[Show abstract][Hide abstract] ABSTRACT: Acinetobacter baumannii is increasingly becoming a major nosocomial pathogen. This opportunistic pathogen secretes outer membrane vesicles (OMVs) that interact with host cells. The aim of this study was to investigate the ability of A. baumannii OMVs to elicit a pro-inflammatory response in vitro and the immunopathology in response to A. baumannii OMVs in vivo. OMVs derived from A. baumannii ATCC 19606(T) induced expression of pro-inflammatory cytokine genes, interleukin (IL)-1β and IL-6, and chemokine genes, IL-8, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1, in epithelial cells in a dose-dependent manner. Disintegration of OMV membrane with ethylenediaminetetraacetic acid resulted in low expression of pro-inflammatory cytokine genes, as compared with the response to intact OMVs. In addition, proteinase K-treated A. baumannii OMVs did not induce significant increase in expression of pro-inflammatory cytokine genes above the basal level, suggesting that the surface-exposed membrane proteins in intact OMVs are responsible for pro-inflammatory response. Early inflammatory processes, such as vacuolization and detachment of epithelial cells and neutrophilic infiltration, were clearly observed in lungs of mice injected with A. baumannii OMVs. Our data demonstrate that OMVs produced by A. baumannii elicit a potent innate immune response, which may contribute to immunopathology of the infected host.
PLoS ONE 08/2013; 8(8):e71751. DOI:10.1371/journal.pone.0071751 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Emergence and spread of specific carbapenem-resistant Acinetobacter baumannii (CRAB) clones cause a serious therapeutic problem. This study was aimed to investigate the clonal diversity and genetic basis of antimicrobial resistance among the 69 CRAB isolates from 2009 to 2010 in a Korean hospital. All CRAB isolates were found to be sequence type (ST) 2 using the Institute Pasteur's multilocus sequence typing (MLST) scheme, but classified into two sequence groups and nine pulsotypes. Fifty-six CRAB isolates belonging to two main pulsotypes were found to be ST191 using the Bartual's MLST scheme. All CRAB isolates showed an extensively drug-resistant phenotype. The blaOXA-51/blaOXA-23, blaAmpC/blaPER-1 and armA genes were largely responsible for resistance to carbapenems, extended-spectrum β-lactams and aminoglycosides, respectively. The first CRAB strains identified in 2005 in this hospital were found to be ST2 using the Institute Pasteur's MLST scheme, but showed ST353 using the Bartual's MLST scheme and different pulsotypes from the CRAB isolates from 2009 to 2010. In conclusion, this is the first report of emergence and spread of A. baumannii ST191 in Korea, as well of the genetic basis of its antimicrobial resistance.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 07/2013; 19. DOI:10.1016/j.meegid.2013.07.016 · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs) produced by gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.
PLoS ONE 11/2011; 6(11):e27958. DOI:10.1371/journal.pone.0027958 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Of the 100 multidrug-resistant Pseudomonas aeruginosa isolates from a Korean hospital, 14 isolates that were resistant to all aminoglycosides tested carried 16S rRNA methylase gene armA. Fourteen armA-positive isolates were classified into 8 pulsotypes. Seven armA-positive isolates cocarried bla(IMP-1). This study is the first report of occurrence of armA in P. aeruginosa.
[Show abstract][Hide abstract] ABSTRACT: Bacteremia is a common systemic disease caused by Acinetobacter baumannii, an important hospital-acquired pathogen among critically ill patients. The complement system is central to innate immune defense against invading bacteria in the blood. The present study investigated the susceptibility of clinical A. baumannii isolates to normal human sera (NHS), and determined the resistance mechanism of A. baumannii against complement-mediated lysis. The survival of A. baumannii isolates from bacteremic patients was significantly decreased in undiluted NHS, but they were resistant to 40% NHS. The alternative complement pathway was responsible for the direct killing of bacteria. The main regulator of the alternative complement pathway, factor H, bound to the surface of live A. baumannii treated with NHS. Factor H interacted with the outer membrane proteins with molecular sizes of 38 (AbOmpA), 32, and 24 kDa. The isogenic AbOmpA(-) mutant was highly susceptible to NHS in comparison with the wild-type A. baumannii strain, suggesting that AbOmpA was an important complement regulator-acquiring surface protein. These results indicate that A. baumannii evades complement attack through the acquisition of factor H to their surface.
[Show abstract][Hide abstract] ABSTRACT: Mutations in DNA gyrase and topoisomerase IV genes are the main mechanisms of resistance to quinolones. In this study, we determined mutations in gyrA, gyrB, parC and parE among 57 ciprofloxacin-resistant Escherichia coli isolates from a South Korean hospital and analysed the relationship between the minimal inhibitory concentrations (MICs) of fluoroquinolones and mutations in the topoisomerase IV gene. All ciprofloxacin-resistant E. coli isolates carried double mutations in gyrA and at least a single mutation in parC; some isolates also carried a single mutation in parE. The most common mutations were S83L and D87N in gyrA, S80I in parC and S458A in parE, which accounted for 25% of isolates. Single mutations in parE at L445I, S458P and S458W were identified for the first time. Double mutations in parC and a combination of single mutations in parC and parE significantly increased the MIC values of fluoroquinolones. In vitro induction of resistance to ciprofloxacin showed that double mutations in gyrA were a prerequisite to conferring a resistant phenotype to fluoroquinolones, and an additional mutation in the topoisomerase IV gene increased the MIC values of ciprofloxacin. In conclusion, emergence of a new mutation in parC and parE and its accumulation induces high levels of resistance to fluoroquinolones in E. coli.
International journal of antimicrobial agents 09/2009; 35(1):76-9. DOI:10.1016/j.ijantimicag.2009.08.003 · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A total of 75 Acinetobacter isolates resistant to all available aminoglycosides obtained from 2 Korean hospitals were studied for the genetic basis of resistance to aminoglycosides. The MIC(50) and MIC(90) of Acinetobacter baumannii isolates (n = 61) to amikacin, gentamicin, isepamycin spectinomycin, streptomycin, and tobramycin were higher than those of Acinetobacter genomic species 13TU isolates (n = 14). Genes encoding aminoglycoside-modifying enzymes, ant(3")-Ia, aac(6')-Ib, aph(3')-1a, aac(3)-Ia, and aph(3')-VI, and 16S ribosomal RNA (rRNA) methylase armA were detected. ant(3")-Ia and aac(6')-Ib were commonly detected in both Acinetobacter spp., but armA and aph(3")-Ia were only detected in A. baumannii. armA was located on the plasmids. A. baumannii isolates carrying armA were classified into 7 pulsotypes but belonged to sequence group 1. The combination of aminoglycoside-modifying enzymes is responsible for the moderate-level resistance to aminoglycosides in Acinetobacter genomic species 13TU, whereas armA is responsible for the high-level resistance to aminoglycosides in A. baumannii sequence group 1.
[Show abstract][Hide abstract] ABSTRACT: In this study, we identified extended-spectrum beta-lactamase (ESBL) and plasmid-mediated AmpC beta-lactamase which were associated with 16S rRNA methylase gene on the conjugative plasmid. Among 82 clinical isolates of Enterobacteriaceae that carry 16S rRNA methylase gene (64 strains, armA, and 18 strains, rmtB), bla(SHV-12) was detected either alone or combined with bla(DHA-1), bla(CTX-M-3), and bla(CTX-M-14) in 30 strains carrying armA and 6 strains carrying rmtB. The bla(CTX-M-3) was detected in 13 of 64 strains carrying armA but no strains carrying rmtB. Whereas bla(CTX-M-14) was detected in 15 of 18 strains carrying rmtB but only 2 of 64 strains carrying armA. Overall, bla(SHV-12) and bla(CTX-M-14) was the most common ESBL gene which was associated with armA and rmtB, respectively. In addition, we found that bla(CTX-M-3) localized with armA on the same IncL/M plasmid and bla(CTX-M-14) localized with rmtB on the same IncA/C plasmid. Restriction fragment length polymorphism of conjugative plasmids and pulsed-field gel electrophoresis of genomic DNAs revealed that intercellular horizontal transfer of conjugative plasmid and clonal transmission have been occurred at the same time.
The Journal of Microbiology 03/2009; 47(1):68-75. DOI:10.1007/s12275-008-0158-3 · 1.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acinetobacter baumannii is a nosocomial pathogen of increasing importance, but the pathogenic mechanism of this microorganism has not been fully explored. This study investigated the potential of A. baumannii to invade epithelial cells and determined the role of A. baumannii outer membrane protein A (AbOmpA) in interactions with epithelial cells.
A. baumannii invaded epithelial cells by a zipper-like mechanism, which is associated with microfilament- and microtubule-dependent uptake mechanisms. Internalized bacteria were located in the membrane-bound vacuoles. Pretreatment of recombinant AbOmpA significantly inhibited the adherence to and invasion of A. baumannii in epithelial cells. Cell invasion of isogenic AbOmpA- mutant significantly decreased as compared with wild-type bacteria. In a murine pneumonia model, wild-type bacteria exhibited a severe lung pathology and induced a high bacterial burden in blood, whereas AbOmpA- mutant was rarely detected in blood.
A. baumannii adheres to and invades epithelial cells. AbOmpA plays a major role in the interactions with epithelial cells. These findings contribute to the understanding of A. baumannii pathogenesis in the early stage of bacterial infection.
[Show abstract][Hide abstract] ABSTRACT: Background: Enterobacter cloacae have become increasingly important as a nosocomial pathogen and are found to cause biofilm-related infections such as catheter-associated urinary tract infections and biliary tract infections. In this study, 14 strains of E. cloacae isolated from patients were investigated for the ability to form biofilm and the association of curli fimbriae with the biofilm forming ability of these isolates. Methods: Biofilm-forming ability of the isolates was examined by crystal violet staining and SEM analysis. Expression of curli fimbriae was examined by a congo-red (CR) straining method for morphotype pattern analysis, TEM analysis, PCR screening of csgBA(C) operon for the presence of curli gene and RT-PCR anlaysis for csgA (major structure gene for curli fimbriae) and csgD (positive regulator gene for curli fimbriae production) mRNA expression. Results: csgBA(C) operon was detected in 11 of the 14 strains of E. cloacae and the coding region sequence of csgA was identified by a further sequencing analysis. csgBA(C) operon-positive isolates showed a dark-brown color on CR agar plate indicating the expression of curli fimbriae, which was observed by TEM analysis. csgBA(C) operon-positive isolates had the increased ability on the biofilm formation compared with csgBA(C) operon-negative isolates and biofilm forming ability was positively correlated with the mRNA expression of csgA and csgD. Conclusions: Our data suggested a strong association between the biofilm forming ability and the expression of curli genes (csgA and csgD) in clinical isolates of E. cloacae. Therefore, curli fimbriae may play an important role in biofilm formation of E. cloacae in a hospital environment.
Infectious Diseases Society of America 2008 Annual Meeting; 10/2008
[Show abstract][Hide abstract] ABSTRACT: Acinetobacter baumannii outer membrane protein A (AbOmpA) is a potential virulence factor that induces host cell death. Based on previous findings that AbOmpA translocated into the nuclei of host cells, the cell-death mechanism of AbOmpA through the nuclear targeting was investigated. Acinetobacter baumannii secreted AbOmpA in in vitro culture. The recombinant AbOmpA (rAbOmpA) was internalized by the host cells. The intracellular rAbOmpA was degraded into several forms of subfragments in the cytosol and then two subfragments of rAbOmpA translocated into the nuclei. The rAbOmpA exhibited the divalent cation-dependent endonuclease activity. In an in vivo assay with microinjection of rAbOmpA into the nucleus of fertilized Xenopus laevis eggs, rAbOmpA degraded chromosomal DNA with the characteristic DNA ladders and induced degeneration of the embryos. These results suggest that AbOmpA translocates into the nuclei of host cells and degrades chromosomal DNA by DNAse I-like enzymatic activity, which is a new pathogenic strategy of A. baumannii.
[Show abstract][Hide abstract] ABSTRACT: Screening of 368 consecutive nonreplicate clinical isolates of Enterobacteriaceae resistant to nalidixic acid and at least one extended-spectrum beta-lactam revealed the presence of qnrA, qnrB, and qnrS determinants, and identified novel qnrB variants, in Citrobacter freundii isolates. This study also revealed, for the first time, the linkage of qnrB, armA, and extended-spectrum and/or AmpC-type beta-lactamase genes on large conjugative plasmids.
[Show abstract][Hide abstract] ABSTRACT: The distribution of conjugative-plasmid-mediated 16S rRNA methylase genes among amikacin-resistant Enterobacteriaceae collected between 1995 and 1998 and between 2001 and 2006 at a university hospital in South Korea was examined, and conjugative plasmids carrying the 16S rRNA methylase genes were characterized by PCR-based replicon typing and by determination of their antimicrobial resistance pattern. Among the 7,127 isolates, 463 isolates showed a high level of resistance to amikacin, and 218 of the 463 isolates transferred amikacin resistance by conjugation. Among the 218 isolates, armA was detected in 153 isolates (88 Klebsiella pneumoniae, 28 Escherichia coli, 19 Enterobacter cloacae, and 6 Serratia marcescens isolates and 12 isolates of other organisms), and rmtB was detected in 51 isolates (32 K. pneumoniae isolates, 18 E. coli isolates, and 1 Citrobacter freundii isolate). The first appearance of armA was in 1997. The armA gene was carried by conjugative plasmids of replicon groups IncL/M, IncFIIAs, IncF, IncA/C, IncHI2, and Inc(unidentified) in 38, 20, 7, 9, 4, and 75 strains, respectively. The rmtB gene was carried by conjugative plasmids of groups IncA/C, IncF, and IncI1-Igamma in 43 strains, 7 strains, and 1 strain, respectively. Transconjugants that received the IncL/M plasmid carrying armA or the IncA/C plasmid carrying rmtB showed an additional resistance to cefotaxime. Transconjugants that received the IncFIIA plasmid or Inc(unidentified) plasmid carrying the armA gene showed an additional resistance to cefoxitin and a high MIC(50) (0.25 mg/liter) of ciprofloxacin. In conclusion, this study demonstrated that the dissemination of 16S rRNA methylase genes among the Enterobacteriaceae is mediated by conjugative plasmids of various incompatibility groups that confer resistance to multiple drugs, including aminoglycosides, extended-spectrum beta-lactams, and/or quinolones.
[Show abstract][Hide abstract] ABSTRACT: A total of 58 vancomycin-resistant E. faecium (VREF) was isolated from 3 hospitals located in Daegu, Korea. The VREF isolates were evaluated for the antimicrobial susceptibility pattern and resistance determinants against vancomcin, aminoglycosides, and macrolides. The multilocus sequence types (MLST) were determined to characterize the clonal diversity of the VREF isolates. The VREF isolates were highly resistance to teicoplanin, erythromycin, ciprofloxacin, gentamicin, and streptomycin, whereas quinupristin-dalfopristin and linezolid were the most susceptible drugs. All isolates carried the vanA gene. The aac6'-aph2" (n=53) and aadE (n=27) genes were detected in the high-level aminoglycoside resistant (HLAR) isolates. The aac6'-aph2" gene was located in the conjugally transferable plasmids. The ermB and ermA genes were detected in the 54 and 3 VREF isolates, respectively. The VREF isolates showed 11 different sequence types (ST). The VREF isolates belonging to ST192 was the most prevalent (n=19), but detected in one hospital, whereas the isolates belonging to ST203 (n=11) were detected in 3 hospitals. These results suggest that the VREF isolates resistant to aminoglycosides and erythromycin are originated from different clones and specific VREF clones are spread in the study hospitals.
Journal of Bacteriology and Virology 01/2008; 38(1). DOI:10.4167/jbv.2008.38.1.19
[Show abstract][Hide abstract] ABSTRACT: In this study, we compared the phenotypic and genotypic characteristics of 138 MRSA isolates obtained from adult and pediatric patients (adult, 50; children, 88). The resistance rates against gentamicin, clindamycin, and ciprofloxacin were much higher in the adult MRSA isolates than in the pediatric MRSA isolates. The ermC gene, which is responsible for inducible clindamycin resistance, was detected in 52(59.1%) of the 88 pediatric MRSA isolates but in only 5(10.0%) of the 50 adult MRSA isolates. MRSA isolates of clonal type ST5 with an integration of SCCmec type II/II variants was the most predominant clone among the adult isolates, while clonal type ST72 with an integration of SCCmec IV/IVA was the most predominant clone among the pediatric MRSA isolates. Staphylococcal enterotoxin A and toxic shock syndrome toxin-1 were prevalent among the adult MRSA isolates but not among the pediatric MRSA isolates. The results of this study demonstrated remarkable differences between adult and pediatric MRSA isolates in terms of their antimicrobial susceptibility profiles, SCCmec type, multilocus sequence type, staphylococcal toxin genes, and erythromycin resistance genes.
The Journal of Microbiology 11/2007; 45(5):447-52. · 1.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A total of 121 Salmonella enterica serovars Typhi and Paratyphi A isolated from enteric fever patients at a university hospital in Nepal between February 2004 and January 2006 were tested for their antimicrobial susceptibility. The occurrence and cassette content of integrons as well as the molecular mechanisms of resistance among the multidrug-resistant (MDR) S. Typhi were evaluated. Thirty-nine percent of the isolates were susceptible to all the antimicrobial agents tested. Seven of the S. Typhi strains were MDR. None of the 121 S. enterica isolates were resistant to ciprofloxacin, cefazolin, rifampicin or kanamycin. All MDR S. Typhi isolates contained a class 1 integron with a single cassette, dfrA7, conferring resistance to trimethoprim. Pulsed-field gel electrophoresis (PFGE) of XbaI-generated genomic restriction fragments yielded 12 different patterns. Five of the seven MDR isolates containing class 1 integrons had an identical PFGE pattern. Resistance to sulfamethoxazole, streptomycin, ampicillin, tetracycline and chloramphenicol was mediated by sul1, strA-strB, blaTEM-like, tetB and catA genes, respectively. To the best of our knowledge, this is the first report of integron-associated multidrug resistance as well as the first molecular characterisation of the mechanism of resistance of S. Typhi isolated from Nepal. This study indicates the spread of integron-associated multidrug resistance in S. Typhi in Nepal.
International Journal of Antimicrobial Agents 11/2007; 30(4):330-5. DOI:10.1016/j.ijantimicag.2007.05.009 · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multi-drug resistant Pseudomonas aeruginosa has been implicated in a variety of serious therapeutic problems in clinical environments. Among the 968 P. aeruginosa isolates obtained from two hospitals in Daegu, Korea, we acquired 17 isolates that were resistant to all available tested antimicrobial agents, with the exception of colistin (colistin-only sensitive). We characterized the antimicrobial susceptibilities, metallo-beta-lactamases, and epidemiological relatedness among the colistin-only sensitive P. aeruginosa isolates. All colistin-only sensitive isolates were positive in the modified Hodge test and imipenem-EDTA synergy test, thereby indicating the production of metallo-beta-lactamases. 11 isolates from the secondary hospital and six isolates from the tertiary teaching hospital harbored blaVIM-2 and blaIMP-1, respectively. The pulsed-field gel electrophoretic analysis of the SpeI-digested DNA from P. aeruginosa isolates indicated that two different clones of colistin-only sensitive P. aeruginosa originated from each hospital, and had spread within the hospital environment. Overall, colistin-only sensitive P. aeruginosa was detected in Korea for the first time, but no pan-drug resistant bacteria were identified. Nationwide surveillance is required in order to monitor the emergence of colistin-only sensitive or pan-drug resistant bacteria.
The Journal of Microbiology 09/2007; 45(4):358-63. · 1.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the differences in antimicrobial susceptibility and resistance mechanisms against imipenem between Acinetobacter baumannii and Acinetobacter genomic species 13TU.
A total of 232 non-duplicate Acinetobacter species were consecutively collected from two Korean hospitals in Daegu, Republic of Korea, between November 2004 and November 2005. Antimicrobial susceptibility was determined by agar dilution methods. Resistance to imipenem was characterized by a carbapenemase activity test and PCR amplification. PFGE was performed to determine the clonal relatedness of imipenem-resistant Acinetobacter species.
A. baumannii was the most prevalent species (61.2%), followed by Acinetobacter genomic species 13TU (25.9%). The resistance rates of A. baumannii to most antimicrobial agents were higher than those of other Acinetobacter species, while the resistance rate to imipenem was the highest in Acinetobacter genomic species 13TU. Imipenem-resistant Acinetobacter genomic species 13TU isolates produced VIM-2 metallo-beta-lactamase, while imipenem-resistant A. baumannii isolates produced OXA-23 and/or OXA-51 beta-lactamase. Imipenem-resistant Acinetobacter strains originated from different clones in each hospital.
Two prevalent Acinetobacter species, A. baumannii and Acinetobacter genomic species 13TU, possess distinct phenotypic and genotypic traits against antimicrobials.
[Show abstract][Hide abstract] ABSTRACT: Stenotrophomonas maltophilia is a multi-drug resistant pathogen that has been isolated with increasing frequency from the hospitalized patients. A total of 202 S. maltophilia was isolated from three university hospitals and analysed by molecular typing for an epidemiologic investigation. All isolates were tested by antimicrobial susceptibility, random amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). The RAPD and PFGE patterns were recorded and analysed by the unweighted-pair group method with arithmetic average method. Two or more isolates were considered to be clonally related if their PFGE pattern exhibited ≥80% similarity. Trimethoprim/ sulfamethoxazole and ciprofloxacin were the most active antimicrobial agents tested. The majority of the isolates found to be genetically unrelated by PFGE. The genetically related isolates were recovered from the same patient. The result demonstrates a high genetic diversity of S. maltophilia isolates from clinical specimens. The clonal diversity of S. maltophilia from the hospitalized patients is partly due to the strains originated from the hospital environments, but not horizontal transfer between the patients.
Journal of Bacteriology and Virology 01/2007; 37(2). DOI:10.4167/jbv.2007.37.2.79