To investigate the role of Lcl in adhesion and invasion, the experiment was repeated with Sirolimus supplier bacteria (5 × 107 bacteria mL−1) preincubated

with Lcl-specific antibodies (20 μg mL−1 bacteria culture) at 37 °C for 1 h before they were placed in contact with the eukaryotic cells. As a control, experiments were repeated with a xylanase C (XlnC) antibody. XlnC is a Streptomyces lividans secreted protein (Faury et al., 2004) and the XlnC antibodies were of the same isotype and produced under the same conditions as the Lcl-specific antibodies. Alternatively, for measuring adhesion to host cells, experiments were performed with immobilized purified, refolded Lcl protein. Lcl and BSA (negative control) were immobilized as films on flat-bottomed microtiter 96-well plates (Nunclon) at a concentration of 5 μg per well overnight at 4 °C. Films were blocked with 1% BSA, washed with phosphate-buffered saline (PBS), followed by addition of 100 μL of eukaryotic cell suspension (5 × 105 cells mL−1) to each well and incubation at room temperature for 1 h. Nonadherent cells were removed by two washes with PBS, and those that adhered to the films were stained with crystal violet. Plates were read at A595 nm. Additionally, the immobilized films were preincubated with Lcl-specific antibodies

(20, 2, Fluorouracil manufacturer 0.2 μg per well) for 30 min on ice before adding the eukaryotic cells. Coimmunoprecipitation experiments were carried out using a host cell lysate in combination with refolded Lcl protein. First, pelleted A549 cells or macrophage-like cells were resuspended in solubilization buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 0.2% Triton X-100) and sonicated. Samples (500 μL) of the lysate (0.5 μg μL−1) were incubated with refolded Lcl protein (10 μg in total) for 1 h at 4 °C, rotating end over end. Sepharose A powder (10 mg) was added to the 500 μL mixture and further rotated for 1 h at 4 °C, followed by centrifugation (5 min, Astemizole 1000 g). The supernatant was subsequently incubated with Lcl-specific antibodies or complement component C1q receptor

(C1qR)-specific antibodies rotating for 1 h at 4 °C. This incubation step was followed by addition of 10 mg sepharose A powder again. After 1 h at 4 °C, the immunoprecipitates were isolated by centrifugation (5 min, 1000 g) and washed four times with 150 μL solubilization buffer. After resuspension in 2 × SDS loading dye, the samples were boiled and the immunoprecipitated proteins were visualized by immunodetection with Lcl-specific antibodies. As a control, samples containing only lysate and Lcl protein without antibodies and samples only containing antibodies were also incubated with the protein A sepharose powder. Statistical analyses were performed using the standard Student t-test with equal variances.

Grading: 1D Studies in Africa have included both ART given to the mother and ART given as prophylaxis to the infant during breastfeeding. While serious adverse events were not reported in the infants given nevirapine Entinostat chemical structure for up to 6 months [297], there are currently insufficient safety data to advocate this approach given the particular safety concerns regarding the use of nevirapine in adults uninfected by HIV. The use of nevirapine for longer than the 2–4 weeks currently recommended for PEP is not advised [306]. 8.4.4 Intensive support and monitoring of the mother

and infant are recommended during any breastfeeding period, including monthly measurement of maternal HIV plasma VL, and monthly testing of the infant for HIV by PCR for HIV DNA or RNA (VL). Grading: 1D Where a woman chooses to breastfeed against the medical advice in Recommendation 8.4.2, she and the baby should

be monitored regularly for maternal adherence to ART; VL monitoring of the mother and diagnostic testing of the baby should be performed regularly (monthly). If the mother’s adherence is suboptimal or she has detectable viraemia or an intercurrent illness that affects her ability to take or absorb ART, or buy Bleomycin she develops mastitis, she should be advised again to stop breastfeeding. 8.4.5 All infants born to mothers infected with HIV should have an antibody test at age 18 months. Grading: 1C The potential for breastfeeding emphasizes the possibility of late transmission of HIV after the standard 3-month PCR test. Babies known to be breastfed should be tested monthly by PCR as above, but not all Phosphoprotein phosphatase breastfeeding will be disclosed, and all babies born to HIV-positive women should have a negative HIV antibody test documented

at age 18 months (see Section 8.5: Infant testing below). 8.5.1 HIV DNA PCR (or HIV RNA testing) should be performed on the following occasions (Grading: 1C): During the first 48 h and before hospital discharge. 2 weeks post infant prophylaxis (6 weeks of age). 2 months post infant prophylaxis (12 weeks of age). On other occasions if additional risk (e.g. breastfeeding). HIV antibody testing for seroreversion should be checked at age 18 months. The gold standard test for HIV infection in infancy was HIV DNA PCR on peripheral blood lymphocytes, although a number of studies, including the large French perinatal cohort have now demonstrated equal or increased early sensitivity with amplification of viral RNA with no false positives [307]. Infants infected intrapartum may have low peripheral blood HIV levels, so HIV DNA/RNA may not be amplified from all infected infants at birth. Indeed a positive HIV DNA PCR result within 72 h of birth is taken as presumptive evidence of intrauterine transmission.

The lists of all community pharmacies in Alberta and Northern Ireland were obtained from the Alberta College of Pharmacists’ website (http://www.pharmacists.ab.ca), and the Ulster Chemist Association Diary respectively. All registered community AC220 pharmacies in Northern Ireland and Alberta were placed in a numbered list and called in a random order (using a random-number generator) until the desired sample size of community pharmacists was obtained. Pharmacy type (independent or

chain for Alberta and independent, small chain (two to five pharmacies) or multiple (six pharmacies or more) for Northern Ireland) and location (urban or rural) were also recorded. For the purpose of sample size calculation it was estimated that 35% (±10%) of participants would use language related to patient-centred care to describe what a pharmacist does. Using EPI INFO v6. (CDC, Atlanta, Georgia, USA), Stat Calc for population surveys it was determined that 85 pharmacists from each jurisdiction were required to achieve the previous estimate

at a confidence level of 95%. This figure was rounded to a total of 100 pharmacists per jurisdiction. The present study methodology, which involved short telephone interviews with community pharmacists as the data collection vehicle, has been outlined elsewhere.[34] Community pharmacists were interviewed by telephone. The interviewer introduced himself as a researcher who was examining Verteporfin datasheet how various health professionals use language to describe what they do and then asked the interview questions. The interview was composed of two questions: Linifanib (ABT-869) (a) How many years have you been practising pharmacy? (b) In three or four words (or phrases), from your perspective, could you please tell me ‘What does a pharmacist do?’ The brevity of the telephone conversations enabled the researcher to document participants’ responses by hand. The intention of using this methodology was to prevent pharmacists from thinking too much about their answer, thereby eliciting a ‘top of mind’ or automatic response. This approach was used because it engages certain unconscious

mental processes which affect and influence the judgements, feelings and behaviours of the person.[35] In the literature it has been reported that individuals’ automatic response does not usually match their self-reported attitudes.[36] The slight deception and restriction of response were intended to remove some of the effects of social desirability bias.[37] The first phase of data analysis involved two researchers independently coding the responses using qualitative content analysis. The definitions of product-focused (dispensing) and patient-centred care, obtained from the Canadian Pharmacist Association’s Blueprint for Pharmacy: Implementation Plan[38] (see Table 1 for definitions), were applied to further refine the analysis.

, 2009). Other rhizosphere bacterial species benefit plant growth through indirect effects, which are mainly associated with reduction of damage caused by plant pathogens (Van Loon, 2007; Weller, 2007). The Azospirillum genus belongs to the alphaproteobacteria class and comprises free-living, nitrogen-fixing, vibrio- or spirillum-shaped rods, which produce polar

and peritrichous flagella (Baldani et al., 2005) (Fig. 1). Azospirilla exert beneficial effects on plant growth and yield of many agronomically see more important crops (Okon, 1985; Spaepen et al., 2009; Helman et al., 2011). Commercial inoculants of azospirilla have been tested and applied in hundreds of thousands of hectares, mainly in Latin America (Fuentes-Ramirez & Caballero-Mellado, 2005; Cassan & Garcia de Salamone,

2008; Hungria et al., 2010; Helman et al., 2011). About 16 Azospirillum species have been described so far; however, Azospirillum brasilense and Azospirillum lipoferum have been studied in more detail than the others (Baldani et al., 2005). Draft genomic sequences of A. brasilense Sp245 and A. lipoferum CRT1 have been obtained, but the check details full annotations of these genomes have not yet been published (I. Zhulin and F. Wisniewsky-Dye, pers. commun.). Preliminary data from the A. brasilense genome sequencing project are available at http://genomics.ornl.gov/research/azo. Azospirilla are able to fix nitrogen in association with plants, but apparently, nitrogen fixation does not play a major role in plant growth promotion in most systems evaluated so far (Spaepen et al., 2009; Helman et al., 2011). On the other hand, azospirilla

are able to produce and secrete plant growth regulators (phytohormones) such as auxins (indole-3-acetic acid; IAA), cytokinins, and gibberellins, as well as nitric oxide (NO), which likely are key signals and components of plant growth promotion effects (Dobbelaere & Okon, 2007; Spaepen et al., 2007, 2009; Molina-Favero et al., 2008; Bashan & de-Bashan, 2010; Helman et al., 2011). Basic knowledge about Sclareol the physiological properties of PGPRs is crucial for understanding diverse aspects related to rhizosphere performance and successful interactions with plant roots. For instance, this knowledge might help understanding the modes of colonization of plant surfaces by PGPRs, their interactions with other microorganisms, and the modes of action by which these microorganisms benefit plants. In addition, this knowledge might stimulate ideas about how to improve the production and application of PGPR inoculants. Here we focus on recent advances on the understanding of A. brasilense physiological properties that are important for rhizosphere performance and successful interactions with plant roots (Table 1).

These findings are consistent with previously published data in which 7.1% of the X. bovienii-SF43 genome and 4.6% of the X. nematophila genome match with ORFs identified as phage related (Ogier et al., 2010). All of the described phage clusters Akt inhibitor in vivo match with phagic modules previously identified with the RGPFinder web tool, which identifies large regions of genomic plasticity. The xbp1 cluster encodes the main tail synthesis proteins including the tail sheath (XbpS1), tube (XbpT1), and tail fiber (XbpH1) proteins. To determine whether

the xbp1 phage gene cluster encoded a mitomycin C-inducible xenorhabdicin, the xbpS1 sheath gene was inactivated. PEG-precipitated tail structures prepared from the SF43 and SF70 (xbpS1) strains were analyzed by SDS-PAGE gel electrophoresis. Figure 3 shows that the preparation from SF43 contained

43 and 98 kDa proteins that were identified by N-terminal sequence analysis as XbpS1 and XbpH1, respectively (lane 1). We were unable to resolve the identity of the 65-kDa protein by N-terminal sequencing. In the xbpS1 strain, the level of the 43-kDa protein band was dramatically reduced but not completely eliminated (lane 2). The identity of this additional 43-kDa protein could not be resolved by MALDI-TOF MS analysis. TEM analysis showed that phage tail structures were not produced check details in the SF70 strain (data not shown). Interestingly, inactivation of xbpS1 did not reduce XbpH1 production (Fig. 3), suggesting that xbpH1 was expressed, and XbpH1 fiber and baseplate structures were assembled in the xbpS1 strain. This result was similar to that observed with the ΔxnpS1 strain of X. nematophila (Morales-Soto & Forst, 2011). Phage tail preparations were PEG-precipitated from the SF43 and SF70 strains and assayed for antimicrobial activity against Photorhabdus luminescens Protein kinase N1 TT01 and Xenorhabdus bovieni SF31 (Table 1). Preparations derived from the SF43 strain displayed a high level of activity against P. luminescens TT01 and X. bovienii-SF31 (Table 2), while antimicrobial activity was dramatically reduced in the preparations derived from SF70. These findings indicate

that Xbp1 xenorhabdicin was responsible for antimicrobial activity against susceptible competitors. The tail synthesis genes of xnp1 and xbp1 are highly conserved. The sequence identity for the 12 proteins encoded by the genes located between the cI and gpI genes ranges from 77% to 95%. The respective sheath proteins, XnpS1 and XbpS1, share 94% identity and the XnpT1 and XbpT1 tube proteins share 98% identity. The 5.9 kb insertion in the region neighboring fun(Z) contains 11 ORFs and is located between the tail fiber gene xbpH1 and the tail sheath gene xbpS1. It contains three truncated tail fiber ORFs (Ff–Fh) and three tandem transposase genes. As described previously, the xnp1 remnant P2 prophage encodes the tail sheath (XnpS1), tube (XnpT1), and tail fiber (XnpH1).

These findings are consistent with previously published data in which 7.1% of the X. bovienii-SF43 genome and 4.6% of the X. nematophila genome match with ORFs identified as phage related (Ogier et al., 2010). All of the described phage clusters LDK378 manufacturer match with phagic modules previously identified with the RGPFinder web tool, which identifies large regions of genomic plasticity. The xbp1 cluster encodes the main tail synthesis proteins including the tail sheath (XbpS1), tube (XbpT1), and tail fiber (XbpH1) proteins. To determine whether

the xbp1 phage gene cluster encoded a mitomycin C-inducible xenorhabdicin, the xbpS1 sheath gene was inactivated. PEG-precipitated tail structures prepared from the SF43 and SF70 (xbpS1) strains were analyzed by SDS-PAGE gel electrophoresis. Figure 3 shows that the preparation from SF43 contained

43 and 98 kDa proteins that were identified by N-terminal sequence analysis as XbpS1 and XbpH1, respectively (lane 1). We were unable to resolve the identity of the 65-kDa protein by N-terminal sequencing. In the xbpS1 strain, the level of the 43-kDa protein band was dramatically reduced but not completely eliminated (lane 2). The identity of this additional 43-kDa protein could not be resolved by MALDI-TOF MS analysis. TEM analysis showed that phage tail structures were not produced Lapatinib in the SF70 strain (data not shown). Interestingly, inactivation of xbpS1 did not reduce XbpH1 production (Fig. 3), suggesting that xbpH1 was expressed, and XbpH1 fiber and baseplate structures were assembled in the xbpS1 strain. This result was similar to that observed with the ΔxnpS1 strain of X. nematophila (Morales-Soto & Forst, 2011). Phage tail preparations were PEG-precipitated from the SF43 and SF70 strains and assayed for antimicrobial activity against Photorhabdus luminescens Galeterone TT01 and Xenorhabdus bovieni SF31 (Table 1). Preparations derived from the SF43 strain displayed a high level of activity against P. luminescens TT01 and X. bovienii-SF31 (Table 2), while antimicrobial activity was dramatically reduced in the preparations derived from SF70. These findings indicate

that Xbp1 xenorhabdicin was responsible for antimicrobial activity against susceptible competitors. The tail synthesis genes of xnp1 and xbp1 are highly conserved. The sequence identity for the 12 proteins encoded by the genes located between the cI and gpI genes ranges from 77% to 95%. The respective sheath proteins, XnpS1 and XbpS1, share 94% identity and the XnpT1 and XbpT1 tube proteins share 98% identity. The 5.9 kb insertion in the region neighboring fun(Z) contains 11 ORFs and is located between the tail fiber gene xbpH1 and the tail sheath gene xbpS1. It contains three truncated tail fiber ORFs (Ff–Fh) and three tandem transposase genes. As described previously, the xnp1 remnant P2 prophage encodes the tail sheath (XnpS1), tube (XnpT1), and tail fiber (XnpH1).

Clinicians should consider NCC in patients from Burma with epilepsy, chronic headache, or unexplained neurologic symptoms. Clinicians should also be aware of stigma and cultural interpretations related to epilepsy which may preclude patient disclosure of seizures. The primary tools for diagnosis of NCC include neuroimaging and serology assays. However, additional clinical and

epidemiologic criteria are usually required to establish the diagnosis per consensus guidelines.6 selleck kinase inhibitor Occasionally, a definitive diagnosis is possible with neuroimaging by demonstration of a visible scolex within a cyst, or with histopathologic confirmation of an excised or biopsied cyst. Head CT readily identifies most forms of NCC and can facilitate detection of small calcifications. The fine resolution possible with MRI aids in detection of smaller cysts, as well as cysts near bony structures or within the ventricles. The EITB LLGP serologic assay is highly specific (∼100%) and sensitive (∼98%) for detection of NCC involving more than one cyst.7 However, false-negative results frequently occur in NCC involving only calcified cysts, or in cases involving a single parenchymal cyst. Recently developed assays detect T solium cyst antigens or DNA in serum, cerebrospinal fluid, or urine, but these are not yet routinely available and their contribution

to clinical diagnosis remains unclear. Further detail regarding diagnosis, treatment, and outcome of NCC is available in recent reviews.1,8,9 Consideration of the health of the patient’s family is important Screening Library when NCC is diagnosed as there may be additional infections within the household. In addition to NCC acquired in the country of origin,

transmission can occur after resettlement as an adult intestinal tapeworm can live for several years. Exposure may also be maintained through travel and visiting friends and relatives. Stool ADAMTS5 examination of the index NCC case and household members can identify taeniasis and treatment may prevent additional NCC cases.10–12 Stool screening is accomplished preferentially by ELISA for Taenia sp. coproantigens or otherwise by light microscopy for eggs and proglottids. A combination of symptom screening, serology, and neuroimaging may identify additional cases of NCC. Finally, in the case we present here as well as in the case described by Hewagama and colleagues, neurologic symptoms first appeared within days of treatment with albendazole or praziquantel for presumed intestinal helminth infection. Both medications penetrate the CNS well and are used in the treatment of NCC, typically in conjunction with corticosteroids to control resulting inflammation. The Food and Drug Administration recently updated labels for both drugs to warn clinicians of the possibility of precipitating inflammatory reactions in patients with occult asymptomatic NCC. Multiple suspected adverse reactions of this type have been reported.

Neurological examination revealed selective strength loss and decreased muscle activity in the dorsal interossei of the hand, flexor digitorum, extensor carpi radialis brevis and longus,

and abnormalities of the triceps and ulnar reflexes. This patient had no evidence of alcohol abuse, recent exposure to toxins, sarcoidosis, malignancy, vitamin B12 deficiency, malnutrition, renal or liver disease, diabetes mellitus, or thyroid dysfunction. During hospitalization, laboratory findings did not reveal abnormalities except for lymphopenia (480/mm3), hypoalbuminemia (33 g/L), and hypogammaglobulinemia (4.7 g/L). Cerebrospinal fluid (CSF) examination showed 1 cell/mm3, a total protein concentration Selleckchem GSK3 inhibitor of 0.33 g/L, and a glucose concentration of 4.3 mmol/L (serum glucose concentration of 6.7 mmol/L). Cranial, chest, abdomen, and pelvis computed tomography did not reveal abnormalities. Magnetic resonance imaging of the cervical

spine showed C5-T1 disc degeneration without disc herniation or other anomalies that could explain the neurologic deficit. Intravenous Ceftriaxone was administered for 3 weeks in association with physiotherapy treatment due to suspicion of neuroborreliosis. Acute and convalescent-phase sera and CSF were sent to the WHO Collaborative Center for Rickettsial Diseases and Arthropod-Borne Bacterial Diseases, Marseille. Indirect immunofluorescence selleck products (IF) for rickettsial antigens of spotted fever group[5] (SFG) was negative. The sensitivity of IF for R africae infection is 83% in this laboratory.[6] Quantitative polymerase chain reaction (qPCR) for all SFG Rickettsiae targeting the RC0338 gene[7] on a CSF DNA sample was negative. As the clinical picture was associated with a tick-bite, Oxalosuccinic acid other bacteria transmitted by ticks were tested. Specific qPCR for Borrelia targeting the 16 S rRNA gene[8] in CSF DNA sample collected on February 26, 2010 (4 weeks after tick-bite) was positive. A sequence of 149 bp was obtained after sequencing of the qPCR amplicon of 16 S rRNA gene,[8] with 100% similarity with Borrelia microti

(JF803950); Borrelia latyschewii (JF681793); Borrelia crocidurae (GU350713); Borrelia duttonii (GU350712); Borrelia hispanica (GU350710); Borrelia turicatae (CP000049); Borrelia parkeri (AY604975); and with 98% (147/150) homology with Borrelia burgdorferi strain CS4 (HQ433694). Subsequently, this DNA sample was subjected to a regular PCR in automated DNA thermal cyclers to amplify the portion of the flaB (flagellin) gene of Borrelia spp.[9] but it remained negative. IF assay with B crocidurae, B duttonii, and Borrelia recurentis was negative.[10] However, the enzyme-linked immunosorbent assay (ELISA) assay with B burgdorferi antigen showed positive bands of IgM (0.295) and IgG (1.211). WB analysis was positive with IgG (VLSE, p100, p58, p41, p30, OspC, p17) and IgM (OspC) bands.

“
“Three soil bacterial strains were identified as Chryseobacterium sp. TFB on the basis of their 16S rRNA gene sequences. Conidia of Arthrobotrys oligospora produced a few mycelial traps (MT) and conidial traps (CT) when cultured with bacterial cells that they

did not produce when cultured with a bacterial cell-free culture filtrate. However, co-culture of A. oligospora with bacterial cells and bacteria-free filtrate simultaneously induced MT and CT in large amounts. With the increased concentration of bacteria-free filtrate, the number of typical CT increased, but conidial germination was progressively inhibited. Scanning electron GSK-3 inhibitor microscopy of A. oligospora co-cultured with bacteria revealed that bacterial attachment to hyphae was a prerequisite to trap formation and that bacteria-free filtrate facilitated bacterial attachments to hyphae. The results that the addition of nutrients in co-culture medium decreased the number

of traps suggest that this type of trap formation may be favoured at a low nutrient status. Eight fungi tested were able to form MT and CT when co-cultured with bacterial cells and bacteria-free culture filtrate, check details but the abilities varied among species. This study provides novel evidence that under laboratory conditions, soil bacteria attaching to hyphae could induce traps in nematode-trapping fungi. Over 200 species of predacious fungi develop specific morphological structures called traps that adhere to, penetrate, kill and digest free-living nematodes in the soil (Li et al., 2000). Among the nematode-trapping fungi, differentiated structures such as adhesive nets, branches and knobs as well as mechanical traps called constricting or nonconstricting rings are well known and typical of particular species (Nordbring-Hertz et al., 2002). The formation of traps is very important for these fungi. These

fungi thus enter the parasitic phase see more and capture nematodes on the surface of these structures. The traps can develop from hyphal branches and these are termed mycelial traps (MT). Alternatively, they can also form directly upon spore germination without an intermediate mycelial phase or on the germination hyphae, forming conidial traps (CT). MT can be formed either spontaneously or be induced in response to signals from the environment, including certain amino acids, valyl peptides and nemin that were secreted by host nematodes (Dijksterhuis et al., 1994). CTs were formed when conidia were allowed to germinate in cow dung (Dackman & Nordbring-Hertz, 1992), fungistatic soil (Mankau, 1962), rhizosphere soil or soil extracts (Persmark & Nordbring-Hertz, 1997), and the formation of CT was believed to be a response to nutrient deprivation due to strong nutrient competition between soil microorganisms. Fungi and bacteria coexist in a myriad of different environments.

aureus 8325-4 and DU 1090 were cultured in TSB at 37 °C to an optical density at 600 nm of 0.5. Fifty-milliliter culture aliquots were centrifuged, NVP-BKM120 datasheet washed with PBS, and resuspended in 1 mL PBS (2 × 108 CFU per 30 μL)

for histopathology experiments. For cytotoxicity studies, 5 mL of culture described above was resuspended in 10 mL of Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, CA). The minimal inhibitory concentrations (MICs) of apigenin for S. aureus were evaluated using the broth microdilution method according to CLSI guidelines (CLSI, 2005). Briefly, apigenin was diluted in a 96-well plate over the concentration range of 4-1024 μg mL−1 using double dilution method. Following inoculation with 5 × 105 CFU mL−1 of overnight broth cultures in each well, the plate was inoculated at 37 °C for 24 h. The MIC was defined as the lowest concentration at which the growth of S. aureus was inhibited. Staphylococcus aureus strain 8325-4 was cultured in TSB medium at 37 °C, shaken at 200 r.p.m. to an optical density (OD600 nm) of 0.3, and aliquoted into five 250-mL flasks in a volume of 100 mL. Apigenin dissolved in DMSO was added to the four cultures to obtain final concentrations of 1, 4, 16, and 64 μg mL−1. 1% DMSO was added to the control culture. The bacteria were cultured at 37 °C with constant shaking, and cell growth

was measured by reading the OD600 nm values every 30 min. Hemolytic activity was measured as described previously (Worlitzsch et al., 2001; Qiu et al., 2010a) using rabbit Erastin erythrocytes. Briefly, S. aureus cultures with different concentrations of apigenin were harvested when grown to the postexponential growth phase by centrifugation

(5500 g, 4 °C, 1 min), and the residual cells were removed using a 0.2-μm filter. A 0.1 mL volume of bacterial culture supernatants were brought up to 1 mL with the addition of PBS and 25 μL defibrinated rabbit erythrocytes for 30 min at 37 °C. The unlysed blood cells were removed by centrifugation (5500 g, room temperature, selleckchem 1 min). Following centrifugation, the hemolytic activity of the supernatants was detected by measuring the optical density at 543 nm. Medicine-free culture supernatant served as the 100% hemolysis control. The percent of hemolysis was calculated by comparison with the control culture supernatant. The culture supernatants collected previously were used in Western blot analysis. Samples were boiled with Laemmli sample buffer for 10 min, and then 25 μL of the sample was fractionated by SDS-PAGE (12% polyacrylamide gels; Laemmli, 1970). The Western blot protocol was performed as described previously (Qiu et al., 2010a, b). Proteins were transferred onto polyvinylidene fluoride membranes (Roche, Basel, Switzerland) using a semi-dry transfer cell (Bio-Rad, Munich, Germany). The membrane was blocked for 2 h with 5% bovine serum albumin (Amresco) at room temperature.