According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.

SummaryCampylobacter jejuni is a highly prevalent food-borne pathogen that causes diarrhoeal disease in humans. A natural zoonotic, it must overcome significant stresses both in vivo and during transmission despite the absence of several traditional stress response genes. Although relatively little is understood about its mechanisms of pathogenesis, its ability to interact with and invade human intestinal epithelial cells closely correlates with virulence. A C. jejuni microarray-based screen revealed that several known virulence genes and several uncharacterized genes, including spoT , were rapidly upregulated during infection of human epithelial cells. spoT and its homologue relA have been shown in other bacteria to regulate the stringent response, an important stress response that to date had not been demonstrated for C. jejuni or any other epsilon-proteobacteria. We have found that C. jejuni mounts a stringent response that is regulated by spoT .

The genome sequence of the enteric bacterial pathogen Campylobacter jejuni NCTC 11168 (11168-GS) was published in 2000, providing a valuable resource for the identification of C. jejuni-specific colonization and virulence factors. Surprisingly, the 11168-GS clone was subsequently found to colonize 1-day-old chicks following oral challenge very poorly compared to other strains. In contrast, we have found that the original clinical isolate from which 11168-GS was derived, 11168-O, is an excellent colonizer of chicks. Other marked phenotypic differences were also identified: 11168-O invaded and translocated through tissue culture cells far more efficiently and rapidly than 11168-GS, was significantly more motile, and displayed a different morphology. Serotyping, multiple high-resolution molecular genotyping procedures, and subtractive hybridization did not yield observable genetic differences between the variants, suggesting that they are clonal. However, microarray transcriptional profiling of these strains under microaerobic and severely oxygen-limited conditions revealed dramatic expression differences for several gene families. Many of the differences were in respiration and metabolism genes and operons, suggesting that adaptation to different oxygen tensions may influence colonization potential. This correlates biologically with our observation that anaerobically priming 11168-GS or aerobically passaging 11168-O caused an increase or decrease, respectively, in colonization compared to the parent strain. Expression differences were also observed for several flagellar genes and other less well-characterized genes that may participate in motility. Targeted sequencing of the sigma factors revealed specific DNA differences undetected by the other genomic methods. These observations highlight the capacity of C. jejuni to adapt to multiple environmental niches, the likelihood that this adaptation involves genetic evolution, and provides the first whole-genome molecular exploration of the effect of laboratory culture and storage on colonization and virulence properties of this pathogen.

SummaryCampylobacter jejuni, a prevalent cause of bacterial gastroenteritis, must adapt to different environments to be a successful pathogen. We previously identified a C. jejuni two-component regulatory system (Cj1226/ 7c) as upregulated during cell infections. Analyses described herein led us to designate the system CprRS (Campylobacter planktonic growth regulation). While the response regulator was essential, a cprS sensor kinase mutant was viable. The DcprS mutant displayed an apparent growth defect and formed dramatically enhanced and accelerated biofilms independent of upregulation of previously characterized surface polysaccharides. DcprS also displayed a striking dose-dependent defect for colonization of chicks and was modestly enhanced for intracellular survival in INT407 cells. Proteomics analyses identified changes consistent with modulation of essential metabolic genes, upregulation of stress tolerance proteins, and increased expression of MOMP and FlaA. Consistent with expression profiling, we observed enhanced motility and secretion in DcprS, and decreased osmotolerance and oxidative stress tolerance. We also found that C. jejuni biofilms contain a DNase I-sensitive component and that biofilm formation is influenced by deoxycholate and the metabolic substrate fumarate. These results suggest that CprRS influences expression of factors important for biofilm formation, colonization and stress tolerance, and also add to our understanding of C. jejuni biofilm physiology.

Summary A Campylobacter jejuni two-component signal transduction system (TCSTS), designated dccR-dccSMicroarray analysis identified several genes encoding putative periplasmic and membrane proteins as being regulated by this two-component system; binding of purified His-tagged DccR to the promoter region of two of these genes supports a direct protein-DNA interaction. A conserved repeat sequence was identified in the promoter regions of these genes and in three other promoter regions in the genome, including that of an operon encoding a putative type I secretion system. Two of the regulated target genes were found to be essential for optimal colonization. Both the two-component system and the putative regulated genes have uncharacterized homologues in other Campylobacter and Helicobacter spp., suggesting that they may perform an important function in colonization among a variety of related pathogenic species.

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Bartonella bacteria adhere to erythrocytes and persistently infect the mammalian bloodstream. We previously identified four highly conserved Bartonella quintana adhesin genes that undergo phase variation during prolonged bloodstream infection. The variably expressed outer membrane proteins (Vomp) encoded by these genes are members of the trimeric autotransporter adhesin family. Each B. quintana Vomp appears to contribute a different adhesion phenotype, likely mediated by the major variable region at the adhesive tip of each Vomp. Although studies document that the Vomp adhesins confer virulence phenotypes in vitro, little is known about in vivo virulence strategies of Bartonella. We sought to determine whether the B. quintana Vomp adhesins are necessary for infection in vivo by using a vomp null mutant. It first was necessary to develop a system to generate in-frame deletions of defined genes by allelic exchange in a wild-type Bartonella background, which had not been achieved previously. We utilized sacB negative selection to generate a targeted, in-frame, markerless deletion of the entire vomp locus in B. quintana. We also recently developed the first animal model for B. quintana infection, and using this model, we demonstrate here that the deletion of the entire vomp locus, but not the deletion of two vomp genes, results in a null mutant strain that is incapable of establishing bloodstream infection in vivo. The Vomp adhesins therefore represent critical virulence factors in vivo, warranting further study. Finally, our allelic exchange strategy provides an important advance in the genetic manipulation of all Bartonella species and, combined with the animal model that recapitulates human disease, will facilitate pathogenesis studies of B. quintana.Bartonella species are fastidious, gram-negative bacteria that persistently infect the bloodstream of many mammals. The three major Bartonella pathogens infecting humans are Bartonella quintana, B. henselae, and B. bacilliformis. B. quintana is transmitted by the human body louse and causes relapsing fever ("trench fever"), endocarditis, and the highly vascular lesions of bacillary angiomatosis (13). Bacteremia can persist for months, and unsuspected bloodstream infection with Bartonella can be detected in 5 to 14% of asymptomatic individuals in certain geographic regions (6, 27). B. quintana can cause debilitating, even fatal, illness in immunocompromised individuals with cancer, transplanted organs, or AIDS.Phase and antigenic variation are immune response-evading virulence strategies exploited by microbial pathogens to persist in a host (3). We identified a family of B. quintana proteins that appears to undergo phase variation (28). These surface-localized adhesins, designated Vomp (variably expressed outer membrane proteins), are variably expressed over the course of prolonged bloodstream infection in vivo and are encoded by four highly conserved, tandemly arranged genes (28). These genes, vompD, vompA, vompB, and vompC, are located on a 12.8-kb region of the B. ...