(4)/H+ symport system of the organism. The coupled excretion of MeHPO(4) and H+ in A. johnsonii 210A can generate a proton motive force. In membrane vesicles and deenergized cells, a membrane potential of about -70 mV and transmembrane pH gradient of about -8 mV were formed in response to an imposed outwardly directed MeHPO, concentration gradient of 120 mV (initial value). The MeHPO(4) efflux-induced proton motive force could drive energy-requiring processes, such as the accumulation of L-proline and L-lysine and the synthesis of ATP via the membrane-bound F0F1 H+-ATPase. In vivo P-31 NMR studies of polyphosphate degradation in anaerobic cell suspensions revealed the presence of a considerable outwardly directed phosphate gradient across the cytoplasmic membrane corresponding to a MgHPO4 concentration gradient of at least 100 mV. This MgHPO4 concentration gradient was maintained for several hours. Thus, energy recycling by MeHPO(4)/H+ efflux will contribute significantly to the overall production of metabolic energy from the degradation of polyphosphate in A. johnsonii 210A.

Two types of Enterococcus species were isolated from femoral head necrosis lesions of chickens. A total of 150 femoral articular tissues, of which 121 were from commercial broilers, 18 from broiler breeders, and 11 from layer breeders, were sampled. Out of 48 Enterococcus isolates of these clinical samples, 37 and 11 isolates were identified as Enterococcus faecalis and Enterococcus faecium, respectively. Additionally, 42 (28%) Escherichia coli and 33 (22%) Staphylococcus aureus isolates were obtained either alone or together with 1 of the 2 Enterococcus species from the same clinical samples, while no bacteria were detected from the remaining 56 samples. None of the Enterococcus isolates showed gelatinase activity, while only 1 Enterococcus faecalis isolate was found to be positive for both aggregation substance and cytolysin production. Resistance rates to tetracycline, erythromycin, ciprofloxacin, chloramphenicol, and ampicillin in Enterococcus faecalis were 94.6%, 43.2%, 37.8%, 17.8%, and 2.8%, respectively, while they were 81.8%, 45.5%, 18.2%, 9.8%, and 9.8% for the Enterococcus faecium isolates. All of the Enterococcus isolates were susceptible to both vancomycin and gentamicin. Resistance rate to 3 or more antibiotics was 18.9% and 18.1% for Enterococcus faecalis and Enterococcus faecium isolates, respectively. Bu çalışmada 121 ticari broyler, 18 broyler damızlık, 11 yumurtacı damızlıktan elde edilen toplam 150 femur başı nekrozu incelendi ve iki farklı Enterococcus türü izole edildi. Elde edilen 48 Enterococcus izolatından 37’si Enterococcus faecalis, 11’i Enterococcus faecium olarak identifiye edildi. Ayrıca aynı örneklerden iki Enterococcus türünden biri ile birlikte ya da ayrı olarak 42 (% 28) Escherichia coli ve 33 (% 22) Staphylococcus aureus izole edilirken, 56 örnekten hiçbir bakteri saptanmadı. Enterococcus izolatlarından hiçbiri jelatinaz aktivitesi göstermezken, bir Enterococcus faecalis izolatı agregasyon maddesi ve sitolizin üretimi yönünden pozitif bulundu. Enterococcus faecalis ve Enterococcus faecium’un tetrasiklin, eritromisin, siprofloksasin, kloramfenikol ve ampisilin direnç yüzdeleri sırasıyla % 94,6, % 43,2, % 37,8, % 17,8, % 2,8, ve % 81,8, % 45,5, % 18,2, % 9,8, % 9,8 olarak bulundu. Sonuç olarak; tüm Enterococcus izolatlarının vankomisin ve gentamisine duyarlı olduğu, Enterococcus faecalis ve Enterococcus faecium izolatlarında 3 ya da daha fazla antibiyotiğe çoklu dirençlilik oranının sırasıyla % 18,9 ve % 18,1 olduğu belirlendi.

Enterococcus faecalis, a gram-positive member of the mammalian gastrointestinal flora, emerged as an important contributor to nosocomial infections and antibiotic resistance gene transfer. Lipoteichoic acid (LTA), a vital component of gram-positive cell walls, has been reported to function in numerous cellular processes, ranging from maintenance of cation homeostasis and virulence to modulating function and presentation of wall proteins such as adhesins and autolysins. Interestingly, LTA can be covalently modified by the addition of D-alanyl ester residues, which appear to help regulate its function by altering surface charge. In E. faecalis the process of esterification is catalyzed by four proteins encoded by the dlt operon. Mutants lacking a functional dlt operon display the inability to incorporate D-alanyl residues on LTA and are thus deficient in their ability to regulate the anionic charge of the outer envelope in response to extracellular cues. Recent evidence suggests that two-component systems are responsible for sensing environmental conditions and regulating dlt operon expression. Utilizing a reporter construct with the upstream promoter region of dlt fused to lacZ, we were able to determine how extracellular stimuli affect transcription of this operon by measuring [Beta]-galactosidase activity. Furthermore, we were able to identify specific response regulators important for bile salt, magnesium and polymyxin B signaling.