Therefore, polysaccharide intercellular adhesin, also called the slime exopolysaccharide component, and accumulation-associated protein selleck kinase inhibitor have been described as factors playing an essential role in biofilm formation (Cramton et al., 1999; Mack, 1999; O’Gara & Humphreys, 2001; Götz, 2002). A number of methods

are available to detect the capability of staphylococci to colonize the biomedical devices. The Congo red agar (CRA) assay described by Freeman et al. (1989) and/or the microtiter plate (MtP) test devised by Christensen et al. (1985) were most commonly used as the phenotypical methods for slime and/or biofilm production. We would like to point out that the slime-positive strain (measured by the CRA test) does not necessarily indicate the ability of this strain to form a biofilm (by the Everolimus solubility dmso MtP test). Therefore, ‘slime’ and ‘biofilm’ terms cannot be used alternatively. In this study, a collection of 146 nasopharyngeal S. epidermidis strains was screened for the presence of genetical biofilm markers (icaAD and aap genes), the ability of slime secretion using the CRA test and biofilm formation by the MtP method. The aim of our work was to evaluate the relationship between these phenotypic data and the genotypic pattern of the screened strains. The collection of 146 S. epidermidis strains isolated from the nasopharynx of lung cancer patients was included

in the present study. These strains were collected during patients’ hospitalization at The Department of Thoracic Surgery of Medical University of Lublin. The patients with resectable lung cancer received a preoperative antimicrobial prophylaxis according to hospital policy (piperacillin, cefuroxime alone or in combination with amikacin). At the time of sampling, none of the patients had clinical symptoms of airway infections. The study has been

approved by the Ethical Committee of the Medical University of selleck inhibitor Lublin. Informed consent was obtained from all patients. Clumping factor detection using the Slidex Staph Kit (BioMerieux, France), a coagulase-test tube and the ID32Staph system (BioMerieux) was performed for species identification of staphylococcal isolates. Staphylococcus epidermidis strain ATCC 12228 and S. epidermidis ATCC 35984 were used in biofilm assays as a negative and a positive control, respectively. Biofilm formation in vitro was carried out as described by Christensen et al. (1985) and Mack et al. (1992), with a slight modification. All strains were grown overnight at 35 °C in Trypticase soy broth (TSB; Biocorp, Poland) as well as in a medium supplemented with 0.5% glucose and 4% NaCl. The cultures were diluted 1 : 200 in the appropriate medium (TSB as standard conditions and TSB supplemented with 0.5% glucose plus 4% NaCl as inducing conditions), and 200 μL of cell suspensions per well were used to inoculate sterile 96-well polystyrene microtitrate plates (Nunc, Denmark).

, 2009). Mesorhizobium loti induced small white ‘tumor-like’ growth on Leucaena leucocephala, but a mutant in a conserved structural component of T3SS (the Galunisertib nmr M. loti rhcJ mutant strain) formed large pink nodules (Hubber et al., 2004). Little is known about the role of each of the putative M. loti T3SS effectors. The protein encoded by mlr6316 has been described to have a partial negative

effect on Le. leucocephala nodulation (Hubber et al., 2004), whereas the protein encoded by mlr6361 has been described to have a negative role in Lo. halophilus nodulation (Okazaki et al., 2010). However, it has not yet been determined which of the proteins secreted by the M. loti T3SS are involved in the positive nodulation effects observed in some Lotus spp. The aim of this work

was to determine whether the N-terminal regions of proteins encoded by mlr6316 and mlr6331 are able to direct the protein secretion via M. loti T3SS and to determine the involvement of the different T3SS putative effectors in the symbiosis with two different Lotus species. Bacterial strains and plasmids used in this study are listed in Supporting Information, Table S1. MAFF303099 strains were grown at 28 °C in AB minimal medium (Douglas et al., 1985) supplemented with sucrose (0.5% w/v). When necessary, antibiotics were added to the following final concentrations (μg mL−1): gentamicin (Gm), 30; ampicillin (Amp), 100; neomycin Alectinib datasheet (Nm), 100; spectinomycin (Sp), 100; and tetracycline (Tc), 10 for Escherichia coli or 1 for M. loti. For T3SS induction, naringenin was added to cultures at an optical density 600 nm (OD600 nm) of 0.1 to a final concentration of 1 μM. pBAD-mlr6331 was constructed as previously described (Sánchez et al., 2009). The oligonucleotide primer pairs used are described in Table S1. Sequences encoding the N-terminal portion of the protein, together with the upstream

promoter region, were cut from pBAD-mlr6361 (59 aa), pBAD-mlr6316 (160 aa), pBAD-mlr6358 (160 aa) (Sánchez et al., 2009), and pBAD-mlr6331 (177 aa), respectively, and cloned into the pK18mobTc vector (Sánchez et al., 2009). The same plasmids were also P-type ATPase introduced by biparental mating into an M. loti rhcN pMP2112 mutant strain. Supernatant protein extractions were carried out by direct TCA precipitation as previously described (Sánchez et al., 2009). Supernatant was concentrated approximately 2000 times. For total (intracellular) bacterial protein extractions, 1 ml of the bacterial cultures used above was centrifuged and the pellets dissolved in cracking buffer. Proteins were separated using SDS-PAGE and then stained using silver nitrate. For immunoblotting, anti-NGR234 strain NopA (Marie et al., 2004) or a commercially available anti-FLAG M2 monoclonal antibody (Sigma) was used. Analyzed mutants and oligonucleotides pairs used for their construction are described in Table S1.

, 2009). Mesorhizobium loti induced small white ‘tumor-like’ growth on Leucaena leucocephala, but a mutant in a conserved structural component of T3SS (the http://www.selleckchem.com/btk.html M. loti rhcJ mutant strain) formed large pink nodules (Hubber et al., 2004). Little is known about the role of each of the putative M. loti T3SS effectors. The protein encoded by mlr6316 has been described to have a partial negative

effect on Le. leucocephala nodulation (Hubber et al., 2004), whereas the protein encoded by mlr6361 has been described to have a negative role in Lo. halophilus nodulation (Okazaki et al., 2010). However, it has not yet been determined which of the proteins secreted by the M. loti T3SS are involved in the positive nodulation effects observed in some Lotus spp. The aim of this work

was to determine whether the N-terminal regions of proteins encoded by mlr6316 and mlr6331 are able to direct the protein secretion via M. loti T3SS and to determine the involvement of the different T3SS putative effectors in the symbiosis with two different Lotus species. Bacterial strains and plasmids used in this study are listed in Supporting Information, Table S1. MAFF303099 strains were grown at 28 °C in AB minimal medium (Douglas et al., 1985) supplemented with sucrose (0.5% w/v). When necessary, antibiotics were added to the following final concentrations (μg mL−1): gentamicin (Gm), 30; ampicillin (Amp), 100; neomycin Selleckchem Bortezomib (Nm), 100; spectinomycin (Sp), 100; and tetracycline (Tc), 10 for Escherichia coli or 1 for M. loti. For T3SS induction, naringenin was added to cultures at an optical density 600 nm (OD600 nm) of 0.1 to a final concentration of 1 μM. pBAD-mlr6331 was constructed as previously described (Sánchez et al., 2009). The oligonucleotide primer pairs used are described in Table S1. Sequences encoding the N-terminal portion of the protein, together with the upstream

promoter region, were cut from pBAD-mlr6361 (59 aa), pBAD-mlr6316 (160 aa), pBAD-mlr6358 (160 aa) (Sánchez et al., 2009), and pBAD-mlr6331 (177 aa), respectively, and cloned into the pK18mobTc vector (Sánchez et al., 2009). The same plasmids were also 17-DMAG (Alvespimycin) HCl introduced by biparental mating into an M. loti rhcN pMP2112 mutant strain. Supernatant protein extractions were carried out by direct TCA precipitation as previously described (Sánchez et al., 2009). Supernatant was concentrated approximately 2000 times. For total (intracellular) bacterial protein extractions, 1 ml of the bacterial cultures used above was centrifuged and the pellets dissolved in cracking buffer. Proteins were separated using SDS-PAGE and then stained using silver nitrate. For immunoblotting, anti-NGR234 strain NopA (Marie et al., 2004) or a commercially available anti-FLAG M2 monoclonal antibody (Sigma) was used. Analyzed mutants and oligonucleotides pairs used for their construction are described in Table S1.