In Vitro and In Vivo Testing of Microbe Growth on Antimicrobial Nursing Scrubs.

Around 5% to 10% of hospitalized patients develop a hospital-acquired infection (HAI). Scrubs are a potential vector of HAIs. To compare the antimicrobial characteristics of scrubs with and without an antimicrobial fabric coating, as tested in the laboratory (in vitro) and hospital (in vivo) environments. Two protocols were conducted to address the purpose. The in vitro protocol was a laboratory study that involved observing the microbe growth after inoculating coated and uncoated scrub fabric swatches with S. aureus and then processing them in moist and dry environments. The in vivo protocol was a clinical trial that measured microbe growth on coated and uncoated scrubs prior to and following nursing staff completing a 12-hr shift on an acute care unit, as measured by colony forming units (CFUs). For high-humidity environments, the in vitro study indicated that swatches treated with an antimicrobial coating exhibited minimal microbe growth, while untreated swatches exhibited significant microbe growth. For low-humidity environments, coated and uncoated swatches were all found to exhibit minimal microbe growth. In the in vivo study, the CFUs increased on scrubs worn by nurses over a 12-hr shift with no significant difference in CFUs for coated and uncoated scrubs. For bacteria in a warm and moist environment, the antimicrobial coating was found to be important for inhibiting growth. For bacteria in a warm and dry environment, both coated and uncoated fabrics performed similarly as measured at 24 hr, with minimal bacterial growth observed. In a hospital environment, microbe growth was observed, but no significant difference was detected when comparing coated and uncoated scrubs. This may have been due to the short time between exposure and culturing the scrubs for analysis immediately at the end of the shift not allowing for enough time to kill or inhibit growth. Contact time between the bacteria and scrub fabric (coated or uncoated) in the in vivo study more directly correlated with the 0-hr observations for the in vitro study, suggesting that the ineffectiveness of the treated scrubs in the clinical results may be due in part to short residence times before collection.

Stress Responses in Pathogenic Vibrios and Their Role in Host and Environmental Survival.

Vibrio is a genus of bacteria commonly found in estuarine, marine, and freshwater environments. Vibrio species have evolved to occupy diverse niches in the aquatic ecosystem, with some having complex lifestyles. About a dozen of the described Vibrio species have been reported to cause human disease, while many other species cause disease in other organisms. Vibrio cholerae causes epidemic cholera, a severe dehydrating diarrheal disease associated with the consumption of contaminated food or water. The human pathogenic non-cholera Vibrio species, Vibrio parahaemolyticus and Vibrio vulnificus, cause gastroenteritis, septicemia, and other extra-intestinal infections. Infections caused by V. parahaemolyticus and V. vulnificus are normally acquired through exposure to sea water or through consumption of raw or undercooked contaminated seafood. The human pathogenic Vibrios are exposed to numerous different stress-inducing agents and conditions in the aquatic environment and when colonizing a human host. Therefore, they have evolved a variety of mechanisms to survive in the presence of these stressors. Here we discuss what is known about important stress responses in pathogenic Vibrio species and their role in bacterial survival.

Characterization of Vibrio cholerae isolates from freshwater sources in northwest Ohio.

Vibrio cholerae is a natural inhabitant of aquatic ecosystems worldwide, typically residing in coastal or brackish water. While more than 200 serogroups have been identified, only serogroups O1 and O139 have been associated with epidemic cholera. However, infections other than cholera can be caused by nonepidemic, non-O1/non-O139 V. cholerae strains, including gastroenteritis and extraintestinal infections. While V. cholerae can also survive in freshwater, that is typically only observed in regions of the world where cholera is endemic. We recently isolated V. cholerae from several locations in lakes and rivers in northwest Ohio. These isolates were all found to be non-O1/non-O139 V. cholerae strains, that would not cause cholera. However, these isolates contained a variety of virulence genes, including ctxA, rtxA, rtxC, hlyA, and ompU. Therefore, it is possible that some of these isolates have the potential to cause gastroenteritis or other infections in humans. We also investigated the relative motility of the isolates and their ability to form biofilms as this is important for V. cholerae survival in the environment. We identified one isolate that forms very robust biofilms, up to 4x that of our laboratory strains. Finally, we investigated the susceptibility of these isolates to a panel of antibiotics. We found that many of the isolates showed decreased susceptibility to some of the antibiotics tested, which could be of concern. While we do not know if these isolates are pathogenic to humans, increased surveillance to better understand the public health risk to the local community should be considered.

Regulated Proteolysis in Vibrio cholerae Allowing Rapid Adaptation to Stress Conditions.

The lifecycle of the causative agent of the severe secretory diarrheal disease cholera, Vibrio cholerae, is characterized by the transition between two dissimilar habitats, i.e., as a natural inhabitant of aquatic ecosystems and as a pathogen in the human gastrointestinal tract. Vibrio cholerae faces diverse stressors along its lifecycle, which require effective adaptation mechanisms to facilitate the survival fitness. Not surprisingly, the pathogen's transcriptome undergoes global changes during the different stages of the lifecycle. Moreover, recent evidence indicates that several of the transcription factors (i.e., ToxR, TcpP, and ToxT) and alternative sigma factors (i.e., FliA, RpoS, and RpoE) involved in transcriptional regulations along the lifecycle are controlled by regulated proteolysis. This post-translational control ensures a fast strategy by the pathogen to control cellular checkpoints and thereby rapidly respond to changing conditions. In this review, we discuss selected targets for regulated proteolysis activated by various stressors, which represent a key feature for fast adaptation of V. cholerae.

Characterization of the Vibrio cholerae Phage Shock Protein Response.

The phage shock protein (Psp) system is a stress response pathway that senses and responds to inner membrane damage. The genetic components of the Psp system are present in several clinically relevant Gram-negative bacteria, including Vibrio cholerae However, most of the current knowledge about the Psp response stems from in vitro studies in Escherichia coli and Yersinia enterocolitica In fact, the Psp response in V. cholerae has remained completely uncharacterized. In this study, we demonstrate that V. cholerae does have a functional Psp response system. We found that overexpression of GspD (EpsD), the type II secretion system secretin, induces the Psp response, whereas other V. cholerae secretins do not. In addition, we have identified several environmental conditions that induce this stress response. Our studies on the genetic regulation and induction of the Psp system in V. cholerae suggest that the key regulatory elements are conserved with those of other Gram-negative bacteria. While a psp null strain is fully capable of colonizing the infant mouse intestine, it exhibits a colonization defect in a zebrafish model, indicating that this response may be important for disease transmission in the environment. Overall, these studies provide an initial understanding of a stress response pathway that has not been previously investigated in V. choleraeIMPORTANCEVibrio cholerae leads a dual life cycle, as it can exist in the aquatic environment and colonize the human small intestine. In both life cycles, V. cholerae encounters a variety of stressful conditions, including fluctuating pH and temperature and exposure to other agents that may negatively affect cell envelope homeostasis. The phage shock protein (Psp) response is required to sense and respond to such insults in other bacteria but has remained unstudied in V. cholerae Interestingly, the Psp system has protein homologs, principally, PspA, in a number of bacterial clades as well as in archaea and plants. Therefore, our findings not only fill a gap in knowledge about an unstudied extracytoplasmic stress response in V. cholerae, but also may have far-reaching implications.

A Periplasmic Antimicrobial Peptide-Binding Protein Is Required for Stress Survival in Vibrio cholerae.

Vibrio cholerae must sense and respond appropriately to stresses encountered in the aquatic environment and the human host. One stress encountered in both environments is exposure to antimicrobial peptides (AMPs), produced as a part of the innate immune response by all multicellular organisms. Previous transcriptomic analysis demonstrated that expression of Stress-inducible protein A (SipA) (VCA0732), a hypothetical protein, was highly induced by AMP exposure and was dependent on a specific uncharacterized two-component system. In order to better understand role of this protein in stress relief, we examined whether it shared any of the phenotypes reported for its homologs. SipA is required for survival in the presence of two other stressors, cadmium chloride and hydrogen peroxide, and it localizes to the bacterial periplasm, similar to its homologs. We also found that SipA physically interacts with OmpA. Importantly, we found that SipA binds AMPs in the bacterial periplasm. This suggests a model where SipA may act as a molecular chaperone, binding AMPs that enter the periplasm and delivering them to OmpA for removal from the cell. While El Tor V. cholerae strains lacking SipA do not show a survival defect in the presence of AMPs, we found that Classical sipA mutants are less able to survive in the presence of AMPs. This phenotype is likely masked in the El Tor background due to a functional lipid A modification system that increases AMP resistance in these strains. In summary, we have identified a protein that contributes to a novel mechanism of stress relief in V. cholerae.

Genome Sequence of Vibrio cholerae Strain D1, Isolated from the Maumee River in Toledo, Ohio.

Vibrio cholerae is a human bacterial pathogen and an inhabitant of aquatic environments. It is endemic to many regions of the world but is typically found in warm climates in saltwater. Here, we present the sequence of a V. cholerae strain isolated from a freshwater river in Ohio.

Vibrio responses to extracytoplasmic stress.

A critical factor for bacterial survival in any environment is the ability to sense and respond appropriately to any stresses encountered. This is especially important for bacteria that inhabit environments that are constantly changing, or for those that inhabit more than one biological niche. Vibrio species are unique in that they are aquatic organisms, and must adapt to ever-changing temperatures, salinity levels and nutrient concentrations. In addition, many species of Vibrio colonize other organisms, and must also deal with components of the host immune response. Vibrio infections of humans and other organisms have become more common in recent years, due to increasing water temperatures in many parts of the world. Therefore, understanding how these ubiquitous marine bacteria adapt to their changing environments is of importance. In this review, we discuss some of the ways that Vibrios sense and respond to the variety of stresses that negatively affect the bacterial cell envelope. Specifically, we will focus on what is currently known about the σE response, the Cpx response and the contributions of OmpU to extracytoplasmic stress relief.

Preparation of Vibrio cholerae Samples for RNA-seq Analysis.

Massively parallel cDNA sequencing (RNA-seq) is a powerful tool for providing an unbiased approach to assess transcript abundance under a variety of conditions. In comparison to microarrays, this technique provides increased resolution and sensitivity and the ability to identify rare transcripts and sRNAs. Here, we describe the sample preparation (based on Illumina technology) used for transcriptomic analysis of V. cholerae cDNA libraries. We describe the entire process from RNA isolation through to the generation of barcoded cDNA libraries ready for sequencing.

Random Transposon Mutagenesis of Vibrio cholerae.

Transposon-based random mutagenesis of bacterial genomes has proven to be a powerful genetic tool for the identification of genes and regulatory elements that contribute to specific phenotypes. One such approach that has been used in Vibrio cholerae for many years is the introduction of mariner transposons to generate random libraries of mutants. These libraries have been successfully used for a wide variety of genetic screens and selections in this important bacterial pathogen. Here we present a detailed protocol for the use of plasmid pFD1 (containing the mariner transposon magellan3) to create mutant libraries in V. cholerae.

Infant Mouse Model of Vibrio cholerae Infection and Colonization.

Cholera is an epidemic diarrheal disease caused by Vibrio cholerae that continues to cause significant morbidity and mortality in many parts of the world. Several different animal models have been used by scientists over the years to study the pathogenesis of cholera. However, the most commonly used is the infant (suckling) mouse model, which has been found to replicate important aspects of human intestinal colonization. Here we present a detailed protocol for using the infant mouse model to assess the colonization of V. cholerae strains using a competition assay.

RAIL: A new tool for defining bacterial promoter regions.

In this issue of the Journal of Bacteriology, Hustmyer and colleagues describe a new method for rapidly generating reporter libraries (Hustmyer citation). This RAIL technique (Rapid Arbitrary PCR Insertion Libraries) uses arbitrary PCR and isothermal DNA assembly to insert random fragments of promoter regions into reporter plasmids, resulting in libraries that can be screened to identify regions required for gene expression. This technique will likely be useful for a number of different genetic applications.

Sterilization of Biofilm on a Titanium Surface Using a Combination of Nonthermal Plasma and Chlorhexidine Digluconate.

Nosocomial infections caused by opportunistic bacteria pose major healthcare problem worldwide. Out of the many microorganisms responsible for such infections, Pseudomonas aeruginosa is a ubiquitous bacterium that accounts for 10-20% of hospital-acquired infections. These infections have mortality rates ranging from 18 to 60% and the cost of treatment ranges from $20,000 to $80,000 per infection. The formation of biofilms on medical devices and implants is responsible for the majority of those infections. Only limited progress has been made to prevent this issue in a safe and cost-effective manner. To address this, we propose employing jet plasma to break down and inactivate biofilms in vitro. Moreover, to improve the antimicrobial effect on the biofilm, a treatment method using a combination of jet plasma and a biocide known as chlorhexidine (CHX) digluconate was investigated. We found that complete sterilization of P. aeruginosa biofilms can be achieved after combinatorial treatment using plasma and CHX. A decrease in biofilm viability was also observed using confocal laser scanning electron microscopy (CLSM). This treatment method sterilized biofilm-contaminated surfaces in a short treatment time, indicating it to be a potential tool for the removal of biofilms present on medical devices and implants.

A putative Vibrio cholerae two-component system controls a conserved periplasmic protein in response to the antimicrobial peptide polymyxin B.

The epidemic pathogen Vibrio cholerae senses and responds to different external stresses it encounters in the aquatic environment and in the human host. One stress that V. cholerae encounters in the host is exposure to antimicrobial peptides on mucosal surfaces. We used massively parallel cDNA sequencing (RNA-Seq) to quantitatively identify the transcriptome of V. cholerae grown in the presence and absence of sub-lethal concentrations of the antimicrobial peptide polymyxin B. We evaluated the transcriptome of both wild type V. cholerae and a mutant carrying a deletion of vc1639, a putative sensor kinase of an uncharacterized two-component system, under these conditions. In addition to many previously uncharacterized pathways responding with elevated transcript levels to polymyxin B exposure, we confirmed the predicted elevated transcript levels of a previously described LPS modification system in response to polymyxin B exposure. Additionally, we identified the V. cholerae homologue of visP (ygiW) as a regulatory target of VC1639. VisP is a conserved periplasmic protein implicated in lipid A modification in Salmonellae. This study provides the first systematic analysis of the transcriptional response of Vibrio cholerae to polymyxin B, raising important questions for further study regarding mechanisms used by V. cholerae to sense and respond to envelope stress.

Assay development and high-throughput screening for small molecule inhibitors of a Vibrio cholerae stress response pathway.

Antibiotics are important adjuncts to oral rehydration therapy in cholera disease management. However, due to the rapid emergence of resistance to the antibiotics used to treat cholera, therapeutic options are becoming limited. Therefore, there is a critical need to develop additional therapeutics to aid in the treatment of cholera. Previous studies showed that the extracytoplasmic stress response (σE) pathway of Vibrio cholerae is required for full virulence of the organism. The pathway is also required for bacterial growth in the presence of ethanol. Therefore, we exploited this ethanol sensitivity phenotype in order to develop a screen for inhibitors of the pathway, with the aim of also inhibiting virulence of the pathogen. Here we describe the optimization and implementation of our high-throughput screening strategy. From a primary screen of over 100,000 compounds, we have identified seven compounds that validated the growth phenotypes from the primary and counterscreens. These compounds have the potential to be developed into therapeutic agents for cholera and will also be valuable probes for uncovering basic molecular mechanisms of an important cause of diarrheal disease.

Evolution of a global regulator: Lrp in four orders of γ-Proteobacteria.

BACKGROUND: Bacterial global regulators each regulate the expression of several hundred genes. In Escherichia coli, the top seven global regulators together control over half of all genes. Leucine-responsive regulatory protein (Lrp) is one of these top seven global regulators. Lrp orthologs are very widely distributed, among both Bacteria and Archaea. Surprisingly, even within the phylum γ-Proteobacteria (which includes E. coli), Lrp is a global regulator in some orders and a local regulator in others. This raises questions about the evolution of Lrp and, more broadly, of global regulators. RESULTS: We examined Lrp sequences from four bacterial orders of the γ-Proteobacteria using phylogenetic and Logo analyses. The orders studied were Enterobacteriales and Vibrionales, in which Lrp plays a global role in tested species; Pasteurellales, in which Lrp is a local regulator in the tested species; and Alteromonadales, an order closely related to the other three but in which Lrp has not yet been studied. For comparison, we analyzed the Lrp paralog AsnC, which in all tested cases is a local regulator. The Lrp and AsnC phylogenetic clusters each divided, as expected, into subclusters representing the Enterobacteriales, Vibrionales, and Pasteuralles. However the Alteromonadales did not yield coherent clusters for either Lrp or AsnC. Logo analysis revealed signatures associated with globally- vs. locally- acting Lrp orthologs, providing testable hypotheses for which portions of Lrp are responsible for a global vs. local role. These candidate regions include both ends of the Lrp polypeptide but not, interestingly, the highly-conserved helix-turn-helix motif responsible for DNA sequence specificity. CONCLUSIONS: Lrp and AsnC have conserved sequence signatures that allow their unambiguous annotation, at least in γ-Proteobacteria. Among Lrp orthologs, specific residues correlated with global vs. local regulatory roles, and can now be tested to determine which are functionally relevant and which simply reflect divergence. In the Alteromonadales, it appears that there are different subgroups of Lrp orthologs, one of which may act globally while the other may act locally. These results suggest experiments to improve our understanding of the evolution of bacterial global regulators.

Regulated intramembrane proteolysis of the virulence activator TcpP in Vibrio cholerae is initiated by the tail-specific protease (Tsp).

Vibrio cholerae uses a multiprotein transcriptional regulatory cascade to control expression of virulence factors cholera toxin and toxin-co-regulated pilus. Two proteins in this cascade are ToxR and TcpP - unusual membrane-localized transcription factors with relatively undefined periplasmic domains and transcription activator cytoplasmic domains. TcpP and ToxR function with each other and two other membrane-localized proteins, TcpH and ToxS, to activate transcription of toxT, encoding the direct activator of toxin and pilus genes. Under some conditions, TcpP is degraded in a two-step proteolytic pathway known as regulated intramembrane proteolysis (RIP), thereby inactivating the cascade. The second step in this proteolytic pathway involves the zinc metalloprotease YaeL; V. cholerae cells lacking YaeL accumulate a truncated yet active form of TcpP termed TcpP*. We hypothesized that a protease acting prior to YaeL degrades TcpP to TcpP*, which is the substrate of YaeL. In this study, we demonstrate that a C-terminal protease called Tsp degrades TcpP to form TcpP*, which is then acted upon by YaeL. We present evidence that TcpH and Tsp serve to protect full-length TcpP from spurious proteolysis by YaeL. Cleavage by Tsp occurs in the periplasmic domain of TcpP and requires residues TcpPA172 and TcpPI174 for wild-type activity.

Single-molecule tracking in live Vibrio cholerae reveals that ToxR recruits the membrane-bound virulence regulator TcpP to the toxT promoter.

Vibrio cholerae causes the human disease cholera by producing a potent toxin. The V. cholerae virulence pathway involves an unusual transcription step: the bitopic inner-membrane proteins TcpP and ToxR activate toxT transcription. As ToxT is the primary direct transcription activator in V. cholerae pathogenicity, its regulation by membrane-localized activators is key in the disease process. However, the molecular mechanisms by which membrane-localized activators engage the transcription process have yet to be uncovered in live cells. Here we report the use of super-resolution microscopy, single-molecule tracking, and gene knockouts to examine the dynamics of individual TcpP proteins in live V. cholerae cells with < 40 nm spatial resolution on a 50 ms timescale. Single-molecule trajectory analysis reveals that TcpP diffusion is heterogeneous and can be described by three populations of TcpP motion: one fast, one slow, and one immobile. By comparing TcpP diffusion in wild-type V. cholerae to that in mutant strains lacking either toxR or the toxT promoter, we determine that TcpP mobility is greater in the presence of its interaction partners than in their absence. Our findings support a mechanism in which ToxR recruits TcpP to the toxT promoter for transcription activation.

Genome Sequence of Klebsiella pneumoniae Respiratory Isolate IA565.

Klebsiella pneumoniae is a clinically significant opportunistic bacterial pathogen as well as a normal member of the human microbiota. K. pneumoniae strain IA565 was isolated from a tracheal aspirate at the University of Iowa Hospitals and Clinics. Here, we present the genome sequence of K. pneumoniae IA565.

Imaging live cells at the nanometer-scale with single-molecule microscopy: obstacles and achievements in experiment optimization for microbiology.

Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane.