Clonal evolution and antimicrobial resistance of Acinetobacter baumannii isolates from Korean hospitals over the last decade.

The wide-spread of drug-resistant Acinetobacter baumannii is a global health problem. This study investigated the clonal distribution and antimicrobial resistance of 167 A. baumannii isolates from two Korean university hospitals from 2009 to 2019 by analyzing the sequence types (STs), antimicrobial resistance, and resistance determinants of carbapenems and aminoglycosides. Twenty STs, including 16 pre-existing STs and four unassigned STs, were identified in A. baumannii isolates using the Oxford multilocus sequence typing scheme. Two STs, ST191 (n = 77) and ST451 (n = 40), were prevalent, and majority (n = 153) of the isolates belonged to clonal complex 208. The ST191 isolates were detected during the study period, whereas ST451 isolates were detected after 2016. One hundred forty-seven (87%) of 167 A. baumannii isolates were non-susceptible to carbapenems. The ST191 and ST451 isolates exhibited higher resistance to antimicrobial agents than that of the sporadic ST isolates. Interestingly, ST451 isolates exhibited lower susceptibility to minocycline and tigecycline than the other ST isolates. All carbapenem-non-susceptible A. baumannii isolates, except four, carried the ISAbaI-blaOXA-23 structure. armA was detected in all amikacin-non-susceptible isolates (n = 128) except for one isolate. Five aminoglycoside-modifying enzyme (AME) genes were detected, but their carriage varied between STs; ant(3″)-Ia and aac(6')-Ib were more common in ST191 than in ST451, while aph(3')-Ia was more common in ST451 than in ST191. This study demonstrated the clonal evolution related to antimicrobial resistance in A. baumannii.

Clonal change of carbapenem-resistant Acinetobacter baumannii isolates in a Korean hospital.

The expansion of specific carbapenem-resistant Acinetobacter baumannii (CRAB) clones is a global concern due to its therapeutic difficulty and epidemicity. To understand the prevalence of CRAB isolates in a Korean hospital, we investigated the epidemiological characteristics of 96 CRAB isolates between 2016 and 2018, including the sequence types (STs), antimicrobial susceptibility, and genetic background of resistance to carbapenems and aminoglycosides. Six STs were identified using the Oxford multilocus sequence typing scheme; ST191 (n = 8), ST208 (n = 12), ST229 (n = 11), and ST369 (n = 21) were previously identified clones in the study hospital, whereas gpi variants of ST208, ST451 (n = 34) and ST784 (n = 10), were emerging clones. ST208 isolates exhibited higher resistance rates to minocycline than other ST isolates, whereas ST369 isolates exhibited lower resistance rates to aminoglycosides and trimethoprim/sulfamethoxazole than other ST isolates. All CRAB isolates previously isolated in the study hospital carried ISAbaI-blaOXA-23 for carbapenem resistance, but 10 ST229 isolates carried only ISAbaI-blaOXA-51. The carriage of armA was lower in ST369 isolates (38%) than in other ST isolates (≥83%). The frequency and diversity of aminoglycoside-modifying enzyme genes were decreased among the CRAB isolates between 2016 and 2018 compared with CRAB isolates between 2013 and 2015 at the study hospital. In conclusion, clonal complex 208 CRAB isolates are predominant in the study hospital. This study demonstrates the evolutionary change of CRAB isolates in the study hospital in relation to the emergence of new STs and selection of resistant genes.

Synergy between Florfenicol and Aminoglycosides against Multidrug-Resistant Escherichia coli Isolates from Livestock.

The increasing prevalence of antimicrobial resistance and the laborious development of novel antimicrobial agents have limited the options for effective antimicrobial therapy. The combination of previously used antimicrobial agents represents an alternative therapy for multidrug-resistant (MDR) pathogens. The objective of this study was to investigate the synergistic effect of a florfenicol (FFL)-based combination with other antimicrobial agents against MDR Escherichia coli isolates from livestock using checkerboard assays and murine infection models. The FFL/amikacin (AMK) and FFL/gentamicin (GEN) combinations showed synergy against 10/11 and 6/11 MDR E. coli isolates in vitro, respectively. The combination of FFL with aminoglycosides (AMK or GEN) exhibited a better synergistic effect against MDR E. coli isolates than the cephalothin (CEF)/GEN or FFL/CEF combinations. The combination of FFL with AMK or GEN could reduce the emergence of resistant mutants in vitro. The FFL/AMK combination showed a higher survival rate of mice infected with MDR E. coli isolates than FFL or AMK alone. In summary, the combination of FFL with aminoglycosides (AMK or GEN) is highly effective against MDR E. coli isolates both in vitro and in vivo. Our findings may contribute to the discovery of an effective combination regimen against MDR E. coli infections in veterinary medicine.

Inhibitory effects of thymol on the cytotoxicity and inflammatory responses induced by Staphylococcus aureus extracellular vesicles in cultured keratinocytes.

Staphylococcus aureus extracellular vesicles (EVs) deliver effector molecules to host cells and induce host cell pathology. This study investigated the disruption of S. aureus EVs by thymol along with its inhibitory effects on the cytotoxicity and inflammatory responses induced by EVs derived from two different S. aureus strains in cultured keratinocytes. Membrane disruption of the S. aureus EVs treated with thymol was determined using transmission electron microscopy. Human keratinocyte HaCaT cells were incubated with either intact or thymol-treated S. aureus EVs and then analyzed for cytotoxicity and pro-inflammatory cytokine gene expression. Thymol inhibited the growth of S. aureus strains and disrupted the membranes of the S. aureus EVs. The cytotoxicity and the expression levels of the pro-inflammatory cytokine genes towards HaCaT cells differed between the EVs derived from two S. aureus strains. Thymol-treated S. aureus EVs inhibited the cytotoxicity and the expression of the pro-inflammatory cytokine genes when compared to intact S. aureus EVs. Thymol-treated S. aureus EVs delivered lesser amounts of the EV component to host cells than intact EVs. Our results suggest that the thymol-induced disruption of the S. aureus EVs inhibits the delivery of effector molecules to host cells, resulting in the suppression of cytotoxicity and inflammatory responses in keratinocytes. Thymol may attenuate the host cell pathology induced by an S. aureus infection via both the antimicrobial activity against the bacteria and the disruption of the secreted EVs.

Different epithelial cell response to membrane vesicles produced by Listeria monocytogenes cultured with or without salt stress.

We have previously shown that Listeria monocytogenes, a causative agent of listeriosis, can produce membrane vesicles (MVs) during in vitro culture. The aim of this study was to investigate the ability of MVs from L. monocytogenes cultured with or without salt stress to induce cytotoxicity and pro-inflammatory responses in colon epithelial Caco-2 cells. MVs were purified from wild-type L. monocytogenes 10403S strain and an isogenic ΔsigB mutant strain. MVs from both wild-type and ΔsigB mutant strains increased viability of Caco-2 cells regardless of salt stress. Both MVs from wild-type and ΔsigB mutant strains stimulated expression of pro-inflammatory cytokine and chemokine genes in Caco-2 cells. Expression levels of pro-inflammatory cytokine genes in cells treated with MVs from bacteria cultured without salt stress were significantly higher than those in cells treated with MVs from bacteria cultured with salt stress. However, expression levels of chemokine genes in cells treated with MVs from bacteria cultured with salt stress were significantly higher than those in cells treated with MVs from bacteria cultured without salt stress. In addition, expression levels of interleukin (IL)-1ß and IL-8 genes were partially inhibited by either lysozyme-treated MVs or ethylenediaminetetraacetic acid-treated MVs compared to those after treatment with intact MVs. Our results suggest that salt stress can affect the production of L. monocytogenes MVs, thus causing different pro-inflammatory responses in host cells.

Thymol attenuates the worsening of atopic dermatitis induced by Staphylococcus aureus membrane vesicles.

Staphylococcus aureus membrane vesicles (MVs) aggravate atopic dermatitis (AD) through the delivery of bacterial effector molecules to host cells and the stimulation of inflammatory responses. This study investigated the inhibitory effect of thymol, a phenolic monoterpene found in essential oils derived from plants, on the worsening of AD induced by S. aureus MVs both in vitro and in vivo. The sub-minimal inhibitory concentrations of thymol disrupted S. aureus MVs. Intact S. aureus MVs induced the expression of pro-inflammatory cytokine (interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α) and chemokine (IL-8 and monocyte chemoattractant protein-1) genes in cultured keratinocytes, whereas thymol-treated S. aureus MVs did not stimulate the expression of these genes. Topical application of thymol-treated S. aureus MVs or treatment with thymol after intact S. aureus MVs to AD-like skin lesions diminished the pathology of AD. This included decreases in epidermal/dermal thickness and infiltration of eosinophils/mast cells, and inhibited expression of pro-inflammatory cytokine and chemokine genes in mouse AD model. Moreover, thymol significantly suppressed the Th1, Th2, and Th17-mediated inflammatory responses in AD-like skin lesions induced by S. aureus MVs, and reduced the serum levels of immunoglobulin (Ig) G2a, mite-specific IgE, and total IgE. In summary, thymol disrupts S. aureus MVs and suppresses inflammatory responses in AD-like skin lesions aggravated by S. aureus MVs. Our results suggest that thymol is a possible candidate for the management of AD aggravation induced by S. aureus colonization or infection in the lesions.

Salt stress affects global protein expression profiles of extracellular membrane-derived vesicles of Listeria monocytogenes.

Our previous study has suggested that Listeria monocytogenes produces extracellular membrane vesicles (MVs) and its general stress transcription factor sigma B (σB) affects the production of MVs under energy stress. The objective of this study was to evaluate the production of MVs and perform global protein profiling for MVs with or without salt stress to understand the function of MVs in the pathogenesis of L. monocytogenes. When cells of L. monocytogenes were grown under 0.5 M salt stress, protein concentrations of MVs derived from wild-type strain and its isogenic ΔsigB mutant were approximately doubled compared to those of MVs derived from cells without salt stress. Proteomic analyses showed that the number of MV proteins expressed in wild-type strain was similar to that in ΔsigB mutant under salt stress. However, global protein expression profiles were dramatically changed under salt stress compared to those without salt stress. Fifteen σB dependent proteins were expressed in MVs of wild-type strain under salt stress, including osmolyte transporter OpuCABCD. In addition, MVs produced by salt stressed wild-type and ΔsigB mutant inhibited biofilm formation abilities of both strains. Taken together, our results suggest that salt stress can promote the production of MVs involved in carnitine transporter proteins, with σB playing a pivotal role in biological event.

Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells.

Staphylococcus aureus secretes membrane-derived vesicles (MVs), which can deliver virulence factors to host cells and induce cytopathology. However, the cytopathology of host cells induced by MVs derived from different S. aureus strains has not yet been characterized. In the present study, the cytotoxic activity of MVs from different S. aureus isolates on host cells was compared and the proteomes of S. aureus MVs were analyzed. The MVs purified from S. aureus M060 isolated from a patient with staphylococcal scalded skin syndrome showed higher cytotoxic activity toward host cells than that shown by MVs from three other clinical S. aureus isolates. S. aureus M060 MVs induced HEp-2 cell apoptosis in a dose-dependent manner, but the cytotoxic activity of MVs was completely abolished by treatment with proteinase K. In a proteomic analysis, the MVs from three S. aureus isolates not only carry 25 common proteins, but also carry ≥60 strain-specific proteins. All S. aureus MVs contained δ-hemolysin (Hld), γ-hemolysin, leukocidin D, and exfoliative toxin C, but exfoliative toxin A (ETA) was specifically identified in S. aureus M060 MVs. ETA was delivered to HEp-2 cells via S. aureus MVs. Both rETA and rHld induced cytotoxicity in HEp-2 cells. In conclusion, MVs from clinical S. aureus isolates differ with respect to cytotoxic activity in host cells, and these differences may result from differences in the MV proteomes. Further proteogenomic analysis or mutagenesis of specific genes is necessary to identify cytotoxic factors in S. aureus MVs.

Outer membrane Protein A plays a role in pathogenesis of Acinetobacter nosocomialis.

Acinetobacter nosocomialis is an important nosocomial pathogen that causes a variety of human infections. However, the specific virulence factors of this microorganism have not yet been determined. We investigated the role of outer membrane protein A (OmpA) in the pathogenesis of A. nosocomialis. A ΔompA mutant of the A. nosocomialis ATCC 17903(T) strain was constructed using markerless gene deletion. The ΔompA mutant displayed reduced biofilm formation in polystyrene tubes and reduced adherence to A549 cells in comparison to the wild-type strain. These virulence traits of the ΔompA mutant strain were restored when the ompA gene was complemented. Cytotoxicity was not significantly different between the wild-type strain and the ΔompA mutant when A549 cells were infected with bacteria or treated with outer membrane vesicles (OMVs). However, OMVs from the wild-type strain induced cytotoxicity in HEp-2 cells, whereas OMVs from the ΔompA mutant did not induce cytotoxicity. Proteomic analysis of OMVs revealed that OmpA influenced the distribution of envelope and periplasmic proteins. Overall, this study is the first report that links OmpA to A. nosocomialis pathogenesis, and highlights OmpA as a putative target to develop anti-virulence agents or vaccines against A. nosocomialis infection.

Morphological changes in human gastric epithelial cells induced by nuclear targeting of Helicobacter pylori urease subunit A.

Nuclear targeting of bacterial proteins and their pathological effects on host cells are an emerging pathogenic mechanism in bacteria. We have previously reported that urease subunit A (UreA) of Helicobacter pylori targets the nuclei of COS-7 cells through nuclear localization signals (NLSs). This study further investigated whether UreA of H. pylori targets the nuclei of gastric epithelial cells and then induces molecular and cellular changes in the host cells. H. pylori 26695 strain produced and secreted outer membrane vesicles (OMVs). UreA was translocated into gastric epithelial AGS cells through outer membrane vesicles (OMVs) and then targeted the nuclei of AGS cells. Nuclear targeting of rUreA did not induce host cell death, but resulted in morphological changes, such as cellular elongation, in AGS cells. In contrast, AGS cells treated with rUreA?NLS proteins did not show this morphological change. Next generation sequencing revealed that nuclear targeting of UreA differentially regulated 102 morphogenesis- related genes, of which 67 and 35 were up-regulated and down-regulated, respectively. Our results suggest that nuclear targeting of H. pylori UreA induces both molecular and cellular changes in gastric epithelial cells.

Acinetobacter nosocomialis secretes outer membrane vesicles that induce epithelial cell death and host inflammatory responses.

Acinetobacter nosocomialis is an important nosocomial pathogen that causes a variety of opportunistic infections; however, pathogenesis of this microorganism has not yet been characterized. The aim of this study was to investigate the secretion of outer membrane vesicles (OMVs) from A. nosocomialis and to determine their cytotoxic effects and their ability to induce inflammatory responses both in vitro and in vivo by using human epithelial HEp-2 cells and a mouse model, respectively. A. nosocomialis ATCC 17903(T) secreted spherical OMVs when cultured in vitro. Proteomic analysis revealed that 147 different proteins were associated with A. nosocomialis OMVs and virulence-associated proteins, such as outer membrane protein A (OmpA), CsuA, CsuC, CsuD, PilW, hemolysin, and serine protease, were identified. A. nosocomialis OMVs were cytotoxic to HEp-2 cells. These vesicles also induced the expression of pro-inflammatory cytokine genes in the HEp-2 cells. Early inflammatory responses, such as congestion and focal neutrophilic infiltration, were observed in the lungs of mice injected with A. nosocomialis OMVs. In conclusion, A. nosocomialis OMVs are important secretory nanocomplexes that induce cytotoxicity of epithelial cells and host inflammatory responses, which may contribute to the pathogenesis of A. nosocomialis.

Acinetobacter baumannii outer membrane vesicles elicit a potent innate immune response via membrane proteins.

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)-1ß 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.

Prediction and screening of nuclear targeting proteins with nuclear localization signals in Helicobacter pylori.

Host cell pathology induced by nuclear targeting of bacterial proteins has recently been identified as a pathogenic mechanism of bacteria. However, very few bacterial proteins were identified to target the nuclei of host cells. This study was designed to screen nuclear targeting proteins with nuclear localization signals (NLSs) in Helicobacter pylori using a combination of bioinformatic analysis and the Gateway recombinational cloning system. Forty-nine functional or hypothetical proteins were predicted to carry the putative NLSs among 1570 open reading frames (ORFs) of H. pylori 26695. Entire sets of 49 H. pylori ORFs were cloned for the generation of green fluorescent protein-tagged proteins using the Gateway recombinational cloning system. Twenty-six H. pylori proteins with the putative NLSs were found to target in the nuclei of COS-7 cells, whereas 23 were localized in the cytoplasm of host cells. Deletion of NLS sequences from four selected nuclear targeting proteins, urease subunit A, Omp18, secreted protein involved in flagellar motility, and response regulator, resulted in cytoplasmic localization of these mutant proteins. In conclusion, a combination of bioinformatic analysis and the Gateway cloning system was shown to be a useful tool for large-scale screening of nuclear targeting proteins with NLSs in H. pylori, which can be used to better understand the H. pylori-directed host cell pathology.

Klebsiella pneumoniae secretes outer membrane vesicles that induce the innate immune response.

Outer membrane vesicles (OMVs) derived from pathogenic Gram-negative bacteria are an important vehicle for delivery of effector molecules to host cells, but the production of OMVs from Klebsiella pneumoniae, an opportunistic pathogen of both nosocomial and community-acquired infections, and their role in bacterial pathogenesis have not yet been determined. In the present study, we examined the production of OMVs from K. pneumoniae and determined the induction of the innate immune response against K. pneumoniae OMVs. Klebsiella pneumoniae ATCC 13883 produced and secreted OMVs during in vitro culture. Proteomic analysis revealed that 159 different proteins were associated with K. pneumoniae OMVs. Klebsiella pneumoniae OMVs did not inhibit cell growth or induce cell death. However, these vesicles induced expression of proinflammatory cytokine genes such as interleukin (IL)-1ß and IL-8 in epithelial cells. An intratracheal challenge of K. pneumoniae OMVs in neutropenic mice resulted in severe lung pathology similar to K. pneumoniae infection. In conclusion, K. pneumoniae produces OMVs like other pathogenic Gram-negative bacteria and K. pneumoniae OMVs are a molecular complex that induces the innate immune response.