The corresponding flagella-less S. Dublin mutant did not show this phenotype (CI: 0.91) (Table 3). Table 3 Virulence phenotypes of flagella and chemotaxis mutants of S. Dublin (SDu) and S. Typhimurium (STm) in C57/B6 mice Mutant Challenge routea MRT67307 in vivo CIb S.Du CIb STm cheA p.o. 1.03 1.09 cheB p.o. 0.97 1.05 fliC p.o. 0.46** – fliC i.p. 0.91 – fliC/fljB p.o. – 1.12** fliC/fljB i.p. – 1.78*** a: p.o. = per oral challenge; i.p. = intraperitoneal challenge. b:

The competitive index was calculated as the ratio of mutant to wild type in the spleen 4–5 days post infection divided by the ratio of mutants to wild type strain in the input pool. Indexes where the output was significantly different from the input pool are marked with ** (p<0.01) and *** (p<0.001). Discussion In the current study we used chemotaxis and flagella mutants of the host adapted serovar S. Dublin and corresponding mutants of the broad host range serovar S. Typhimurium to study possible serovar differences in the importance of these genes for host pathogen interaction. The studies were based on defined mutants in one strain of each serovar, and we cannot rule out that there may be strain differences within serovar. The constitutively tumbling cheB

S. Dublin mutant, but not the constitutively smooth swimming cheA LY2603618 mutant, was negatively affected in invasion of epithelial cells. Since cheA has previously been shown to be important for S. Typhimurium cell invasion [20], which we also observed in our studies, S. Typhimurium and S. Dublin apparently differ with respect to the role of cheA in epithelial cell invasion. Lack of flagella (fliC mutation) caused reduced adhesion, which is in accordance with previously reported results for the effect of fliC/fljB mutation in S. Typhimurium [17] and our observations

on the role of flagella in this serotype. It has previously been reported that it is the flagella and not motility, which are important for cell adhesion and invasion [17], but it is currently unknown how precisely flagella influence this in a motility independent way, at least in cell culture experiments. Since we used centrifugation to maximize cell contact, it is also unlikely that our results were caused by reduced motility, which would lead to a reduction in number of contacts between bacteria and cells. Flagella Phenylethanolamine N-methyltransferase in S. Typhimurium are expressed inside epithelial cells and can be demonstrated in infected cultured HeLa cells [21]. During in vivo invasion, the stimulation of TLR-5 by flagellin and the following pro-inflammatory response may be important. However, invasion by S. Typhimurium in cell culture experiments happens within 15 minutes [22], and it is unlikely to be influenced by secretion of stimulating factors. A more likely explanation is down-regulation of SPI1 in flagella mutants, as suggested by Kim et al.[23]. This down regulation can be caused by several regulatory systems, which control both flagella and virulence gene expression [24, 25].

Andrews JM: Determination of minimum inhibitory Milciclib concentrations. J Antimicrob Chemother

2001,48(Suppl 1):5–16.PubMedCrossRef Competing interests The authors declare that they have no competing interests. Authors’ contributions Experiments were carried out by YD, AL, JL, SC, SA, YHD. Data analysis was finished by YD and LHZ. The study was designed by YD and LHZ, who also drafted the manuscript. All authors read and approved the final manuscript.”
“Background Vibrio cholerae, a Gram-negative rod-shaped bacterium belonging to the family Vibrionaceae, induces the acute diarrheal disease cholera. Cholera has pandemic properties and appears mainly in third world countries with estimated 3–5 million cases and more than 100,000 deaths per year [1]. The major pathogenic strains belong to the serogroups O1 and O139. Infections are treated by oral or intravenous rehydration therapy, which

is complemented in severe cases with antibiotics to shorten the duration of the clinical symptoms and to reduce the spreading. Long-term and extensive use of antibiotics has led to resistance development. A growing problem is the emergence of multidrug resistant pathogenic V. cholerae strains against which therapeutic options are more and more limited [2]. Due to this development the availability of novel therapeutic options is urgently needed. In the present study we have developed a high-throughput RGFP966 in vivo screening (HTS) assay that utilizes a V. cholerae reporter strain constitutively expressing green fluorescence protein and screened approximately 28,300 compounds from six different chemical structural groups in a growth inhibition assay. Several active molecules were identified which are active in suppressing growth of V. cholerae in vitro. V. cholerae mutants resistant to the most potent molecule were generated. Whole-genome sequencing and comparative analysis of the mutant to the wild type strain was carried out. The apparent target of the most active compound was identified to be the osmosensitive K+-channel sensor histidine kinase Dapagliflozin KdpD that apparently

exerts certain essential function in this pathogen. Results HTS assay for inhibitors of V. cholerae viability Green fluorescence producing plasmid pG13 was electroporated into V. cholerae strain MO10 and the transformants were selected on LB agar plates containing kanamycin (Km, 30 μg/ml). Transfer of the plasmid pG13 conferred green fluorescence phenotype in V. cholerae O139 strain MO10. The screening assay was optimized in 96- and 384-well microtiter plates (MTP). To differentiate between active and non-active compounds and as controls for the functionality of the assay, ciprofloxacin (Cip, 100 μM) and dimethyl sulfoxide (DMSO, 1%) were included on each plate. DMSO had no growth reducing effect at concentrations up to 1%.

Nucleic Acids Res 2005, 33:D294-D296.PubMedCrossRef 14. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM: The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 2009, 37:D141-D145.PubMedCrossRef 15. Seshadri R, Kravitz SA, Smarr L, Gilna P, Frazier FG-4592 solubility dmso M: CAMERA: a community resource for metagenomics. PLoS Biol 2007, 5:394–397.CrossRef 16. Bru D, Martin-Laurent F, Philippot L: Quantification of the detrimental effect of a single primer-template mismatch by real-time PCR using the 16S rRNA gene as an example. Appl Environ Microb 2008,

74:1660–1663.CrossRef 17. Wu JH, Hong PY, Liu WT: Quantitative effects of position and type of single mismatch on single base primer extension. J Microbiol Meth 2009, 77:267–275.CrossRef 18. Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, Olsen GJ: Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appl Environ Microb 2008, 74:2461–2470.CrossRef

19. Humblot C, Guyot J-P: Pyrosequencing of tagged 16S rRNA gene amplicons for rapid deciphering of the microbiomes of fermented foods selleck kinase inhibitor such as pearl millet slurries. Appl Environ Microb 2009, 75:4354–4361.CrossRef 20. Forney LJ, Gajer P, Williams CJ, Schneider GM, Koenig SSK, McCulle SL, Karlebach S, Brotman RM, Davis CC, Ault K, Ravel J: Comparison of self-collected and physician-collected vaginal swabs for microbiome analysis. J Clin Microbiol 2010, 48:1741–1748.PubMedCrossRef 21. Lauber CL, Hamady M, Knight R, Fierer N: Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale. Appl Environ Microb 2009, 75:5111–5120.CrossRef 22. Bai YH, Sun QH, Zhao C, Wen DH, Tang XY: Bioaugmentation treatment for coking wastewater containing pyridine and quinoline in a sequencing batch reactor. Appl Microbiol Biot 2010, 87:1943–1951.CrossRef 23. Tan YF, Ji GD: Bacterial community structure and dominant

bacteria in activated sludge from a 70 degrees C ultrasound-enhanced anaerobic reactor for treating carbazole-containing wastewater. Bioresource Technol PRKACG 2010, 101:174–180.CrossRef 24. Miller W, Hayes VM, Ratan A, Petersen DC, Wittekindt NE, Miller J, Walenz B, Knight J, Qi J, Zhao F, et al.: Genetic diversity and population structure of the endangered marsupialSarcophilus harrisii(Tasmanian devil). P Natl Acad Sci USA 2011, 108:12348–12353.CrossRef 25. Ayyadevara S, Thaden JJ, Reis RJS: Discrimination of primer 3′-nucleotide mismatch by Taq DNA polymerase during polymerase chain reaction. Anal Biochem 2000, 284:11–18.PubMedCrossRef 26. Huang MM, Arnheim N, Goodman MF: Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. Nucleic Acids Res 1992, 20:4567–4573.PubMedCrossRef 27.

Kuroda M, Ohta T, Uchiyama I, Baba T, Yuzawa H, Kobayashi I, Cui L, Oguchi A, Aoki K, Nagai Y, et al.: Whole genome sequencing of meticillin-resistant Staphylococcus aureus . Lancet 2001, 357 (9264) : 1225–1240.PubMedCrossRef 57. Novick R: Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus . Virology 1967, 33 (1) : 155–166.PubMedCrossRef 58. Horsburgh MJ, Aish JL, White IJ, Shaw L, Lithgow JK, Foster SJ: σ B modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325–4. JBacteriol 2002, 184 (19) : 5457–5467.CrossRef Authors’ contributions MT carried out the phospholipid

analyses and molecular genetic studies, and participated in manuscript preparation. RLO performed the high-salinity survival analyses, Anti-infection chemical and YK performed the antimicrobial peptide susceptibility tests. SLT participated selleck products in the molecular genetic studies. YK, RLO, TO, and SS participated in designing the study. HH conceived of the study with KM and helped to coordinate the study. KM carried out molecular genetic studies, participated in the design and coordination of the study, and helped

to draft the manuscript. All authors have read and approved the final manuscript.”
“Background Burkholderia pseudomallei is a facultative intracellular pathogen responsible for melioidosis, an infectious disease of humans prevalent in Southeast Asia and Northern Australia [1]. Infections in humans may result in a wide range of clinical symptoms and manifestations [2, 3] and in some individuals the bacterium is able to persist with symptoms not shown until several years after exposure [4]. B. pseudomallei has been shown to have a broad host range with disease reported

in animals Grape seed extract ranging from kangaroos to dolphins [5, 6]. However, in the laboratory, the mouse is the most commonly used infection model [7]. Different strains of B. pseudomallei vary markedly in their virulence in murine models of disease. When given by the intraperitoneal (i.p) route, the most virulent isolates have an infectious dose of less than 50 colony forming units (cfu), whereas in the least virulent isolates the infectious dose is over 5,000 cfu [7]. It is not clear whether these differences in virulence in mice are associated with the various clinical outcomes observed in humans. Whilst murine models of infection are valuable for understanding mechanisms of virulence, the behaviour of B. pseudomallei in cell culture systems has been used to characterise the intracellular lifestyle of the bacterium. B. pseudomallei has been shown to be taken up by professional phagocytes including mouse macrophage-like cell lines such as J774 and RAW264 [8, 9] and non-phagocytic cells including HeLa and A549 cells [8]. More recently, other members of the Burkholderia genus including B. thailandensis and B. oklahomensis have been described as being closely related to B. pseudomallei [10, 11]. Indeed, until recently, B.