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The glutathione transferase (GST) gene family is divided into 14 classes in photosynthetic organisms. Among them, the Phi class (GSTF) is composed of a large number of genes that are often induced in response to environmental constraints due to their ability to detoxify xenobiotics, to their peroxidase activity and to their involvement in the biosynthesis and/or transport of secondary metabolites. However, the exact functions of GSTFs from many plants including Populus trichocarpa are unknown.Here, following GSTF1 characterization, we have performed a comparative analysis of the seven other GSTFs found in poplar by systematically evaluating the biochemical and enzymatic properties of the corresponding recombinant proteins and of variants mutated for active site residues and by determining the three-dimensional structures of several representatives. Owing to the presence of a cysteine with a pK(a) value around 5 in their active site, GSTF3, F7, and F8 displayed a thiol transferase activity in addition to the usual glutathione transferase and peroxidase activities. From structural analyses, it appeared that these dual biochemical properties originate from the existence of a certain variability in the beta 1-alpha 1 loop. This allows positioning of several active site residues at proximity of the glutathione molecule, which itself remains unchanged in GSTF three-dimensional structures.These results highlight the promiscuity of some GSTFs and that changes of active site residues in some isoforms during evolution generated functional diversity by modifying their activity profile.

Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infection (UTI) in humans. For the successful colonisation of the human urinary tract, UPEC employ a diverse collection of secreted or surface-exposed virulence factors including toxins, iron acquisition systems and adhesins. In this study, a comparative proteomic approach was utilised to define the UPEC pan and core surface proteome following growth in pooled human urine. Identified proteins were investigated for subcellular origin, prevalence and homology to characterised virulence factors. Fourteen core surface proteins were identified, as well as eleven iron uptake receptor proteins and four distinct fimbrial types, including type 1, P, F1C/S and a previously uncharacterised fimbrial type, designated UCA-like (UCL) fimbriae in this study. These pathogenicity island (PAI)-associated fimbriae are related to UCA fimbriae of Proteus mirabilis, associated with UPEC and exclusively found in members of the E. coli B2 and D phylogroup. We further demonstrated that UCL fimbriae promote significant biofilm formation on abiotic surfaces and mediate specific attachment to exfoliated human uroepithelial cells. Combined, this study has defined the surface proteomic profiles and core surface proteome of UPEC during growth in human urine and identified a new type of fimbriae that may contribute to UTI.

Objectives: The objective of this study was to perform a multinational survey of patients' colonization by metallo-beta-lactamase (MBL)-producing Enterobacteriaceae, including their molecular characterization. Methods: Patients in 18 hospital units across Europe and Israel (n = 17945) were screened between mid-2008 and mid-2011. MBL-producing isolates were typed by PFGE and MLST. MBL genes were amplified and sequenced within their integrons. Plasmids with MBL genes were analysed by nuclease S1 plus hybridization profiling, mating and transformation assays, and by PCR-based replicon typing. Results: Ninety-one patients in nine centres (six countries), including 62 patients in two Greek ICUs, carried 94 non-duplicate MBL-producing organisms. Klebsiella pneumoniae isolates from Greece dominated (n = 57) and belonged mainly to ST147, ST36 and ST383. All but one of the isolates expressed VIM-1-type MBLs. Isolates of Greek origins produced five enzymes, including new VIM-39, encoded by class 1 integrons of four types. In-e541-like elements prevailed, comprising six variants located on IncR, IncFII(K), IncR+FIIK, IncR+A/C or non-typeable plasmids. The other group were new In4873 and In4863, being the first In416-like elements identified in Greece, which were present on IncA/C or non-typeable plasmids. Isolates from other countries produced only VIM-1 and the major integron was In916, identified in 16 organisms from France, Italy and Spain. In916 was carried by four plasmid types, including IncA/C, IncFII(K) and IncHI2. Other integrons included a new element, In3103, in Spain and In110 identified only in Latvia. Conclusions: This study provided fully comparable data on the occurrence and molecular characteristics of VIM-producing Enterobacteriaceae in a group of hospital units across Europe, documenting recent changes in their epidemiology.

The non-domesticated Bacillus subtilis strain 3610 displays, over a wide range of humidity, hyper-branched, dendritic, swarming-like migration on a minimal agar medium. At high (70 %) humidity, the laboratory strain 168 sfp + (producing surfactin) behaves very similarly, although this strain carries a frameshift mutation in swrA, which another group has shown under their conditions (which include low humidity) is essential for swarming. We reconcile these different results by demonstrating that, while swrA is essential for dendritic migration at low humidity (30–40 %), it is dispensable at high humidity. Dendritic migration (flagella- and surfactin-dependent) of strains 168 sfp + swrA and 3610 involves elongation of dendrites for several hours as a monolayer of cells in a thin fluid film. This enabled us to determine in situ the spatiotemporal pattern of expression of some key players in migration as dendrites develop, using gfp transcriptional fusions for hag (encoding flagellin), comA (regulation of surfactin synthesis) as well as eps (exopolysaccharide synthesis). Quantitative (single-cell) analysis of hag expression in situ revealed three spatially separated subpopulations or cell types: (i) networks of chains arising early in the mother colony (MC), expressing eps but not hag; (ii) largely immobile cells in dendrite stems expressing intermediate levels of hag; and (iii) a subpopulation of cells with several distinctive features, including very low comA expression but hyper-expression of hag (and flagella). These specialized cells emerge from the MC to spearhead the terminal 1 mm of dendrite tips as swirling and streaming packs, a major characteristic of swarming migration. We discuss a model for this swarming process, emphasizing the importance of population density and of the complementary roles of packs of swarmers driving dendrite extension, while non-mobile cells in the stems extend dendrites by multiplication.

Broad-spectrum β-lactamase genes (coding for extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases) have been frequently demonstrated in the microbiota of food-producing animals. This may pose a human health hazard since these genes may be present in zoonotic bacteria, which would cause a direct problem. They can also be present in commensals, which may act as a reservoir of resistance genes for pathogens causing disease both in humans and animals. Broad-spectrum β-lactamase genes are frequently located on mobile genetic elements, such as plasmids, transposons and integrons, which often also carry additional resistance genes. This could limit treatment options for infections caused by broad-spectrum β-lactam-resistant microorganisms. This review addresses the growing burden of broad-spectrum β-lactam resistance among Enterobacteriaceae isolated from food, companion and wild animals worldwide. To explore the human health hazard, the diversity of broad-spectrum β-lactamases among Enterobacteriaceae derived from animals is compared with respect to their presence in human bacteria. Furthermore, the possibilities of the exchange of genes encoding broad-spectrum β-lactamases – including the exchange of the transposons and plasmids that serve as vehicles for these genes – between different ecosystems (human and animal) are discussed.

Background Swarming motility allows microorganisms to move rapidly over surfaces. The Gram-positive bacterium Paenibacillus vortex exhibits advanced cooperative motility on agar plates resulting in intricate colonial patterns with geometries that are highly sensitive to the environment. The cellular mechanisms that underpin the complex multicellular organization of such a simple organism are not well understood. Results Swarming by P. vortex was studied by real-time light microscopy, by in situ scanning electron microscopy and by tracking the spread of antibiotic-resistant cells within antibiotic-sensitive colonies. When swarming, P. vortex was found to be peritrichously flagellated. Swarming by the curved cells of P. vortex occurred on an extremely wide range of media and agar concentrations (0.3 to 2.2% w/v). At high agar concentrations (> 1% w/v) rotating colonies formed that could be detached from the main mass of cells by withdrawal of cells into the latter. On lower percentage agars, cells moved in an extended network composed of interconnected "snakes" with short-term collision avoidance and sensitivity to extracts from swarming cells. P. vortex formed single Petri dish-wide "supercolonies" with a colony-wide exchange of motile cells. Swarming cells were coupled by rapidly forming, reversible and non-rigid connections to form a loose raft, apparently connected via flagella. Inhibitors of swarming (p-Nitrophenylglycerol and Congo Red) were identified. Mitomycin C was used to trigger filamentation without inhibiting growth or swarming; this facilitated dissection of the detail of swarming. Mitomycin C treatment resulted in malcoordinated swarming and abortive side branch formation and a strong tendency by a subpopulation of the cells to form minimal rotating aggregates of only a few cells. Conclusion P. vortex creates complex macroscopic colonies within which there is considerable reflux and movement and interaction of cells. Cell shape, flagellation, the aversion of cell masses to fuse and temporary connections between proximate cells to form rafts were all features of the swarming and rotation of cell aggregates. Vigorous vortex formation was social, i.e. required > 1 cell. This is the first detailed examination of the swarming behaviour of this bacterium at the cellular level.

An association between swarming and hemolysin BL secretion was observed in a collection of 42 Bacillus cereus isolates ( P = 0.029). The highest levels of toxin were detected in swarmers along with swarm cell differentiation ( P = 0.021), suggesting that swarming B. cereus strains may have a higher virulence potential than nonswarming strains.

Objectives: The purpose of this work was to study the genetic determinants responsible for extended-spectrum beta-lactamase (ESBL) resistance of Salmonella isolated from Dutch poultry, poultry meat and hospitalized humans. Methods: Thirty-four ESBL-resistant Salmonella isolates from The Netherlands were tested towards 21 antimicrobial agents. PCR and sequencing were used to determine the underlying genetic determinants responsible for the ESBL phenotypes. The transferability of the ESBL phenotypes was tested by conjugation to a susceptible Salmonella enterica serovar Dublin and plasmid purification, restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) were employed to further characterize a subset of the isolates. Results: A great genetic diversity was seen among the isolates. The bla(TEM-52) gene was most predominant and was found among Salmonella enterica serovars Blockley, Thomson, London, Enteritidis phage type 14b, Paratyphi B, Virchow and Typhimurium phage types 11 and 507. We also found the bla(TEM-20) gene in S. Paratyphi B var. Java and the bla(TEM-63) gene in S. Isangi. Furthermore, we detected the bla(CTX-M-28) gene in S. Isangi and the bla(CTX-M-3) gene in S. Typhimurium phage type 507. The bla(CTX-M-2) gene was identified in S. Virchow, which also contained a copy of the bla(SHV-2) gene and a copy of the bla(TEM-1) gene. The bla(SHV-12) gene was found alone in S. Concord and together with the bla(TEM-52) gene in S. Typhimurium. Finally, the bla(ACC-1) gene was cloned from a S. Bareilly isolate and was found to be present on indistinguishable plasmids in all S. Bareilly isolates examined as well as in a S. Braenderup isolate and a S. Infantis isolate. Conclusions: Our data underscore the diversity of ESBL genes in Salmonella enterica isolated from animals, food products and human patients.