Filled blue squares represent the relative expression Protein Tyrosine Kinase inhibitor of vjbR and the open light blue squares represent the OD600 of corresponding cultures. The exponential growth stage for microarray analysis corresponds to OD600 = 0.4 (14 hrs) and the stationary growth phase corresponds to OD600

= 1.5 (28 hrs). VjbR and C12-HSL alter expression of a common set of genes To examine the relationship between VjbR and C12-HSL gene regulation, the significantly altered genes from the VjbR regulon were compared to the significantly altered genes from the C12-HSL regulon (Tables 2, 3, 4 and Additional File 3, Table S3). In all, 72 genes were found to be co-regulated during the exponential growth phase and 55 genes at the stationary growth phase, representing approximately 20% of the total number of altered genes identified by microarray analysis. The majority of the common, differently expressed transcripts (124 out of 127) were found to be altered in the same direction by both the vjbR mutant and in response to C12-HSL administration, implying that VjbR and C12-HSL exert inverse effects on gene expression. In addition to the T4SS and flagella operons being inversely co-regulated, T4SS-dependent effector proteins VceA and VceC were also found to be inversely regulated by the vjbR deletion mutant and addition of C12-HSL to

wildtype cells, as well as exopolysaccharide production, proteases, peptidases and a universal stress protein (Table 4). Flagellar and exopolysaccharide selleck compound synthesis genes have (-)-p-Bromotetramisole Oxalate been implicated in the Y-27632 solubility dmso intracellular survival of Brucella in mice and macrophages [4, 41]. The down-regulation of these factors in vjbR mutants and in response to C12-HSL suggests that VjbR promotes Brucella virulence; while conversely, C12-HSL represses such gene expression, either through the same regulatory pathway or independently. These results expand on earlier findings that C12-HSL represses transcription of the T4SS through interactions with the response domain of VjbR [17, 42]. The genes identified as co-regulated between VjbR and C12-HSL may be

the result of C12-HSL reducing VjbR transcriptional activity through the AHL binding domain. Additionally, the observation that the expression of vjbR itself was down-regulated at the stationary growth phase in response to C12-HSL administration further supports a non-cooperative relationship between VjbR and C12-HSL, (2.9-fold by qRT-PCR and 1.2-fold by microarray analysis, Table 1). Physiological characterization of VjbR and C12-HSL transcriptomes Virulence. Microarray results confirmed alteration of the previously identified T4SS and flagellar genes, both virulence-associated operons found to be regulated by VjbR and/or C12-HSL, as well as genes with homology to the recently identified T4SS effector proteins in B. abortus and B. suis [14, 27]. Furthermore, many putative virulence factors not previously correlated with VjbR or C12-HSL regulation in Brucella spp.

Plant Ecol 149:181–193CrossRef Kessler M (2001a) Pteridophyte species richness in Andean forests in Bolivia. Biodivers Conserv 10:1473–1495CrossRef Kessler M (2001b) Patterns of diversity and range size of selected plant groups along an elevational transect in the Bolivian Andes. Biodivers Conserv 10:1897–1921CrossRef Kessler M, Keßler PJA, Gradstein SR et al (2005) Tree diversity in primary forest and different land use systems in Central Sulawesi, Indonesia. Biodivers Conserv 14:547–560CrossRef Kessler M, Abrahamczyk S, Bos M et al (2009) Alpha and beta diversity

of plants and animals along a tropical land-use gradient. Ecol Appl 19:2142–2156PubMedCrossRef Kluge J, Kessler M, Dunn RR (2006) What drives elevational patterns of diversity? drug discovery A test of geometric constraints, climate and species pool effects for pteridophytes on an elevational gradient in Costa Rica. Inhibitor Library Global Ecol Biogeogr 15:358–371CrossRef Kluge J, Bach K, Kessler M (2008) Elevational distribution and zonation of tropical pteridophyte assemblages in Costa Rica. Basic Appl Ecol 9:35–43CrossRef Legendre P, Legendre L (1998) Numerical Ecology, vol 2. Elsevier, Amsterdam, pp 557–558 McCain CM (2009) Global analysis of bird elevational diversity. Global Ecol Biogeogr 18:346–360CrossRef McCune B,

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p-type Si Protein Tyrosine Kinase inhibitor wafers with resistivity of 15 to 25 Ω cm are used, which are previously pre-structured with a quadratic array of pits with 3-μm pitch Selleck Lazertinib by contact lithography,

reactive ion etching, and chemical anisotropic etching. The electrolyte consists of 5 wt% hydrofluoric acid (HF) in N,N′-dimethylformamide (DMF) and 8.2 g polyethyleneglycol (PEG) 3400 per liter electrolyte. The electrolyte temperature is kept constant at 17°C, while it is pumped through the etching cell at a rate of 600 mL/min. (b) After their production, the pores are over-etched to produce the desired wires. A common etchant is composed of 100 mL of a 0.45 wt% aqueous solution of KOH and 2 g of PEG 3400. The temperature is kept at 50°C. (c) The solution for the chemical deposition of Cu is prepared with 2 mL HF 48%, 98 mL H2O, and 1.9 g CuSO4 · 5H2O. The deposition is performed at 30°C. (d) The electrochemical Cu deposition is performed using a solution find more composed of 2.5 g CuSO4, 9.6 mL H2SO4, and 100 mL H2O. The deposition is done with a constant current of 5 mA/cm2 at 20°C. Standard anodes have Si microwires with quadratic

cross section of 1 μm × 1 μm and length of 70 μm [2]. Figure 2 Current profile used for the electrochemical etching of pores to produce wires. The solid line indicates the profile used for the fabrication of the ‘standard’ wires of 70 μm in length. The dashed line indicates the case for producing longer wires. Battery cycling tests were performed using half-cells, with Li metal as counting and reference electrode. The separator was a glass fiber filter from Whatman (Piscataway, NJ, USA), with pores of 1 μm. The electrolyte was LP-30, consisting of dimethyl carbonate and ethylene carbonate (1:1) plus 1 mol/L of LiPF6. The tests were

done with a BatSMALL battery charging system from Astrol Electronic AG (Othmarsingen, Switzerland). The anodes were cycled in a galvanostatic/potentiostatic mode, for which the voltage limits 0.11 V for lithiation and 0.7 V for delithiation were set. By this mode, when the voltage limit is reached, the cycling is switched to potentiostatic mode, and this mode finishes when the current has decreased to 10% of its initial value or when the capacity limit is reached. SEM observations were performed with an Ultra Plus SEM from Zeiss (Oberkochen, PD184352 (CI-1040) Germany). Results and discussion Scalable processing Aiming to prove that the previously described method is scalable to produce anodes with longer microwires or larger areas, different anodes were prepared. To prepare anodes with different wire lengths, the main parameter to be varied is the electro-chemical etching time between the two narrow sections of the pores. The current profile of Figure  2, in dashed line, is used to prepare larger wires than the standard ones; for this purpose, the etching time has been extended. It is clear that additionally the current density has to be reduced in depth in order to take into account the diffusion limitation of etchant.

Conclusion In this study we show that siderophore-mediated iron uptake is important for the virulence of P. luminescens to insect larvae. This is similar to what has been reported for other pathogens and further highlights the relevance of Photorhabdus as a model for studying bacteria-host interactions [43]. Moreover, in contrast to what we previously reported in another species of Photorhabdus (P. temperata K122) [11], we show that siderophore-mediated

selleck chemical iron uptake in P. luminescens TT01 is not required for the growth and development of the nematode. Therefore it appears that different Photorhabdus-Heterorhabditis complexes have specific requirements for iron. In addition we show that the yfeABCD operon (encoding the Yfe divalent cation transporter)

is required for virulence in some, but not all, insect hosts. Although the Yfe transporter can mediate the uptake of either Fe2+ or Mn2+ we have shown that this transporter is involved in iron uptake during pathogenicity. On the other hand we present data that suggests that the Yfe transporter may be involved in Mn2+-uptake during growth in the gut lumen of the IJ nematode. Therefore, the substrate specificity of the Yfe www.selleckchem.com/CDK.html transporter in P. luminescens TT01 appears to be dependent on the invertebrate host colonized by the bacteria. Methods Bacterial strains and growth conditions Strains used in this study are listed in Table 3. Photorhabdus temperata K122,

Photorhabdus luminescens subsp laumondii TT01 and Escherichia coli strains were routinely cultured in Luria-Bertani (LB) broth or on LB agar and were incubated at 30°C or 37°C respectively. CAS agar, for the detection of siderophores, was prepared by adding CAS solution (1:10 (v:v)) into the LB agar just before pouring. CAS solution was prepared as described previously [11]. When required antibiotics were added at the following final concentrations: kanamycin (Km) 50 μg/ml, ampicilin (Amp) 100 μg/ml, chloramphenicol (Cm) 20 μg/ml and rifampicin Anidulafungin (LY303366) (Rif) 100 μg/ml. Table 3 Bacterial strains used in this study Strain Genotype Reference Photorhabdus     P. temperata (Pt) K122 Spontaneous RifRmutant Joyce and www.selleckchem.com/products/R406.html Clarke, 2003 P. luminescens (Pl) TT01 Spontaneous RifRmutant Bennett and Clarke, 2005 BMM417 K122 exbD::Km Watson and Clarke, 2005 BMM430 TT01 ΔexbD This study BMM431 (Δyfe) TT01 ΔyfeABCD This study BMM432 (Δfeo) TT01 ΔfeoABC This study BMM433 TT01 ΔexbD Δyfe This study BMM434 TT01 ΔexbD Δfeo This study BMM435 TT01 Δfeo Δyfe This study BMM436 TT01 ΔexbD Δfeo Δyfe This study E.coli     S17-1(λpir) lysogenised with λpir, replication of ori R6K Laboratory stock Construction of deletions in exbD, feoABC and yfeABCD Targeted deletion mutants were constructed as previously described [10].

Nevertheless, very few strains have been analyzed for some of these serogroups (O2, O14, O18, O25, O159, and O166) due to the nature of the strains isolated from the intestinal

mucosa, thus no robust conclusions can be extracted for them. Distribution of virulence-associated genes and phylogroups within biofilm producers Of the 65 E. coli strains used in this study, 45 (69.2%) harboured more than two virulence-associated genes in addition to fimH; thus, these strains are considered an extraintestinal pathogenic E. coli according to the definition of Johnson et al [21]. Virulence-associated gene distribution was similar between biofilm producers (moderate-strong) and non-biofilm producers (weak), with the exception of adherence factor sfa/focDE (S or F1C fimbriae) and the invasion-associated Proteases inhibitor see more gene ibeA (Table 4), which were more prevalent in mTOR inhibitor biofilm-forming strains (P = 0.003 and P = 0.017, respectively). Table 4 Comparison of virulence gene prevalence and phylogroup between weak and moderate-strong biofilm producers.       Biofilm formation category     Total (N = 65) Moderate-Strong

(N = 26) Weak (N = 39) P Virulence gene N (%) N (%) N (%)   Adhesin-encoding genes papC 32 (49.2) 11 (42.3) 21 (53.8) 0.255 sfa/focDE 13 (20.0) 10 (38.5) 3 (7.7) 0.003 afa/draBC 8 (12.3) 2 (7.7) 6 (15.4) 0.301 fimH 62 (95.4) 26 (100) 36 (92.3) 0.209 fimAv MT78 14 (21.5) 6 (23.1) 8 (20.5) 0.520 Protectin/invasion-encoding genes ibeA 9 (13.8) 7 (26.9) 2 (5.1) 0.017 K1 neuC 9 (13.8) 3 (11.5) Telomerase 6 (15.4) 0.478 Siderophore-related genes iucD 37

(56.9) 13 (50.0) 24 (61.5) 0.253 Toxin-encoding genes hlyA 15 (23.1) 9 (34.6) 6 (15.4) 0.067 cnf1 15 (23.1) 9 (34.6) 6 (15.4) 0.067 cdtB 5 (7.7) 3 (11.5) 2 (5.1) 0.312 Phylogroup A 9 (13.8) 1 (3.8) 8 (21.1) 0.052 B1 8 (12.3) 3 (11.5) 5 (13.2) 0.583 B2 34 (52.3) 21 (80.8) 13 (34.2) < 0.001 D 13 (20.0) 1 (3.8) 12 (31.6) 0.006 Although the E. coli collection studied was mainly composed of B2 (52.3%) and D (20%) phylotypes, significant differences were observed between the two categories of biofilm producers. As shown in Table 4, the B2 phylogroup was more frequent in moderate-strong biofilm forming strains (80.8% vs. 34.2%; P < 0.001), whereas A and D phylogroups were more frequent within weak biofilm producers. Discussion In this work, we describe the biofilm formation capacity of a recently described pathovar, adherent-invasive E. coli (AIEC), which is associated with Crohn’s disease. The main result was that AIEC strains have stronger biofilm formation abilities than other E. coli strains isolated from the intestinal mucosa (non-AIEC).

2005a,

b). In particular coffee and cacao agroforestry, two globally important agricultural systems, receive growing attention for their potential in conservation of biodiversity (Perfecto et al. 1996; Klein et al. 2002; Tylianakis et al. 2006; Perfecto et al. 2007; Steffan-Dewenter et al. 2007). They can provide appropriate surrogate click here habitats for many forest species, but the composition of these habitats is crucial for the maintenance of a native species community (Dietsch et al. 2007). Agroforestry systems include a range of different land-use intensities, from a diverse shade tree community containing primary forest tree species and a dense canopy cover to plantations with only a few planted shade tree species and low canopy cover (Perfecto et al. 2007). High biodiversity in agricultural selleck products landscapes is particularly important

for the maintenance of ecosystem services, such as pollination (Kremen et al. 2002; Klein et al. 2003a; Ricketts et al. 2008) and the most important taxon performing this ecosystem service is the family Apidae (Klein et al. 2007). As the European honeybee (Apis mellifera L.) is declining world wide, there is an increasing reliance on diverse wild bee communities for pollinating Tariquidar cash crops (Kearns et al. 1998; Klein et al. 2003a; Kremen et al. 2004; Klein et al. 2007). Studies relating the influence of disturbance and land-use intensity in different habitats to bee species composition apparently reach opposite conclusions. Agricultural intensification leads to reduced species richness and abundance of the native bee community in North American watermelon Isotretinoin fields (Kremen et al. 2002), and high anthropogenic disturbance lowered species richness of stingless bees in tropical forest habitats (Cairns et al. 2005). In contrast, bee species richness increased with decreasing forest cover in the landscape and was highest in agricultural fields compared to extensive forest, which resemble the natural habitat in a pine oak heath in a study of Winfree

et al. (2007). Similarly, bee species richness was higher in disturbed forests, compared to primary forest, in tropical Southeast Asia (Liow et al. 2001). Comparative studies of a broad range of habitats along a land-use intensification gradient from primary forests, managed agroforestry systems differing in land-use intensity to openland, and their relative importance for bee species richness are missing, but required to clarify these mixed results. In this study, we hypothesized agroforestry systems to increase species richness and density of bees compared to primary forest due to increased floral density of herbs (including cash crops) and high management diversity. Furthermore, agroforestry systems might maintain higher species richness and density compared to openland, because forested habitats with open canopy offer both floral rewards and suitable nesting sites for wood-nesting bee species (Klein et al. 2003b).

The two other groups included either two distinct COI groups learn more of B. tabaci ASL and AnSL or individuals from two different host check details species : B. tabaci (with Ms genetic group individuals from Madagascar, Tanzania and Reunion) and T. vaporariorum (Tables

3, 4). Comparative analysis of the genetic divergence of these groups at the three loci (Tables 3, 4) revealed that the group composed of ASL and AnSL individuals is the most polymorphic (π = 0.0068), while the Q2 group is highly homogeneous despite several sampling origins (Table 1). Overall, DNA polymorphism was rather low with an average value of group π means of 0.002. Phylogenetic relatedness of Arsenophonus strains from other insects species The Arsenophonus isolates observed in our B. tabaci samples proved to be phylogenetically very close to the Arsenophonus strains found in other insect species (Figure 3). One clade, composed of T. vaporariorum, B. afer, the B. tabaci groups Ms, Q2, and some individuals belonging to ASL, fell into the Aphis sp. and Triatoma sp. Arsenophonus clade described by Duron et al. [17]. The other clade was comprised mainly Arsenophonus infecting Hymenoptera (Nasonia vitripennis, Pachycrepoideus vindimmiae, Muscidifurax uniraptor) and the dipteran Protocalliphora azurea. Discussion In this paper we report on a survey

of the Arsenophonus bacterial symbiont in whitefly species, and in particular in B. tabaci. The data revealed considerable within-genus diversity at this fine host taxonomic level. Previous studies conducted in several arthropod species have found Selleck MX69 Arsenophonus to be one of the richest and most widespread symbiotic bacteria in arthropods [9, 15]. However, those studies were performed with 16S rRNA, which is present in multiple copies

in the genome of the bacterium [25] and has proven to be a marker that is highly sensitive to methodological artifacts, leading to an overestimation of the diversity [15]. The phylogenetic analyses performed on concatenated sequences of three Arsenophonus genes from whiteflies identified two well-resolved clades corresponding to the two clades obtained in the MLST study performed by Duron et al. on a larger insect species scale [17]. One clade was composed of Arsenophonus lineages from three B. tabaci genetic groups Decitabine nmr (Ms, ASL, Q2), T. vaporariorum and B. afer, and strains found in other Hemiptera. The other clade, initially clustering Arsenophonus strains found in Hymenoptera and Diptera, also contained whitefly symbionts of the AnSL, ASL and Q3 genetic groups of the B. tabaci species complex. This clade thus combines insect hosts from phylogenetically distant taxa. The lineages of Arsenophonus from this clade were most likely acquired by whiteflies more recently through lateral transfers from other insect species. The genetic groups of B.

We expect that our results will facilitate further experimental studies of the tunable MMs and make this technique suitable for tuning of plasmon resonance in the optical regime. Acknowledgements We acknowledge the financial support from MAPK Inhibitor Library manufacturer National

Natural Science Foundation of China (grant nos. 61172059, 51302026), PhD Programs Foundation of the Ministry of Education of China (grant no. 20110041120015), Postdoctoral Gathering Project of Liaoning Province (grant no. 2011921008), and The Fundamental Research for the Central University (grant no. DUT12JB01). References 1. Pendry JB: Negative refraction makes a perfect lens. Phys Rev Lett 2000, 61:3966–3969.CrossRef 2. Qiu CW, Gao L: Resonant light scattering by small coated nonmagnetic spheres: magnetic resonances, negative refraction and

prediction. J Opt Soc Am B 2008, 25:1728–1737.CrossRef 3. Shalaev VM: Optical negative-index metamaterials. Nat Photonics 2007, 1:41–48.CrossRef 4. Soukoulis CM, Wegener M: Past achievements and future challenges in the development of three-dimensional photonic metamaterials. Nat Photonics 2011, 5:523–530. 5. Zheludev N: The road ahead for metamaterials. Science 2010, 328:5582–5583.CrossRef 6. Zhou S, Huang X, Li Q, Xie YM: A study of shape optimization HDAC inhibitor drugs on the metallic nanoparticles for thin-film solar cells. Nanoscale Res Lett 2013, 8:447.CrossRef 7. Liaw JW, Chen HC, Kuo MK: Plasmonic Fano resonance and dip of Au-SiO 2 -Au nanomatryoshka. Nanoscale Res Lett 2013, 8:468.CrossRef 8.

Zhang S, Fan W, Panoiu NC, Malloy KJ, Akt inhibitor review Osgood RM, Brueck SRJ: Experimental demonstration of near-infrared negative-index metamaterials. Phys Rev Lett 2005, 95:137404.CrossRef 9. Li T, Li JQ, Wang FM, Wang QJ, Liu H, Zhu SN, Zhu YY: Exploring magnetic plasmon polaritons in optical transmission through hole arrays perforated in trilayer structures. Appl Phys Lett 2007, 90:251112.CrossRef 10. Valentine J, Zhang S, Zentgraf T, Ulin-Avila E, Genov DA, Bartal G, Zhang X: Three-dimensional optical metamaterial with a negative refractive index. Nature 2008, 455:376–379.CrossRef 11. Minovich A, Neshev DN, Powell DA, Shadrivov IV, Lapine M, Hattori those HT, Tan HH, Jagadish C, Kivshar YS: Tilted response of fishnet metamaterials at near-infrared optical wavelengths. Phys Rev B 2010, 81:115109.CrossRef 12. Zhang S, Fan W, Panoiu NC, Malloy KJ, Osgood RM, Brueck SRJ: Demonstration of metal-dielectric negative-index metamaterials with improved performance at optical frequencies. J Opt Soc Am B 2006, 23:434–438.CrossRef 13. Cao T, Cryan MJ: Study of incident angle dependence for dual-band double negative-index material using elliptical nanohole arrays. J Opt Soc Am A 2012, 29:209–215.CrossRef 14. Pendry JB, Holden A, Robbins D, Stewart W: Magnetism from conductors and enhanced nonlinear phenomena. IEEE Trans Microw Theory Tech 1999,47(11):2075–2084.CrossRef 15.

These cells did not appear to be true pseudohyphae, as they had a highly BMS-907351 aberrant and variable morphology, similar

to that seen in Candida albicans strains defective in cell cycle progression. The numbers of cells with normal and abnormal morphology were quantitated and are shown in Table 1 and Figure 2c and 2d. When compared to wildtype, log phase cultures of the rad54Δ/rad54Δ strain had far fewer normal budding yeast cells, and a large increase in the number of cells exhibiting the abnormal morphology shown in Figure 2b. The elongated pseudohyphal cells displayed an aberrant nuclear morphology with a preponderance of the pseudohyphal cells having an elongated single DAPI staining body stuck in the neck between the two

cell bodies (Figure 2c). Additional nuclear morphologies included apparent anucleate cells (two check details cells with only one nucleus), cells with a nucleus Selleckchem SB431542 in each bud where one nucleus is elongated, and cells with multiple nuclei (Figure 2c). Regarding the pseudohyphal cells, in the single nucleate cells, 9/14 had an elongated single nucleus, and in the cells with two nuclei, 10/20 had one or two elongated nuclei. Table 1 Log phase morphology of Candida albicans mutants Strain Unbudded Budded Abnormal/Pseudohyphae Total Wildtype 108 191 1 300 rdh54Δ/RDH54 111 187 2 300 rdh54Δ/rdh54Δ 78 221 1 300 rad54Δ/RAD54 71 227 1 300 rad54Δ/rad54Δ-1 92 143 65 300 rad54Δ/RAD54(+) 108 191 1 300 DAPI staining of cells also showed additional defects in chromosome segregation in the rad54Δ/rad54Δ strain. There was an increase in G2 doublet cells that have a single nucleus at the neck (Figure 2d). This morphology is suggestive of a DNA damage checkpoint arrest in Saccharomyces cerevisiae [25] and could apply to Candida albicans [26]. These phenotypes were not seen in the rdh54Δ/rdh54Δ strain, showing that these Cediranib (AZD2171) two genes have

different roles in vivo. Additionally, neither the wildtype strain nor the RAD54 reintegration strain showed these aberrant nuclear morphologies. Sensitivity to DNA damage is increased in the Candida albicans rad54Δ/rad54Δ mutant In Saccharomyces cerevisiae, deletion of RDH54 and RAD54 leads to increased sensitivity to DNA damage. The Saccharomyces cerevisiae haploid rad54Δ is highly sensitive to methyl methanesulfonate (MMS) [19], but the Saccharomyces cerevisiae RDH54 gene does not appear to have as strong of a role in haploid cells, as deletion of RDH54 only increases MMS sensitivity in diploids at normal MMS concentrations [27]. To test the effect of deletion of Candida albicans RAD54 and RDH54 on MMS and menadione sensitivity, spot dilution assays were performed on YPD agar plates containing a range of MMS concentrations from 0.0025% to 0.02%, or menadione concentrations from 0.05 mM to 0.5 mM.

Clin Tariquidar cell line Microbiol Infect 2007,13(11):1048–1057.CrossRefPubMed 18. Zaiß NH, Weile J, Ackermann G, Kuijper E, Witte W, Nübel U: A case of Clostridium difficile-associated disease due to the highly virulent clone of Clostridium difficile CX-6258 cell line PCR ribotype 027, March 2007 in Germany. Euro Surveill 2007,12(11):E071115.1.PubMed 19. van Belkum A, Tassios PT, Dijkshoorn L, Haeggman

S, Cookson B, Fry NK, Fussing V, Green J, Feil E, Gerner-Smidt P, et al.: Guidelines for the validation and application of typing methods for use in bacterial epidemiology. Clin Microbiol Infect 2007,13(Suppl 3):1–46.CrossRefPubMed 20. Berg RJ, Schaap I, Templeton KE, Klaassen CH, Kuijper EJ: Typing and subtyping of Clostridium difficile isolates by using multiple-locus variable-number Hydroxylase inhibitor tandem-repeat analysis. J Clin Microbiol 2007,45(3):1024–1028.CrossRefPubMed 21. Marsh JW, O’Leary MM, Shutt KA, Pasculle AW, Johnson S, Gerding DN, Muto CA, Harrison LH: Multilocus variable-number tandem-repeat analysis for investigation of Clostridium difficile transmission in Hospitals. J Clin Microbiol 2006,44(7):2558–2566.CrossRefPubMed 22. Fawley WN, Freeman

J, Smith C, Harmanus C, Berg RJ, Kuijper EJ, Wilcox MH: Use of highly discriminatory fingerprinting to analyze clusters of Clostridium difficile infection cases due to epidemic ribotype 027 strains. J Clin Microbiol 2008,46(3):954–960.CrossRefPubMed 23. Killgore G, Thompson A, Johnson S, Brazier J, Kuijper E, Pepin J, Frost EH,

Savelkoul P, Nicholson B, Berg RJ, et al.: Comparison of seven techniques for typing international epidemic strains of Clostridium difficile: restriction endonuclease analysis, PtdIns(3,4)P2 pulsed-field gel electrophoresis, PCR-ribotyping, multilocus sequence typing, multilocus variable-number tandem-repeat analysis, amplified fragment length polymorphism, and surface layer protein A gene sequence typing. J Clin Microbiol 2008,46(2):431–437.CrossRefPubMed 24. Gal M, Northey G, Brazier JS: A modified pulsed-field gel electrophoresis (PFGE) protocol for subtyping previously non-PFGE typeable isolates of Clostridium difficile polymerase chain reaction ribotype 001. J Hosp Infect 2005,61(3):231–236.CrossRefPubMed 25. Stubbs SL, Brazier JS, O’Neill GL, Duerden BI: PCR targeted to the 16S–23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes. J Clin Microbiol 1999,37(2):461–463.PubMed 26. Bidet P, Barbut F, Lalande V, Burghoffer B, Petit JC: Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing. FEMS Microbiol Lett 1999,175(2):261–266.CrossRefPubMed 27. Bidet P, Lalande V, Salauze B, Burghoffer B, Avesani V, Delmee M, Rossier A, Barbut F, Petit JC: Comparison of PCR-ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis for typing Clostridium difficile. J Clin Microbiol 2000,38(7):2484–2487.PubMed 28.