Identification of the general stress stimulon related to colonization in Enterococcus faecalis.

Enterococcus faecalis has to cope with major stress conditions during colonization. To understand the effects of stress encountered during infection, the present study assessed the transcriptomic response of the bacteria facing exposure to serum, urine, bile salts, acid pH, or oxidative stress. Compared to non-stressed culture, 30% of the E. faecalis genes were differentially expressed. The transcriptome analysis reveals common but also specific responses, depending on stresses encountered: thus, urine exposure has the most important impact, and the highest number of genes with modified expression is involved in transport and metabolism. The results also pinpoint many stress-related sRNA or intergenic regions not yet characterized. This study identified the general stress stimulon related to infection: when the commensal bacterium initiates its response to stress related to infection, it increases its ability to survive to rough conditions for colonization, rather than promoting expression of virulence factors, and becomes this opportunistic pathogen that thrives in hospital settings.

Study of key RNA metabolism proteins in Enterococcus faecalis.

The control of mRNA turnover is essential in bacteria to allow rapid adaptation, especially in opportunistic pathogen like Enterococcus faecalis. This mechanism involves RNase and DEAD-box helicases that are key elements in RNA processing and their associations form the degradosome with accessory proteins. In this study, we investigated the function of four RNases (J1, J2, Y and III) and three DEAD-box helicases (CshA, CshB, CshC) present in most Enterococci. The interactions of all these RNA metabolism actors were investigated in vitro, and the results are in accordance with a degradosome structure close to the one of Bacillus subtilis. At the physiological level, we showed that RNase J1 is essential, whereas RNases J2 and III have a role in cold, oxidative and bile salts stress response, and RNase Y in general fitness. Furthermore, RNases J2, Y and III mutants are affected in virulence in the Galleria mellonella infection model. Concerning DEAD-box helicases, all of them are involved in cold shock response. Since the ΔcshA mutant was the most stress impacted strain, we studied this DEAD-box helicase CshA in more detail. This showed that CshA autoregulates its own expression by binding to its mRNA 5'Unstranslated Region. Interestingly, CshC is also involved in the expression control of CshA by a hitherto unprecedented mechanism.

The role of the CroR response regulator in resistance of Enterococcus faecalis to D-cycloserine is defined using an inducible receiver domain.

Enterococcus faecalis is an opportunistic multidrug-resistant human pathogen causing severe nosocomial infections. Previous investigations revealed that the CroRS two-component regulatory pathway likely displays a pleiotropic role in E. faecalis, involved in virulence, macrophage survival, oxidative stress response as well as antibiotic resistance. Therefore, CroRS represents an attractive potential new target for antibiotherapy. In this report, we further explored CroRS cellular functions by characterizing the CroR regulon: the 'domain swapping' method was applied and a CroR chimera protein was generated by fusing the receiver domain from NisR to the output domain from CroR. After demonstrating that the chimera CroR complements a croR gene deletion in E. faecalis (stress response, virulence), we conducted a global gene expression analysis using RNA-Seq and identified 50 potential CroR targets involved in multiple cellular functions such as cell envelope homeostasis, substrate transport, cell metabolism, gene expression regulation, stress response, virulence and antibiotic resistance. For validation, CroR direct binding to several candidate targets was demonstrated by EMSA. Further, this work identified alr, the gene encoding the alanine racemase enzyme involved in E. faecalis resistance to D-cycloserine, a promising antimicrobial drug to treat enterococcal infections, as a member of the CroR regulon.

Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci.

UNLABELLED: Enterococci are naturally tolerant to typically bactericidal cell wall-active antibiotics, meaning that their growth is inhibited but they are not killed even when exposed to a high concentration of the drug. The molecular reasons for this extraordinary tolerance are still incompletely understood. Previous work showed that resistance to killing collapsed specifically in mutants affected in superoxide dismutase (Sod) activity, arguing that bactericidal antibiotic treatment led to induction of a superoxide burst. In the present work, we show that loss of antibiotic tolerance in ΔsodA mutants of pathogenic enterococci is dependent on the energy source present during antibiotic treatment. Hexoses induce greater killing than the pentose ribose, and no killing was observed with glycerol as the energy source. These results point to glycolytic reactions as crucial for antibiotic-mediated killing of ΔsodA mutants. A transposon mutant library was constructed in Enterococcus faecalis ΔsodA mutants and screened for restored tolerance of vancomycin. Partially restored tolerance was observed in mutants with transposon integrations into intergenic regions upstream of regulators implicated in arginine catabolism. In these mutants, the arginine deiminase operon was highly upregulated. A model for the action of cell wall-active antibiotics in tolerant and nontolerant bacteria is proposed. IMPORTANCE: Antibiotic tolerance is a serious clinical concern, since tolerant bacteria have considerably increased abilities to resist killing by bactericidal drugs. Using enterococci as models for highly antibiotic-tolerant pathogens, we showed that tolerance of these bacteria is linked to their superoxide dismutase (Sod), arguing that bactericidal antibiotics induce generation of reactive oxygen species inside cells. Wild-type strains are tolerant because they detoxify these deleterious molecules by the activity of Sod, whereas Sod-deficient strains are killed. This study showed that killing depends on the energy source present during treatment and that an increase in arginine catabolism partially restored tolerance of the Sod mutants. These results are used to propose a mode-of-action model of cell wall-active antibiotics in tolerant and nontolerant bacteria.

Virulence phenotypes differ between toxigenic Vibrio parahaemolyticus isolated from western coasts of Europe.

Vibrio parahaemolyticus is the leading bacterial cause of gastroenteritis associated with seafood consumption worldwide. Not all members of the species are thought to be pathogenic, thus identification of virulent organisms is essential to protect public health and the seafood industry. Correlations of human disease and known genetic markers (e.g. thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH)) appear complex. Some isolates recovered from patients lack these factors, while their presence has become increasingly noted in isolates recovered from the environment. Here, we used whole-genome sequencing in combination with mammalian and insect models of infection to assess the pathogenic potential of V. parahaemolyticus isolated from European Atlantic shellfish production areas. We found environmental V. parahaemolyticus isolates harboured multiple virulence-associated genes, including TDH and/or TRH. However, carriage of these factors did not necessarily reflect virulence in the mammalian intestine, as an isolate containing TDH and the genes coding for a type 3 secretion system (T3SS) 2α virulence determinant, appeared avirulent. Moreover, environmental V. parahaemolyticus lacking TDH or TRH could be assigned to groups causing low and high levels of mortality in insect larvae, with experiments using defined bacterial mutants showing that a functional T3SS1 contributed to larval death. When taken together, our findings highlight the genetic diversity of V. parahaemolyticus isolates found in the environment, their potential to cause disease and the need for a more systematic evaluation of virulence in diverse V. parahaemolyticus to allow better genetic markers.

Redox balance via lactate dehydrogenase is important for multiple stress resistance and virulence in Enterococcus faecalis.

Enterococcus faecalis is a highly stress resistant opportunistic pathogen. The intrinsic ruggedness of this bacterium is supposed to be the basis of its capacity to colonize the hostile environments of hospitals and to cause several kinds of infections. We show in this work that general resistance to very different environmental stresses depends on the ability of E. faecalis to maintain redox balance via lactate dehydrogenase (LDH). Furthermore, LDH-deficient mutants are less successful than the wild type at colonizing host organs in a murine model of systemic infection. Taken together, our results, as well as those previously published for Staphylococcus aureus (A. R. Richardson, S. J. Libby, and F. C. Fang, Science 319:1672-1676, 2008), identify LDH as an attractive drug target. These drugs may have additional applications, as in the fight against glycopeptide antibiotic-resistant bacteria and even cancer.

Analysis of the tolerance of pathogenic enterococci and Staphylococcus aureus to cell wall active antibiotics.

OBJECTIVES: Tolerance refers to the phenomenon that bacteria do not significantly die when exposed to bactericidal antibiotics. Enterococci are known for their high tolerance to these drugs, but the molecular reasons why they resist killing are not understood. In a previous study we showed that the superoxide dismutase (SOD) is implicated in this tolerance. This conclusion was based on the results obtained with one particular strain of Enterococcus faecalis and therefore the objective of the present communication was to analyse whether dependence of tolerance on active SOD is a general phenomenon for enterococci and another Gram-positive pathogen, Staphylococcus aureus. METHODS: Mutants deficient in SOD activity were constructed in pathogenic enterococci. The wild-type sodA gene was cloned into an expression vector and transformed into SOD-deficient strains for complementation with varying levels of SOD activity. Previously constructed SOD-deficient strains of S. aureus were also included in this study. Tolerance to vancomycin and penicillin was then tested. RESULTS: We demonstrated that the dependence on SOD of tolerance to vancomycin and penicillin is a common trait of antibiotic-susceptible pathogenic enterococci. By varying the levels of expression we could also show that tolerance to vancomycin is directly correlated to SOD activity. Interestingly, deletion of the sodA gene in a non-tolerant Enterococcus faecium strain did not further sensitize the mutant to bactericidal antibiotics. Finally, we showed that the SOD enzymes of S. aureus are also implicated in tolerance to vancomycin. CONCLUSION: High tolerance of enterococci to cell wall active antibiotics can be reversed by SOD deficiency.

Comparative Analysis of Campylobacter jejuni and C. coli Isolated from Livestock Animals to C. jejuni and C. coli Isolated from Surface Water Using DNA Sequencing and MALDI-TOF.

This study evaluated the contribution of cattle, sheep, poultry and pigs to the contamination of surface water from rivers by Campylobacter jejuni and C. coli using MLST, cgMLST and considered MALDI-TOF MS as an alternative technique. The 263 strains isolated from cattle (n = 61), sheep (n = 42), poultry (n = 65), pigs (n = 60) and surface water (n = 35) were distributed across 115 sequence types (STs), 49 for C. jejuni and 66 for C. coli. Considering MLST data, 14.2%, 11.4% and 2.8% of the surface water strains could be attributed to cattle, poultry and sheep, respectively, none to pigs, and 85.7% were non-attributed. Analysis of cg-MLST data with STRUCTURE indicated that C. jejuni strains from water were predominantly attributed to poultry (93.5%), weakly to sheep (<1%) and 6.3% non-attributed, and that conversely, C. coli strains from water were predominantly non-attributed (94.3%) and 5.7% attributed to poultry. Considering the protein profiles with a threshold of 94% and 97% of similarity, respectively, strains from surface water could be attributed to poultry (31.4% and 17.1%), and to cattle (17.1% and 5.7%); 54.1% and 77.1% were non-attributed. This study confirmed these livestock animals might contribute to the contamination of surface water, with a level of contribution depending on the typing technique and the method of analysis. MALDI-TOF could potentially be an alternative approach for source attribution.

Involvement of peptidylprolyl cis/trans isomerases in Enterococcus faecalis virulence.

Peptidylprolyl cis/trans isomerases (PPIases) are enzymes involved in protein folding. Analysis of the genome sequence of Enterococcus faecalis V583 allowed for identification of 3 PPIases carrying genes. ef2898 encodes an intracellular PPIase which was not shown to be important for the E. faecalis stress response or virulence. The other two PPIases, the parvulin family rotamase EF0685 and the cyclophilin family member EF1534, are expected to be surface-exposed proteins. They were shown to be important for virulence and resistance to NaCl. A Δef0685 Δef1534 mutant was also more resistant to oxidative stress, was able to grow under a high manganese concentration, and showed altered resistance to ampicillin and quinolone antibiotics.

Aerobic glycerol dissimilation via the Enterococcus faecalis DhaK pathway depends on NADH oxidase and a phosphotransfer reaction from PEP to DhaK via EIIADha.

Two pathways for glycerol dissimilation are present in Enterococcus faecalis. Either glycerol is first phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase with molecular oxygen as the electron acceptor (GlpO/GlpK pathway), or it is first oxidized by glycerol dehydrogenase with NAD(+) as the acceptor of the reduction equivalents and then phosphorylated by dihydroxyacetone kinase (GldA/DhaK pathway). The final end product in both cases is dihydroxyacetone phosphate (DHAP). The genes of the GldA/DhaK pathway are present in a four-gene operon structure encoding GldA, a small hypothetical protein (EF1359), and two subunits of dihydroxyacetone kinase (DhaK and DhaL). We demonstrate in this study that protein EF1359 is part of a phosphorylation cascade which phosphorylates dihydroxyacetone in a phosphoenolpyruvate (PEP)-dependent reaction via EI, HPr, EF1359 and DhaLK. Furthermore we show that aerobic dissimilation of glycerol via the GldA/DhaK pathway is dependent on active NADH oxidase to regenerate NADH in Ent. faecalis. A refined model of the aerobic metabolism of glycerol via the GldA/DhaK pathway is presented.

The prolipoprotein diacylglyceryl transferase (Lgt) of Enterococcus faecalis contributes to virulence.

Enterococcus faecalis is an opportunistic pathogen responsible for nosocomial infections. Lipoproteins in Gram-positive bacteria are translocated across the plasma membrane and anchored by the fatty acid group. They perform critical roles, with some described as virulence determinants. The aim of this study was to explore the roles of E. faecalis lipoproteins in the stress response and virulence. We constructed a mutant affected in the predicted prolipoprotein diacylglyceryl transferase gene lgt, and examined the role of Lgt in membrane anchoring, growth, the stress response and virulence. Inactivation of lgt enhanced growth in a high concentration of Mn(2+) or under oxidative stress in vitro, and significantly decreased virulence.

Development of a microsphere-based immunoassay for the serological diagnosis of equine trypanosomosis.

Trypanozoon infections in equids are caused by three parasite species in the Trypanozoon subgenus: Trypanosoma equiperdum, T. brucei and T. evansi. They are respectively responsible for infectious diseases dourine, nagana and surra. Due to the threat that Trypanozoon infection represents for international horse trading, accurate diagnostic tests are crucial. Current tests suffer from poor sensitivity and specificity, due in the first case to the transient presence of parasites in the blood and in the second, to antigenic cross-reactivity among Trypanozoon subspecies. This study was designed to develop a microsphere-based immunoassay for diagnosing equine trypanosomosis. We tested beads coated with eight Trypanosoma spp. recombinant antigens: enolase, GM6, PFR1, PFR2, ISG65, VSGat, RoTat1.2 and JN2118HU. Of these, GM6 was identified as the best candidate for the serological diagnosis of Trypanozoon infections in equids. Using a receiver operating characteristic (ROC) analysis on 349 equine sera, anti-GM6 antibodies were detected with an AUC value of 0.994 offering a sensitivity of 97.9% and a specificity of 96.0%. Our findings show that the GM6 antigen is a good target for diagnosing equine trypanosomosis using a microsphere-based immunoassay. This promising assay could be a useful alternative to the official diagnostic tool for equine trypanosomosis.

The NagY regulator: A member of the BglG/SacY antiterminator family conserved in Enterococcus faecalis and involved in virulence.

Enterococcus faecalis is a commensal bacterium of the gastrointestinal tract but also a major nosocomial pathogen. This bacterium uses regulators like BglG/SacY family of transcriptional antiterminators to adapt its metabolism during host colonization. In this report, we investigated the role of the BglG/SacY family antiterminator NagY in the regulation of the nagY-nagE operon in presence of N-acetylglucosamine, with nagE encoding a transporter of this carbohydrate, as well as the expression of the virulence factor HylA. We showed that this last protein is involved in biofilm formation and glycosaminoglycans degradation that are important features in bacterial infection, confirmed in the Galleria mellonella model. In order to elucidate the evolution of these actors, we performed phylogenomic analyses on E. faecalis and Enterococcaceae genomes, identified orthologous sequences of NagY, NagE, and HylA, and we report their taxonomic distribution. The study of the conservation of the upstream region of nagY and hylA genes showed that the molecular mechanism of NagY regulation involves ribonucleic antiterminator sequence overlapping a rho-independent terminator, suggesting a regulation conforming to the canonical model of BglG/SacY family antiterminators. In the perspective of opportunism understanding, we offer new insights into the mechanism of host sensing thanks to the NagY antiterminator and its targets expression.

Lipoproteins of Enterococcus faecalis: bioinformatic identification, expression analysis and relation to virulence.

Enterococcus faecalis is a ubiquitous bacterium that is capable of surviving in a broad range of natural environments, including the human host, as either a natural commensal or an opportunistic pathogen involved in severe hospital-acquired infections. How such opportunistic pathogens cause fatal infections is largely unknown but it is likely that they are equipped with sophisticated systems to perceive external signals and interact with eukaryotic cells. Accordingly, being partially exposed at the cell exterior, some surface-associated proteins are involved in several steps of the infection process. Among them are lipoproteins, representing about 25 % of the surface-associated proteins, which could play a major role in bacterial virulence processes. This review focuses on the identification of 90 lipoprotein-encoding genes in the genome of the E. faecalis V583 clinical strain and their putative roles, and provides a transcriptional comparison of microarray data performed in environmental conditions including blood and urine. Taken together, these data suggest a potential involvement of lipoproteins in E. faecalis virulence, making them serious candidates for vaccine production.

Molecular detection of 7SL-derived small RNA is a promising alternative for trypanosomosis diagnosis.

Equine trypanosomosis comprises different parasitic diseases caused by protozoa of the subgenus Trypanozoon: Trypanosoma equiperdum (causative agent of dourine), Trypanosoma brucei (nagana) and Trypanosoma evansi (surra). Due to the absence of a vaccine and the lack of efficacy of the few available drugs, these diseases represent a major health and economic problem for international equine trade. Development of affordable, sensitive and specific diagnostic tests is therefore crucial to ensure the control of these diseases. Recently, it has been shown that a small RNA derived from the 7SL gene (7SL-sRNA) is produced in high concentrations in sera of cattle infected with Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei. Our objective was to determine whether 7SL-sRNA could serve as a marker of active infection in equids experimentally infected with Trypanosoma equiperdum by analysing the sensitivity, specificity and stability of the 7SL-sRNA. Using a two-step RT-qPCR, we were able to detect the presence of 7SL-sRNA between 2 and 7 days post-infection, whereas seroconversion was detected by complement fixation test between 5 and 14 days post-infection. There was a rapid loss of 7SL-sRNA signal from the blood of infected animals one day post-trypanocide treatment. The 7SL-sRNA RT-qPCR allowed an early detection of a treatment failure revealed by glucocorticoid-induced immunosuppression. In addition, the 7SL-sRNA remains detectable in positive sera after 7 days of storage at either 4°C, room temperature or 30°C, suggesting that there is no need to refrigerate serum samples before analysis. Our findings demonstrate continual detection of 7SL-sRNA over an extended period of experimental infection, with signals detected more than six weeks after inoculation. The detection of a strong and consistent 7SL-sRNA signal even during subpatent parasitemia and the early detection of treatment failure highlight the very promising nature of this new diagnostic method.

Occurrence of Bacterial Pathogens and Human Noroviruses in Shellfish-Harvesting Areas and Their Catchments in France.

During a 2-year study, the presence of human pathogenic bacteria and noroviruses was investigated in shellfish, seawater and/or surface sediments collected from three French coastal shellfish-harvesting areas as well as in freshwaters from the corresponding upstream catchments. Bacteria isolated from these samples were further analyzed. Escherichia coli isolates classified into the phylogenetic groups B2, or D and enterococci from Enterococcus faecalis and E. faecium species were tested for the presence of virulence genes and for antimicrobial susceptibility. Salmonella members were serotyped and the most abundant serovars (Typhimurium and its monophasic variants and Mbandaka) were genetically characterized by high discriminative subtyping methods. Campylobacter and Vibrio were identified at the species level, and haemolysin-producing Vibrio parahaemolyticus were searched by tdh- and trh- gene detection. Main results showed a low prevalence of Salmonella in shellfish samples where only members of S. Mbandaka were found. Campylobacter were more frequently isolated than Salmonella and a different distribution of Campylobacter species was observed in shellfish compared to rivers, strongly suggesting possible additional inputs of bacteria. Statistical associations between enteric bacteria, human noroviruses (HuNoVs) and concentration of fecal indicator bacteria revealed that the presence of Salmonella was correlated with that of Campylobacter jejuni and/or C. coli as well as to E. coli concentration. A positive correlation was also found between the presence of C. lari and the detection of HuNoVs. This study highlights the importance of simultaneous detection and characterization of enteric and marine pathogenic bacteria and human noroviruses not only in shellfish but also in catchment waters for a hazard assessment associated with microbial contamination of shellfish.

Validation of host-specific Bacteriodales 16S rRNA genes as markers to determine the origin of faecal pollution in Atlantic Rim countries of the European Union.

The recent implementation of the Revised Bathing Water Directive in the European Union has highlighted the need for development of effective methods to differentiate between sources of faecal contamination. It had previously been shown that amplification of 16S rRNA genes of host-specific Bacteriodales species using the HF183F and CF128F primers could be used as markers for human and bovine faecal contamination in the United States. This paper determined the sensitivity and specificity of these markers in four Atlantic Rim countries (France, Ireland, Portugal and the United Kingdom) to evaluate their usefulness in determining the origin of faecal contamination. It was shown that the HF183F marker displayed high sensitivity (80-100%) and specificity (91-100%), and is reliable as an indication of human faecal contamination. The CF128F marker displayed 100% sensitivity in all four countries. However, strong regional variations in specificity (41-96%) were observed, highlighting the need for local validation before this marker is employed in source tracking of faecal contamination.

Implication of hypR in the virulence and oxidative stress response of Enterococcus faecalis.

HypR has recently been described as the first transcriptional regulator involved in the oxidative stress response and in the intracellular survival of Enterococcus faecalis within macrophages. In order to characterize the HypR regulon, real-time quantitative RT-PCR experiments were performed. The expression of four genes involved in the oxidative stress response encoding catalase, glutathione reductase, and the two subunits of alkyl hydroperoxide reductase were down regulated in the hypR background under H(2)O(2) condition. These findings show that HypR acts as a transcriptional activator, especially during oxidative stress. In addition, DNAse I footprinting assays allowed us to identify the HypR-protected DNA regions corresponding to the "HypR box" in the hypR promoter. Moreover, the effect of the hypR mutation on the virulence of E. faecalis was evaluated in comparison with the wild-type JH2-2 strain using a mouse peritonitis model. Our results revealed that HypR appears to be an important virulence factor in E. faecalis.

Prevalence and Characterization of Shiga Toxin-Producing and Enteropathogenic Escherichia coli in Shellfish-Harvesting Areas and Their Watersheds.

more strains formed a strong biofilm at 18 than at 30°C. Finally, more than 85% of analyzed strains were found to be sensitive to the 16 tested antibiotics. These data suggest the low risk of human infection by STEC if shellfish from these shellfish-harvesting areas were consumed.

Physiological and molecular aspects of bile salt response in Enterococcus faecalis.

Analysis of the susceptibility and the acquisition of tolerance in Enterococus faecalis towards bile salts showed a nearly instantaneous killing effect and yielded evidence for homologous tolerance as well as for cross-protections. Two-dimensional (2-D) electrophoresis revealed 45 proteins which are amplified in response to the bile salt treatment. These include a set of seven proteins, the synthesis of which is increased not only with the bile salts but also with multiple sublethal stresses of various nature. Characterisation of the latter (called general stress proteins) showed that at least five of them are related to resistance to bile salts, heat, ethanol, oxidative and alkaline pH stresses and are probably involved in cross-protection development. On the other hand, random mutagenesis of E. faecalis allowed the isolation of 10 bile salt-sensitive mutants. Their characterisation revealed that the mutation loci corresponded to genes related to DNA repair, oxidative response, transcriptional regulation, dGTP hydrolysis, membrane composition or cell wall synthesis. Further characterisation of one mutant revealed that the insertion within the E. faecalis sagA gene led to morphology changes, to perturbations of cell division and to a decrease of the resistance towards several independent physicochemical stresses.