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Staphylococcus aureus is a common pathogen causing both hospital and community-acquired infections. Hemolysin is one of the important virulence factors for S. aureus and causes the typical β-hemolytic phenotype which is called complete hemolytic phenotype as well. Recently, Staphylococcus aureus with an incomplete hemolytic phenotype (SIHP) was isolated from clinical samples. To study the microbiologic characteristics of SIHP, SIHP was inoculated on the sheep blood agar plates supplied by different manufacturers to compare their hemolytic phenotype. Expression of hemolysin genes hla, hlb, hlgC and hld of SIHP was detected by qRT-PCR. In addition, the alpha-hemolysin encoded by gene hla was analyzed by western blot. At the same time, the antimicrobial susceptibility of SIHP was tested using the broth dilution method. The main antibiotic resistance gene mecA and virulence genes tst were detected by PCR in SIHP strains. Furthermore, the virulence of SIHP strains was detected through comparing their intracellular survival in macrophage. The cytokines and chemokines secreted by macrophage were measured by flow cytometry. Finally, the genotyping of SIHP was performed by multilocus sequence typing (MLST) analysis. The results showed that the incomplete hemolytic phenotype of SIHP could be observed on the sheep blood agar plates from different suppliers. The relative mRNA expression of hlb in SIHP was obviously increased compared to the control Staphylococcus aureus strains, while the expression of hla, hlgC and hld in SIHP was significantly decreased. In addition, it was shown that the alpha-hemolysin of SIHP was less than that of control strains as well. All sixty SIHP strains were identified to be the methicillin resistant Staphylococcus aureus (MRSA), and moreover these SIHP strains all contains mecA gene. The virulence gene tst were all present in SIHP, and the intracellular survival ability of SIHP was much greater than that of the gene tst negative Staphylococcus aureus. We also found that IL-2, IL-6 and IL-17A secreted in the supernatant of SIHP infected macrophages increased significantly compared to tst negative control strains infected ones. MLST analysis showed that all of SIHP strains were classified into ST5 clone. To our knowledge, this study firstly showed that SIHP strains are

Bacterial antimicrobial resistance has been associated with the up regulation of genes encoding efflux pumps and the down regulation of genes encoding outer membrane proteins (OMPs). Gene expression in bacteria is primarily initiated by sigma factors (σ factors) such as RpoE, which plays an important role in responding to many environmental stresses. Here, we report the first observation that RpoE serves as an antibiotic resistance regulator in Salmonella enteric serovar Typhi (S. Typhi). In this study, we found that the rpoE mutant (ΔrpoE) of S. Typhi GIFU10007 has elevated resistance to several antimicrobial agents, including β-lactams, quinolones, and aminoglycosides. Genomic DNA microarray analysis was used to investigate the differential gene expression profiles between a wild type and rpoE mutant in response to ampicillin. The results showed that a total of 57 genes displayed differential expression (two-fold increase or decrease) in ΔrpoE versus the wild-type strain. The expressions of two outer membrane protein genes, ompF and ompC, were significantly down-regulated in ΔrpoE (six and seven-fold lower in comparison to wild-type strain) and RamA, a member of the efflux pump AraC/XylS family, was up-regulated about four-fold in the ΔrpoE. Our results suggest RpoE is a potential antimicrobial regulator in S. Typhi, controlling both the down regulation of the OMP genes and up-regulating the efflux system.

Staphylococcus aureus (S. aureus) is an important human pathogen, which commonly causes the acquired infectious diseases in the hospital and community. Effective and simple antibiotic treatment against S. aureus-related disease becomes increasingly difficult. Developing a safe and effective vaccine against S. aureus has become one of the world's hot spots once again. The key issue of developing the vaccine of S. aureus is how to find an ideal key pathogenic gene of S. aureus. It was previously suggested that EsxA might be a very important factor in S. aureus abscess formation in mice, but clinical experimental evidence was lacking. We therefore expressed EsxA protein through prokaryotic expression system and purified EsxA protein by Ni-affinity chromatography. ELISA was used to detect the anti-EsxA antibodies in sera of 78 patients with S. aureus infection and results showed that the anti-EsxA antibodies were positive in the sera of 19 patients. We further analyzed the EsxA positive antibodies related strains by antimicrobial susceptibility assay and found that all of the corresponding strains were multi-drug resistant. Among those multi-drug resistant strains, 73.7% were resistant to MRSA. The results indicated EsxA is very important in the pathogenesis of S. aureus. We suggested that the EsxA is very valuable as vaccine candidate target antigens for prevention and control of S. aureus infection.

Background Tumor cell expression of Toll-like receptors (TLRs) can promote inflammation and cell survival in the tumor microenvironment. Toll-like receptor 4 (TLR4) signaling in tumor cells can mediate tumor cell immune escape and tumor progression, and it is regarded as one of the mechanisms for chronic inflammation in tumorigenesis and progression. The expression of TLR4 in human breast cancer cell line MDA-MB-231 and its biological function in the development and progression of breast cancer have not been investigated. We sought to characterize the expression of TLR1-TLR10 in the established human breast cancer cell line MDA-MB-231, and to investigate the biological roles of TLR4 in breast cancer cells growth, survival, and its potential as a target for breast cancer therapy. Methods TLRs mRNA and protein expressions were detected in human breast cancer cell line MDA-MB-231 by RT-PCR, real-time PCR and flow cytometry (FCM). RNA interference was used to knockdown the expression of TLR4 in MDA-MB-231. MDA-MB-231 transfected with the vector pGenesil-1 and the vector containing a scrambled siRNA were as controls. Recombinant plasmids named TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA specific to TLR4 were transfected into human breast cancer cell line MDA-MB-231 with Lipfectamine™2000 reagent. TLR4 mRNA and protein expressions were investigated by RT-PCR, real-time PCR, FCM and immunofluorescence after silence. MTT analysis was performed to detect cell proliferation and FCM was used to detect the secretion of inflammatory cytokines in supernatant of transfected cells. Results The human breast cancer cell line MDA-MB-231 was found to express TLR1-TLR10 at both the mRNA and protein levels. TLR4 was found to be the highest expressed TLR in MDA-MB-231. TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA were found to significantly inhibit TLR4 expression in MDA-MB-231 at both mRNA and protein levels as compared to vector control(vector transfected cells). TLR4AsiRNA mediated the strongest effect. Knockdown of TLR4 gene in MDA-MB-231 resulted in a dramatic reduction of breast cancer cell viability. The cytokines which were secreted by the TLR4 silenced cells, such as IL-6 and IL-8, also decreased significantly as compared with vector control. No significant difference was observed in siRNA control (Recombinant plasmid named ScrambledsiRNA transfected cells) compared to vector control. Conclusions These studies identified the expression levels of multiple TLRs in human breast cancer cell line MDA-MB-231 and demonstrated that knockdown of TLR4 could actively inhibit proliferation and survival of breast cancer cells. Taken together, our results suggest RNAi-directed targeting of TLR4 may be a beneficial strategy for breast cancer therapy.