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.

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.

The objective of this study was to determine the prevalence of Acinetobacter spp. in bulk tank milk (BTM) samples from different provinces of Korea and to analyze their antimicrobial susceptibility. Altogether, 2,287 BTM samples were investigated. Among them, Acinetobacter spp. were isolated from 176 BTM samples. Out of 176 Acinetobacter spp., 57 isolates were identified as Acinetobacter baumannii. None of the isolates were resistant to cefepime, imipenem, meropenem, ciprofloxacin, levofloxacin, or colistin. Resistance to amikacin, gentamicin, piperacillin, and cefotaxime was 2.3, 7.4, 2.3, and 4.0%, respectively. Acinetobacter spp. were least susceptible to tetracycline (17.6%), followed by trimethoprim-sulfamethoxazole (15.9%), ceftazidime (10.8%), and ampicillin-sulbactam (10.2%). Overall, A. baumannii strains were susceptible to most of the antimicrobial agents tested compared with other Acinetobacter spp. The Acinetobacter isolates showed 17 different patterns of antimicrobial resistance. The most frequent resistance profile observed was ampicillin-sulbactam (n=13), followed by tetracycline (n=9), ceftazidime-tetracycline (n=8), and trimethoprim-sulfamethoxazole-tetracycline (n=8). The results of this study confirmed that Acinetobacter, including A. baumannii strains, are present in BTM, which clearly showed the importance of examining BTM not only for foodborne pathogens but also for Acinetobacter spp., which could be of public health concern. To the best of our knowledge, this is the first report of Acinetobacter spp. in BTM samples from Korea.

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A total of 84 extended-spectrum ␤-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 farmer's 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.

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