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The purpose of the present study was to examine the inactivation of Salmonella enterica (50 μL; 109 CFU g−1), Listeria monocytogenes (50 μL; 109 CFU g−1), and murine norovirus (MNV-1; 50 μL; 107 50% tissue culture infectious dose (TCID50) mL−1) on whole and fresh-cut strawberries after 2 min disinfection treatments (water (H2O), chlorine 200 mg L−1 (NaClO), water-assisted ultraviolet-C (UV-C) (WUV), and the combination WUV and 40 mg L−1 of PA (WUV + PA)) in a water tank (15 L) equipped with 4 UV-C lamps (17.2 W each), and after 7 days of cold storage (4 and 10 °C). For MNV-1, dry UV-C treatment (DUV) was also tested. For all UV-C treatments, an irradiation dose of 1.3 kJ m−2 was used. RESULTS: When strawberries were washed with WUV, L. monocytogenes and S. enterica were reduced by 2.8 and 2.2 log CFU g−1, respectively. The addition of 40 mg L−1 of PA to WUV (WUV + PA) increased the reduction range of L. monocytogenes and S. enterica by 1.9 and 0.8 log, respectively. Regarding the wash water, no pathogens were recovered after the WUV + PA treatment (detection limit 50 CFU mL−1). Depending on storage conditions (7 days at 4 or 10 °C), reductions observed were 0.5 to 2.0 log for S. enterica and 0.5 to 3.0 log for L. monocytogenes. The reductions in MNV-1 titer after disinfection treatments ranged from 1.3 to 1.7 log. No significant differences between storage conditions were observed for MNV-1: titers did not decline or were reduced up to 0.3 log after 7 days of cold storage. CONCLUSION: The three-way action for disinfecting strawberries by UV-C irradiation and PA, plus the physical removal of the microorganisms by agitated water, are effective against foodborne pathogens on strawberries and water wash. During storage, WUV had a larger impact on the inactivation kinetics of S. enterica. Storage had little impact on MNV-1 inactivation. The authors are grateful to the Spanish government (Ministerio deEconomía y Competitividad, research project AGL2016-78086-R)and the Centres de Recerca de Catalunya (CERCA) Programmeof‘Generalitat de Catalunya’for itsfinancial support. J. Ortiz-Solàthanks the University of Lleida for its PhD grant (BOU186-243/2017 UdL) and M. Anguera thanks them for their techni-cal support. I. Nicolau-Lapeña is in receipt of a predoctoral grantawarded by the‘Ministerio de Economía, Industria y Competitivi-dad’(grant number BES-2017-079779). The authors gratefullyacknowledged the assistance of Gloria Sánchez and Susana Guixfor teaching them the methodologies related to MNV-1 andRAW 264.7 assays.

By comparison of the Proteus mirabilis HI4320 genome with known lipopolysaccharide (LPS) phosphoethanolamine transferases, three putative candidates (PMI3040, PMI3576, and PMI3104) were identified. One of them, eptC (PMI3104) was able to modify the LPS of two defined non-polar core LPS mutants of Klebsiella pneumoniae that we use as surrogate substrates. Mass spectrometry and nuclear magnetic resonance showed that eptC directs the incorporation of phosphoethanolamine to the O-6 of l-glycero-d-mano-heptose II. The eptC gene is found in all the P. mirabilis strains analyzed in this study. Putative eptC homologues were found for only two additional genera of the Enterobacteriaceae family, Photobacterium and Providencia. The data obtained in this work supports the role of the eptC (PMI3104) product in the transfer of PEtN to the O-6 of l,d-HepII in P. mirabilis strains.

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

A group of virulent Aeromonas hydrophila, A. sobria, and A. veronii biovar sobria strains isolated from humans and fish have been described, these strains classified to serotype O11 are serologically related by their lipopolysaccharide (LPS) O-antigen (O-polysaccharide), and the presence of an S-layer consisting of multiple copies of a crystalline surface array protein with a molecular weight of 52 kDa in the form of a crystalline surface array which lies peripheral to the cell wall. A. hydrophila strain AH-1 is one of them. We isolated the LPS from this strain and determined the structure of the O-polysaccharide, which was similar to that previously described for another strain of serotype O11. The genetics of the O11-antigen showed the genes (wbO11 cluster) in two sections separated by genes involved in biosynthesis and assembly of the S-layer. The O11-antigen LPS is an example of an ABC-2-transporter-dependent pathway for O-antigen heteropolysaccharide (disaccharide) assembly. The genes involved in the biosynthesis of the LPS core (waaO11 cluster) were also identified in three different chromosome regions being nearly identical to the ones described for A. hydrophila AH-3 (serotype O34). The genetic data and preliminary chemical analysis indicated that the LPS core for strain AH-1 is identical to the one for strain AH-3.

In this work, we used the eel (Anguilla anguilla) as an animal model to test the hypothesis of Barr et al. (2013a,b) about the putative role of the epidermal mucosa as a phage enrichment layer. To this end, we analyzed the microbial content of the skin mucus of wild and farmed eels by using a metagenomic approach. We found a great abundance of replicating phage genomes (concatemers) in all the samples. They were assembled in four complete genomes of three Myovirus and one Podovirus. We also found evidences that ΦKZ and Podovirus phages could be part of the resident microbiota associated to the eel mucosal surface and persist on them over the time. Moreover, the viral abundance estimated by epiflorescent counts and by metagenomic recruitment from eel mucosa was higher than that of the surrounding water. Taken together, our results support the hypothesis that claims a possible role of phages in the animal mucus as agents controlling bacterial populations, including pathogenic species, providing a kind of innate immunity.

Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle secreted by the Antarctic bacterium Shewanella vesiculosa M7T. Their formation is characterized by the protrusion of both outer and plasma membranes, which pulls cytoplasmic components into the vesicles. To demonstrate that this is not a singular phenomenon in a bacterium occurring in an extreme environment, the identification of O-IMVs in pathogenic bacteria was undertaken. With this aim, a structural study by Transmission Electron Microscopy (TEM) and Cryo-transmission electron microscopy (Cryo-TEM) was carried out, confirming that O-IMVs are also secreted by Gram-negative pathogenic bacteria such as Neisseria gonorrhoeae, Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii AB41, in which they represent between 0.23% and 1.2% of total vesicles produced. DNA and ATP, which are components solely found in the cell cytoplasm, were identified within membrane vesicles of these strains. The presence of DNA inside the O-IMVs produced by N. gonorrhoeae was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. A proteomic analysis of N. gonorrhoeae-derived membrane vesicles identified proteins from the cytoplasm and plasma membrane. This confirmation of O-IMV extends the hitherto uniform definition of membrane vesicles in Gram-negative bacteria and explains the presence of components in membrane vesicles such as DNA, cytoplasmic and inner membrane proteins, as well as ATP, detected for the first time. The production of these O-IMVs by pathogenic Gram-negative bacteria opens up new areas of study related to their involvement in lateral gene transfer, the transfer of cytoplasmic proteins, as well as the functionality and role of ATP detected in these new vesicles.

The human respiratory tract pathogen M. pneumoniae is one of the best characterized minimal bacterium. Until now, two main groups of clinical isolates of this bacterium have been described (types 1 and 2), differing in the sequence of the P1 adhesin gene. Here, we have sequenced the genomes of 23 clinical isolates of M. pneumoniae. Studying SNPs, non-synonymous mutations, indels and genome rearrangements of these 23 strains and 4 previously sequenced ones, has revealed new subclasses in the two main groups, some of them being associated with the country of isolation. Integrative analysis of in vitro gene essentiality and mutation rates enabled the identification of several putative virulence factors and antigenic proteins; revealing recombination machinery, glycerol metabolism and peroxide production as possible factors in the genetics and physiology of these pathogenic strains. Additionally, the transcriptomes and proteomes of two representative strains, one from each of the two main groups, have been characterized to evaluate the impact of mutations on RNA and proteins levels. This study has revealed that type 2 strains show higher expression levels of CARDS toxin, a protein recently shown to be one of the major factors of inflammation. Thus, we propose that type 2 strains could be more toxigenic than type 1 strains of M. pneumoniae. This work was supported by the European Research Council (ERC). This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 634942.

Medical records of 101 blood culture-confirmed bacteremic foals were reviewed to determine whether foals with Actinobacillus sp. bacteremia are affected at an earlier age, have more severe signs of disease, and have a worse prognosis than do foals with bacteremia of other causes. Thirty percent (30/101) of bacteremic foals had Actinobacillus sp. cultured, and these were 2 times more likely to die (crude odds ratio [ORCR] 0.8, 4; P 5 .14), with a survival rate of 43% (13/30) compared to the overall survival rate of 55% (56/101). When compared to other bacteremic foals, foals with actinobacillosis were 7 times more likely to have been sick from birth (adjusted odds ratio [ORADJ] 2, 26; P 5 .003) and 6 times more likely to have diarrhea (ORADJ 1, 22; P 5 .009). By bivariate analysis, foals with Actinobacillus sp. bacteremia were 5 times more likely to have a sepsis score .11 (ORCR 1, 18; P 5 .007), 6 times more likely to be obtunded (ORCR 2, 20; P 5 .005), and 3 times more likely to have pneumonia (ORCR 1, 7; P 5 .03). Furthermore, Actinobacillus sp. bacteremic foals were 27 times more likely to have a segmented neutrophil count ,3.3 3 109 cells/L (ORADJ 4, 166; P , .0001) and were 4.5 times more likely to have a band neutrophil count .0.46 3 109 cells/L (ORADJ 1, 17; P 5 .02) when compared to foals that had bacteremia caused by either gram-negative enteric or gram-positive organisms. Sepsis score was #11 in 49% (29/59) of bacteremia foals aged ,13 days for which a discernible sepsis score was calculable. Results of this study should improve the diagnostic sensitivity of clinical examinations of neonatal foals, thereby facilitating treatment decisions

Vibrio vulnificus is a heterogeneous bacterial species that comprises virulent and avirulent strains from environmental and clinical sources that have been grouped into three biotypes. To validate the typing methods proposed to distinguish clinical from environmental isolates, we performed phenotypic (API 20E, API 20NE, and BIOLOG tests) and genetic (ribotyping and DNA polymorphism at several loci) studies with a large strain collection representing different biotypes, origins, and host ranges. No phenotypic method was useful for biotyping or grouping strains with regard to the origin of an isolate, and only the BIOLOG system was reliable for identifying the strains at the species level. DNA polymorphisms divided the population into three major profiles. Profile 1 strains were vcg type C, 16S rRNA type B, and vvh type 1 and included most of the biotype 1 human septicemic isolates; profile 2 strains were vcg type E, 16S rRNA type A, and vvh type 2 and included all biotype 2 isolates together with biotype 1 isolates from fish and water and some human isolates; and profile 3 strains were vcg type E, 16S rRNA type AB, and vvh type 2 and included biotype 3 strains. Ribotyping divided the species into two groups: one group that included profile 1 biotype 1 isolates and one group that included isolates of all three biotypes with the three profiles described above. In conclusion, no genotyping system was able to distinguish either clinical strains from environmental strains or biogroups within the species V. vulnificus , which suggests that new typing methodologies useful for public health have to be developed for this species.

BACKGROUND: Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. RESULTS: To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. CONCLUSIONS: This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population. Grant support for this work was from FIS PI10/00165, FUNCIS 26/03 from the Gobierno de Canarias “Diagnóstico directo de rickettsiosis prevalentes en nuestro medio (fiebre Q y tifus murino)”, from the “Departamento de Agricultura y Pesca, Gobierno Vasco” “Ensayo de control de la fiebre Q en la cabaña ovina lechera de la CAPV”, INIA FAU2006-00002-C04-01 to -04 “Ecología y control de la fiebre Q: Epidemiología molecular de Coxiella burnetii”, and AGL2010-21273-C03-01-GAN from CICYT “Interacciones-inmuno endocrinas materno-fetal y con Coxiella burnetii en vacas lecheras de alta producción”. Sí

Background Many computational methods are available for assembly and annotation of newly sequenced microbial genomes. However, when new genomes are reported in the literature, there is frequently very little critical analysis of choices made during the sequence assembly and gene annotation stages. These choices have a direct impact on the biologically relevant products of a genomic analysis - for instance identification of common and differentiating regions among genomes in a comparison, or identification of enriched gene functional categories in a specific strain. Here, we examine the outcomes of different assembly and analysis steps in typical workflows in a comparison among strains of Vibrio vulnificus. Results Using six recently sequenced strains of V. vulnificus, we demonstrate the "alternate realities" of comparative genomics, and how they depend on the choice of a robust assembly method and accurate ab initio annotation. We apply several popular assemblers for paired-end Illumina data, and three well-regarded ab initio genefinders. We demonstrate significant differences in detected gene overlap among comparative genomics workflows that depend on these two steps. The divergence between workflows, even those using widely adopted methods, is obvious both at the single genome level and when a comparison is performed. In a typical example where multiple workflows are applied to the strain V. vulnificus CECT 4606, a workflow that uses the Velvet assembler and Glimmer gene finder identifies 3275 gene features, while a workflow that uses the Velvet assembler and the RAST annotation system identifies 5011 gene features. Only 3171 genes are identical between both workflows. When we examine 9 assembly/ annotation workflow scenarios as input to a three-way genome comparison, differentiating genes and even differentially represented functional categories change significantly from scenario to scenario. Conclusions Inconsistencies in genomic analysis can arise depending on the choices that are made during the assembly and annotation stages. These inconsistencies can have a significant impact on the interpretation of an individual genome's content. The impact is multiplied when comparison of content and function among multiple genomes is the goal. Tracking the analysis history of the data - its analytic provenance - is critical for reproducible analysis of genome data.

Background In a previous study, we demonstrated that Vibrio scophthalmi, the most abundant Vibrio species among the marine aerobic or facultatively anaerobic bacteria inhabiting the intestinal tract of healthy cultured turbot (Scophthalmus maximus), contains at least two quorum-sensing circuits involving two types of signal molecules (a 3-hydroxy-dodecanoyl-homoserine lactone and the universal autoinducer 2 encoded by luxS). The purpose of this study was to investigate the functions regulated by these quorum sensing circuits in this vibrio by constructing mutants for the genes involved in these circuits. Results The presence of a homologue to the Vibrio harveyi luxR gene encoding a main transcriptional regulator, whose expression is modulated by quorum–sensing signal molecules in other vibrios, was detected and sequenced. The V. scophthalmi LuxR protein displayed a maximum amino acid identity of 82% with SmcR, the LuxR homologue found in Vibrio vulnificus. luxR and luxS null mutants were constructed and their phenotype analysed. Both mutants displayed reduced biofilm formation in vitro as well as differences in membrane protein expression by mass-spectrometry analysis. Additionally, a recombinant strain of V. scophthalmi carrying the lactonase AiiA from Bacillus cereus, which causes hydrolysis of acyl homoserine lactones, was included in the study. Conclusions V. scophthalmi shares two quorum sensing circuits, including the main transcriptional regulator luxR, with some pathogenic vibrios such as V. harveyi and V. anguillarum. However, contrary to these pathogenic vibrios no virulence factors (such as protease production) were found to be quorum sensing regulated in this bacterium. Noteworthy, biofilm formation was altered in luxS and luxR mutants. In these mutants a different expression profile of membrane proteins were observed with respect to the wild type strain suggesting that quorum sensing could play a role in the regulation of the adhesion mechanisms of this bacterium.