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1.
PLoS One ; 12(3): e0174716, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28355266

RESUMO

Clostridium difficile is one of the main etiological agents causing antibiotic-associated diarrhea. This study investigated the genetic diversity of 70 toxigenic C. difficile isolates from two Korean hospitals by employing toxinotyping, ribotyping, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Toxin gene amplification resulted in 68 A⁺B⁺ and two A-B+ isolates. Most isolates (95.7-100%) were susceptible to daptomycin, metronidazole, and vancomycin. Seventy C. difficile isolates were classified into five toxinotypes, 19 ribotypes, 16 sequence types (STs), and 33 arbitrary pulsotypes. All C. difficile isolates of ribotype 018 (n = 38) were classified into ST17, which was the most prevalent ST in both hospitals. However, C. difficile isolates of ST17 (ribotype 018) exhibited pulsotypes that differed by hospital. ST2 (ribotype 014/020), 8 (ribotypes 002), 17 (ribotype 018), and 35 (ribotypes 015) were detected in both hospitals, whereas other STs were unique to each hospital. Statistical comparison of the different typing methods revealed that ribotyping and PFGE were highly predictive of STs. In conclusion, our epidemiological study indicates that C. difficile infections in both hospitals are associated with the persistence of endemic clones coupled with the emergence of many unique clones. A combination of MLST with PFGE or ribotyping could be useful for monitoring epidemic C. difficile strains and the emergence of new clones in hospitals.


Assuntos
Antibacterianos/farmacologia , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/genética , Infecções por Clostridium/microbiologia , Infecção Hospitalar/microbiologia , Testes de Sensibilidade Microbiana/métodos , Povo Asiático , Clostridioides difficile/classificação , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/etnologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/etnologia , Daptomicina/farmacologia , Diarreia/epidemiologia , Diarreia/etnologia , Diarreia/microbiologia , Eletroforese em Gel de Campo Pulsado/métodos , Fezes/microbiologia , Genes Bacterianos/genética , Hospitais , Humanos , Metronidazol/farmacologia , Epidemiologia Molecular/métodos , Tipagem de Sequências Multilocus , Reprodutibilidade dos Testes , República da Coreia/epidemiologia , Ribotipagem , Vancomicina/farmacologia
2.
Microb Pathog ; 107: 6-11, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28284851

RESUMO

Clostridium difficile is the most common etiological agent of antibiotic-associated diarrhea in hospitalized and non-hospitalized patients. This study investigated the secretion of membrane vesicles (MVs) from C. difficile and determined the expression of pro-inflammatory cytokine genes and cytotoxicity of C. difficile MVs in epithelial cells in vitro. C. difficile ATCC 43255 and two clinical isolates secreted spherical MVs during in vitro culture. Proteomic analysis revealed that MVs of C. difficile ATCC 43255 contained a total of 262 proteins. Translation-associated proteins were the most commonly identified in C. difficile MVs, whereas TcdA and TcdB toxins were not detected. C. difficile ATCC 43255-derived MVs stimulated the expression of pro-inflammatory cytokine genes, including interleukin (IL)-1ß, IL-6, IL-8, and monocyte chemoattractant protein-1 in human colorectal epithelial Caco-2 cells. Moreover, these extracellular vesicles induced cytotoxicity in Caco-2 cells. In conclusion, C. difficile MVs are important nanocomplexes that elicit a pro-inflammatory response and induce cytotoxicity in colonic epithelial cells, which may contribute, along with toxins, to intestinal mucosal injury during C. difficile infection.


Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Clostridioides difficile/metabolismo , Colo/patologia , Citocinas/efeitos dos fármacos , Citocinas/genética , Enterotoxinas/toxicidade , Células Epiteliais/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CACO-2/efeitos dos fármacos , Técnicas de Cultura de Células , Quimiocina CCL2/efeitos dos fármacos , Quimiocina CCL2/genética , Clostridioides difficile/genética , Enterocolite Pseudomembranosa/metabolismo , Enterotoxinas/genética , Enterotoxinas/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2/efeitos dos fármacos , Humanos , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-6/genética , Interleucina-8/efeitos dos fármacos , Interleucina-8/genética , Mucosa Intestinal/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Proteômica
3.
Int J Antimicrob Agents ; 49(1): 107-111, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27908580

RESUMO

The aim of this study was to screen lead compounds exhibiting potent in vitro antimicrobial activity against multidrug-resistant (MDR) Acinetobacter baumannii strains from a library of chemical compounds. In a high-throughput screening analysis of 7520 compounds representative of 340,000 small molecules, two 4H-4-oxoquinolizine compounds were the most active against A. baumannii ATCC 17978. Subsequent selection and analysis of 70 4H-4-oxoquinolizine compounds revealed that the top 7 compounds were extremely active against extensively drug-resistant (XDR) A. baumannii isolates. These compounds commonly carried a 1-cyclopropyl-7-fluoro-4-oxo-4H-quinolizine-3-carboxylic acid core structure but had different C-8 and/or C-9 moieties. Minimum inhibitory concentrations (MICs) of the seven compounds against fluoroquinolone-resistant A. baumannii isolates were found to be in the range of 0.02-1.70 µg/mL regardless of the mutation types in the quinolone resistance-determining region (QRDR) of GyrA and ParC. Cytotoxicity of the seven compounds was observed in HeLa and U937 cells at a concentration of 50 µg/mL, which was >32.5- to 119-fold higher than the MIC90 for A. baumannii isolates. In conclusion, novel 4H-4-oxoquinolizine compounds represent a promising scaffold on which to develop antimicrobial agents against drug-resistant A. baumannii strains.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Quinolizinas/farmacologia , Antibacterianos/química , Antibacterianos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Quinolizinas/química , Quinolizinas/toxicidade
4.
Pathog Dis ; 74(8)2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27756813

RESUMO

Stenotrophomonas maltophilia has become one of the most prevalent opportunistic pathogens in hospitalized patients. This microorganism secretes outer membrane vesicles (OMVs), but the pathogenesis of S. maltophilia as it relates to OMVs has not been characterized. This study investigated the cytotoxic activity of S. maltophilia OMVs and their ability to induce inflammatory responses both in vitro and in vivo Stenotrophomonas maltophilia ATCC 13637 and two clinical isolates were found to secrete spherical OMVs during in vitro culture. OMVs from S. maltophilia ATCC 13637 were cytotoxic to human lung epithelial A549 cells. Stenotrophomonas maltophilia OMVs stimulated the expression of proinflammatory cytokine and chemokine genes, including interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor-α and monocyte chemoattractant protein-1, in A549 cells. Early inflammatory responses such as congestion and neutrophilic infiltrations and profound expression of proinflammatory cytokine and chemokine genes were observed in the lungs of mice injected with S. maltophilia OMVs, and were similar to responses elicited by the bacteria. Our data demonstrate that S. maltophilia OMVs are important secretory nanocomplexes that elicit a potent inflammatory response that might contribute to S. maltophilia pathogenesis during infection.


Assuntos
Membrana Celular/metabolismo , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/microbiologia , Inflamação/metabolismo , Inflamação/microbiologia , Stenotrophomonas maltophilia/fisiologia , Vesículas Transportadoras/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Feminino , Expressão Gênica , Infecções por Bactérias Gram-Negativas/genética , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Vesículas Secretórias/metabolismo , Stenotrophomonas maltophilia/patogenicidade
5.
PLoS One ; 11(9): e0162576, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27622249

RESUMO

Understanding the biology behind the epidemicity and persistence of Acinetobacter baumannii in the hospital environment is critical to control outbreaks of infection. This study investigated the contributing factors to the epidemicity of carbapenem-resistant A. baumannii (CRAB) sequence type (ST) 191 by comparing the differences in biofilm formation, expression of biofilm-associated genes, and resistance to desiccation between major epidemic (n = 16), minor epidemic (n = 12), and sporadic (n = 12) clones. Biofilm mass was significantly greater in the major epidemic than the minor epidemic and sporadic clones. Major and minor epidemic clones expressed biofilm-associated genes, abaI, bap, pgaABCD, and csuA/BABCDE, higher than the sporadic clones in sessile conditions. The csuC, csuD, and csuE genes were more highly expressed in the major epidemic than minor epidemic clones. Interestingly, minor epidemic clones expressed more biofilm-associated genes than the major epidemic clone under planktonic conditions. Major epidemic clones were more resistant to desiccation than minor epidemic and sporadic clones on day 21. In conclusion, the epidemic CRAB ST191 clones exhibit a higher capacity to form biofilms, express the biofilm-associated genes under sessile conditions, and resist desiccation than sporadic clones. These phenotypic and genotypic characteristics of CRAB ST191 may account for the epidemicity of specific CRAB ST191 clones in the hospital.


Assuntos
Infecções por Acinetobacter/epidemiologia , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Carbapenêmicos/farmacologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Resistência beta-Lactâmica/genética , Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/fisiologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Infecção Hospitalar/tratamento farmacológico , Dessecação , Epidemias , Genes Bacterianos , Humanos , República da Coreia/epidemiologia
6.
Microb Pathog ; 93: 185-93, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26924795

RESUMO

Staphylococcus aureus secretes membrane-derived vesicles (MVs), which can deliver virulence factors to host cells and induce cytopathology. However, the cytopathology of host cells induced by MVs derived from different S. aureus strains has not yet been characterized. In the present study, the cytotoxic activity of MVs from different S. aureus isolates on host cells was compared and the proteomes of S. aureus MVs were analyzed. The MVs purified from S. aureus M060 isolated from a patient with staphylococcal scalded skin syndrome showed higher cytotoxic activity toward host cells than that shown by MVs from three other clinical S. aureus isolates. S. aureus M060 MVs induced HEp-2 cell apoptosis in a dose-dependent manner, but the cytotoxic activity of MVs was completely abolished by treatment with proteinase K. In a proteomic analysis, the MVs from three S. aureus isolates not only carry 25 common proteins, but also carry ≥60 strain-specific proteins. All S. aureus MVs contained δ-hemolysin (Hld), γ-hemolysin, leukocidin D, and exfoliative toxin C, but exfoliative toxin A (ETA) was specifically identified in S. aureus M060 MVs. ETA was delivered to HEp-2 cells via S. aureus MVs. Both rETA and rHld induced cytotoxicity in HEp-2 cells. In conclusion, MVs from clinical S. aureus isolates differ with respect to cytotoxic activity in host cells, and these differences may result from differences in the MV proteomes. Further proteogenomic analysis or mutagenesis of specific genes is necessary to identify cytotoxic factors in S. aureus MVs.


Assuntos
Proteoma/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Transporte Proteico , Proteoma/genética , Proteômica , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Vesículas Transportadoras/genética , Virulência
7.
Infect Genet Evol ; 36: 1-7, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26341860

RESUMO

This study investigated the genetic basis of antimicrobial resistance and the epidemiological characteristics of 125 carbapenem-resistant Acinetobacter baumannii (CRAB) isolates collected from 2011 to 2012 in a Korean hospital. All CRAB isolates showed an extensively drug-resistant phenotype, but were susceptible to tigecycline. The blaOXA-23 and armA genes were mainly responsible for resistance to carbapenems and aminoglycosides, respectively. Four colistin-resistant CRAB isolates with different pulsotypes were identified. All four colistin-resistant isolates had a deletion at nucleotide 776 in lpxA, while one also had an insertion at nucleotide 732 in lpxA. All CRAB isolates belonged to three sequence types (STs): ST191 (n=118), ST208 (n=6), and ST436 (n=1), but were classified into 33 arbitrary pulsotypes. Of the CRAB ST191 isolates, two main arbitrary pulsotypes 5 (n=20) and 18 (n=17) emerged sequentially, but were not clonally related to CRAB isolates collected from 2009 to 2010 in the same hospital. Furthermore, of the two main pulsotypes identified among CRAB ST191 isolates from 2009 to 2010, one was clonally related to sporadic CRAB ST191 isolates from 2011 to 2012, but the other was not related to any CRAB isolate from 2011 to 2012. In conclusion, this study shows the clonal dynamics of CRAB ST191 isolates in a Korean hospital during the last four years.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Carbapenêmicos/farmacologia , Infecção Hospitalar , Genótipo , Resistência beta-Lactâmica/genética , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/epidemiologia , Acinetobacter baumannii/classificação , Acinetobacter baumannii/isolamento & purificação , Aciltransferases/genética , Análise por Conglomerados , Hospitais , Humanos , Testes de Sensibilidade Microbiana , Tipagem Molecular , Mutação , Fenótipo , República da Coreia , beta-Lactamases/genética
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