Involvement of curli fimbriae in the biofilm formation of Enterobacter cloacae.

In this study, we examined the biofilm forming ability, the mRNA expression of curli genes and the morphologies of curli fimbriae and biofilms in clinical isolates of Enterobacter cloacae. The csgBA operon was found in 11 (78.6%) of the 14 isolates. The ability of E. cloacae isolates to form biofilms was significantly correlated with the mRNA expression level of the csgA and csgD genes. The curli protein fimbriae appeared as tangled fibers and the curli-proficient strain formed mature biofilms. Our data suggest that the expression of the curli fimbriae play an important role in biofilm formation in E. cloacae.

Staphylococcus aureus produces membrane-derived vesicles that induce host cell death.

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.

A simple colorimetric method for testing antimicrobial susceptibility of biofilmed bacteria.

This study introduces a simple colorimetric method which can measure the antimicrobial susceptibility of bacteria in biofilms using trimethyl tetrazolium chloride (TTC) as an indicator of viable bacteria. The new method was utilized for the evaluation of antibiotic susceptibility of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus biofilms.

Emergence of 16S rRNA methylase gene armA and cocarriage of bla(IMP-1) in Pseudomonas aeruginosa isolates from South Korea.

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.

Emergence of a new mutation and its accumulation in the topoisomerase IV gene confers high levels of resistance to fluoroquinolones in Escherichia coli isolates.

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.

Serum resistance of Acinetobacter baumannii through the binding of factor H to outer membrane proteins.

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.

Characterization of conjugative plasmids carrying antibiotic resistance genes encoding 16S rRNA methylase, extended-spectrum beta-lactamase, and/or plasmid-mediated AmpC beta-lactamase.

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.

Nuclear translocation and DNAse I-like enzymatic activity of Acinetobacter baumannii outer membrane protein A.

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.

Plasmid-mediated quinolone resistance determinants qnrA, qnrB, and qnrS among clinical isolates of Enterobacteriaceae in a Korean hospital.

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.

Distribution of conjugative-plasmid-mediated 16S rRNA methylase genes among amikacin-resistant Enterobacteriaceae isolates collected in 1995 to 1998 and 2001 to 2006 at a university hospital in South Korea and identification of conjugative plasmids mediating dissemination of 16S rRNA methylase.

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.

A comparison of adult and pediatric methicillin-resistant Staphylococcus aureus isolates collected from patients at a university hospital in Korea.

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.

Emergence of multidrug-resistant Salmonella enterica serovar Typhi associated with a class 1 integron carrying the dfrA7 gene cassette in Nepal.

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.

Differences in phenotypic and genotypic traits against antimicrobial agents between Acinetobacter baumannii and Acinetobacter genomic species 13TU.

OBJECTIVES: To investigate the differences in antimicrobial susceptibility and resistance mechanisms against imipenem between Acinetobacter baumannii and Acinetobacter genomic species 13TU. METHODS: 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. RESULTS: 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. CONCLUSIONS: Two prevalent Acinetobacter species, A. baumannii and Acinetobacter genomic species 13TU, possess distinct phenotypic and genotypic traits against antimicrobials.

Acanthamoeba: could it be an environmental host of Shigella?

Shigellosis is a serious public health problem in Korea, because large outbreaks of Shigella sonnei infections were recorded in many parts of the country during the period 1998-2000. However, the epidemiological features of shigellosis are not well known. In this study, we devised conditions suitable for the growth and replication of Shigella in an amoebic intracellular environment, and investigate whether medium conditions affect the survival and replication of Shigella within Acanthamoeba. We evaluated the uptake rates of invasive and non invasive S. sonnei strains by three Acanthamoeba species, namely, A. castellanii Neff, A. astronyxis Ray & Hayes, and A. healyi OC-3A. When A. castellanii Neff was infected with S. sonnei 99OBS1 or 80DH248, shigellae was maintained for a longer time in cytoplasms than in other Acanthamoeba species. S. sonnei 99OBS1 strain (a virulent strain) was recovered in higher numbers than the non-virulent S. sonnei 80DH248 strain in all experiments. Moreover, S. sonnei was more easily engulfed by Acanthamoeba at 18 degrees C. The shigellae uptake rates of Neff strain, which was cultured in free-media (less nutrition), were higher (>10-fold) than those observed in original amoeba culture media (PYG medium) in all time points. S. sonnei 99OBS1 was localized, with an intact membrane, to the vacuoles of Acanthamoeba. We conclude that free-living amoebae more likely act as environmental hosts for shigellae, and thus, may have contributed to outbreaks of shigellosis in Korea.

VanB-vanA incongruent VRE isolated from animals and humans in 1999.

16 chicken isolates and four clinical isolates of VanB-vanA incongruent vancomycinresistant Enterococcus faecium strains without vanS were isolated in 1999. Pulsed-field gel electrophoresis revealed only a peripheral relationship between the chicken isolates and clinical isolates, but suggested clonal spread in the chicken isolates.

Molecular characterization of antimicrobial resistance in Shigella sonnei isolates in Korea.

The antimicrobial resistance of 122 Shigella sonnei isolates obtained in Korea during the period 1991-2000 was characterized. These isolates were highly resistant to traditional antibiotics such as trimethoprim (100 %), streptomycin (100 %), sulfamethoxazole (94 %), tetracycline (93 %) and nalidixic acid (90 %). All S. sonnei isolates carried Tn7 in their chromosomes. The 8.4 kb non-transferable resistance (R) plasmid carrying tetA, strA-strB and sul1 was found in 93 % of the S. sonnei isolates. Resistance to nalidixic acid first appeared in a S. sonnei isolate in 1997, and then in all S. sonnei isolates from 1998 and 1999. Resistance to commonly prescribed antibiotics such as ampicillin was increased in S. sonnei isolates during the outbreak period 1998-2000. Resistance to ampicillin was mediated by the conjugative R plasmids carrying blaTEM-1. In conclusion, S. sonnei acquired antimicrobial resistance to commonly prescribed antibiotics through the horizontal transfer of conjugative R plasmids, while the genetic stability of transposon and non-transferable R plasmids was responsible for resistance to traditional antibiotics.

Risk factors for specific methicillin-resistant Staphylococcus aureus clones in a Korean hospital.

OBJECTIVES: To analyse the risk factors for nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infections caused by different clonal types. METHODS: A total of 134 non-duplicate nosocomial MRSA isolates were analysed for clonal types by molecular typing techniques. The medical records of 90 patients who had documented MRSA infection were evaluated retrospectively. RESULTS: Two predominant MRSA clones of sequence types (STs) ST239 (n = 75) and ST5 (n = 39) accounted for 85% of the isolates. Management of patients in the departments of orthopaedic surgery, neurosurgery and plastic surgery was identified as a risk factor for infection with MRSA of ST239, while the presence of intravascular catheters was a risk factor for infection with ST5. Pulmonary infection was significantly higher in the patients infected with ST239 strains than in the patients infected with ST5 strains (P < 0.05). The overall mean duration of antimicrobial therapy for the patients with ST239 infection was significantly more than that for the patients with ST5 infection (P < 0.05). CONCLUSIONS: ST239 and ST5 were the predominant MRSA clones in the study hospital. Risk factors were significantly different between ST239 and ST5 strains. The results of this study will be of use in designing larger prospective epidemiological studies for MRSA infection based on clonal types.

Isolation of quinolone-resistant Escherichia coli found in major rivers in Korea.

Twenty isolates resistant to seven quinolones were isolated from major rivers in Korea. All isolates had three mutations, Ser83-->Leu and Asp87-->Asn in GyrA and Ser80-->Ile or Ser80-->Arg in ParC and three isolates had an additional mutation Glu84-->Gly or Glu84-->Val in ParC. In addition, a clonal spread was not found in these isolates.

Prevalence of the ST239 clone of methicillin-resistant Staphylococcus aureus and differences in antimicrobial susceptibilities of ST239 and ST5 clones identified in a Korean hospital.

A total of 188 nonduplicate methicillin-resistant Staphylococcus aureus (MRSA) isolates obtained between 2001 and 2004 in a university hospital in Daegu, Korea, were analyzed for their clonal types by molecular typing techniques, including multilocus sequence typing, spaA typing, staphylococcal chromosomal cassette mec (SCCmec) typing, and pulsed-field gel electrophoresis (PFGE). They were examined for their antimicrobial susceptibilities. The majority (87%) of MRSA isolates belonged to sequence type 239 (ST239; n = 100; 53%) and ST5 (n = 63, 34%) on the basis of sequence typing. MRSA isolates belonging to ST239 were genotypically homogeneous, while those belonging to ST5 showed variations in spaA type, SCCmec type, and PFGE patterns. The rates of resistance of the MRSA isolates belonging to ST239 to trimethoprim, sulfamethoxazole, tobramycin, gentamicin, erythromycin, and tetracycline were significantly higher than those of the isolates belonging to ST5 (P < 0.05). This study demonstrated that the ST239 clone, while rarely detected in Korea, was prevalent and that the antimicrobial susceptibility of the ST239 clone was significantly different from that of the ST5 clone.