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

Identification of a DNA region from lactococcal phage sk1 protecting phage 712 from the abortive infection mechanism AbiF

International audienceThe partial nucleotide sequence of a Lactococcus lactis subsp. lactis ADRIA 85LO30 bacteriocin-producing operon was determined. The first two open reading frames of the operon are necessary to get bacteriocin expression in L. lactis IL1403R

IS1675, a Novel Lactococcal Insertion Element, Forms a Transposon-Like Structure Including the Lacticin 481 Lantibiotic Operon

Two copies of IS1675, a novel lactococcal insertion element from the IS4 family, are present on a 70-kb plasmid, where they frame the lantibiotic lacticin 481 operon. The whole structure could be a composite transposon designated Tn5721. This study shows that the lacticin 481 operon does not include any regulatory gene and provides a new example of a transposon-associated bacteriocin determinant. We identified five other IS1675 copies not associated with the lacticin 481 operon. The conservation of IS1675 flanking sequences suggested a 24-bp target site

pBLA8, from Brevibacterium linens, belongs to a Gram-positive subfamily of ColE2-related plasmids

International audienceSUMMARY: A 3.1 kb DNA fragment from pBLA8, a Brevibacterium linens cryptic plasmid, containing all the information required for autonomous replication was cloned and sequenced. Using deletion analysis, the fragment essential and sufficient for autonomous replication was delimited to 1.5 kb. This fragment is characterized by the presence of an ori site located upstream of an operon encoding two proteins, RepA and RepB, both essential for replication. Based on structural similarities and a strong conservation of ori, RepA and RepB, pBLA8 was assigned to a new subfamily of the ColE2 plasmid family. This subfamily is distinguished by the requirement for two Rep proteins and the location of an ori site upstream of the mpAB operon. RepA is thought to encode primase activity, whereas RepB could be a DNA-binding protein. An Escherichia coli-B. linens shuttle vector, derived from pBLA8, was constructed. Its host spectrum was extended to Arthrobacter species

Isolation and Characterization of Bile Salts-Sensitive Mutants of Enterococcus faecalis

International audienceA library of insertional mutants of Enterococcus faecalis was constructed; it allowed the isolation and the characterization of 10 mutants affected in resistance to bile salts. Insertion loci of two mutants corresponded to genes of unknown function, while the amino acid sequences deduced from the other loci were homologous to proteins related to DNA repair, oxidative response, transcriptional regulation, dGTP hydrolysis, membrane composition, or cell wall synthesis. Further characterization of one mutant revealed that the insertion within the E. faecalis sagA gene led to a decrease of the resistance towards numerous independent physicochemical stresses, to modifications of the cell wall integrity, and to perturbations of cell division with septation anomalies

Lacticin 481: an antimicrobial peptide of the lantibiotic family produced by Lactococcus lactis.

Lacticin 481 is an antimicrobial peptide secreted by some strains of the lactic acid bacterium Lactococcus lactis. This peptide contains two lanthionines, one 3-methyllanthionine and one 2,3-didehydrobutyrine. Such unusual residues are specifically found in lantibiotics and are created by enzymatic modification of some amino acids included into a precursor peptide. The latter is encoded by a structural gene. Several groups of lantibiotics have been distinguished and lacticin 481 is representative the so-called lacticin 481 group. Here, we present an overview of the data accumulated on lacticin 481, including its properties and spectrum of action, its genetic determinants, its maturation pathway, the immunity system protecting the producer strains from their own lantibiotic, and potential applications

New Insights into the Enterococcus faecalis CroRS Two-Component System Obtained Using a Differential-Display Random Arbitrarily Primed PCR Approach▿

Using a modified random arbitrarily primed PCR approach, the operon encoding the Enterococcus faecalis JH2-2 CroRS two-component regulatory system was shown to be repressed during stationary phase, and a CroRS-regulated operon (glnQHMP) was identified. Gel retardation assays showed that the CroR regulator binds specifically to the glnQHMP promoter

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

Enterococcus faecalisis an opportunistic multidrug-resistant human pathogen causing severe nosoco-mial infections. Previous investigations revealed thatthe CroRS two-component regulatory pathway likelydisplays a pleiotropic role inE. faecalis, involved invirulence, macrophage survival, oxidative stressresponse as well as antibiotic resistance. Therefore,CroRS represents an attractive potential new targetfor antibiotherapy. In this report, we further exploredCroRS cellular functions by characterizing the CroRregulon: the ‘domain swapping’ method was appliedand a CroR chimera protein was generated by fusingthe receiver domain from NisR to the output domainfrom CroR. After demonstrating that the chimeraCroR complements acroRgene deletion inE. faeca-lis(stress response, virulence), we conducted aglobal gene expression analysis using RNA-Seq andidentified 50 potential CroR targets involved in multi-ple cellular functions such as cell envelope homeo-stasis, substrate transport, cell metabolism, geneexpression regulation, stress response, virulenceand antibiotic resistance. For validation, CroR directbinding to several candidate targets was demon-strated by EMSA. Further, this work identifiedalr, thegene encoding the alanine racemase enzymeinvolved inE. faecalisresistance to D-cycloserine, apromising antimicrobial drug to treat enterococcalinfections, as a member of the CroR regulon

Identification of new genes associated with intermediate resistance of Enterococcus faecalis to divercin V41, a pediocin-like bacteriocin

International audienceIt has been suggested that resistance to class IIa bacteriocins occurs at either a low or a high level. In listerial strains, low-level resistance (2-4-fold) to class IIa bacteriocins is attributed to alterations in membrane lipid composition. In Listeria monocytogenes and Enterococcus faecalis, high-level resistance (1000-fold) correlates with inactivation of the mptACD operon, which encodes the EII(Man)(t) mannose permease of the phosphotransferase system (PTS). Previous studies reported that in L. monocytogenes, high-level resistance involved the sigma(54) factor and the ManR activator. In this investigation, three genes associated with the resistance of Ent. faecalis JH2-2 to divercin V41, a pediocin-like bacteriocin from Carnobacterium divergens V41, were clearly identified by screening an insertional mutant library of Ent. faecalis JH2-2. These genes correspond to the well-known rpoN gene, which encodes sigma(54) factor, and to genes encoding a glycerophosphoryl diester phosphodiesterase (GlpQ) and a protein with a putative phosphodiesterase function (PDE). Resistance of the three mutants defective in the aforementioned genes appeared to be graduated: the rpoN mutant was more resistant than the glpQ mutant, which was more resistant than the pde mutant. Moreover, this resistance was specific to class IIa bacteriocins

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

International audienceObjectives: 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