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Central venous access devices (CVADs) are an essential component of modern health care. However, their prolonged use commonly results in microbial colonization, which carries the potential risk of hospital-acquired bloodstream infections. These infections complicate the treatment of already sick individuals and cost the existing health care systems around the world millions of dollars. The microbes that colonize CVADs typically form multicellular biofilms that are difficult to dislodge and are resistant to antimicrobial treatments. Clinicians are searching for better ways to extend the working life span of implanted CVADs, by preventing colonization and reducing the risk of bloodstream infections. In this study, we analyzed 210 bacterial and fungal isolates from colonized CVADs or human bloodstream infections from two hospitals geographically separated in the east and west of Canada and screened the isolates for biofilm formation in vitro Twenty isolates, representing 12 common, biofilm-forming species, were exposed to 4% tetrasodium EDTA, an antimicrobial lock solution that was recently approved in Canada for use as a medical device. The EDTA solution was effective at eradicating surface-attached biofilms from each microbial species, indicating that it could likely be used to prevent biofilm growth within CVADs and to eliminate established biofilms. This new lock solution fits with antibiotic stewardship programs worldwide by sparing the use of important antibiotic agents, targeting prevention rather than the expensive treatment of hospital-acquired infections. IMPORTANCE The colonization of catheters by microorganisms often precludes their long-term use, which can be a problem for human patients that have few body sites available for new catheters. The colonizing organisms often form biofilms, and increasingly these organisms are resistant to multiple antibiotics, making them difficult to treat. In this article, we have taken microorganisms that are associated with biofilm formation in catheters from two Canadian hospitals and tested them with tetrasodium EDTA, a new antimicrobial catheter lock solution. Tetrasodium EDTA was effective at eliminating Gram-positive, Gram-negative, and fungal species and represents a promising alternative to antibiotic treatment with less chance of the organisms developing resistance. We expect that our results will be of interest to researchers and clinicians and will lead to improved patient care., (Copyright © 2018 Liu et al.)

The fhuACDB operon, present in a number of Enterobacteriaceae , encodes components essential for the uptake of ferric hydroxamate type siderophores. FhuA acts not only as a transporter for physiologically important chelated ferric iron but also as a receptor for various bacteriophages, toxins, and antibiotics, which are pathogenic to bacterial cells. In this research, fhuA gene distribution and sequence diversity were investigated in Enterobacteriaceae , especially Salmonella and Escherichia Comparative sequence analysis resulted in a fhuA phylogenetic tree that did not match the expected phylogeny of species or trees of the fhuCDB genes. The fhuA sequences showed a unique mosaic clustering pattern. On the other hand, the gene sequences showed high conservation for strains from the same serovar or serotype. In total, six clusters were identified from FhuA proteins in Salmonella and Escherichia , among which typical peptide fragment variations could be defined. Six fragmental insertions/deletions and two substitution fragments were discovered, for which the combination of polymorphism patterns could well classify the different clusters. Structural modeling demonstrated that all the six featured insertions/deletions and one substitution fragment are located at the apexes of the long loops present as part of the FhuA external pocket. These frequently mutated regions are likely under high selection pressure, with bacterial strains balancing escape from phage infection or toxin/antibiotics attack via fhuA gene mutations while maintaining the siderophore uptake activity essential for bacterial survival. The unusual fhuA clustering suggests that high-frequency exchange of fhuA genes has occurred between enterobacterial strains after distinctive species were established., (Copyright © 2018 American Society for Microbiology.)

Pathogenic bacteria often need to survive in the host and the environment, and it is not well understood how cells transition between these equally challenging situations. For the human and animal pathogen Salmonella enterica serovar Typhimurium, biofilm formation is correlated with persistence outside a host, but the connection to virulence is unknown. In this study, we analyzed multicellular-aggregate and planktonic-cell subpopulations that coexist when S. Typhimurium is grown under biofilm-inducing conditions. These cell types arise due to bistable expression of CsgD, the central biofilm regulator. Despite being exposed to the same stresses, the two cell subpopulations had 1,856 genes that were differentially expressed, as determined by transcriptome sequencing (RNA-seq). Aggregated cells displayed the characteristic gene expression of biofilms, whereas planktonic cells had enhanced expression of numerous virulence genes. Increased type three secretion synthesis in planktonic cells correlated with enhanced invasion of a human intestinal cell line and significantly increased virulence in mice compared to the aggregates. However, when the same groups of cells were exposed to desiccation, the aggregates survived better, and the competitive advantage of planktonic cells was lost. We hypothesize that CsgD-based differentiation is a form of bet hedging, with single cells primed for host cell invasion and aggregated cells adapted for persistence in the environment. This allows S. Typhimurium to spread the risks of transmission and ensures a smooth transition between the host and the environment., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Salmonella enterica subsp. enterica serovar Enteritidis is a leading causative agent of gastroenteritis in humans. This pathogen also colonizes the intestinal tracts of poultry and can spread systemically in chickens. Transfer to humans usually occurs through undercooked or improperly handled poultry meat or eggs. The bacterial twin-arginine transport (Tat) pathway is responsible for the translocation of folded proteins across the cytoplasmic membrane. In order to study the role of the Tat system in the infection and colonization of chickens by Salmonella Enteritidis, we constructed chromosomal deletion mutants of the tatB and tatC genes, which are essential components of the Tat translocon. We observed that the tat mutations affected bacterial cell morphology, motility, and sensitivity to albomycin, sodium dodecyl sulfate (SDS), and EDTA. In addition, the mutant strains showed reduced invasion of polarized Caco-2 cells. The wild-type phenotype was restored in all our Salmonella Enteritidis tat mutants by introducing episomal copies of the tatABC genes. When tested in chickens by use of a Salmonella Enteritidis Delta tatB strain, the Tat system inactivation did not substantially affect cecal colonization, but it delayed systemic infection. Taken together, our data demonstrated that the Tat system plays a role in Salmonella Enteritidis pathogenesis.

Salmonella enterica subsp. enterica serovar Enteritidis is a leading cause of human food-borne illness that is mainly associated with the consumption of contaminated poultry meat and eggs. To cause infection, S. Enteritidis is known to use two type III secretion systems, which are encoded on two salmonella pathogenicity islands, SPI-1 and SPI-2, the first of which is thought to play a major role in invasion and bacterial uptake. In order to study the role of SPI-1 in the colonization of chicken, we constructed deletion mutants affecting the complete SPI-1 region (40 kb) and the invG gene. Both DeltaSPI-1 and DeltainvG mutant strains were impaired in the secretion of SipD, a SPI-1 effector protein. In vitro analysis using polarized human intestinal epithelial cells (Caco-2) revealed that both mutant strains were less invasive than the wild-type strain. A similar observation was made when chicken cecal and small intestinal explants were coinfected with the wild-type and DeltaSPI-1 mutant strains. Oral challenge of 1-week-old chicken with the wild-type or DeltaSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection of the liver and spleen was delayed in birds that were challenged with the DeltaSPI-1 strain. These data demonstrate that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of the organism in chickens.

Cells of Escherichia coli take up vitamin B(12) (cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon. The open reading frame termed yadT in the E. coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in Salmonella. The E. coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His(6) tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation in btuF, but not in the flanking genes pfs and yadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC, btuD, or btuF mutants. Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.