Whereas the EcoSim analysis suggests an overall signature of negative co-occurrence, Fisher’s Exact test indicates negative and positive co-occurrences for certain species pairings. It is noteworthy that none of the three additional species exhibited negative co-occurrence with M. bolleyi and M. phragmitis in the total data set. Instead, M. bolleyi generally co-occurred significantly more frequently with Ms7Mb4 and Ms43Mb21 than expected

by chance. Such a positive co-occurrence may appear when the conditions that are conducive for one species are also favorable for another species. Alternatively, positive co-occurrence may result MLN8237 in vivo from synergism. On the other hand, there existed an overall negative co-occurrence between Stagonospora sp. and Ms7Mb4, significantly preferring leaves [17] and roots [15], respectively. This could

have resulted from strongly contrasting niche OICR-9429 molecular weight preferences, severe competition for the same substrates or from the secretion of toxins (antagonism). Our results suggest that it is rather unlikely that antagonism by any of the other three fungi is responsible for the differential colonization of roots by Microdochium spp. Since the fungal community on common reed is larger than addressed here, we cannot rule out that other endophytes may SIS3 exert such influences. Conclusions This study supports the concept that niche partitioning allows for differential colonization of common reed by the fungal species investigated. Therefore, Montelukast Sodium a purely neutral model is unlikely to explain the assembly of the mycoflora of common reed. Nonetheless, it remains to be shown to what extent stochastic factors could also contribute to variations in the composition, distribution and diversity of this fungal community. Acknowledgements This work was financially supported by the Deutsche Forschungsgemeinschaft through SFB 454 (Bodenseelitoral). We thank Dr. Jan Nechwatal

(Universität Konstanz) for providing the temperature data for Lake Constance and for discussion of the data. We gratefully acknowledge Dr. Willi Nagl (Universität Konstanz) for advice on statistics, Dr. Ulrike Damm (CBS, Utrecht) for advice on taxonomy, and Michael Koch (Universität Konstanz) for technical help. Electronic supplementary material Additional file 1: Details of isolates studied. This file provides a list of 21 Microdochium isolates used in this study, including accession numbers of ITS sequences and information about their origins. (PDF 11 KB) Additional file 2: Specificity of nested-PCR assays targeting Microdochium spp. This file documents the specificity of the assays employed. A) First PCR step using primers ITS1F and ITS4. M = 100 bp size standard, water: no template DNA included, P. australis: genomic DNA of axenically grown reed plants, genomic DNAs from fungal isolates 4/97-9 (Humicola sp.), 6/97-38 (Chaetomium sp.), 6/97-54 (Fusarium sp.), A4 (Fusarium sp.), 5/97-16 (Microdochium phragmitis), 5/97-54 (M.

HPLC analysis of benzylpenicillin was performed in an Agilent 1100 HPLC system with an analytical 4.6 × 150 mm (5 μm) ZORBAX Eclipse XDB-C18 column (Agilent Technologies), a flow rate of 1 ml/min and a detector wavelength of 214 nm. Samples (20 μl) were injected and eluted using as mobile phase Buffer A (30 mM ammonium formate pH 5.0 and 5% acetonitrile) and Buffer B (same as Buffer

A plus acetonitrile 20:80, v/v) with an isocratic method (85% of A). Benzylpenicillin showed a retention time of 8.69 ± 0.14 min and its detection limit was 0.1 μg/ml. NMR analyses of penicillin from filtrates Analysis of CA-4948 ic50 β-lactams produced by the ial null mutant was done by quantitative 1H NMR at 600 MHz on a Bruker Avance 600 spectrometer. To a known quantity of filtrate, a known AZD1390 clinical trial quantity of internal standard (maleic acid), dissolved in phosphate buffer was added prior to lyophilisation. The residue

was dissolved in D2O and measured at 300 K. The delay between scans (30 s) was more than 5 times T1 of all compounds, so the ratio between the integrals of the compounds of interest and the integral of the internal standard is an exact measure for the quantity of the β-lactams. Tideglusib cost Overexpression of the penDE and ial genes in E. coli and SDS-PAGE of the aminophylline proteins The penDE and ial genes were overexpressed in E. coli JM109 (DE3) cells using 0.5 mM IPTG for 6 h at 26°C. Protein samples to be analysed by SDS-PAGE were diluted in loading buffer (60 mM Tris-HCl

pH 6.8, 2% SDS, 100 mM DTT, 10% glycerol and 0.1% bromophenol blue), boiled for 5 min, and run in a 12% acrylamide gel. The “”Precision Plus Protein All Blue Standards”" (Bio-Rad, Hercules, CA, USA), was used as molecular mass marker. Proteins were stained using Coomassie Brilliant Blue R250 dying. Determination of the in vitro phenylacetyl-CoA: 6-APA acyltransferase activity Measurement of the phenylacetyl-CoA: 6-APA acyltransferase activity in vitro was carried out using soluble extracts obtained from E. coli strains overexpressing either the penDE or the ial genes. Briefly, 72 μl of cell extracts were mixed with 48 μl of the reaction mixture (0.1 M Tris-HCl pH 8.0, 0.05 M DTT, 0.2 mM 6-APA and 0.2 mM phenylacetyl-CoA) and incubated at 26°C for 15 minutes. The reaction was stopped with 120 μl of methanol, centrifuged at 10,000 × g for 5 minutes and biossayed using Micrococcus luteus as test microorganism. Biossays were performed as previously described [26]. Appendix Primers used in this work.

Conclusions We could show that the phage JG024 belongs to the PB1-like phages and shares several characteristic features of this group. These phages are widespread in nature and very successful. A new member of this group, phage JG024, was isolated and characterized. General growth characteristics as well as the genome were investigated, showing that JG024 is able to pass one infection cycle in approximately 50 min. Genome analysis revealed the strong relatedness to the PB1-like phages.

Moreover, we could show that JG024 has broad spectrum activity with a prevalence to clinical isolates. Also, infection of the host P. aeruginosa was even possible under challenging conditions like the ASM medium which mimics the CF lung. High viscosity and microcolony growth of the host were only small obstacles for JG024 to infect and multiply under these conditions. These Blasticidin S supplier results show that this group of https://www.selleckchem.com/products/bindarit.html bacteria could be an important contribution to phage therapy. Moreover, we established a method to investigate the possibility of a phage to lyse bacteria under infection conditions prior to use for phage therapy in vivo. Methods Bacterial strains and growth conditions

Table 1 shows the Selleck Dactolisib genotype and phenotypes of the bacteria and phage JG024 used in this study. The 100 environmental Pseudomonas aeruginosa strains used in this study origin from a comprehensive screen of approx. 400 environmental river strains. These were genetically characterized using the ArrayTube hybridization chip [37]. The 100 strains used here are all different in their core genomic SNP pattern and were chosen such to represent the entire population genetic diversity currently known for P. aeruginosa. Details of the comprehensive screen

will be published elsewhere. P. aeruginosa strains were routinely propagated in Luria Bertani (LB) broth medium aerobically at 37°C. The composition of the artificial sputum medium (ASM) is described elsewhere [12]. Phage Isolation Phages were isolated Cetuximab in vitro from sewage following a simple enrichment procedure. Samples from a sewage plant Steinhof in Braunschweig, Germany were centrifuged for 5 min at 4100 × g (Biofuge fresco). Ten ml of the supernatant were mixed with 5 ml of a P. aeruginosa overnight culture and incubated in 50 ml LB broth at room temperature. After an incubation of 48 h, the cells were sedimented by centrifugation at 4100 × g (Biofuge fresco) for 10 min and the supernatant was transferred to a clean tube. To kill remaining bacteria, several drops of chloroform were added to the supernatant and the emulsion was mixed for 30 s. To separate the phages, appropriate dilutions of the phage lysate were spotted onto bacterial lawns of top-agar plates. Top-agar plates were produced by adding approximately 5*108 cells/ml of P. aeruginosa from an overnight LB broth to 3.

Currently, Hadrospora selleck screening library includes two species, i.e. H. fallax and H. clarkii (Sivan.) Boise differentiated by ascospore size. Phylogenetic study None. Concluding remarks Hadrospora seems not closely related to Phaeosphaeriaceae. Halotthia Kohlm., Nova Hedwigia 6: 9 (1963). (?Zopfiaceae) Generic description Habitat marine, saprobic. Ascomata large, solitary, gregarious or confluent, broadly conical to subglobose, flattened at the base, carbonaceous, immersed to erumpent, ostiolate, epapillate. Peridium plectenchymatous. Hamathecium of dense, long, cellular pseudoparaphyses, septate, branching.

Asci 8-spored, SHP099 solubility dmso bitunicate, cylindrical, with a short pedicel. Ascospores uniseriate, ellipsoidal, subcylindrical or obtuse-fusoid, dark brown, 1-septate, constricted at the septum. Anamorphs reported for genus: none. Literature: Kohlmeyer 1963; Suetrong et al. 2009. Type species Halotthia posidoniae (Durieu & Mont.) Kohlm., Nova Hedwigia 6: 9 (1963). (Fig. 34) Fig. 34 Halotthia posidoniae (from S, isotype of Sphaeria posidoniae). a Ascomata gregarious on the host surface. EPZ5676 nmr b–d Mature or

immature cylindrical asci. e–h Ellipsoidal, dark-brown, 1-septate ascospores. Scale bars: a = 1 mm, b–d = 50 μm, e–h = 5 μm ≡ Sphaeria posidoniae Durieu & Mont. Exploration scientifique de l’Algérie, pp. 502–503, Taf. 25, Abb. 8a-i, 1849. Ascomata 0.8–1.1 mm high × 1.5–2.1 mm diam., solitary, gregarious or confluent, broadly conical to subglobose, flattened at the base, carbonaceous, immersed to erumpent, ostiolate, epapillate (Fig. 34a). Peridium enough 165–275 μm thick at sides, thicker near the apex, plectenchymatous. Hamathecium of dense, long cellular pseudoparaphyses, 1.5–2 μm broad, septate, branching. Asci 275–290 × 25–35 μm, 8-spored, bitunicate, cylindrical, with a short pedicel (Fig. 34b, c and d). Ascospores 37–60.5 × 16.5–26 μm, uniseriate, ellipsoidal, subcylindrical or obtuse-fusoid, dark brown, 1-septate, constricted at the septum (Fig. 34e, f, g and h) (adapted from Kohlmeyer and Kohlmeyer 1979). Anamorph: none reported. Material examined: ITALY, in rhizomes

of Posidonia oceanica (Posidoniaceae), 1861, Caldesi (S, isotype of Sphaeria posidoniae) Notes Morphology Halotthia was introduced to accommodate the marine fungus, H. posidoniae (as Sphaeria posidoniae), which is characterized by immersed to erumpent, large, carbonaceous ascomata, thick peridium, bitunicate, 8-spored, cylindrical asci, ellipsoidal, 1-septate, and dark brown ascospores (Kohlmeyer 1963). Morphologically, Halotthia is most comparable with Bicrouania maritima, but the conical ascomata with flattened base of H. posidoniae can be readily distinguished from B. maritima. Phylogenetic study Phylogenetically, Halotthia posidoniae, Pontoporeia biturbinata and Mauritiana rhizophorae form a robust clade, which may represent a potential family (Suetrong et al. 2009).

(data not shown). The selected mutant strains, named TSE and TSN, contained transposon insertions into hrpE and hrcN, respectively. Light and transmission electron microscopy Leaves were taken at 21 days after inoculation, washed twice in phosphate buffer (50 mM, pH 7.0) and fixed in 2.5% (v/v) glutaraldehyde (in 50 mM phosphate buffer, pH 7.0). Leaf sections were prepared for light and transmission electron microscopy according click here to James et al. [67]. Acknowledgements This work was supported by the Brazilian agencies CAPES and INCT-FBN/CNPq. The authors thank Roseli Prado and Julieta Pie for technical assistance. Electronic supplementary material

Additional file 1: Table S1. Aminoacids sequence homology between Hrp/Hrc proteins of H. rubrisubalbicans and H. seropedicae. These data show FRAX597 purchase the identity and similarity between the T3SS proteins from H. rubrisubalbicans and H. seropedicae. (DOC 53 KB) References 1. Olivares FL, James EK, Baldani JI, Dobereiner J: Infection of mottled stripe disease-susceptible and resistant sugar cane varieties by the endophytic diazotroph Herbaspirillum

. New Phytol 1997, 135:723–737.CrossRef 2. James EK, Olivares FL: Infection and colonization of sugarcane and other graminaceous plants by endophytic diazotrophs. Crit Rev Plant Sci 1998, 17:77–119.CrossRef 3. Pimentel JP, Olivares FL, Pitard RM, Urquiaga S, Akiba F, Döbereiner J: Dinitrogen fixation and infection of Grass leaves by Pseudomonas rubrisubalbicans and Herbaspirillum seropedicae . Plant Soil 1991, 137:61–65.CrossRef 4. Hale CN, Wilkie JP: A comparative study of Pseudomonas species pathogenic to sorghum. New Zeal J Agr Res 1972, 15:448–456.CrossRef 5. James EK, Olivares FL, Baldani Tyrosine-protein kinase BLK JI, Dobereiner J: Herbaspirillum , an endophytic diazotroph colonizing vascular tissue in leaves of Sorghum bicolor L. Moench. J Exp Bot 1997, 48:785–797.CrossRef 6. Christopher WN, Edgerton CW: Bacterial stripe diseases of sugarcane in Louisiana. J Agric Res 1932, 41:259–267. 7. Oliveira ALM, Urquiaga S, Döbereiner J, Baldani

JI: The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants. Plant Soil 2002, 242:205–215.CrossRef 8. Oliveira ALM, Canuto EL, Urquiaga S, Reis VM, Baldani JI: Yield of micropropagated sugarcane varieties in different soil types following inoculation with NCT-501 nmr diazotrophic bacteria. Plant Soil 2006, 284:23–32.CrossRef 9. Oliveira ALM, Stoffels M, Schmid M, Reis VM, Baldani JI, Hartmann A: Colonization of sugarcane plantlets by mixed inoculations with diazotrophic bacteria. Eur J Soil Biol 2009, 45:106–111.CrossRef 10. Reis VM, Oliveira ALMM, da Silva F, Olivares FL, Baldani JI, Boddey RM, Urquiaga S: Inoculants for Sugar Cane: The Scientific Bases for the Adoption of the Technology for Biofuel Production. Biological Nitrogen Fixation: Towards Poverty Alleviation through Sustainable Agriculture. Curr Plant Sci Biotechnol Agric 2008, 42:67–68.

The available literature on RTW and sick leave has been focused mainly on the determinants selleck inhibitor of the return to work of employees on short-term sick leave, while largely ignoring the importance of the determinants of long-term sick leave. Literature shows that there is no international

consensus about the definition of long-term sick leave and short-term sick leave. In the present study, we define long-term sick leave as sickness absence during at least 1.5 years. A systematic review showed that most studies on sick leave are based on sickness absence periods of 6 weeks or less, and there is much less literature about sick leave periods longer than 6 weeks (Dekkers-Sánchez et al. 2008). The importance of early work resumption for employees on sick leave has been highlighted by several previous studies (e.g. Bernacki et al. 2000; Tveito et al. 2004). The literature suggests that the impact of factors related to sick leave and absence from work can vary through the different stages of illness (Krause et al. 2001; Burton et al. 2003). The initial onset of absence from work is almost always due to medical reasons. Sufficient evidence suggests that both medical and non-medical factors play a role in the maintenance of sick leave (Dekkers-Sánchez et al. 2008). This diversity of factors could explain why the resumption of work is increasingly difficult as the time absent from work increases

(WHO selleck 2003). Despite the importance of long-term sickness absence, previous Luminespib research has shown that there is a lack of scientific knowledge on Acadesine the factors associated with long-term sick leave (Dekkers-Sánchez et al. 2008). Literature shows that the causes of long-term sick leave and complex may involve medical, psychosocial, financial, organisational and work-related factors (Alexanderson

2004). Therefore, a proper workability assessment should take into account all factors that seem responsible for the maintenance of the sickness absence. After 2 years of sick leave, these complex conditions require a multifactorial analysis, including the medical situation, work situation and personal situation of the claimant. This implies that the assessment of workability should include not only the medical factors, but also the non-medical factors responsible for a decreased ability to perform work. With better knowledge about the factors associated with sickness absence, IPs can make useful recommendations to achieve RTW, which is in concordance with the Dutch legislation, aiming at improving RTW outcomes. Despite the important role of physicians in the RTW process, little is known about the views of physicians on the factors that should be addressed in the evaluation of the work ability of employees on long-term sick leave. Therefore, enhancing the knowledge of physicians regarding these relevant factors is warranted.

M*: 100 Base-Pair Ladder (GE Healthcare). Within the hoxE and xisH promoter regions the following regions are indicated: Selleck Birinapant putative LexA binding sites, putative IHF binding sites (boxed with the mismatching nucleotide shaded), the -10 and -35 boxes and the ribosome binding site – RBS (underlined), the transcription start point (+1, bold and underlined), and the start codons of hoxE and xisH (bold and underlined).

Cotranscription of hoxEFUYH and hoxW, and hupSL and hupW To assess TPX-0005 in vivo the cotranscription of hox genes and to clarify if the genes encoding the hydrogenases-specific endopeptidases (hoxW and hupW) are cotranscribed with the respective structural genes, RT-PCR experiments were performed with RNA collected from Lyngbya majuscula cells grown in conditions in which the transcript levels were selleck demonstrated to be high (for details see Material and Methods, [1, 2]). The cDNAs were synthesized using a hoxH-, a hoxW- or a hupW-specific antisense primer, and amplifications were performed with primer pairs that covered regions between hoxEF, hoxF-hcp, hoxUY, hoxYH, ORF16-hoxW, hupSL and hupL-W. In all cases, PCR products were obtained (Fig. 1A, B, 2A and

2B). These data indicate that all the structural genes encoding the bidirectional hydrogenase, and the gene putatively encoding the hybrid cluster protein (hcp), can be transcribed as a single operon in L. majuscula. Sirolimus The results also show that hoxW is cotranscribed with ORF16 (Fig. 1B), ORF15, xisI and xisH (data not shown). The ORF14 is in the opposite direction in relation to the hox genes, and no PCR product was detected using the cDNA generated with hoxW-specific primer and ORF14 specific primers. In order

to assess the transcription of ORF14, RT-PCR was performed using cDNA synthesized with random primers, the only PCR product obtained was generated using a ORF14 internal primer pair suggesting that ORF14 is indeed transcribed as a monocistronic unit (data not shown). Concerning the uptake hydrogenase it has been previously demonstrated that the structural genes hupSL were cotranscribed [2], however until now the transcription of the gene encoding the putative specific endopeptidase -hupW – was not accessed. In this work, we demonstrated that hupW can be transcribed together with hupSL, although a promoter region upstream hupW was also identified (see Fig. 2C and text below). Figure 2 hup genes physical map, hupW promoter, and analysis of cotranscription in Lyngbya majuscula CCAP 1446/4. (A) Physical map of L. majuscula hup genes (adapted from [3], accession number GenBank:AF368526), (B) analysis of the hup genes cotranscription by RT-PCR, and (C) nucleotide sequence of the promoter region upstream of hupW. A schematic representation of the cDNA and the products generated in the RT-PCRs is depicted below the physical map.

Conserv Biol 2009, in press. 49. Van Doninck K, Schon I, Martens K, Backeljau T: Clonal diversity in the ancient asexual ostracod Darwinula stevensoni assessed by RAPD-PCR. Heredity 2004,93(2):154–160.CrossRefPubMed 50. Drouin G, de Sa MM: The concerted evolution learn more of 5S ribosomal

genes linked to the repeat units of other multigene families. Mol Biol Evol 1995,12(3):481–493.PubMed 51. Vralstad T, Knutsen AK, Tengs T, Holst-Jensen A: A quantitative TaqMan MGB real-time polymerase chain reaction based assay for detection of the causative agent of crayfish plague Aphanomyces astaci. Vet Microbiol 2009,137(1–2):146–155.CrossRefPubMed 52. Betsou F, Beaumont K, Sueur JM, Orfila J: Construction and evaluation of internal control DNA for PCR amplification of Chlamydia trachomatis DNA from urine samples. J Clin Microbiol 2003,41(3):1274–1276.CrossRefPubMed 53. Gregory JB, Litaker RW, Noble RT: Rapid one-step quantitative reverse transcriptase PCR assay with competitive internal positive control for detection of enteroviruses in environmental samples. Appl Avapritinib Environ Microbiol 2006,72(6):3960–3967.CrossRefPubMed 54. Bart A, Heijden HM, Greve S, Speijer D, Landman WJ,

van Gool T: Intragenomic variation in the internal transcribed spacer 1 region of Dientamoeba fragilis as a molecular epidemiological marker. J Clin Microbiol 2008,46(10):3270–3275.CrossRefPubMed 55. Worheide G, Nichols SA, Goldberg J: Intragenomic variation of the rDNA internal transcribed spacers in sponges (phylum Porifera ): S63845 in vitro implications for phylogenetic studies. Mol Phylogenet Evol 2004,33(3):816–830.CrossRefPubMed

Dipeptidyl peptidase 56. Papin JF, Vahrson W, Dittmer DP: SYBR Green-based real-time quantitative PCR assay for detection of West Nile virus circumvents false-negative results due to strain variability. J Clin Microbiol 2004,42(4):1511–1518.CrossRefPubMed 57. Anderson TP, Werno AM, Beynon KA, Murdoch DR: Failure to genotype herpes simplex virus by real-time PCR assay and melting curve analysis due to sequence variation within probe binding sites. J Clin Microbiol 2003,41(5):2135–2137.CrossRefPubMed 58. Lind K, Ståhlberg A, Zoric N, Kubista M: Combining sequence-specific probes and DNA binding dyes in real-time PCR for specific nucleic acid quantification and melting curve analysis. Biotechniques 2006,40(3):315–319.CrossRefPubMed 59. Reischer GH, Kasper DC, Steinborn R, Mach RL, Farnleitner AH: Quantitative PCR method for sensitive detection of ruminant fecal pollution in freshwater and evaluation of this method in alpine karstic regions. Appl Environ Microbiol 2006,72(8):5610–5614.CrossRefPubMed 60. Blazer VS, Vogelbein WK, Densmore CL, May EB, Lilley JH, Zwerner DE:Aphanomyces as a cause of ulcerative skin lesions of menhaden from chesapeake bay tributaries. J Aquat Anim Health 1999,11(4):340–349.CrossRef 61.

Forty Chk inhibitor (50%) of the 80 serotypes encompassing Erastin solubility dmso atypical EPEC were associated with strains carrying one or more of the EHEC-plasmid genes ehxA, katP, etpD, espP. EHEC-plasmid genes etpD (p < 0.01), ehxA (p < 0.001) and espP (p < 0.001) were significantly more frequent among strains (89/129 = 69%) and serotypes (28/40 = 70%) belonging to Cluster 1 than in strains (32/106 = 30.2%) and serotypes (15/46

= 32.6%) of Cluster 2 (data not shown). Presence of virulence genes in STEC and apathogenic E. coli strains The 52 STEC strains investigated in this study belonged to 20 different serotypes (Table 2). Twelve of these (O113:H4, O113:H21, O118:H12, O146:H28, O153:H25, O174:H8, O22:H8, O22:H16, O76:H19, O8:H19, O91:H10 and O91:H21) were previously described from isolates of human origin [3]. Apart from

stx-genes, 33 (63.5%) of 52 STEC were positive for one or more of EHEC-plasmid associated genes ehxA, espP and katP. None of the STEC was positive for the plasmid etpD gene as for all other nle-genes investigated in this study (Table 1). The 21 apathogenic E. coli strains belonged to 18 different serotypes (Table 2) and were negative TPCA-1 for all virulence markers investigated in this study (Table 1). Discussion The concept of molecular risk assessment [24] has been successfully employed for grouping STEC strains into those that are associated with outbreaks and life-threatening disease in humans and those which cause less severe or are not implicated in human disease. The presence of non-LEE effector

Interleukin-3 receptor genes encoded by O-islands OI-122, OI-71 and OI-57 has been shown to be highly associated with EHEC strains that were frequently involved in outbreaks and severe disease in humans [4, 16, 17, 24, 28, 29]. In a previous work, we were able to associate the presence of OI-122 and OI-71 encoded genes with an “”EHEC-Cluster”" comprising forty-four EHEC strains as well as eight of twenty-one EPEC strains investigated [17]. This finding indicates that some EPEC strains are more related to EHEC in their virulence patterns, than others. In order to explore this relationship between EPEC and EHEC more closely, we investigated larger numbers of strains and serotypes of typical and atypical EPEC for thirteen virulence genes associated with EHEC O157 O-islands OI-122, OI-71, OI-57, the EHEC-plasmid and prophage CP-933N. Genes for nleG5-2 and nleG6-2 were included since OI-57 specific genes were previously found to be associated with classical EHEC and also with some EPEC strains [24, 28].

A) Cytochalasin D; B) Colchicine. Monolayers were infected for 6 h (aEPEC) and 3 h (tEPEC). S. enterica sv Typhimurium and S. flexneri were used as controls and monolayers were infected for 4 h and

6 h, respectively. Results as percent invasion are means ± standard error from at least three independent experiments performed in duplicate. * P < 0.05 by an unpaired, two-tailed t test. HeLa cells are derived Enzalutamide cost from a human uterine cervix carcinoma. They are widely used to study bacterial interactions with epithelial cells yet they do not represent an adequate host cell type to mimic human gastrointestinal infections. To examine whether aEPEC strains would also invade intestinal epithelial cells, we infected T84 cells (derived from a colonic adenocarcinoma), cultivated for 14 days for polarization and differentiation, with all 6 aEPEC strains. The ability of these strains to promote A/E lesions in T84 cells was confirmed by FAS (Table 1). In the gentamicin protection assays performed with these cells, NVP-HSP990 purchase 5 of 6 strains were significantly more invasive than the prototype tEPEC strain E2348/69 (Fig. 1B). The exception was aEPEC 4051-6 (1.5% ± 1.2) that showed similar invasion index as tEPEC E2348/69 (0.5% ± 0.2). The invasion indexes of the 5 aEPEC strains

varied from 5.8% ± 1.7 (aEPEC 4281-7) to 17.8% ± 3.1 (aEPEC 1632-7). These results demonstrate that besides invading HeLa cells, aEPEC strains carrying distinct intimin subtypes invade epithelial cells of human intestinal origin to different levels. Interestingly, the aEPEC invasion indexes were significantly higher than that of tEPEC E2348/69, but this comparison

should be made with caution since the incubation-periods used were different. Nonetheless, it has already been demonstrated that tEPEC is unable to efficiently invade fully differentiated intestinal epithelial cells [42]. To confirm invasiveness, we examined T84 cells infected with aEPEC strains by transmission electron microscopy (TEM). This approach confirmed that 5 out of 6 aEPEC strains tested promoted A/E lesion formation and were also internalized (Fig. 3A and 3B). Under the conditions used, although some tEPEC E2348/69 cells were intra-cellular, most Selleckchem NU7026 remained extra-cellular and intimately attached to the epithelial cell surface (Fig. 3C). Except for aEPEC Tenoxicam strains 4281-7 in HeLa cells and 4051-6 in T84 cells, the remaining four strains tested were more invasive than tEPEC E2348/69 and showed heterogeneous invasion index in both HeLa and T84 cells. Figure 3 Transmission electron microscopy of infected polarized and differentiated T84. A) aEPEC 1551-2, B) aEPEC 0621-6 and C) prototype tEPEC E2348/69. Monolayers were infected for 6 h (aEPEC) and 3 h (tEPEC). aEPEC 1551-2 and 0621-6 were selected because, according to the data in Fig. 1B, they presented an average invasion index as compared to the other strains studied. Arrows indicate bacterial-containing vacuoles.