In this Review, Schwechheimer and Kuehn describe recent developments in elucidating the mechanisms of biogenesis and cargo selection of the outer-membrane vesicles (OMVs) produced by Gram-negative bacteria. They also discuss the functions of OMVs in bacterial physiology and during pathogenesis. Outer-membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content and are commonly produced by Gram-negative bacteria. The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, enabling bacterial survival during stress conditions and regulating microbial interactions within bacterial communities. Additionally, because of this functional versatility, researchers have begun to explore OMVs as a platform for bioengineering applications. In this Review, we discuss recent advances in the study of OMVs, focusing on new insights into the mechanisms of biogenesis and the functions of these vesicles.

Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions

Staphylococcal cassette chromosome mec (SCCmec) typing, in combination with genotyping of the Staphylococcus aureus chromosome, has become essential for defining methicillin-resistant S. aureus (MRSA) clones in epidemiological studies. We have developed a convenient system for SCCmec type assignment. The system consists of six multiplex PCRs (M-PCRs) for identifying the ccr gene complex (ccr), the mec gene complex (mec), and specific structures in the junkyard (J) regions: M-PCR with primer set 1 (M-PCR 1) identified five types of ccr genes; M-PCR 2 identified class A to class C mec; M-PCRs 3 and 4 identified specific open reading frames in the J1 regions of type I and IV and of type II, III, and V SCCmec elements, respectively; M-PCR 5 identified the transposons Tn554 and ΨTn554 integrated into the J2 regions of type II and III SCCmec elements; and M-PCR 6 identified plasmids pT181 and pUB110 integrated into J3 regions. The system was validated with 99 MRSA strains carrying SCCmec elements of different types. The SCCmec types of 93 out of the 99 MRSA strains could be assigned. The SCCmec type assignments were identical to those made with a PCR system that uses numerous primer pairs to identify genes or gene alleles. Our system of six M-PCRs is thus a convenient and reliable method for typing SCCmec elements.

https://aac.asm.org/content/aac/51/1/264.full.pdf

Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions.

Extracellular vesicles (EVs) are produced by all domains of life In Gram-negative bacteria, EVs are produced by the pinching off of the outer membrane; however, how EVs escape the thick cell walls of Gram-positive bacteria, mycobacteria and fungi is still unknown Nonetheless, EVs have been described in a variety of cell-walled organisms, including Staphylococcus aureus, Mycobacterium tuberculosis and Cryptococcus neoformans These EVs contain varied cargo, including nucleic acids, toxins, lipoproteins and enzymes, and have important roles in microbial physiology and pathogenesis In this Review, we describe the current status of vesiculogenesis research in thick-walled microorganisms and discuss the cargo and functions associated with EVs in these species

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Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi

SUMMARY Staphylococcus aureus, a major human pathogen, has a collection of virulence factors and the ability to acquire resistance to most antibiotics. This ability is further augmented by constant emergence of new clones, making S. aureus a “superbug.” Clinical use of methicillin has led to the appearance of methicillin-resistant S. aureus (MRSA). The past few decades have witnessed the existence of new MRSA clones. Unlike traditional MRSA residing in hospitals, the new clones can invade community settings and infect people without predisposing risk factors. This evolution continues with the buildup of the MRSA reservoir in companion and food animals. This review focuses on imparting a better understanding of MRSA evolution and its molecular characterization and epidemiology. We first describe the origin of MRSA, with emphasis on the diverse nature of staphylococcal cassette chromosome mec (SCCmec). mecA and its new homologues (mecB, mecC, and mecD), SCCmec types (13 SCCmec types have been discovered to date), and their classification criteria are discussed. The review then describes various typing methods applied to study the molecular epidemiology and evolutionary nature of MRSA. Starting with the historical methods and continuing to the advanced whole-genome approaches, typing of collections of MRSA has shed light on the origin, spread, and evolutionary pathways of MRSA clones.

Methicillin-Resistant Staphylococcus aureus: Molecular Characterization, Evolution, and Epidemiology

Pseudomonas aeruginosa is intrinsically resistant to a variety of antimicrobials and can develop resistance during anti-pseudomonal chemotherapy both of which compromise treatment of infections caused by this organism. Resistance to multiple classes of antimicrobials (multidrug resistance) in particular is increasingly common in P. aeruginosa, with a number of reports of pan-resistant isolates treatable with a single agent, colistin. Acquired resistance in this organism is multifactorial and attributable to chromosomal mutations and the acquisition of resistance genes via horizontal gene transfer. Mutational changes impacting resistance include upregulation of multidrug efflux systems to promote antimicrobial expulsion, derepression of ampC, AmpC alterations that expand the enzyme's substrate specificity (i.e., extended-spectrum AmpC), alterations to outer membrane permeability to limit antimicrobial entry and alterations to antimicrobial targets. Acquired mechanisms contributing to resistance in P. aeruginosa include β-lactamases, notably the extended-spectrum β-lactamases and the carbapenemases that hydrolyze most β-lactams, aminoglycoside-modifying enzymes, and 16S rRNA methylases that provide high-level pan-aminoglycoside resistance. The organism's propensity to grow in vivo as antimicrobial-tolerant biofilms and the occurrence of hypermutator strains that yield antimicrobial resistant mutants at higher frequency also compromise anti-pseudomonal chemotherapy. With limited therapeutic options and increasing resistance will the untreatable P. aeruginosa infection soon be upon us?

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Pseudomonas aeruginosa: resistance to the max.

Objectives Antimicrobial resistance and class 1 integrons found in Escherichia coli isolates from humans and animals in Korea were characterized. Methods E. coli isolates were examined for susceptibility to antimicrobial agents. Integrase genes were amplified. Gene cassette regions for classes 1 and 2 integrons were amplified and sequenced. Conjugal transfer and Southern hybridization were performed to determine the genetic localization of class 1 integrons. The clonal relationship of E. coli isolates carrying an identical cassette array was analysed by PFGE. Results Commensal E. coli isolates from animals were highly resistant to commonly used antimicrobial agents such as tetracycline, sulfamethoxazole, streptomycin, ampicillin and carbenicillin. Integrons were most prevalent in commensal E. coli isolates from poultry (44%), followed by clinical isolates from humans (33%), commensal isolates from swine (23%) and humans (13%). dfrA17-aadA5, dfrA12-orfF-aadA2 and aadA1 were found most frequently in E. coli isolates from humans, poultry and swine, respectively. Class 1 integrons were mostly located in conjugative plasmids. E. coli isolates carrying an identical cassette array were phylogenetically unrelated. Conclusions The use of antibiotics is strongly associated with antimicrobial resistance. E. coli isolates from different sources may select a specific gene cassette by antibiotic selective pressure, which results in differences in class 1 integrons. The horizontal transfer of class 1 integrons through conjugative plasmids seems to be responsible for wide dissemination of a particular type of class 1 integron.

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Characterization of antimicrobial resistance and class 1 integrons found in Escherichia coli isolates from humans and animals in Korea.

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.

Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles.

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.

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

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.

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

Acinetobacter baumannii secretes outer membrane vesicles (OMVs) during both in vitro and in vivo growth, but the biogenesis mechanism by which A. baumannii produces OMVs remains undefined. Outer membrane protein A of A. baumannii (AbOmpA) is a major protein in the outer membrane and the C-terminus of AbOmpA interacts with diaminopimelate of peptidoglycan. This study investigated the role of AbOmpA in the biogenesis of A. baumannii OMVs. Quantitative and qualitative approaches were used to analyze OMV biogenesis in A. baumannii ATCC 19606T and an isogenic ΔAbOmpA mutant. OMV production was significantly increased in the ΔAbOmpA mutant compared to wild-type bacteria as demonstrated by quantitation of proteins and lipopolysaccharides (LPS) packaged in OMVs. LPS profiles prepared from OMVs from wild-type bacteria and the ΔAbOmpA mutant had identical patterns, but proteomic analysis showed different protein constituents in OMVs from wild-type bacteria compared to the ΔAbOmpA mutant. In conclusion, AbOmpA influences OMV biogenesis by controlling OMV production and protein composition.

Dong Chan Moon

Papers

Characterization of antimicrobial resistance and class 1 integrons found in Escherichia coli isolates from humans and animals in Korea.

Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles.

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

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

Acinetobacter baumannii outer membrane protein a modulates the biogenesis of outer membrane vesicles