Staphylococcus aureus foldase PrsA contributes to the folding and secretion of protein A.

BACKGROUND: Staphylococcus aureus secretes a variety of proteins including virulence factors that cause diseases. PrsA, encoded by many Gram-positive bacteria, is a membrane-anchored lipoprotein that functions as a foldase to assist in post-translocational folding and helps maintain the stability of secreted proteins. Our earlier proteomic studies found that PrsA is required for the secretion of protein A, an immunoglobulin-binding protein that contributes to host immune evasion. This study aims to investigate how PrsA influences protein A secretion. RESULTS: We found that in comparison with the parental strain HG001, the prsA-deletion mutant HG001ΔprsA secreted less protein A. Deleting prsA also decreased the stability of exported protein A. Pulldown assays indicated that PrsA interacts with protein A in vivo. The domains in PrsA that interact with protein A are mapped to both the N- and C-terminal regions (NC domains). Additionally, the NC domains are essential for promoting PrsA dimerization. Furthermore, an immunoglobulin-binding assay revealed that, compared to the parental strain HG001, fewer immunoglobulins bound to the surface of the mutant strain HG001ΔprsA. CONCLUSIONS: This study demonstrates that PrsA is critical for the folding and secretion of protein A. The information derived from this study provides a better understanding of virulent protein export pathways that are crucial to the pathogenicity of S. aureus.

Exoproteome Profiling Reveals the Involvement of the Foldase PrsA in the Cell Surface Properties and Pathogenesis of Staphylococcus aureus.

Staphylococcus aureus is a bacterial pathogen that produces and exports many virulence factors that cause diseases in humans. PrsA, a membrane-bound foldase, is expressed ubiquitously in Gram-positive bacteria and required for the folding of exported proteins into a stable and active structure. To understand the involvement of PrsA in posttranslocational protein folding in S. aureus, a PrsA-deficient mutant of S. aureus HG001 was constructed. Using isobaric tags for relative and absolute quantification (iTRAQ)-based mass spectrometry analyses, the exoproteomes of PrsA mutant and wild type S. aureus were comparatively profiled, and 163 cell wall-associated proteins and 67 exoproteins with altered levels have been identified in the PrsA-deficient mutant. Bioinformatics analyses further reveal that prsA deletion altered the amounts of proteins that are potentially involved in the regulation of cell surface properties and bacterial pathogenesis. To determine the relevancy of our findings, we investigated the functional consequence of prsA deletion in S. aureus. PrsA deficiency can enhance bacterial autoaggregation and increase the adhesion ability of S. aureus to human lung epithelial cells. Moreover, mice infected with PrsA-deficient S. aureus had a better survival rate compared with those infected with the wild-type S. aureus. Collectively, our findings reveal that PrsA is required for the posttranslocational folding of numerous exported proteins and critically affects the cell surface properties and pathogenesis of S. aureus.

Development of carbapenem resistance in Pseudomonas aeruginosa is associated with OprD polymorphisms, particularly the amino acid substitution at codon 170.

Objectives: Pan-susceptible Pseudomonas aeruginosa (PSPA) clinical isolates carrying an OprD with loop 7 shortening (the group-1A allele) were found to rapidly develop carbapenem resistance under continuous selection pressure. We further studied whether OprD polymorphisms are associated with the potential to develop carbapenem resistance. Methods: OprD amino acid sequences of 126 PSPA clinical isolates were analysed to determine their STs using P. aeruginosa strain PAO1 as the control strain. Site-directed mutagenesis was performed in PAO1 to generate polymorphisms of interest. A disc diffusion method was used to select carbapenem-resistant variants from the mutant strains. Expression levels of oprD were determined by quantitative RT-PCR. MICs of carbapenems were determined by Etest. Results: Forty-eight (38.1%) of the tested isolates carried the group-1A allele. Another two major STs, C1 and C2, both of which harboured an F170L polymorphism, were found in 21 (16.7%) and 39 (31.0%) isolates, respectively. The PAO1 type was also found in 14 (11.1%) isolates. Under continuous selective pressure, isolates of most STs developed carbapenem resistance at different numbers of passaging events; only those belonging to the PAO1 type remained susceptible. However, PAO1 mutants carrying either the oprD group-1A allele or the OprD-F170L polymorphism were able to develop carbapenem resistance. Reduced oprD expression triggered by continuous imipenem challenge was found in PAO1 mutants, but not in the PAO1 WT strain. Conclusions: OprD polymorphisms, particularly the F170L substitution and the specific shortening in loop 7, appear to determine the potential for P. aeruginosa to develop carbapenem resistance.

Temporal regulation of σB by partner-switching mechanism at a distinct growth stage in Bacillus cereus.

The alternative transcription factor σB in Bacillus cereus governs the transcription of a number of genes that confer protection against general stress. This transcription factor is regulated by protein-protein interactions among RsbV, RsbW, σB, RsbY, RsbM and RsbK, all encoded in the sigB cluster. Among these regulatory proteins, RsbV, RsbW and σB comprise a partner-switching mechanism. Under normal conditions, σB remains inactive by associating with anti-sigma factor RsbW, which prevents σB from binding to the core RNA polymerase. During environmental stress, RsbK activates RsbY to hydrolyze phosphorylated RsbV, and the dephosphorylated RsbV then sequesters RsbW to liberate σB from RsbW. Although the σB partner-switching module is thought to be the core mechanism for σB regulation, the actual protein-protein interactions among these three proteins in the cell remain to be investigated. In the current study, we show that RsbW and RsbV form a long-lived complex under transient stress treatment, resulting in high persistent expression of RsbV, RsbW and σB from mid-log phase to stationary phase. Full sequestration of RsbW by excess RsbV and increased RsbW:RsbV complex stability afforded by cellular ADP contribute to the prolonged activation of σB. Interestingly, the high expression levels of RsbV, RsbW and σB were dramatically decreased beginning from the transition stage to the stationary phase. Thus, protein interactions among σB partner-switching components are required for the continued induction of σB during environmental stress in the log phase and significant down-regulation of σB is observed in the stationary phase. Our data show that σB is temporally regulated in B. cereus.

The expression of fibronectin is significantly suppressed in macrophages to exert a protective effect against Staphylococcus aureus infection.

BACKGROUND: Fibronectin (Fn) plays a major role in the attachment of Staphylococcus aureus to host cells by bridging staphylococcal fibronectin-binding proteins (FnBPs) and cell-surface integrins. A previous study demonstrated that the phagocytosis of S. aureus by macrophages is enhanced in the presence of exogenous Fn. We recently found that FnBPs overexpression also enhances phagocytic activity. The effect of S. aureus infection on the expression of macrophage Fn was investigated. RESULT: The level of Fn secreted by monocytes (THP-1), macrophages, human lung adenocarcinoma (A549) cells, and hepatocellular carcinoma (HepG2) cells in response to S. aureus infection was determined by Western blotting and it was significantly suppressed only in macrophages. The activation of signaling pathways associated with Fn regulation in macrophages and HepG2 cells was also investigated by Western blotting. Erk was activated in both macrophages and HepG2 cells, whereas Src-JNK-c-Jun signaling was only activated in macrophages. A significant decrease in macrophage viability was observed in response to S. aureus infection in the presence of exogenous Fn. CONCLUSION: The Src-JNK-c-Jun signaling pathway was activated in macrophages in response to S. aureus infection and resulted in the suppression of Fn expression. This suppression may play a protective role in macrophages against S. aureus infection. This study provides the first demonstration that Fn is suppressed in macrophages by S. aureus infection.

Reduced susceptibility to vancomycin in isogenic Staphylococcus aureus strains of sequence type 59: tracking evolution and identifying mutations by whole-genome sequencing.

OBJECTIVES: Vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) phenotypes are increasingly reported in methicillin-resistant S. aureus (MRSA) strains of distinct genetic backgrounds. This study tracked genetic evolution during the development of vancomycin non-susceptibility in a prevalent Asian community-associated MRSA clone of sequence type (ST) 59. METHODS: ST59 strains were consecutively isolated from a patient who failed chemotherapy for a septic knee over 15 months. The genetic mutations associated with the VISA phenotype were identified by whole-genome sequencing of two strains, which had the vancomycin-susceptible S. aureus (VSSA) and VISA phenotypes. The mutations were subsequently screened in other strains. By correlating the accumulated mutations with vancomycin susceptibility, genetic evolution was tracked at the whole-genome scale. RESULTS: Nine non-synonymous mutations and two steps of genetic evolution were identified during the development of the VISA phenotype. The first step involved a nonsense mutation in agrC and point mutations at five other loci, which were associated with the VSSA-to-hVISA conversion. Mutations of rpoB and fusA following the use of rifampicin and fusidic acid were identified in the second step of evolution, which corresponded to the development of dual resistance to rifampicin and fusidic acid and the conversion of hVISA to VISA. CONCLUSIONS: In vivo genetic evolution of S. aureus occurred in stepwise order during the development of incremental vancomycin non-susceptibility and was related to the use of antimicrobial agents.

Gut microbiota dysbiosis-related susceptibility to nontuberculous mycobacterial lung disease.

The role of gut microbiota in host defense against nontuberculous mycobacterial lung disease (NTM-LD) was poorly understood. Here, we showed significant gut microbiota dysbiosis in patients with NTM-LD. Reduced abundance of Prevotella copri was significantly associated with NTM-LD and its disease severity. Compromised TLR2 activation activity in feces and plasma in the NTM-LD patients was highlighted. In the antibiotics-treated mice as a study model, gut microbiota dysbiosis with reduction of TLR2 activation activity in feces, sera, and lung tissue occurred. Transcriptomic analysis demonstrated immunocompromised in lung which were closely associated with increased NTM-LD susceptibility. Oral administration of P. copri or its capsular polysaccharides enhanced TLR2 signaling, restored immune response, and ameliorated NTM-LD susceptibility. Our data highlighted the association of gut microbiota dysbiosis, systematically compromised immunity and NTM-LD development. TLR2 activation by P. copri or its capsular polysaccharides might help prevent NTM-LD.

Differential regulation and activity against oxidative stress of Dps proteins in Bacillus cereus.

In this study, the sequence similarity, structure, ferroxidase activity and efficacy in antagonizing oxidative stress of three Dps-like proteins, Dps1, Dps2 and Dps3, encoded by Bacillus cereus were comparatively analyzed. The three Dps-like proteins are homologous to other bacterial Dps proteins that exhibit ferroxidase activity. Both Dps1 and Dps2 have a typical Dps spherical structure, but Dps3 has a unique filamentous structure. Several dps mutant strains were generated to investigate the functional role of dps genes in cell protection. The dps1 null strain was the most labile to oxidative stress in the stationary phase, and the loss of dps2 resulted in greater sensitivity to peroxide exposure compared with the other mutant strains in the log phase. Interestingly, after simultaneous deletion of dps1 and dps2, the survival rate was dramatically reduced by approximately 5 log in the stationary phase. Immunoblotting analysis demonstrated that Dps1 and Dps2 in the wild-type strain were induced by oxidative stress, and Dps3 responded to general stress in the log phase. Constitutively high expression of Dps2 in a perR null mutant and PerR-specific binding of the promoter region of dps2 confirmed Dps2 as a member of the PerR regulon. In addition, the expression of Dps1 and Dps2, absent any stress, was initiated in the log phase and was abundant in the stationary phase, suggesting that the expression of Dps1 and Dps2 was dependent on the bacterial growth stage. In summary, the three Dps proteins conferred cellular protection, particularly from oxidative stress, and were differentially regulated in response to varied stress conditions.

Interplay of RsbM and RsbK controls the σ(B) activity of Bacillus cereus.

The alternative transcription factor σ(B) of Bacillus cereus controls the expression of a number of genes that respond to environmental stress. Four proteins encoded in the sigB gene cluster, including RsbV, RsbW, RsbY (RsbU) and RsbK, are known to be essential in the σ(B)-mediated stress response. In the context of stress, the hybrid sensor kinase RsbK is thought to phosphorylate the response regulator RsbY, a PP2C serine phosphatase, leading to the dephosphorylation of the phosphorylated RsbV. The unphosphorylated RsbV then sequesters the σ(B) antagonist, RsbW, ultimately liberating σ(B). The gene arrangement reveals an open reading frame, bc1007, flanked immediately downstream by rsbK within the sigB gene cluster. However, little is known about the function of bc1007. In this study, the deletion of bc1007 resulted in high constitutive σ(B) expression independent of environmental stimuli, indicating that bc1007 plays a role in σ(B) regulation. A bacterial two-hybrid analysis demonstrated that BC1007 interacts directly with RsbK, and autoradiographic studies revealed a specific C(14)-methyl transfer from the radiolabelled S-adenosylmethionine to RsbK when RsbK was incubated with purified BC1007. Our data suggest that BC1007 (RsbM) negatively regulates σ(B) activity by methylating RsbK. Additionally, mutagenic substitution was employed to modify 12 predicted methylation residues in RsbK. Certain RsbK mutants were able to rescue σ(B) activation in a rsbK-deleted bacterial strain, but RsbK(E439A) failed to activate σ(B), and RsbK(E446A) only moderately induced σ(B). These results suggest that Glu439 is the preferred methylation site and that Glu446 is potentially a minor methylation site. Gene arrays of the rsbK orthologues and the neighbouring rsbM orthologues are found in a wide range of bacteria. The regulation of sigma factors through metylation of RsbK-like sensor kinases appears to be widespread in the microbial world.

Cooperation of TLR2 with MyD88, PI3K, and Rac1 in lipoteichoic acid-induced cPLA2/COX-2-dependent airway inflammatory responses.

Lipoteichoic acid (LTA) plays a role in the pathogenesis of severe inflammatory responses induced by Gram-positive bacterial infection. Cytosolic phospholipase A(2) (cPLA(2)), cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)), and interleukin (IL)-6 have been demonstrated to engage in airway inflammation. In this study, LTA-induced cPLA(2) and COX-2 expression and PGE(2) or IL-6 synthesis were attenuated by transfection with siRNAs of TLR2, MyD88, Akt, p42, p38, JNK2, and p65 or pretreatment with the inhibitors of PI3K (LY294002), p38 (SB202190), MEK1/2 (U0126), JNK1/2 (SP600125), and NF-kappaB (helenalin) in human tracheal smooth muscle cells (HTSMCs). LTA also induced cPLA(2) and COX-2 expression and leukocyte count in bronchoalveolar lavage fluid in mice. LTA-regulated PGE(2) or IL-6 production was inhibited by pretreatment with the inhibitors of cPLA(2) (AACOCF(3)) and COX-2 (NS-398) or transfection with cPLA(2) siRNA or COX-2 siRNA, respectively. LTA-stimulated NF-kappaB translocation or cPLA(2) phosphorylation was attenuated by pretreatment with LY294002, SB202190, U0126, or SP600125. Furthermore, LTA could stimulate TLR2, MyD88, PI3K, and Rac1 complex formation. We also demonstrated that Staphylococcus aureus could trigger these responses through a similar signaling cascade in HTSMCs. It was found that PGE(2) could directly stimulate IL-6 production in HTSMCs or leukocyte count in bronchoalveolar lavage fluid in mice. These results demonstrate that LTA-induced MAPKs activation is mediated through the TLR2/MyD88/PI3K/Rac1/Akt pathway, which in turn initiates the activation of NF-kappaB, and ultimately induces cPLA(2)/COX-2-dependent PGE(2) and IL-6 generation.

Identification of prophage gene z2389 in Escherichia coli EDL933 encoding a DNA cytosine methyltransferase for full protection of NotI sites.

Pulsed-field gel electrophoresis (PFGE) analysis revealed that the genomes of some pathogenic Escherichia coli O157:H7 strains, including EDL933, were resistant to NotI digestion. An amino acid sequence comparison suggested that the z2389 gene carried on prophage CP-933R in strain EDL933 is likely to encode a C(5)-cytosine methyltransferase. The z2389-equivalent gene was found in the NotI-resistant strains tested, but it was not detected in the NotI-susceptible strains. PFGE analysis of the wild-type EDL933 strain and of a z2389 null mutant revealed that z2389 was associated with full genome protection against NotI digestion and partial protection against EagI digestion. In vitro methylation experiments with purified recombinant protein demonstrated that Z2389 is capable of methylating NotI and EagI sites. Sequencing of bisulfite-treated DNA indicated that the methylation occurred at the first cytosine residue of the NotI recognition sequence, whereas EagI sites remained unmethylated or were methylated at the first cytosine residue. Thus, z2389 encodes a DNA cytosine methyltransferase that confers full protection to NotI sites.

Impaired inflammasome activation and bacterial clearance in G6PD deficiency due to defective NOX/p38 MAPK/AP-1 redox signaling.

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway that modulates cellular redox homeostasis via the regeneration of NADPH. G6PD-deficient cells have a reduced ability to induce the innate immune response, thus increasing host susceptibility to pathogen infections. An important part of the immune response is the activation of the inflammasome. G6PD-deficient peripheral blood mononuclear cells (PBMCs) from patients and human monocytic (THP-1) cells were used as models to investigate whether G6PD modulates inflammasome activation. A decreased expression of IL-1ß was observed in both G6PD-deficient PBMCs and PMA-primed G6PD-knockdown (G6PD-kd) THP-1 cells upon lipopolysaccharide (LPS)/adenosine triphosphate (ATP) or LPS/nigericin stimulation. The pro-IL-1ß expression of THP-1 cells was decreased by G6PD knockdown at the transcriptional and translational levels in an investigation of the expression of the inflammasome subunits. The phosphorylation of p38 MAPK and downstream c-Fos expression were decreased upon G6PD knockdown, accompanied by decreased AP-1 translocation into the nucleus. Impaired inflammasome activation in G6PD-kd THP-1 cells was mediated by a decrease in the production of reactive oxygen species (ROS) by NOX signaling, while treatment with hydrogen peroxide (H2O2) enhanced inflammasome activation in G6PD-kd THP-1 cells. G6PD knockdown decreased Staphylococcus aureus and Escherichia coli clearance in G6PD-kd THP-1 cells and G6PD-deficient PBMCs following inflammasome activation. These findings support the notion that enhanced pathogen susceptibility in G6PD deficiency is, in part, due to an altered redox signaling, which adversely affects inflammasome activation and the bactericidal response.

Regulation of swarming motility and flhDC(Sm) expression by RssAB signaling in Serratia marcescens.

Serratia marcescens cells swarm at 30 degrees C but not at 37 degrees C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37 degrees C reduced the expression levels of flhDC(Sm) and hag(Sm) in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a two-component system, also resulted in precocious swarming phenotypes at 37 degrees C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDC(Sm) and synthesis of flagella are significantly increased in the rssA mutant strain at 37 degrees C. Primer extension analysis for determination of the transcriptional start site(s) of flhDC(Sm) revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB approximately P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB approximately P, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssB approximately P binding site is located between base pair positions -341 and -364 from the translation start codon ATG in the flhDC(Sm) promoter region. The binding site overlaps with the P2 "-35" promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB-P binds to the flhDC(Sm) promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDC(Sm) promoter activity at 37 degrees C. This inhibitory effect was comparatively alleviated at 30 degrees C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37 degrees C.

Vancomycin-resistant Enterococcus faecium at a university hospital in Taiwan, 2002-2015: Fluctuation of genetic populations and emergence of a new structure type of the Tn1546-like element.

BACKGROUND/PURPOSES: Vancomycin resistance increased significantly to 31.3% among Enterococcus faecium in 2006 and remained high thereafter at a university hospital in Taiwan. A longitudinal study was retrospectively conducted to characterize these vancomycin-resistant E. faecium (VRE-fm). METHODS: A total of 378 non-repetitive VRE-fm blood isolates collected during 2002-2015 were studied. Multilocus sequence typing, pulsed-field gel electrophoresis, analysis of van genes and the Tn1546 structure, and conjugation experiments were performed. RESULTS: The majority (78.0%) of the isolates were associated with hospital-acquired infections. Molecular typing revealed nine major pulsotypes and five predominant sequence types (STs): ST17 (33.9%), ST78 (18.3%), ST414 (14.6%), ST18 (10.6%), and ST203 (7.4%). Fluctuation of these prevailing STs among the study years in association with some major pulsotypes was noted. All isolates carried vanA genes, except that in four isolates vanB genes were found. Among the vanA-carrying Tn1546-like elements, one predominant structure type (Type I, 55.9%) was noted throughout the study years. Since 2009, another predominant structure type (Type II, 40.1%) has emerged firstly in ST414 and gradually spread to other 11 STs in subsequent years. Isolates carrying these Type II Tn1546-like elements have become the most predominant population since 2014, majorly found in ST78 and ST17. Preliminary experiments indicated that plasmids carrying the Type II Tn1546-like elements demonstrated ten-fold higher efficiency than those carrying the Type I Tn1546-like elements. CONCLUSION: Dissemination of some major STs and horizontal transfer of plasmids carrying two major structure types of Tn1546-like elements may have together contributed to the increase of VRE-fm infection.

Prevalence and clinical consequences of cytomegalovirus DNA in the aqueous humour and corneal transplants.

AIM: To determine the prevalence and clinical consequences of cytomegalovirus (CMV) DNA in the aqueous and corneal tissues obtained at the time of corneal transplantation to evaluate the diagnostic value of PCR analysis in identifying patients at risk of postkeratoplasty CMV endotheliitis. METHODS: Thirty patients who underwent corneal transplantation were included in 2011. The aqueous, excised recipient corneas and donor corneoscleral rims were analysed by PCR for the presence of CMV DNA. The medical records of the patients were retrospectively reviewed and linked with PCR results. RESULTS: CMV DNA was detected in three (10%) aqueous, eight (26.7%) recipient corneas and six (20.0%) donor corneas obtained during keratoplasty from the 30 patients. Postoperatively, four patients, who had CMV DNA in either aqueous (3) or recipient cornea (1), were diagnosed with CMV endotheliitis based on clinical features and repeat aqueous tapping for real-time PCR analysis. At the median 60.5 months follow-up, 8 (72.7%), including 4 with postkeratoplasty CMV endotheliitis, of the 11 patients with CMV positivity in any one sample had graft failure, while 9 (47.3%) of the 19 patients without evidence of CMV DNA experienced graft failure. CONCLUSIONS: We found a relatively high prevalence of CMV DNA in the aqueous and corneas obtained during keratoplasty. All the patients who had CMV positivity in aqueous developed CMV endotheliitis postoperatively and experienced graft failure eventually. Aqueous tapping at the time of corneal transplantation for PCR analysis may help to improve the diagnosis and follow-up management of postkeratoplasty CMV endotheliitis.

Risk factors of treatment failure and 30-day mortality in patients with bacteremia due to MRSA with reduced vancomycin susceptibility.

Bacteremia caused by MRSA with reduced vancomycin susceptibility (MRSA-RVS) frequently resulted in treatment failure and mortality. The relation of bacterial factors and unfavorable outcomes remains controversial. We retrospectively reviewed clinical data of patients with bacteremia caused by MRSA with vancomycin MIC = 2 mg/L from 2009 to 2012. The significance of bacterial genotypes, agr function and heterogeneous vancomycin-intermediate S. aureus (hIVSA) phenotype in predicting outcomes were determined after clinical covariates adjustment with multivariate analysis. A total of 147 patients with mean age of 63.5 (±18.1) years were included. Seventy-nine (53.7%) patients failed treatment. Forty-seven (31.9%) patients died within 30 days of onset of MRSA bacteremia. The Charlson index, Pitt bacteremia score and definitive antibiotic regimen were independent factors significantly associated with either treatment failure or mortality. The hVISA phenotype was a potential risk factor predicting treatment failure (adjusted odds ratio 2.420, 95% confidence interval 0.946-6.191, P = 0.0652). No bacterial factors were significantly associated with 30-day mortality. In conclusion, the comorbidities, disease severity and antibiotic regimen remained the most relevant factors predicting treatment failure and 30-day mortality in patients with MRSA-RVS bacteremia. hIVSA phenotype was the only bacterial factor potentially associated with unfavorable outcome in this cohort.

Formulation of two-layer dissolving polymeric microneedle patches for insulin transdermal delivery in diabetic mice.

Dissolving microneedles (MNs) display high efficiency in delivering poorly permeable drugs and vaccines. Here, two-layer dissolving polymeric MN patches composed of gelatin and sodium carboxymethyl cellulose (CMC) were fabricated with a two-step casting and centrifuging process to localize the insulin in the needle and achieve efficient transdermal delivery of insulin. In vitro skin insertion capability was determined by staining with tissue-marking dye after insertion, and the real-time penetration depth was monitored using optical coherence tomography. Confocal microscopy images revealed that the rhodamine 6G and fluorescein isothiocyanate-labeled insulin (insulin-FITC) can gradually diffuse from the puncture sites to deeper tissue. Ex vivo drug-release profiles showed that 50% of the insulin was released and penetrated across the skin after 1 h, and the cumulative permeation reached 80% after 5 h. In vivo and pharmacodynamic studies were then conducted to estimate the feasibility of the administration of insulin-loaded dissolving MN patches on diabetic mice for glucose regulation. The total area above the glucose level versus time curve as an index of hypoglycemic effect was 128.4 ± 28.3 (% h) at 0.25 IU/kg. The relative pharmacologic availability and relative bioavailability (RBA) of insulin from MN patches were 95.6 and 85.7%, respectively. This study verified that the use of gelatin/CMC MN patches for insulin delivery achieved a satisfactory RBA compared to traditional hypodermic injection and presented a promising device to deliver poorly permeable protein drugs for diabetic therapy. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 84-93, 2017.

Antibiotic susceptibility profiles of ocular and nasal flora in patients undergoing cataract surgery in Taiwan: an observational and cross-sectional study.

OBJECTIVE: To investigate the conjunctival and nasal flora and the antibiotic susceptibility profiles of isolates from patients undergoing cataract surgery. DESIGN: Observational and cross-sectional study. SETTING: A single-centre study in Taiwan. PARTICIPANTS: 128 consecutive patients precataract surgery. PRIMARY AND SECONDARY OUTCOME MEASURES METHODS: Conjunctival and nasal cultures were prospectively obtained from 128 patients on the day of cataract surgery before instillation of ophthalmic solutions in our hospital. Isolates and antibiotic susceptibility profiles were identified through standard microbiological techniques. Participants were asked to complete a questionnaire on healthcare-associated factors. RESULTS: The positive culture rate from conjunctiva was 26.6%, yielding 84 isolates. Coagulase-negative Staphylococci were the most commonly isolated organisms (45.2%), and 35% of staphylococcal isolates were methicillin-resistant. Among staphylococcal isolates, all were susceptible to vancomycin, and 75%-82.5% were susceptible to fluoroquinolones. Methicillin-resistant isolates were significantly less susceptible than their methicillin-sensitive counterparts to tobramycin, the most commonly used prophylactic antibiotic in our hospital (28.6% vs 69.2%; p=0.005). The positive culture rate from nares for Staphylococcus aureus was 21.9%, and six isolates were methicillin-resistant. No subjects had S. aureus colonisation on conjunctiva and nares simultaneously. There were no associated risk factors for colonisation of methicillin-resistant Staphylococci. CONCLUSION: The most common conjunctival bacterial isolate of patients undergoing cataract surgery was coagulase-negative Staphylococci in Taiwan. Because of predominant antibiotic preferences and selective antibiotic pressures, Staphylococci were more susceptible to fluoroquinolones but less to tobramycin than in other reports. Additionally, methicillin-resistant Staphylococci exhibited co-resistance to tobramycin but not to fluoroquinolones.

Nucleotide Sequence Variations in Autolysis Genes of ST59 Methicillin-Resistant Staphylococcus aureus Isolates.

Biofilm formation is a virulence factor of bacteria. The goal of this study was to understand the mechanisms of biofilm formation by methicillin-resistant Staphylococcus aureus (MRSA). Whole-genome sequencing of eight MRSA strains was performed to identify sequence variations in genes related to biofilm formation. Thirty-one genes involved in MRSA biofilm formation were analyzed and 11 amino acid sequence variations in four genes related to autolysis were found. These variations include E121D and H387 N in ArlS; Q117K, T424S, K428T, A509S, V752E, A754V, and T771A in Atl; T184K in CidC; and D251N in CidR. Among the 26 clinical MRSA isolates studied, 13 isolates were nonbiofilm producers and were found to harbor these mutations. Furthermore, all of these 13 isolates belonged to ST59. Ten of these 13 ST59 isolates became able to produce biofilms when they were incubated with extracellular DNA from MRSA N315. Results of this study suggest that sequence variations in arlS, atl, cidC, and cidR genes may render MRSA unable to produce biofilms. Further investigations are needed to correlate these sequence variations with the biofilm-forming ability of MRSA isolates.

Studies of SpoIIAB mutant proteins elucidate the mechanisms that regulate the developmental transcription factor sigmaF in Bacillus subtilis.

SigmaF, the first compartment-specific sigma factor of sporulation, is regulated by an anti-sigma factor, SpoIIAB (AB) and its antagonist SpoIIAA (AA). AB can bind to sigmaF in the presence of ATP or to AA in the presence of ADP; in addition, AB can phosphorylate AA. The ability of AB to switch between its two binding partners regulates sigmaF. Early in sporulation, AA activates sigmaF by releasing it from its complex with AB. We have previously proposed a reaction scheme for the phosphorylation of AA by AB which accounts for AA's regulatory role. A crucial feature of this scheme is a conformational change in AB that accompanies its switch in binding partner. In the present study, we have studied three AB mutants, all of which have amino-acid replacements in the nucleotide-binding region; AB-E104K (Glu104-->Lys) and AB-T49K (Thr49-->Lys) fail to activate sigmaF, and AB-R105A (Arg105-->Ala) activates it prematurely. We used techniques of enzymology, surface plasmon resonance and fluorescence spectroscopy to analyse the defects in each mutant. AB-E104K was deficient in binding to AA, AB-T49K was deficient in binding to ADP and AB-R105A bound ADP exceptionally strongly. Although the release of sigmaF from all three mutant proteins was impaired, and all three failed to undergo the wild-type conformational change when switching binding partners, the phenotypes of the mutant cells were best accounted for by the properties of the respective AB species in forming complexes with AA and ADP. The behaviour of the mutants enables us to propose convincing mechanisms for the regulation of sigmaF in wild-type bacteria.