J Appl Ecol 2007, 44:302–311.CrossRef 49. Singh SV, Singh PK, Singh AV, Sohal JS, Gupta VK, Vihan VS: Comparative efficacy of an indigenous ‘inactivated vaccine’ using highly pathogenic field strain of Mycobacterium avium subspecies paratuberculosis ‘Bison type’ with a commercial vaccine for the control of Capri-paratuberculosis in India. Vaccine 2007, 25:7102–7110.CrossRefPubMed 50. Pavlik I, Horvathova A, Dvorska L, Bartl J, Svastova P, du Maine R, Rychlik I: Standardisation of restriction fragment length polymorphism

analysis for Mycobacterium avium subspecies paratuberculosis. J Microbiol Methods 1999, 38:155–167.CrossRefPubMed 51. MRI Mycobacteria Pulsed-Field Gel Electrophoresis Captisol nmr Database[http://​www.​moredun.​ac.​uk/​PFGE-mycobacteria] 52. Selander RK, Caugant DA, Ochman H, Musser JM, Gilmour MN, Whittam TS: Methods of multilocus enzyme electrophoresis for bacterial population genetics and

systematics. Appl Environ Microbiol buy TPCA-1 1986, 51:873–884.PubMed 53. Mazars E, Lesjean S, Banuls AL, Gilbert M, Vincent V, Gicquel B, Tibayrenc M, Locht C, Supply P: High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology. Proc Natl Acad Sci USA 2001, 98:1901–1906.CrossRefPubMed 54. Hunter PR, Gaston MA: Numerical index of the discriminatory ability of typing systems: an application of Simpson’s Index of Diversity. J Clin Microbiol 1988, 26:2465–2466.PubMed 55. Grundmann H, Hori S, Tanner G: Determining confidence intervals when measuring genetic diversity and the discriminatory abilities of typing methods for microorganisms. J Clin Microbiol Interleukin-3 receptor 2001, 39:4190–4192.CrossRefPubMed 56. Thibault VC, Grayon M, Boschiroli ML, Hubbans C, Overduin P, Stevenson K, Gutierrez MC, Supply P, Biet F: New variable-number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: Comparison with IS 900 and IS 1245 restriction fragment length

polymorphism typing. J Clin Microbiol 2007, 45:2404–2410.CrossRefPubMed Authors’ contributions KS conceived of the study, participated in its design and coordination, collated and analysed the data and drafted the manuscript. JA, SD, ZD, KD, IH, LDJ, MK, LM, IP, VT, PW participated in the laboratory and field work. FB, IH, PW and RZ participated in analyzing the data. GFG, DB, JMS, AG participated in the RO4929097 clinical trial conception, design and coordination of the study. All authors read, criticized and approved the final manuscript.”
“Background Staphylococcus aureus is a versatile opportunistic pathogen that causes a variety of infections ranging from superficial skin infections to severe, invasive diseases such as bacteraemia and necrotising pneumonia [1]. Its success as a human pathogen is highlighted by the fact that despite the development of antibiotic therapy, the frequency of staphylococcal bacteraemias is increasing [2].

The level of resistance genes however was differentially affected by antimicrobial treatment. tet (B) in feces from A44 and AS700 were greater than control and T11 treatments, suggesting that chlortetracycline in the diets of animals selected for this determinant.

selleck In contrast, the concentration of tet (C) was greatest in deposited feces from the AS700 treatment. We have previously reported that tet (C) was most prevalent in ampicillin-resistant E. coli isolated from the feces of cattle fed AS700 as compared to A44 and control treatments [12]. The reasons for why the AS700 selects for greater levels of tet (C) are unknown, but may be related to the sulfamethazine in the AS700 treatment. Of the correlations between tet (C) and either sul 1 or su l2, the strongest was observed for the AS700 GW-572016 in vitro treatment, providing support for this theory. Levels of tet (C) in feces from both A44 and T11 were greater than the control, highlighting that tylosin can also select for tet (C), likely through a linkage with a gene conferring resistance to macrolides. It is noteworthy however that there were only weak correlations between tet (C) and the erm genes examined AR-13324 datasheet in our study, perhaps indicating that linkage

was with an additional gene providing resistance to tylosin. Concentrations of tet (M) and tet (W) were clearly higher in feces as compared to the other tetracycline resistance genes. Both tet (M) and tet (W) provide resistance through ribosome protection, a mechanism of resistance 3-oxoacyl-(acyl-carrier-protein) reductase generally attributed to gram positive bacteria [29]. Gram positive bacteria account for the majority of bacteria in the colon [30, 31] offering an explanation as to why tet (M) and tet (W) were detected at higher levels. Previous studies have shown these determinants to be the most abundant in fecal deposits [9, 10, 32]. Interestingly, fecal deposits from cattle fed tylosin had higher concentrations of tet (W). There is evidence that some

erm genes are linked with tet genes [33]. In our study, tet (W) had the strongest correlation to erm (T) and erm (X) in feces from cattle fed tylosin, suggesting that these determinants are linked in certain bacteria. For all fecal treatments, the concentrations of tet (W) declined from initial levels. A previous report found tet (W) to be mainly associated with obligate anaerobes [10], which may explain why there was a constant decline in this determinant in our study. The sulfonamide resistance genes were present in higher numbers in feces from all treatments, increasing over time and in some instances being present at greater concentrations upon completion (day 175) than at initiation (day 7) of the study. Like tetracycline resistance, sulfonamide resistance is also prevalent in many E. coli isolated from agricultural matrices [34]. Surprisingly, levels of sul 1 and sul 2 were greater in A44 feces up to day 14, when compared to the other antibiotic treatments and control samples.

Most GDC-0994 of the strains in Focus F were clustered together, MI-503 order including 14 strains for MT76 and the other six strains presenting in 6 MTs. On the other hand, strains from the same focus were dispersed in the cluster tree. For example, strains isolated from Focus G were dispersed in complex 1, 3 and 4, and strains from Focus C were scattered in complex 1 and 4. MLVA comparison of Yersinia pestis in Yulong and

the adjacent foci Five strains isolated from Yulong, Yunnan had the same MT (MT17: 2-2-2-4-4-7-7-6-2-4-3-3-3-5). Three MTs with a difference in only one locus from MT17 were as follows: MT18 (2-2-2-4-4-7-7-7-2-4-3-3-3-5), including the strains from Foci C and G, had one copy difference on locus M58 with MT17; MT16 (2-2-2-4-4-7-7-6-2-4-3-2-3-5), including a strain which was isolated from Focus H, had one copy difference on locus M51 with MT17; MT29 (2-2-2-4-4-7-7-6-2-4-3-3-3-4), including a strain which was isolated from Focus C, had one copy difference on locus M37 with

MT17. The geographic locations of the natural plague foci adjacent to Yulong were C, E, and F (Figure 3). All the strains from Focus F were Orientalis, and the strains from Foci C, E and Yulong (Focus P) were Antiqua. A further MT comparisons between the Yulong strains and the strains isolated from Foci C and E were as follows: compared with Focus C, It was found that the five Yulong strains and five Focus C strains (belonging to MT29 to MT 33,) were clustered into group D (Figure 1); compared with Focus E, we found one copy selleck chemicals llc difference located at three loci (M66, M58, and M54) in MT35 (major MT) and one copy difference located at four loci (M66, M58, M54,

and M49) in MT23 (major MT); The MST analysis (Figure 2) showed that strains from Foci P, C, and E had a close relationship, and almost all strains belonged to one group. Discussion In 2001, Klevytska et al. performed a systematic, whole genome analysis of Y. pestis Protirelin CO92, and found that TRSs were widespread and randomly distributed in the bacterial chromosomes and plasmids [12]. Subsequent studies had shown that MLVA could distinguish Y. pestis isolated from different natural plague foci [13–15, 20]. Our results showed that the loci selected in this study can distinguish the strains from different natural plague foci and even from the same focus. 214 Y. pestis strains used in this study were divided into 85 MTs. Simpson’s diversity index was 0.9790, indicating that the probability of two unrelated strains being characterized as the same type was 2.10% (1 – 0.9790), showing high resolution and the combination of these 14 loci could be used as a typing method for Y. pestis with the generally accepted probability of 5% of type I errors [21].

This enzyme is important for the ability of bacteria to colonize mucosal membranes in the presence of S-IgA antibodies in saliva [22] and might explain high dominance of these phylotypes in these particular samples. Notably, the

cheek sample from S3 still buy GSK2126458 contained one of the highest counts of taxa (234 phylotypes), but obviously at a very low abundance. Dimensional reduction of the OTU data by principal component analysis (PCA) explained 51% of the total variance among the Selumetinib supplier individual samples by the first three components (Figure 7A-B; PCA loadings and respective taxa are listed in Additional file 7). The greatest component (PC1, 29.7% of variance) discriminated between the samples of dental and mucosal origin, especially in individuals S1 and S3. The second

greatest component (PC2, 12.3% of variance) discriminated all samples of volunteer S3 from the samples of S1 and S2. The third component (PC3, 9.1% of variance) increased the separation of the samples of mucosal and dental origin, e.g. all three tongue samples aligning in the Selleck CP673451 vicinity of each other (Figure 7B), supporting the earlier findings that the tongue has a specific microbial profile [20]. Since saliva is easily and non-invasively accessible it is a popular sample in oral epidemiology and microbiome diversity [4, 16] studies. In our study, the profiles of the saliva samples were closer to communities obtained from mucosal than dental sites, which is in line with the results of a large scale survey on 225 healthy subjects where 40 selected bacterial species were followed using DNA-DNA hybridization technique [23]. Figure 7 Principal Component Analysis Bumetanide results on individual samples. Principal Component Analysis (PCA) results on all individual samples at the level of OTUs clustering sequences at a 3% difference: A) the plot of the PCA axis 1 (accounting for 29.7% of intersample variation) and the axis

2 (12.3% of intersample variation); B) the plot of the PCA axis 1 and the axis 3 (9.1% of intersample variation). Blue dots – samples from individual S1, green dots – samples from individual S2, red dots – individual S3. A – approximal, B – buccal, L – lingual surface of i – incisor or m – molar tooth, respectively. Data were normalized to an equal number of reads per sample and log2 transformed. In order to explore if the location in the oral cavity has an effect on the microbiota of the particular niche (lingual, buccal or approximal surface of the tooth), we sampled two distant teeth – the front tooth and the first molar. No pattern could be found among the samples from individual S2. However, both distantly situated lingual samples from individual S1 and S3, as well as both approximal samples from individual S3, showed higher similarity than the buccal samples of the respective individual (Figure 7A-B).

This avoided the problems resulting from suboptimal or unreliable denaturation learn more associated with standard PCR methods. The effectiveness of the re-designed gyrB/parE primers

and the production of ssDNA during the PCR step were assessed using DNA extracts of various bacterial species. Figure 1 shows the production of ssDNA and the same or even improved sensitivity for bacteria included in the assay panel. Figure 1 Comparison of the amplification efficacy between the gyrB/parE primer pairs of this study (lanes 1, 3, 5, and 7) and those of Roth et al ., (2004) [4] (lanes Blebbistatin clinical trial 2, 4, 6, and 8). The production of ssDNA during the PCR program are shown with the species of E. faecalis (lane 1 and 2), E. faecium (lane 3 and 4). K. pneumoniae (lane 5 and 6), and N. meningitidis (lane 7 and 8) by gel electrophoresis using a 2% agarose gel containing SYBR® Green II. The ssDNA amplicons of gyrB/parE (200 bp) were detected using the primer pair of this study together

with the dsDNA amplicons of gyrB/parE (300 bp). When designing the microarray probes for A. baumannii, E. faecalis, E. faecium, H. influenzae, K. pneumoniae, L. monocytogenes, N. meningitidis, S. aureus, S. epidermidis, S. agalactiae, S. pneumoniae, S. pyogenes, and the ABT-888 order selected SDHB CNS species, we used the gyrB and parE sequences of these bacteria together with those of other clinically

relevant bacteria. The sequence alignments were used to maximize the specific hybridization of the consensus sequences of the targeted bacteria, while minimizing the cross-hybridization of sequences of any non-targeted bacteria. Various in silico parameters were used in the design process to assess the accuracy of the oligonucleotide probes. Annealing potential was predicted by calculating the thermodynamic factors, whereas sequence specificity was evaluated by sequence comparisons and homologue searches of the EBI and NCBI databases using the BLAST algorithm. The oligonucleotide probes for the final microarray layout (Table 1) were chosen from a set of oligonucleotide probes tested in the laboratory. Table 1 Oligonucleotide probes included in the final microarray layout.

Oral Microbiol Immunol 2003,18(4):260–262.PubMedCrossRef 51. Syrjänen SM, Alakuijala L, Alakuijala P, Markkanen SO, Markkanen H: Free amino acid levels in oral fluids of normal subjects and patients with periodontal disease. Arch Oral Biol 1990,35(3):189–193.PubMedCrossRef 52. Steeves CH, Potrykus J, Barnett DA, Bearne SL: Oxidative stress response KPT-8602 order in the opportunistic oral pathogen fusobacterium nucleatum. Proteomics 2011, 11:2027–2037.PubMedCrossRef 53. Zilm PS, Gully N, Rogers A: Growth pH and transient increases in amino acid availability influence polyglucose synthesis by fusobacterium nucleatum grown in continuous culture. FEMS Microbiol Lett 2002,215(2):203–208.PubMedCrossRef

54. White R, Ramezani M, Gharbia S, Seth R, Doherty-Kirby A, Shah H: Stable isotope studies of glutamate catabolism check details in fusobacterium nucleatum. Biotechnol Appl Biochem 1995,22(3):385–396.PubMed 55. Driessen AJM, Rosen BP, Konings WN: Diversity of transport mechanisms: common structural principles. Trends Biochem Sci 2000,25(8):397–401.PubMedCrossRef 56. Lin J, Huang S, Zhang Q: Outer membrane proteins: key players for bacterial adaptation in host niches. Microbes

Infect 2002,4(3):325–331.PubMedCrossRef 57. Epigenetics inhibitor Gelfand MS, Rodionov DA: Comparative genomics and functional annotation of bacterial transporters. Phys Life Rev 2008,5(1):22–49.CrossRef 58. Edwards A, Grossman T, Rudney J: Association of a high-molecular weight arginine-binding protein selleck screening library of fusobacterium nucleatum ATCC 10953 with adhesion to secretory immunoglobulin A and coaggregation with streptococcus cristatus. Oral Microbiol Immunol 2007,22(4):217–224.PubMedCrossRef 59. Kaplan CW, Lux R, Haake SK, Shi W: The fusobacterium nucleatum outer membrane protein RadD Is an arginine-inhibitable adhesin required for inter-species adherence and the structured architecture of multi-species biofilm. Mol Microbiol 2009,71(1):35–47.PubMedCrossRef 60. Liu P-F, Shi

W, Zhu W, Smith JW, Hsieh S-L, Gallo RL, Huang C-M: Vaccination targeting surface FomA of fusobacterium nucleatum against bacterial co-aggregation: implication for treatment of periodontal infection and halitosis. Vaccine 2010,28(19):3496–3505.PubMedCrossRef 61. Shaniztki B, Hurwitz D, Smorodinsky N, Ganeshkumar N, Weiss E: Identification of a fusobacterium nucleatum PK1594 galactose-binding adhesin which mediates coaggregation with periopathogenic bacteria and hemagglutination. Infect Immun 1997,65(12):5231–5237.PubMed 62. Kumar A, Schweizer HP: Bacterial resistance to antibiotics: active efflux and reduced uptake. Adv Drug Deliv Rev 2005,57(10):1486–1513.PubMedCrossRef 63. Saier M, Tam R, Reizer A, Reizer J: Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport. Mol Microbiol 1994,11(5):841–847.PubMedCrossRef 64. Feder ME, Hofmann GE: Heat shock-proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology.

alnea, PS 9 as D. neilliae when using two closely related species, D. citri (PS

11) and D. citrichinenesis (PS 10) as out-group taxa in the combined analysis (Fig. 2). Therefore, the limit of the D. eres species complex was determined to correspond to node 19 in Fig. 2, with nine accepted species, selleckchem and D. citri and D. citrichinensis as basal lineages. Diaporthe pulla (PS 2) and D. helicis (PS 3) appeared to be closely related sister taxa and were closely related to D. eres (PS 1). However, based on the comparison of each single gene tree, these two species diverged from D. eres and each should be recognised as distinct phylogenetic species. Fig. 3 The RAxML phylogram based on combined alignment of 7 genes (ACT, Apn2, CAL, EF1-α, HIS, FG1093 and TUB) of Diaporthe eres species complex. The ML, MP bootstrap values ≥70 %, bayesian PP ≥ 0.75 are indicated above the branches. The tree is rooted with Diaporthe citri (AR3405) and D. citrichinensis (ZJUD034A,

B). Ex-type and ex-epitype cultures are in bold. Epitypes and neotypes designated in this study are indicated with a red squares Phylogenetic EPZ5676 purchase informativeness of each locus The informativeness profiles indicated that the EF1-α, Apn2 and HIS genes are the best markers to resolve the phylogenetic species included in this analysis (Fig. 4). The EF1-α and ACT genes performed the best in terms of phylogenetic informativeness per site. In comparison with the percentage parsimony informative characters of each gene (Table 2), EF1-α (16 %) and ACT (15 %) regions show a congruent result with the phylogenetic informativeness per site. Fig. 4 Profiles of phylogenetic informativeness for the 10 cryptic species compared within D. eres species complex (based on types, epitypes or taxonomically authenticated isolates) and 8 genes included in the study. a) Ultrametric tree generated from the combined analysis of Apn2, ACT, ITS, EF1-α, TUB, learn more CAL, FG1093 and HIS genes b) Net Phylogenetic informativeness c) Phylogenetic informativeness per site. d) key Taxonomy Based on the phylogenetic analyses, the type species of Diaporthe, D. eres, is circumscribed along with eleven closely related but phylogenetically

distinct lineages, each of which is briefly described and illustrated. If a modern description already exists, a reference is given and the species is Apoptosis inhibitor provided with host association, distribution and notes on taxonomy and phylogeny. As listed after the descriptions, type and additional specimens were observed for each species. Epitype specimens were designated for six species. In addition, ex-type, ex-epitype, and additional cultures were observed, if available. Diaporthe eres Nitschke, Pyrenomycetes Germanici 2: 245 (1870), nom. cons. prop. Fig. 5 Fig. 5 Morphology of Diaporthe eres a. Pycnidia on alfalfa stem on WA b. pycnidial necks protruding on alfalfa stem c. conidiophores d, e. α- conidia f. β- conidia g. Ectostroma on the dead twigs of Ulmus sp. h. Perithecia i. Ascomata in section j–q.

04 × MS [105]) and incubated for 3 to 4 hours. For the measurement of the oxidative burst 200 μl aliquots of these suspensions were mixed with phosphate buffer (50 mM potassium phosphate, pH 7.9) and 1.2 mM luminol in the same phosphate buffer. The reaction was started by the addition of 100 μl of 14 mM potassium hexacyanate. The luminescence was measured

with a Luminometer 1250 (BioOrbit, 3-Methyladenine in vitro Turku, Finland). The intensity of luminescence was calibrated for hydrogen peroxide concentrations of 0.01 mM, to 0.05 mM. Chemicals Polygalacturonic acid (sodium salt), pectin and polymyxin B agarose was from Sigma-Aldrich, Taufkirchen, Germany. Unless otherwise specified, other chemicals were obtained from Merck, Darmstadt, Germany. Acknowledgements We gratefully acknowledge Dorothee Steinmann for providing the X. campestris

pv. campestris mutant strain B100-Bac2. Also, we want to thank Dr. Bruno Moerschbacher from the Institut für Biochemie und Biotechnologie in Münster, Germany for the kind permission to use his HPAEC system. At Bielefeld University, the project benefitted from work carried out by, Julia Voß, Sergej Wendler, Anna Köpfer, and Tim Steffens. Jannis Harfmann provided supportive transcriptomics data. Completing the project successfully benefited substantially from oxidative burst measurements carried out by Barbara Samenfeld. This work was financially supported SB-715992 clinical trial by the BMBF program “GenoMik Plus”. We acknowledge support of the publication fee by Deutsche Forschungsgemeinschaft and by the Open Access Publication Funds of Bielefeld University. Electronic supplementary material Additional file

1: Multiple alignment of Xanthomonas exbD2 gene products. (PDF 12 KB) Additional file 2: Figure displaying the recovery of extracellular pectate lyase activities in complemented X. campestris pv. campestris strains originally deficient in genes of the TonB system. (PDF 232 KB) Additional file 3: Table S1 with pectate lyase activity in X. campestris pv. campestris and E. coli strains. (PDF 11 KB) Additional file 4: Figure displaying oxidative burst reactions in heterologous N. tabacum cell suspension cultures upon elicitation with supernatants click here of X. campestris pv. campestris cultures deficient in genes of the TonB system. (PDF 29 KB) Additional file 5: Table S2 with genes of pectin-degrading enzymes in X. campestris pv. campestris B100. (PDF 12 KB) References 1. Jones JD, Dangl JL: The plant immune system. Nature 2006,444(7117):323–329.PubMedCrossRef 2. Boller T, Felix G: A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol 2009, 60:379–406.PubMedCrossRef 3. Bauer Z, Gomez-Gomez L, Boller T, Felix G: Sensitivity of different ecotypes and mutants of Selleck PFT�� Arabidopsis thaliana toward the bacterial elicitor flagellin correlates with the presence of receptor-binding sites. J Biol Chem 2001,276(49):45669–45676.

9 % FM: – 0.5 % Strength: + 2.3 % average Nissen 1996 [7] Untrained SB202190 cost college-aged males Monitored progressive resistance training No Yes 3 weeks, 1.5 or 3 grams per day HMB-Ca No TOBEC for total FFM and FM Strength: Average weight lifted during last 3 working sets of upper and lower body exercises FFM: + 0.6 % FM: No Effect Strength: +2.6 to 17.4 % depending on lift Jowko 2001 [10] Active, college-aged males Monitored progressive resistance training No No 3 weeks, 3 grams per day HMB-Ca 20 grams creatine

per day for 7 days followed by 10 grams per day for 14 days BIA Strength: Cumulative 1-RM of major lifts (Squat, Bench Press, Clean) FFM: + 0.6 % FM: – 0.7 % Strength: + 9 % Kreider 1999[15] Resistance trained, college-aged males males with > 1 year experience Not monitored: Instructed not to change current Go6983 manufacturer individualized training regimens No No 28 days, 3 or 6 grams per day HMB-Ca No DXA for: LBM and FM Strength: Bench Press and Leg Press LBM: No Effect FM: No Effect Strength: No Effect Gallagher 2000[12] Untrained college-aged males

Monitored progressive resistance training No No 8 weeks, 3 or 6 grams per day HMB-Ca No 7 site Skin Fold Isometric and Isokinetic testing, Non-specific to training stimulus FFM: + 3 % FM: – 1.6 % Strength: +2-3.5 % No differences between 3 and 6 g Panton 2000[20] Men and women, divided into untrained and resistance trained (> 6 months), 20–40 yrs of age Monitored high intensity progressive resistance training No No 4 weeks, 3 grams per day HMB-Ca ABT-737 mouse No Underwater

Weighing Bench Press and Leg Press 1-RM FFM: +.5 kg FM: – .6 % Strength: +3-15 % Hoffman 2004[19] 3-oxoacyl-(acyl-carrier-protein) reductase College Football players Football camp, not controlled by investigators No No 10 days, 3 grams per day HMB-Ca No Not Measured Wingate Power No Effects Kraemer 2009[13] Recreationally active, college-aged males periodized resistance training split Yes Yes 12 weeks, 3 grams per day HMB-Ca 14 grams arginine and 14 grams glutamine per day DXA for LBM and FM and Limb Circumference Squat and Bench Press 1RM Vertical Jump LBM: + 40% FM: -40 % Strength: 50 % Power: +85 % Thomson 2009[22] Trained college-aged males Non Monitored Assigned progressive resistance training program with 84 % compliance No No 9 weeks, 3 grams per day HMB-Ca No BIA Bench Press, Preacher Curl, and Leg Extension 1-RM FFM: 0.4 FM: – 3.8 Strength: 1.1-9.0 depending on lift Portal 2011[23] Elite adolescent volleyball players 13.5-18 yrs of age Combination of progressive, resistance, and endurance exercise Not reported No 7 weeks, 3 grams per day HMB-Ca No DXA Power on Wingate Strength of Bench Press and Leg Press Fat: PL = +3.5% Vs. HMB= −6.6% FFM: PL= no change Vs. HMB= +3.

Bacterial strains were grown overnight as shaking cultures in M9 minimal medium. Strains which produced a negative result in this assay were enriched for type 3 fimbriae production by three successive rounds of 48 h static growth in M9 minimal medium and then re-tested. CBL0137 purchase Biofilm study Biofilm formation on polyvinyl chloride (PVC) surfaces was monitored by using 96-well microtitre plates (Falcon) essentially as previously described [16]. Briefly, cells were grown for

24 h in M9 minimal medium (containing 0.2% glucose) or 48 h in synthetic urine at 37°C under shaking conditions, washed to remove unbound cells and stained with crystal violet. Quantification of biofilm TH-302 in vivo mass was performed by addition of acetone-ethanol (20:80) and measurement of the dissolved crystal violet at an optical density of 595 nm. All experiments were performed in a minimum of eight replicates. Immunoblotting and immunogold-labelled electron microscopy Crude cell lysates were prepared from overnight cultures and boiled in acid as previously described [14]. Protein samples were analysed by SDS-PAGE and western blotting as previously described [52] employing a type 3 fimbriae specific antiserum. Immunogold labelling was performed using the same Type 3 fimbriae specific antiserum as previously described

[14]. Cells were examined under a JEOL JEM1010 TEM operated at 80 kV. Images were captured using an mTOR inhibitor analysis Megaview clonidine digital camera. Phylogenetic and sequence analysis PCR products were generated from an internal region of mrkA (416 bp), mrkB (243 bp), mrkC (657 bp) and mrkD (778), respectively, from each of the 33 CAUTI strains and sequenced on both strands. These sequences correspond to nucleotides 112 to 530 of mrkA, 66 to 308 of mrkB, 173 to 829 of mrkC and 157 to 934 of mrkD in the reference strain K. pneumoniae MGH78578 (CP000647). Individual and concatenated gene fragments from the 33 CAUTI strains (and six

additional mrk sequences available at GenBank from strains causing other infections; accession numbers: CP000647, EU682505, CP000964, M55912, CP000822, EU370913) were aligned using ClustalX [53], and subjected to phylogenetic analysis using PHYLIP [54]. Maximum likelihood (ML) trees were built from a concatenated alignment of 2104 nucleotides (comprising 1269 conserved sites and 775 informative sites) using the dnaml algorithm in PHYLIP [54]. A consensus tree of 500 ML bootstrap replicates was prepared using the majority rule method as implemented by Splitstree version 4 [55, 56]. We were unable to amplify mrkD from E. coli M202 and only used the mrkABC concatenated fragments in the analysis. For comparative analysis, the complete mrk cluster (and adjacent regions) from E. coli ECOR28, C. freundii M46 and K. oxytoca M126 were amplified using an inverse PCR strategy and sequenced.