RESUMEN
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.
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Disbiosis , Microbioma Gastrointestinal , Infecciones por Mycobacterium no Tuberculosas , Receptor Toll-Like 2 , Disbiosis/microbiología , Animales , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 2/genética , Humanos , Ratones , Masculino , Femenino , Infecciones por Mycobacterium no Tuberculosas/microbiología , Persona de Mediana Edad , Heces/microbiología , Anciano , Prevotella , Enfermedades Pulmonares/microbiología , Micobacterias no Tuberculosas , Susceptibilidad a Enfermedades , Ratones Endogámicos C57BL , Pulmón/microbiologíaRESUMEN
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.
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Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Proteínas Bacterianas/metabolismo , Proteína Estafilocócica A , Pliegue de Proteína , Proteínas de la Membrana/metabolismo , Proteómica , Infecciones Estafilocócicas/microbiología , Inmunoglobulinas/metabolismoRESUMEN
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-1â¯cells upon lipopolysaccharide (LPS)/adenosine triphosphate (ATP) or LPS/nigericin stimulation. The pro-IL-1ß expression of THP-1â¯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-1â¯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-1â¯cells. G6PD knockdown decreased Staphylococcus aureus and Escherichia coli clearance in G6PD-kd THP-1â¯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.
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Deficiencia de Glucosafosfato Deshidrogenasa/inmunología , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , NADPH Oxidasas/metabolismo , Factor de Transcripción AP-1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Regulación hacia Abajo , Femenino , Técnicas de Silenciamiento del Gen , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Deficiencia de Glucosafosfato Deshidrogenasa/microbiología , Células HEK293 , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/efectos adversos , Masculino , Células THP-1/efectos de los fármacos , Células THP-1/inmunología , Células THP-1/microbiología , Adulto JovenRESUMEN
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.
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Infección Hospitalaria/epidemiología , Elementos Transponibles de ADN/genética , Enterococcus faecium/aislamiento & purificación , Infecciones por Bacterias Grampositivas/epidemiología , Enterococos Resistentes a la Vancomicina/genética , Enterococos Resistentes a la Vancomicina/aislamiento & purificación , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Infección Hospitalaria/microbiología , Enterococcus faecium/clasificación , Enterococcus faecium/genética , Genotipo , Infecciones por Bacterias Grampositivas/microbiología , Hospitales Universitarios , Humanos , Estudios Longitudinales , Plásmidos , Estudios Retrospectivos , Taiwán/epidemiología , Resistencia a la Vancomicina/genética , Enterococos Resistentes a la Vancomicina/clasificaciónRESUMEN
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.
RESUMEN
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.
Asunto(s)
Bacteriemia/diagnóstico , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Resistencia a la Vancomicina/efectos de los fármacos , Vancomicina/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Bacteriemia/tratamiento farmacológico , Bacteriemia/microbiología , Bacteriemia/mortalidad , Femenino , Genotipo , Proteínas Hemolisinas/metabolismo , Humanos , Masculino , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Staphylococcus aureus Resistente a Meticilina/metabolismo , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Oportunidad Relativa , Fenotipo , Estudios Retrospectivos , Factores de Riesgo , Tasa de Supervivencia , Insuficiencia del Tratamiento , Vancomicina/uso terapéuticoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Lipoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Proteoma/análisis , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/metabolismo , Factores de Virulencia/metabolismo , Células A549 , Animales , Adhesión Bacteriana , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Lipoproteínas/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Mutación , Pliegue de Proteína , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Propiedades de Superficie , Factores de Virulencia/genéticaRESUMEN
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.
Asunto(s)
Bacillus cereus/crecimiento & desarrollo , Bacillus cereus/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Regulación Bacteriana de la Expresión Génica , Factor sigma/metabolismo , Unión Proteica , Mapas de Interacción de ProteínasRESUMEN
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.
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Antibacterianos/uso terapéutico , Extracción de Catarata/efectos adversos , Conjuntiva/microbiología , Resistencia a Medicamentos , Nariz/microbiología , Infecciones Estafilocócicas/prevención & control , Staphylococcus/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Catarata/terapia , Estudios Transversales , Femenino , Fluoroquinolonas/uso terapéutico , Humanos , Masculino , Meticilina/uso terapéutico , Resistencia a la Meticilina , Staphylococcus aureus Resistente a Meticilina , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Infecciones Estafilocócicas/etiología , Infecciones Estafilocócicas/microbiología , Staphylococcus/crecimiento & desarrollo , Taiwán , Tobramicina/uso terapéutico , Vancomicina/uso terapéutico , Resistencia a la VancomicinaRESUMEN
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.
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Antibacterianos/farmacología , Carbapenémicos/farmacología , Farmacorresistencia Bacteriana/genética , Polimorfismo Genético , Porinas/genética , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Alelos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Infecciones por Pseudomonas/microbiologíaRESUMEN
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.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Fibronectinas/inmunología , Macrófagos/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/fisiología , Células A549 , Línea Celular , Supervivencia Celular , Fibronectinas/genética , Regulación de la Expresión Génica , Células Hep G2 , Humanos , MAP Quinasa Quinasa 4/genética , Sistema de Señalización de MAP Quinasas , Macrófagos/citología , Monocitos/fisiología , Fagocitosis , Proteínas Proto-Oncogénicas c-jun/genética , Familia-src Quinasas/genéticaRESUMEN
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.
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Secuencia de Bases/genética , Variación Genética/genética , Staphylococcus aureus Resistente a Meticilina/genética , Proteínas Bacterianas/genética , Biopelículas , Pruebas de Sensibilidad Microbiana/métodos , Factores de Virulencia/genética , Secuenciación Completa del Genoma/métodosRESUMEN
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.
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Diabetes Mellitus Experimental/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/instrumentación , Sistemas de Liberación de Medicamentos/métodos , Insulina/farmacología , Agujas , Administración Cutánea , Animales , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Masculino , Ratones , Ratones Endogámicos BALB C , PorcinosRESUMEN
σB, an alternative transcription factor, controls the response of the cell to a variety of environmental stresses in Bacillus cereus. Previously, we reported that RsbM negatively regulates σB through the methylation of RsbK, a hybrid sensor kinase, on a signaling helix (S-helix). However, RsbK comprises a C-terminal receiver (REC) domain whose function remains unclear. In this study, deletion of the C-terminal REC domain of RsbK resulted in high constitutive σB expression independent of environmental stimuli. Thus, the REC domain may serve as an inhibitory element. Mutagenic substitution was employed to modify the putative phospho-acceptor residue D827 in the REC domain of RsbK. The expression of RsbKD827N and RsbKD827E exhibited high constitutive σB, indicating that D827, if phosphorylatable, possibly participates in σB regulation. Bacterial two-hybrid analyses demonstrated that RsbK forms a homodimer and the REC domain interacts mainly with the histidine kinase (HK) domain and partly with the S-helix. In particular, co-expression of RsbM strengthens the interaction between the REC domain and the S-helix. Consistently, our structural model predicts a significant interaction between the HK and REC domains of the RsbK intradimer. Here, we demonstrated that coordinated the methylatable S-helix and the REC domain of RsbK is functionally required to modulate σB-mediated stress response in B. cereus and maybe ubiquitous in microorganisms encoded RsbK-type sensor kinases.
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Bacillus cereus/metabolismo , Bacillus cereus/fisiología , Proteínas Quinasas/metabolismo , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Transducción de Señal/fisiología , Estrés Fisiológico/fisiología , Proteínas Bacterianas/metabolismo , Ambiente , Histidina Quinasa , Metilación , Fosforilación/fisiología , Factores de Transcripción/metabolismoRESUMEN
We previously demonstrated that vancomycin treatment increased acquisition of eDNA and enhanced biofilm formation of drug-resistant Staphylococcus aureus through a cidA-mediated autolysis mechanism. Recently we found that such enhancement became more significant under a higher glucose concentration in vitro. We propose that besides improper antibiotic treatment, increased glucose concentration environment in diabetic animals may further enhance biofilm formation of drug-resistant S. aureus. To address this question, the diabetic mouse model infected by vancomycin-resistant S. aureus (VRSA) was used under vancomycin treatment. The capacity to form biofilms was evaluated through a catheter-associated biofilm assay. A 10- and 1000-fold increase in biofilm-bound bacterial colony forming units was observed in samples from diabetic mice without and with vancomycin treatment, respectively, compared to healthy mice. By contrast, in the absence of glucose vancomycin reduced propensity to form biofilms in vitro through the increased production of proteases and DNases from VRSA. Our study highlights the potentially important role of increased glucose concentration in enhancing biofilm formation in vancomycin-treated diabetic mice infected by drug-resistant S. aureus.
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Biopelículas/efectos de los fármacos , Glucemia/metabolismo , Diabetes Mellitus Experimental/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/fisiología , Vancomicina/farmacología , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Diabetes Mellitus Experimental/sangre , Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Masculino , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/genética , Microscopía Electrónica de Rastreo , Ratas Endogámicas WKY , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones Estafilocócicas/sangre , Staphylococcus aureus/genética , Staphylococcus aureus/ultraestructura , Resistencia a la Vancomicina/genéticaRESUMEN
Staphylococcus aureus is an important pathogen that forms biofilms on the surfaces of medical implants. Biofilm formation by S. aureus is associated with the production of poly N-acetylglucosamine (PNAG), also referred to as polysaccharide intercellular adhesin (PIA), which mediates bacterial adhesion, leading to the accumulation of bacteria on solid surfaces. This study shows that the ability of S. aureus SA113 to adhere to nasal epithelial cells is reduced after the deletion of the ica operon, which contains genes encoding PIA/PNAG synthesis. However, this ability is restored after a plasmid carrying the entire ica operon is transformed into the mutant strain, S. aureus SA113Δica, showing that the synthesis of PIA/PNAG is important for adhesion to epithelial cells. Additionally, S. carnosus TM300, which does not produce PIA/PNAG, forms a biofilm and adheres to epithelial cells after the bacteria are transformed with a PIA/PNAG-expressing plasmid, pTXicaADBC. The adhesion of S. carnosus TM300 to epithelial cells is also demonstrated by adding purified exopolysaccharide (EPS), which contains PIA/PNAG, to the bacteria. In addition, using a mouse model, we find that the abscess lesions and bacterial burden in lung tissues is higher in mice infected with S. aureus SA113 than in those infected with the mutant strain, S. aureus SA113Δica. The results indicate that PIA/PNAG promotes the adhesion of S. aureus to human nasal epithelial cells and lung infections in a mouse model. This study elucidates a mechanism that is important to the pathogenesis of S. aureus infections.
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Adhesión Bacteriana/genética , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Polisacáridos Bacterianos/metabolismo , Infecciones Estafilocócicas/patología , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Animales , Carga Bacteriana/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células Epiteliales/microbiología , Células Epiteliales/patología , Prueba de Complementación Genética , Humanos , Pulmón/microbiología , Pulmón/patología , Ratones , Datos de Secuencia Molecular , Operón , Plásmidos/química , Plásmidos/metabolismo , Eliminación de Secuencia , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/metabolismo , Transformación Bacteriana , beta-Glucanos/metabolismoRESUMEN
Resistance mechanisms in a group of carbapenemase-negative Pseudomonas aeruginosa that were susceptible to all antibiotics except carbapenems (carbapenem resistance-only P. aeruginosa [CROPA]) were studied. Ten genetically nonrelated CROPA isolates and their carbapenem-susceptible counterparts were further investigated. OprD production was demonstrated by protein electrophoresis in only 1 of the 10 carbapenem-susceptible isolates, while the other 9 isolates showed hyperproduction of OprM. DNA sequencing of oprD revealed a shortened loop 7 domain (group 1A allele) in eight carbapenem-susceptible isolates. Various oprD mutations, leading to early terminations, were found in 9 of the 10 CROPA isolates. RNA analysis demonstrated hyperexpression of oprM with normal expression of mexA in eight of the carbapenem-susceptible isolates, while in seven of their CROPA counterparts, the oprM expression was significantly reduced. Deletion of oprM was performed in two pairs of representative isolates. Selection of imipenem resistant variants by a disc assay indicated that the lost-of-function mutations in oprD occurred relatively faster in the ΔoprM mutants compared with their corresponding parent strains. Under selection pressure, reduced production of OprM may promote the selection of spontaneous changes in oprD, resulting in the carbapenem resistance in a group of pan-susceptible P. aeruginosa isolates characterized by harboring an oprD-group 1A allele.
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Proteínas de la Membrana Bacteriana Externa/genética , Farmacorresistencia Bacteriana/genética , Imipenem/farmacología , Proteínas de Transporte de Membrana/genética , Mutación/genética , Porinas/genética , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Alelos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Carbapenémicos/farmacología , ADN Bacteriano/genética , beta-Lactamasas/genéticaRESUMEN
Cases of bacteremia caused by vancomycin-resistant E. faecium (VRE-fm) increased significantly in Taiwan. The present multicenter surveillance study was performed to reveal the associated epidemiological characteristics. In 2012, 134 non-repetitive VRE-fm isolates were prospectively collected from 12 hospitals in Taiwan. Antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and analysis of van genes and Tn1546 structures were investigated. Two isolates carried vanB genes, while all the remaining isolates carried vanA genes. Three isolates demonstrated a specific vanA genotype - vanB phenotype. Nine (6.7%) isolates demonstrated tigecycline resistance, and all were susceptible to daptomycin and linezolid. Molecular typing revealed 58 pulsotypes and 13 sequence types (STs), all belonged to three major lineages 17, 18, and 78. The most frequent STs were ST17 (nâ=â48, 35.8%), ST414 (nâ=â22, 16.4%), and ST78 (nâ=â16, 11.9%). Among the vanA harboring isolates, eight structure types of the Tn1546-like element were demonstrated. Type I (a partial deletion in the orf1 and insertion of IS1251-like between the vanS - vanH genes) and Type II (Type I with an additional insertion of IS1678 between orf2 - vanS genes) were the most predominant, consisted of 60 (45.5%) and 62 (47.0%) isolates, respectively. The increase of VRE-fm bacteremia in Taiwan may be associated with the inter- and intra-hospital spread of some major STs and horizontal transfer of vanA genes mostly carried on two efficient Tn1546-like elements. The prevailing ST414 and widespread of the Type II Tn1546-like elements are an emerging problem that requires continuous monitoring.
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Bacteriemia , Elementos Transponibles de ADN , Enterococcus faecium , Infecciones por Bacterias Grampositivas , Enterococos Resistentes a la Vancomicina , Bacteriemia/epidemiología , Bacteriemia/genética , Bacteriemia/metabolismo , Proteínas Bacterianas/genética , Ligasas de Carbono-Oxígeno/genética , Enterococcus faecium/genética , Enterococcus faecium/aislamiento & purificación , Enterococcus faecium/patogenicidad , Femenino , Infecciones por Bacterias Grampositivas/epidemiología , Infecciones por Bacterias Grampositivas/genética , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Masculino , Taiwán/epidemiología , Enterococos Resistentes a la Vancomicina/genética , Enterococos Resistentes a la Vancomicina/aislamiento & purificación , Enterococos Resistentes a la Vancomicina/patogenicidadRESUMEN
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.
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Evolución Molecular , Estudio de Asociación del Genoma Completo/métodos , Mutación/genética , Staphylococcus aureus/genética , Resistencia a la Vancomicina/genética , Vancomicina/farmacología , Anciano , Antibacterianos/farmacología , Femenino , Humanos , Polimorfismo Genético/genética , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/genética , Staphylococcus aureus/efectos de los fármacos , Resistencia a la Vancomicina/efectos de los fármacosRESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway and provides reducing energy to all cells by maintaining redox balance. The most common clinical manifestations in patients with G6PD deficiency are neonatal jaundice and acute hemolytic anemia. The effects of microbial infection in patients with G6PD deficiency primarily relate to the hemolytic anemia caused by Plasmodium or viral infections and the subsequent medication that is required. We are interested in studying the impact of bacterial infection in G6PD-deficient cells. G6PD knock down A549 lung carcinoma cells, together with the common pathogen Staphylococcus aureus, were employed in our cell infection model. Here, we demonstrate that a lower cell viability was observed among G6PD-deficient cells when compared to scramble controls upon bacterial infection using the MTT assay. A significant increase in the intracellular ROS was detected among S. aureus-infected G6PD-deficient cells by observing dichlorofluorescein (DCF) intensity within cells under a fluorescence microscope and quantifying this signal using flow cytometry. The impairment of ROS removal is predicted to enhance apoptotic activity in G6PD-deficient cells, and this enhanced apoptosis was observed by annexin V/PI staining under a confocal fluorescence microscope and quantified by flow cytometry. A higher expression level of the intrinsic apoptotic initiator caspase-9, as well as the downstream effector caspase-3, was detected by Western blotting analysis of G6PD-deficient cells following bacterial infection. In conclusion, we propose that bacterial infection, perhaps the secreted S. aureus α-hemolysin in this case, promotes the accumulation of intracellular ROS in G6PD-deficient cells. This would trigger a stronger apoptotic activity through the intrinsic pathway thereby reducing cell viability when compared to wild type cells.