Mamata Gurung

Acinetobacter baumannii Secretes Cytotoxic Outer Membrane Protein A via Outer Membrane Vesicles

Acinetobacter baumannii is an important nosocomial pathogen that causes a high morbidity and mortality rate in infected patients, but pathogenic mechanisms of this microorganism regarding the secretion and delivery of virulence factors to host cells have not been characterized. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs) that play a role in the delivery of virulence factors to host cells. A. baumannii has been shown to secrete OMVs when cultured in vitro, but the role of OMVs in A. baumannii pathogenesis is not well elucidated. In the present study, we evaluated the secretion and delivery of virulence factors of A. baumannii to host cells via the OMVs and assessed the cytotoxic activity of outer membrane protein A (AbOmpA) packaged in the OMVs. A. baumannii ATCC 19606T secreted OMVs during in vivo infection as well as in vitro cultures. Potential virulence factors, including AbOmpA and tissue-degrading enzymes, were associated with A. baumannii OMVs. A. baumannii OMVs interacted with lipid rafts in the plasma membranes and then delivered virulence factors to host cells. The OMVs from A. baumannii ATCC 19606T induced apoptosis of host cells, whereas this effect was not detected in the OMVs from the ΔompA mutant, thereby reflecting AbOmpA-dependent host cell death. The N-terminal region of AbOmpA22-170 was responsible for host cell death. In conclusion, the OMV-mediated delivery of virulence factors to host cells may well contribute to pathogenesis during A. baumannii infection.

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.

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.

Molecular Characterization of CTX-M β-Lactamase and Associated Addiction Systems in Escherichia coli Circulating among Cattle, Farm Workers, and the Farm Environment

ABSTRACT A total of 84 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from cattle, farm workers, and the farm environment isolated from February to September 2008 in the Republic of Korea were investigated. All 84 ESBL-producing isolates carried bla CTX-M genes that belonged to the CTX-M-1 (n = 35) or CTX-M-9 (n = 49) family. The most predominant CTX-M type identified was CTX-M-14 (n = 49), followed by CTX-M-32 (n = 26). The bla CTX-M genes were identified most commonly in E. coli isolates from feces (n = 29), teats (n = 25), and milk (n = 14). A bla CTX-M-14 gene was also detected in an E. coli isolate from a farmers hand. Transfer of the bla CTX-M gene from 60 bla CTX-M-positive E. coli isolates to the recipient E. coli J53 strain by conjugation was demonstrated. Plasmid isolation from bla CTX-M-positive transconjugants revealed a large (95- to 140-kb) conjugative plasmid. Almost all (82/84) bla CTX-M genes possessed an insertion sequence, ISEcp1, upstream of the bla CTX-M gene. Only in the case of the CTX-M-14 genes was IS903 downstream of the gene. The bla CTX-M genes were associated with seven kinds of addiction systems. Among them, pndAC, hok-sok, and srnBC were the most frequently identified addiction systems in both wild strains and transconjugants. The spread of bla CTX-M genes was attributed to both clonal expansion and horizontal dissemination. Our data suggest that a combination of multiple addiction systems in plasmids carrying bla CTX-M genes could contribute to their maintenance in the host cells. To our knowledge, the bla CTX-M-32 gene has not previously been reported in animal isolates from the Republic of Korea.

Emergence of 16S rRNA methylase gene armA and cocarriage of blaIMP-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.

Prevalence and characterization of Salmonella in pigs from conventional and organic farms and first report of S. serovar 1,4,[5],12:i:- from Korea.

This study compared the prevalence of Salmonella spp. and their antimicrobial susceptibilities in pigs from conventional and organic farms during 2012-2013 in Korea and characterized them by molecular methods. Altogether, 100 nontyphoid Salmonella were isolated: 47 from 1324 pigs (3.5%) from conventional farms and 53 from 641 pigs (8.3%) from organic farms. The most frequent serovar was Typhimurium (49%) followed by Panama (24%), 1,4,[5],12:i:- (5%), and Virchow (5%). Overall, the isolates were most often resistant to tetracycline (75%) followed by ampicillin (66%), streptomycin (57%), and gentamicin (44%). The prevalence of antimicrobial resistance, multi-drug resistance phenotype, and resistance to tetracycline, ampicillin, and gentamicin were significantly higher in swine Salmonella from conventional farms than those from organic farms. The most common resistance pattern was ampicillin-gentamicin-tetracycline (n=16). All eight ceftiofur-resistant Salmonella identified produced CTX-M-15. Overall, decreased susceptibility to ciprofloxacin was observed in 39 isolates. Among them, a single isolate was positive for qnrS1 gene. An insertion sequence ISEcp1 was detected upstream of blaCTX-M gene in all isolates. The spread of blaCTX-M-15 gene was attributed to combination of clonal expansion and horizontal dissemination mediated by IncHI2 plasmid. Multilocus variable number of tandem repeats analysis demonstrated clonal dissemination of S. Typhimurium and S. 1,4,[5],12:i:- strains in pigs. To our knowledge, this is the first report of blaCTX-M-15 gene in S. Virchow from pigs and qnrS1 gene in S. Rissen from animals. This study also reports the first occurrence of Salmonella serovar 1,4,[5],12:i:- from Korea and CTX-M-15 producing Salmonella from pigs in Korea.

Molecular Basis of Resistance to Selected Antimicrobial Agents in the Emerging Zoonotic Pathogen Streptococcus suis

ABSTRACT Characterization of 227 Streptococcus suis strains isolated from pigs during 2010 to 2013 showed high levels of resistance to clindamycin (95.6%), tilmicosin (94.7%), tylosin (93.8%), oxytetracycline (89.4%), chlortetracycline (86.8%), tiamulin (72.7%), neomycin (70.0%), enrofloxacin (56.4%), penicillin (56.4%), ceftiofur (55.9%), and gentamicin (55.1%). Resistance to tetracyclines, macrolides, aminoglycosides, and fluoroquinolone was attributed to the tet gene, erm(B), erm(C), mph(C), and mef(A) and/or mef(E) genes, aph(3′)-IIIa and aac(6′)-Ie-aph(2″)-Ia genes, and single point mutations in the quinolone resistance-determining region of ParC and GyrA, respectively.

Antimicrobial susceptibility and virulence characteristics of Salmonella enterica Typhimurium isolates from healthy and diseased pigs in Korea.

This study compared the antimicrobial susceptibility and prevalence of virulence genes in Salmonella enterica Typhimurium isolated from healthy and diseased pigs in Korea. A total of 456 Salmonella Typhimurium isolated from healthy (n = 238) and diseased (n = 218) pigs between 1998 and 2011 were investigated. In total, 93.4% of the Salmonella Typhimurium isolates were resistant to at least one antimicrobial agent tested. The isolates were most often resistant to tetracycline (85.7%), followed by streptomycin (83.6%), nalidixic acid (67.3%), ampicillin (49.3%), chloramphenicol (42.8%), and gentamicin (37.1%). Moreover, multidrug resistance phenotype and resistance to ampicillin, florfenicol, gentamicin, nalidixic acid, neomycin, streptomycin, and tetracycline were significantly higher (P < 0.01) among Salmonella Typhimurium isolates from the diseased pigs compared with those from the healthy pigs. The most common resistance pattern observed in both groups of isolates was streptomycin-tetracycline. Overall, more than 96% of the isolates tested possessed invA, spiA, msgA, sipB, prgH, spaN, tolC, lpfC, sifA, sitC, and sopB virulence genes. The prevalence of orgA, pagC, and iroN were 50.2, 74.1, and 91.0%, respectively, whereas isolates carrying cdtB (1.5%), pefA (7.0%), and spvB (14.9%) were identified much less frequently. Furthermore, the prevalence of invA, lpfC, orgA, pagC, and iroN was significantly higher (P < 0.01) among the isolates from the diseased pigs than in isolates from the healthy pigs. Our results demonstrated that, among diseased pigs, there was significantly higher resistance to some antimicrobials and greater prevalence of some virulence genes than in healthy pigs, indicating the role these factors play in pathogenesis. Multidrug-resistant Salmonella isolates that carry virulence-associated genes are potentially more dangerous and constitute a public health concern. Thus, continuous surveillance of antimicrobial resistance and virulence characteristics in Salmonella is essential.

Emergence and spread of carbapenem-resistant Acinetobacter baumannii sequence type 191 in a Korean hospital

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 Pasteurs 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 Bartuals 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 Pasteurs MLST scheme, but showed ST353 using the Bartuals 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.

Screening of nuclear targeting proteins in Acinetobacter baumannii based on nuclear localization signals.

Nuclear targeting of bacterial proteins is an emerging pathogenic mechanism in bacteria. However, due to the absence of an appropriate screening system for nuclear targeting proteins, systematic approaches to nuclear targeting of bacterial proteins and subsequent host cell pathology are limited. In this study, we developed a screening system for nuclear targeting proteins in Acinetobacter baumannii using a combination of bioinformatic analysis based on nuclear localization signal (NLS) and the Gateway(®) recombinational cloning system. Among 3367 open reading frames of A. baumannii ATCC 17978, 34 functional or hypothetical proteins were predicted to carry the putative NLS sequences. Of the 29 clones generated by the Gateway(®) recombinational cloning system, 14 proteins tagged with green fluorescent protein (GFP) were targeted to nuclei of host cells. Among the 14 nuclear targeting proteins, S21, L20, and L32 ribosomal proteins and transposase carried putative nuclear export signal (NES) sequences, but only transposase harbored the functional NES. After translocation to nuclei of host cells, four A. baumannii proteins induced cytotoxicity. In conclusion, we have developed a screening system for nuclear targeting proteins in A. baumannii. This system may open the way to a new field of bacterial pathogenesis.