Isolation, identification, and characterization of thermophilic lactic acid bacteria isolated from whey of Kars Kashar cheeses.

Kars Kashar cheese is an artisanal pasta-filata type cheese and geographically marked in Eastern Anatolia of Turkey. The aims of this research were to determine for the first time thermophilic lactic acid bacteria (LAB) of Kars Kashar cheese and characterize the technological properties of obtained isolates. In our research, a number of 15 samples of whey were collected from the different villages in Kars. These samples were incubated at 45 °C and used as the source material for isolating thermophilic LAB. A total of 250 colonies were isolated from thermophilic whey, and 217 of them were determined to be presumptive LAB based on their Gram staining and catalase test. A total of 170 isolates were characterized by their phenotypic properties and identified using the MALDI-TOF mass spectrometry method. Phenotypic identification of isolates displayed that Enterococcus and Lactobacillus were the predominant microbiota. According to MALDI-TOF MS identification, 89 isolates were identified as Enterococcus (52.35%), 57 isolates as Lactobacillus (33.53%), 23 isolates as Streptococcus (13.53%), and one isolate as Lactococcus (0.59%). All thermophilic LAB isolates were successfully identified to the species level and it has been observed that MALDI-TOF MS can be successfully used for the identification of selected LAB. The acidification and proteolytic activities of the isolated thermophilic LAB were examined, and the isolates designated for use as starter cultures were also genotypically defined.

Enterococcus-derived tyramine hijacks α2A-adrenergic receptor in intestinal stem cells to exacerbate colitis.

Inflammatory bowel disease (IBD) is characterized by dysbiosis of the gut microbiota and dysfunction of intestinal stem cells (ISCs). However, the direct interactions between IBD microbial factors and ISCs are undescribed. Here, we identify α2A-adrenergic receptor (ADRA2A) as a highly expressed GPCR in ISCs. Through PRESTO-Tango screening, we demonstrate that tyramine, primarily produced by Enterococcus via tyrosine decarboxylase (tyrDC), serves as a microbial ligand for ADRA2A. Using an engineered tyrDC-deficient Enterococcus faecalis strain and intestinal epithelial cell-specific Adra2a knockout mice, we show that Enterococcus-derived tyramine suppresses ISC proliferation, thereby impairing epithelial regeneration and exacerbating DSS-induced colitis through ADRA2A. Importantly, blocking the axis with an ADRA2A antagonist, yohimbine, disrupts tyramine-mediated suppression on ISCs and alleviates colitis. Our findings highlight a microbial ligand-GPCR pair in ISCs, revealing a causal link between microbial regulation of ISCs and colitis exacerbation and yielding a targeted therapeutic approach to restore ISC function in colitis.

Lactic acid bacteria modulate the CncC pathway to enhance resistance to ß-cypermethrin in the oriental fruit fly.

The gut microbiota of insects has been shown to regulate host detoxification enzymes. However, the potential regulatory mechanisms involved remain unknown. Here, we report that gut bacteria increase insecticide resistance by activating the cap "n" collar isoform-C (CncC) pathway through enzymatically generated reactive oxygen species (ROS) in Bactrocera dorsalis. We demonstrated that Enterococcus casseliflavus and Lactococcus lactis, two lactic acid-producing bacteria, increase the resistance of B. dorsalis to ß-cypermethrin by regulating cytochrome P450 (P450) enzymes and α-glutathione S-transferase (GST) activities. These gut symbionts also induced the expression of CncC and muscle aponeurosis fibromatosis. BdCncC knockdown led to a decrease in resistance caused by gut bacteria. Ingestion of the ROS scavenger vitamin C in resistant strain affected the expression of BdCncC/BdKeap1/BdMafK, resulting in reduced P450 and GST activity. Furthermore, feeding with E. casseliflavus or L. lactis showed that BdNOX5 increased ROS production, and BdNOX5 knockdown affected the expression of the BdCncC/BdMafK pathway and detoxification genes. Moreover, lactic acid feeding activated the ROS-associated regulation of P450 and GST activity. Collectively, our findings indicate that symbiotic gut bacteria modulate intestinal detoxification pathways by affecting physiological biochemistry, thus providing new insights into the involvement of insect gut microbes in the development of insecticide resistance.

Enterolyin S, a Polythiazole-containing Hemolytic Peptide from Enterococcus caccae.

The ß-hemolytic factor streptolysin S (SLS) is an important linear azol(in)e-containing peptide (LAP) that contributes significantly to the virulence of Streptococcus pyogenes. Despite its discovery 85 years ago, SLS has evaded structural characterizing owing to its notoriously problematic physicochemical properties. Here, we report the discovery and characterization of a structurally analogous hemolytic peptide from Enterococcus caccae, termed enterolysin S (ELS). Through heterologous expression, site-directed mutagenesis, chemoselective modification, and high-resolution mass spectrometry, we found that ELS contains an intriguing contiguous octathiazole moiety. The discovery of ELS expands our knowledge of hemolytic LAPs by adding a new member to this virulence-promoting family of modified peptides.

Reverse metabolomics for the discovery of chemical structures from humans.

Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis1,2, we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn's disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4+ T cells3 and agonism of the pregnane X receptor4. Culture of bacteria belonging to the Bifidobacterium, Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems.

Secrets of getting started: Regulation of the first committed step of peptidoglycan synthesis by protein phosphorylation in Enterococcus and other Gram-positive bacteria.

Regulation of the first committed step of peptidoglycan precursor synthesis by MurA-enzyme homologs has recently taken center stage in many different bacteria. In different low-GC Gram-positive bacteria, regulation of this step has been shown to be regulated by phosphorylation of homologs of the IreB/ReoM regulatory protein by PASTA-domain Ser/Thr-protein kinases. In this issue, Mascari, Little, and Kristich determine this regulatory pathway and its links to resistance to cephalosporin ß-lactam antibiotics in the major human pathogen, Enterococcus faecalis (Efa). Unbiased genetic selections identified MurAA (MurA-family homolog) as the downstream target of IreB regulation in the absence of the IreK Ser/Thr-protein kinase. Physiological and biochemical approaches, including determination of MICs to ceftriaxone, Western blotting of MurAA cellular amounts, isotope incorporation into peptidoglycan sacculi, and thermal-shift binding assays of purified proteins, demonstrated that unphosphorylated IreB, together with proteins MurAB (MurZ-family homolog), and ReoY(Efa) negatively regulate MurAA stability and cellular amount by the ClpCP protease. Importantly, this paper supports the idea that ceftriaxone stimulates phosphorylation of IreB, which leads to increased cellular MurAA amount and precursor pathway flux required for E. faecalis cephalosporin resistance. Overall, findings in this paper significantly contribute to understanding variations of this central regulatory pathway in other low-GC Gram-positive bacteria.

Identification of a novel family B DNA polymerase from Enterococcus phage IME199 and its overproduction in Escherichia coli BL21(DE3).

BACKGROUND: Identification and characterization of novel, faithful and processive DNA polymerases is a driving force in the development of DNA amplification methods. Purification of proteins from natural phages is often time-consuming, cumbersome and low yielding. Escherichia coli is a host bacterium widely used for the production of recombinant proteins, is the cell factory of choice for in vitro studies of phage protein function. RESULTS: We expressed the gene encoding Enterococcus faecium phage IME199 DNA polymerase (IME199 DNAP) in Escherichia coli BL21(DE3), and characterized protein function. IME199 DNAP has 3'-5' exonuclease activity, but does not have 5'-3' exonuclease activity. In addition, IME199 DNAP has dNTP-dependent 5'-3' polymerase activity and can amplify DNA at 15-35 °C and a pH range of 5.5-9.5. The amino acid residues Asp30, Glu32, Asp112 and Asp251 are the 3'-5' exonuclease active sites of IME199 DNAP, while residues Asp596 and Tyr639 are essential for DNA synthesis by IME199 DNAP. More importantly, the IME199 DNAP has strand displacement and processive synthesis capabilities, and can perform rolling circle amplification and multiple displacement amplification with very low error rates (approximately 3.67 × 10-6). CONCLUSIONS: A novel family B DNA polymerase was successfully overproduced in Escherichia coli BL21(DE3). Based on the characterized properties, IME199 DNAP is expected to be developed as a high-fidelity polymerase for DNA amplification at room temperature.

The isolation of rumen enterococci strains along with high potential utilizing cyanide.

Cyanogenic glycosides in forage species and the possibility of cyanide (CN) poisoning can have undesirable effects on ruminants. The literature estimates that unknown rumen bacteria with rhodanese activity are key factors in the animal detoxification of cyanogenic glycosides, as they are capable of transforming CN into the less toxic thiocyanate. Therefore, identifying these bacteria will enhance our understanding of how to improve animal health with this natural CN detoxification process. In this study, a rhodanese activity screening assay revealed 6 of 44 candidate rumen bacterial strains isolated from domestic buffalo, dairy cattle, and beef cattle, each with a different colony morphology. These strains were identified as belonging to the species Enterococcus faecium and E. gallinarum by 16S ribosomal DNA sequence analysis. A CN-thiocyanate transformation assay showed that the thiocyanate formation capacity of the strains after a 12 h incubation ranged from 4.42 to 25.49 mg hydrogen CN equivalent/L. In addition, thiocyanate degradation resulted in the production of ammonia nitrogen and acetic acid in different strains. This study showed that certain strains of enterococci substantially contribute to CN metabolism in ruminants. Our results may serve as a starting point for research aimed at improving ruminant production systems in relation to CN metabolism.

An Overview of the Factors Involved in Biofilm Production by the Enterococcus Genus.

Enterococcus species are known for their ability to form biofilms, which contributes to their survival in extreme environments and involvement in persistent bacterial infections, especially in the case of multi-drug-resistant strains. This review aims to provide a comprehensive understanding of the mechanisms underlying biofilm formation in clinically important species such as Enterococcus faecalis and the less studied but increasingly multi-drug-resistant Enterococcus faecium, and explores potential strategies for their eradication. Biofilm formation in Enterococcus involves a complex interplay of genes and virulence factors, including gelatinase, cytolysin, Secreted antigen A, pili, microbial surface components that recognize adhesive matrix molecules (MSCRAMMs), and DNA release. Quorum sensing, a process of intercellular communication, mediated by peptide pheromones such as Cob, Ccf, and Cpd, plays a crucial role in coordinating biofilm development by targeting gene expression and regulation. Additionally, the regulation of extracellular DNA (eDNA) release has emerged as a fundamental component in biofilm formation. In E. faecalis, the autolysin N-acetylglucosaminidase and proteases such as gelatinase and serin protease are key players in this process, influencing biofilm development and virulence. Targeting eDNA may offer a promising avenue for intervention in biofilm-producing E. faecalis infections. Overall, gaining insights into the intricate mechanisms of biofilm formation in Enterococcus may provide directions for anti-biofilm therapeutic research, with the purpose of reducing the burden of Enterococcus-associated infections.

Enterococcus faecalis OG1RF Evolution at Low pH Selects Fusidate-Sensitive Mutants in Elongation Factor G and at High pH Selects Defects in Phosphate Transport.

Enterococcus bacteria inhabit human and soil environments that show a wide range of pH values. Strains include commensals as well as antibiotic-resistant pathogens. We investigated the adaptation to pH stress in E. faecalis OG1RF by conducting experimental evolution under acidic (pH 4.8), neutral pH (pH 7.0), and basic (pH 9.0) conditions. A serial planktonic culture was performed for 500 generations and in a high-pH biofilm culture for 4 serial bead transfers. Nearly all of the mutations led to nonsynonomous codons, indicating adaptive selection. All of the acid-adapted clones from the planktonic culture showed a mutation in fusA (encoding elongation factor G). The acid-adapted fusA mutants had a trade-off of decreased resistance to fusidic acid (fusidate). All of the base-adapted clones from the planktonic cultures as well as some from the biofilm-adapted cultures showed mutations that affected the Pst phosphate ABC transporter (pstA, pstB, pstB2, pstC) and pyrR (pyrimidine biosynthesis regulator/uracil phosphoribosyltransferase). The biofilm cultures produced small-size colonies on brain heart infusion agar. These variants each contained a single mutation in pstB2, pstC, or pyrR. The pst and pyrR mutants outgrew the ancestral strain at pH 9.2, with a trade-off of lower growth at pH 4.8. Additional genes that had a mutation in multiple clones that evolved at high pH (but not at low pH) include opp1BCDF (oligopeptide ABC transporter), ccpA (catabolite control protein A), and ftsZ (septation protein). Overall, the experimental evolution of E. faecalis showed a strong pH dependence, favoring the fusidate-sensitive elongation factor G modification at low pH and the loss of phosphate transport genes at high pH. IMPORTANCE E. faecalis bacteria are found in dental biofilms, where they experience low pH as a result of fermentative metabolism. Thus, the effect of pH on antibiotic resistance has clinical importance. The loss of fusidate resistance is notable for OG1RF strains in which fusidate resistance is assumed to be a stable genetic marker. In endodontal infections, enterococci can resist calcium hydroxide therapy that generates extremely high pH values. In other environments, such as the soil and plant rhizosphere, enterococci experience acidification that is associated with climate change. Thus, the pH modulation of natural selection in enterococci is important for human health as well as for understanding soil environments.

The LiaFSR-LiaX System Mediates Resistance of Enterococcus faecium to Peptide Antibiotics and to Aureocin A53- and Enterocin L50-Like Bacteriocins.

Multidrug-resistant Enterococcus faecium strains are currently a leading cause of difficult-to-treat nosocomial infections. The emerging resistance of enterococci to last-resort antibiotics, such as daptomycin, prompts a search for alternative antimicrobials. Aureocin A53- and enterocin L50-like bacteriocins are potent antimicrobial agents that form daptomycin-like cationic complexes and have a similar cell envelope-targeting mechanism of action, suggesting their potential as next-generation antibiotics. However, to ensure their safe use, the mechanisms of resistance to these bacteriocins and cross-resistance to antibiotics need to be well understood. Here, we investigated the genetic basis of E. faecium's resistance to aureocin A53- and enterocin L50-like bacteriocins and compared it with that to antibiotics. First, we selected spontaneous mutants resistant to the bacteriocin BHT-B and identified adaptive mutations in the liaFSR-liaX genes encoding the LiaFSR stress response regulatory system and the daptomycin-sensing protein LiaX, respectively. We then demonstrated that a gain-of-function mutation in liaR increases the expression of liaFSR, liaXYZ, cell wall remodeling-associated genes, and hypothetical genes involved in protection against various antimicrobials. Finally, we showed that adaptive mutations or overexpression of liaSR or liaR alone results in cross-resistance to other aureocin A53- and enterocin L50-like bacteriocins, as well as antibiotics targeting specific components of the cell envelope (daptomycin, ramoplanin, gramicidin) or ribosomes (kanamycin and gentamicin). Based on the obtained results, we concluded that activation of the LiaFSR-mediated stress response confers resistance to peptide antibiotics and bacteriocins via a cascade of reactions, eventually leading to cell envelope remodeling. IMPORTANCE Pathogenic enterococci carry virulence factors and a considerable resistome, which makes them one of the most serious and steadily increasing causes of hospital epidemiological risks. Accordingly, Enterococcus faecium is classified into a top-priority ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group of six highly virulent and multidrug-resistant (MDR) bacterial pathogens for which novel antimicrobial agents need to be developed urgently. Alternative measures, such as the use of bacteriocins, separately or in combination with other antimicrobial agents (e.g., antibiotics), could be a potential solution, especially since several international health agencies recommend and support the development of such interventions. Nevertheless, in order to exploit their efficacy, more basic research on the mechanisms of cell killing and the development of resistance to bacteriocins is needed. The present study fills some of the knowledge gaps regarding the genetic basis of the development of resistance to potent antienterococcal bacteriocins, pointing out the common and divergent features regarding the cross-resistance to antibiotics.

Structure based virtual screening, molecular dynamic simulation to identify the oxadiazole derivatives as inhibitors of Enterococcus D-Ala-D-Ser ligase for combating vancomycin resistance.

Vancomycin resistance in enterococci mainly arises due to alteration in terminal peptidoglycan dipeptide. A comprehensive structural analysis for substrate specificity of dipeptide modifying d-Alanine: d-Serine ligase (Ddls) is essential to screen its inhibitors for combating vancomycin resistance. In this study modeled 3D structure of EgDdls from E. gallinarum was used for structure based virtual screening (SBVS) of oxadiazole derivatives. Initially, fifteen oxadiazole derivatives were identified as inhibitors at the active site of EgDdls from PubChem database. Further, four EgDdls inhibitors were evaluated using pharmacokinetic profile and molecular docking. The results of molecular docking showed that oxadiazole inhibitors could bind preferentially at ATP binding pocket with the lowest binding energy. Further, molecular dynamics simulation results showed stable behavior of EgDdls in complex with screened inhibitors. The residues Phe172, Lys174, Glu217, Phe292, and Asn302 of EgDdls were mainly involved in interactions with screened inhibitors. Furthermore, MM-PBSA calculation showed electrostatic and van der Waals interactions mainly contribute to overall binding energy. The PCA analysis showed motion of central domain and omega loop of EgDdls. This is involved in the formation of native dipeptide and stabilized after binding of 2-(1-(Ethylsulfonyl) piperidin-4-yl)-5-(furan-2-yl)-1,3,4-oxadiazole, which could be reason for the inhibition of EgDdls. Hence, in this study we have screened inhibitors of EgDdls which could be useful to alleviate the vancomycin resistance problem in enterococci, involved in hospital-acquired infections, especially urinary tract infections (UTI).

In vitro virulence activity of Pseudomonas aeruginosa, enhanced by either Acinetobacter baumannii or Enterococcus faecium through the polymicrobial interactions.

Microbes within an infection impact neighbors' pathogenicity. This study aimed to address in vitro virulence activity of Pseudomonas aeruginosa under the binary interaction with Acinetobacter baumannii or Enterococcus faecium, co-isolated from two chronic wound infections. The biofilm formation of Pseudomonas was enhanced 1.5- and 1.4-fold when it was simultaneously cultured with Acinetobacter and Enterococcus, respectively. Pseudomonas motility was increased by 1.9- and 1.5-fold (swimming), 3.6- and 1.9-fold (swarming), and 1.5- and 1.5-fold (twitching) in the dual cultures with Acinetobacter and Enterococcus, respectively. The synergistic hemolysis activity of Pseudomonas was observed with the heat-killed Acinetobacter and Enterococcus cells. The minimum inhibitory concentration of ciprofloxacin against Pseudomonas was increased from (µg mL-1) 25 to 400 in the individual and mixed cultures, respectively. The pyocyanin production by Pseudomonas in the single and mixed cultures with Acinetobacter and Enterococcus was (µg/mL) 1.8, 2.3, and 2.9, respectively. The expression of lasI, rhlI, and pqsR genes was up-regulated by 1.0-, 1.9-, and 16.3-fold, and 4.9-, 1.0-, and 9.3-fold when Pseudomonas was incubated with Acinetobacter and Enterococcus, respectively. Considering the entire community instead of a single pathogen may lead to a more effective therapeutic design for persistent infections caused by Pseudomonas.

Enterococci enhance Clostridioides difficile pathogenesis.

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

Response of sensitive and resistant Listeria monocytogenes strains against bacteriocins produced by different Enterococcus spp. strains.

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

Strengthening of enterococcal biofilms by Esp.

Multidrug-resistant (MDR) Enterococcus faecalis are major causes of hospital-acquired infections. Numerous clinical strains of E. faecalis harbor a large pathogenicity island that encodes enterococcal surface protein (Esp), which is suggested to promote biofilm production and virulence, but this remains controversial. To resolve this issue, we characterized the Esp N-terminal region, the portion implicated in biofilm production. Small angle X-ray scattering indicated that the N-terminal region had a globular head, which consisted of two DEv-Ig domains as visualized by X-ray crystallography, followed by an extended tail. The N-terminal region was not required for biofilm production but instead significantly strengthened biofilms against mechanical or degradative disruption, greatly increasing retention of Enterococcus within biofilms. Biofilm strengthening required low pH, which resulted in Esp unfolding, aggregating, and forming amyloid-like structures. The pH threshold for biofilm strengthening depended on protein stability. A truncated fragment of the first DEv-Ig domain, plausibly generated by a host protease, was the least stable and sufficient to strengthen biofilms at pH ≤ 5.0, while the entire N-terminal region and intact Esp on the enterococcal surface was more stable and required a pH ≤ 4.3. These results suggested a virulence role of Esp in strengthening enterococcal biofilms in acidic abiotic or host environments.

Enterococcus faecalis V583 cell membrane protein expression to alkaline stress.

Enterococcus faecalis is able to adapt to alkaline conditions and is commonly recovered from teeth in which endodontic treatment has failed. The role that E. faecalis membrane proteins play in survival strategies to extreme alkaline conditions is unclear. We grew E. faecalis V583 in a chemostat at pH 8 and 11 at one-tenth the organism's relative maximum growth rate. Following membrane shaving, isotope-coding protein labels were added at the peptide level to samples and then combined. The relative proportion of membrane proteins were identified using LC-ESI mass spectrometry and MaxQuant analysis. Ratios of membrane proteins were log2 transformed, with proteins deviating by more than 1 SD of the mean considered to be up- or down-regulated. A total of six proteins were up-regulated in pH 11 including: EF0669 (polysaccharide biosynthesis family); EF1927 (glycerol uptake facilitator), and EF0114 (glycosyl hydrolase). A total of five proteins were down-regulated including: EF0108 (C4-dicarboxylate transporter); EF1838 (PTS system IIC component); EF0456 (PTS system IID component); and EF0022 (PTS mannose-specific IID component). In extreme alkaline conditions, the membrane proteins of E. faecalis seem to be involved in a shift of carbohydrate metabolism from the PTS system to glycerol, which supports the formation of a protective capsule protecting the cell.

Evaluation of inhibitory compounds produced by bacteria isolated from a hydrogen-producing bioreactor during the self-fermentation of wheat straw.

AIMS: The objective of this study was to evaluate the inhibitory activity of compounds secreted by bacteria isolated from a hydrogen-producing bioreactor to understand how these microorganisms interact in this community. METHODS AND RESULTS: In vitro inhibitory assays were performed using samples secreted by bacteria subject to different treatments to determine if their inhibitory effect was due to organic acids, non-proteinaceous compounds or bacteriocin-like inhibitory substances (BLIS). Bacterial isolated were suppressed 43%, 30% and 27% by neutralized, precipitated and non-neutralized cell-free supernatants, respectively. Non-hydrogen producers (non-H2 P) lactic acid bacteria (LAB) (Lactobacillus plantarum LB1, Lactobacillus pentosus LB7, Pediococcus acidilactici LB4) and hydrogen producers (H2 P) LAB (Enterococcus faecium F) were inhibited by the production of organic acids, non-proteinaceous compounds and BLIS. Meanwhile, the obligate anaerobe H2 P (Clostridium beijerinckii B) inhibited by the production of non-proteinaceous compounds and BLIS. The presence of BLIS was confirmed when proteolytic enzymes affected the inhibitory activity of secreted proteins in values ranging from 20% to 42%. The BLIS produced by L. plantarum LB1, P. acidilactici LB4, L. pentosus LB7 and E. faecium F showed molecular masses of ~11, 25, 20 and 11 kDa, respectively. CONCLUSIONS: It was demonstrated antagonistic interactions between Lactobacillus-Enterococcus and Pediococcus-Enterococcus species, generated by the secretion of organic acids, non-proteinaceous compounds and BLIS. SIGNIFICANCE AND IMPACT OF THE STUDY: We report the interactions between LAB isolated from hydrogen-producing bioreactors. These interactions might impact the dynamics of the microbial population during hydrogen generation. Our work lays a foundation for strategies that allow controlling bacteria that can affect hydrogen production.

Kinetics of the Growth of Enterococcus spp. Biofilm Formed by Strains Isolated from Patients with Infectious Complications after Large joint Replacements.

We studied the kinetics of growth of biofilms and plankton forms of Enterococcus spp. strains isolated in implant-associated infection of large joints. The capacity for biofilm formation by enterococci was demonstrated. The following stages of biofilm formation were revealed: biofilm formation and maturation, its degradation, and secondary dissemination. These findings extend our understanding of the wound process caused by enterococci.

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

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