Clonal Distribution and Its Association With the Carbapenem Resistance Mechanisms of Carbapenem-Non-Susceptible Pseudomonas aeruginosa Isolates From Korean Hospitals.

Background: Carbapenem resistance in Pseudomonas aeruginosa is a serious global health problem. We investigated the clonal distribution and its association with the carbapenem resistance mechanisms of carbapenem-non-susceptible P. aeruginosa isolates from three Korean hospitals. Methods: A total of 155 carbapenem-non-susceptible P. aeruginosa isolates collected between 2011 and 2019 were analyzed for sequence types (STs), antimicrobial susceptibility, and carbapenem resistance mechanisms, including carbapenemase production, the presence of resistance genes, OprD mutations, and the hyperproduction of AmpC ß-lactamase. Results: Sixty STs were identified in carbapenem-non-susceptible P. aeruginosa isolates. Two high-risk clones, ST235 (N=41) and ST111 (N=20), were predominant; however, sporadic STs were more prevalent than high-risk clones. The resistance rate to amikacin was the lowest (49.7%), whereas that to piperacillin was the highest (92.3%). Of the 155 carbapenem-non-susceptible isolates, 43 (27.7%) produced carbapenemases. Three metallo-ß-lactamase (MBL) genes, blaIMP-6 (N=38), blaVIM-2 (N=3), and blaNDM-1 (N=2), were detected. blaIMP-6 was detected in clonal complex 235 isolates. Two ST773 isolates carried blaNDM-1 and rmtB. Frameshift mutations in oprD were identified in all isolates tested, regardless of the presence of MBL genes. Hyperproduction of AmpC was detected in MBL gene-negative isolates. Conclusions: Frameshift mutations in oprD combined with MBL production or hyperproduction of AmpC are responsible for carbapenem resistance in P. aeruginosa. Further attention is required to curb the emergence and spread of new carbapenem-resistant P. aeruginosa clones.

Analysis of microbiological tests in patients withholding or withdrawing life-sustaining treatment at the end stage of life in 2 Korean hospitals.

OBJECTIVE: We evaluated the adequacy of microbiological tests in patients withholding or withdrawing life-sustaining treatment (WLST) at the end stage of life. SETTING: The study was conducted at 2 tertiary-care referral hospitals in Daegu, Republic of Korea. DESIGN: Retrospective cross-sectional study. METHODS: Demographic findings, clinical and epidemiological characteristics, statistics of microbiological tests, and microbial species isolated from patients within 2 weeks before death were collected in 2 tertiary-care referral hospitals from January to December 2018. We also reviewed the antimicrobial treatment that was given within 3 days of microbiological testing in patients on WLST. RESULTS: Of the 1,187 hospitalized patients included, 905 patients (76.2%) had WLST. The number of tests per 1,000 patient days was higher after WLST than before WLST (242.0 vs 202.4). Among the category of microbiological tests, blood cultures were performed most frequently, and their numbers per 1,000 patient days before and after WLST were 95.9 and 99.0, respectively. The positive rates of blood culture before and after WLST were 17.2% and 18.0%, respectively. Candida spp. were the most common microbiological species in sputum (17.4%) and urine (48.2%), and Acinetobacter spp. were the most common in blood culture (17.3%). After WLST determination, 70.5% of microbiological tests did not lead to a change in antibiotic use. CONCLUSIONS: Many unnecessary microbiological tests are being performed in patients with WLST within 2 weeks of death. Microbiological testing should be performed carefully and in accordance with the patient's treatment goals.

PmrAB controls virulence-associated traits and outer membrane vesicle biogenesis in Acinetobacter baumannii.

The PmrAB two-component system modulates colistin resistance in Acinetobacter baumannii, but its association with the virulence traits of this bacterium remains uncharacterized. This study explored the role of A. baumannii PmrAB in surface motility, biofilm formation, and outer membrane vesicle (OMV) biogenesis using wild-type (WT) A. baumannii 17978 and ΔpmrA and ΔpmrB mutant strains. The two mutant strains exhibited significantly decreased surface motility compared with that of WT strain by the low expression of abaI, abaR, A1S_0113, A1S_0115, and A1S_0116. Biofilm mass also significantly decreased in the two mutant strains at 12 h of incubation, but restored at 24 h. Under static culture conditions for 12 h, the two mutant strains showed low pgaA expression. However, the other biofilm-associated genes, such as csuC, csuE, ompA, and bap, showed different expression between the two mutant strains. Although the size of OMVs was similar among the three strains, the number of OMVs secreted from the two mutant strains slightly decreased compared with that secreted from the WT strain. Protein concentrations in the OMVs of ΔpmrA mutant significantly decreased compared with those in the OMVs of WT and ΔpmrB strains. Overall, PmrAB modulates virulence traits and OMV biogenesis in A. baumannii.

First Case of Pseudoclavibacter alba Bacteremia in a Patient with Cholangitis.

BACKGROUND: Pseudoclavibacter alba isolated from human urine in culture collection was introduced as a new species, but since then, no other reports on P. alba isolated from the environment or organisms have been published. We thus present the first case report of P. alba bacteremia. METHODS: An 85-year-old female patient was admitted with intermittent abdominal pain and chills that had persisted for one week. She was diagnosed cholangitis with common bile duct stones. RESULTS: Gram-positive bacteria were detected in her peripheral blood culture and identified Pseudoclavibacter species by matrix-assisted laser desorption-ionization-time of flight mass spectrometry. Pseudoclavibacter alba was identified by performing the 16S ribosomal RNA gene sequence. CONCLUSIONS: This is the first case report of P. alba bacteremia in a patient with cholangitis.

Antimicrobial activity of α-mangostin against Staphylococcus species from companion animals in vitro and therapeutic potential of α-mangostin in skin diseases caused by S. pseudintermedius.

Antimicrobial resistance in Staphylococcus species from companion animals is becoming increasingly prevalent worldwide. S. pseudintermedius is a leading cause of skin infections in companion animals. α-mangostin (α-MG) exhibits various pharmacological activities, including antimicrobial activity against G (+) bacteria. This study investigated the antimicrobial activity of α-MG against clinical isolates of Staphylococcus species from companion animals and assessed the therapeutic potential of α-MG in skin diseases induced by S. pseudintermedius in a murine model. Furthermore, the action mechanisms of α-MG against S. pseudintermedius were investigated. α-MG exhibited antimicrobial activity against clinical isolates of five different Staphylococcus species from skin diseases of companion animals in vitro, but not G (-) bacteria. α-MG specifically interacted with the major histocompatibility complex II analogous protein (MAP) domain-containing protein located in the cytoplasmic membrane of S. pseudintermedius via hydroxyl groups at C-3 and C-6. Pretreatment of S. pseudintermedius with anti-MAP domain-containing protein polyclonal serum significantly reduced the antimicrobial activity of α-MG. The sub-minimum inhibitory concentration of α-MG differentially regulated 194 genes, especially metabolic pathway and virulence determinants, in S. pseudintermedius. α-MG in pluronic lecithin organogel significantly reduced the bacterial number, partially restored the epidermal barrier, and suppressed the expression of cytokine genes associated with pro-inflammatory, Th1, Th2, and Th17 in skin lesions induced by S. pseudintermedius in a murine model. Thus, α-MG is a potential therapeutic candidate for treating skin diseases caused by Staphylococcus species in companion animals.

Acinetobacter baumannii under Acidic Conditions Induces Colistin Resistance through PmrAB Activation and Lipid A Modification.

Colistin is a last-resort antimicrobial agent for treating carbapenem-resistant Acinetobacter baumannii infections. The activation of PmrAB by several environmental signals induces colistin resistance in Gram-negative bacteria. This study investigated the molecular mechanisms of colistin resistance in A. baumannii under acidic conditions using wild-type (WT) A. baumannii 17978, ΔpmrA and ΔpmrB mutants, and pmrA-complemented strains. The pmrA or pmrB deletion did not affect the growth of A. baumannii under acidic or aerobic conditions. A. baumannii under acidic (pH 5.5) and high-iron (1 mM) conditions showed 32- and 8-fold increases in the minimum inhibitory concentrations (MICs) of colistin, respectively. The ΔpmrA and ΔpmrB mutants at pH 5.5 showed a significant decrease in colistin MICs compared to the WT strain at pH 5.5. No difference in colistin MICs was observed between WT and mutant strains under high-iron conditions. The pmrCAB expression significantly increased in the WT strain at pH 5.5 compared to the WT strain at pH 7.0. The pmrC expression significantly decreased in two mutant strains at pH 5.5 compared to the WT strain at pH 5.5. The PmrA protein was expressed in the ΔpmrA strain carrying ppmrA_FLAG plasmids at pH 5.5 but not at pH 7.0. Lipid A modification by the addition of phosphoethanolamine was observed in the WT strain at pH 5.5. In conclusion, this study demonstrated that A. baumannii under acidic conditions induces colistin resistance via the activation of pmrCAB operon and subsequent lipid A modification.

Complete genome sequence of the multidrug-resistant clinical isolate Acinetobacter baumannii KBN10P05679: Insights into antimicrobial resistance genotype and potential virulence traits.

OBJECTIVES: Acinetobacter baumannii, a nosocomial pathogen, exhibits multidrug resistance and is a major concern worldwide. We therefore aimed to evaluate the genomic features of the clinical strain A. baumannii KBN10P05679 to elucidate its antibiotic resistance mechanisms and virulence factors. METHODS: In silico multilocus sequence typing, phylogenetic identification, genome annotation, genome analysis, antibiotic susceptibility testing, and biofilm formation assay were performed, and the expression levels of antibiotic resistance- and biofilm-related genes were investigated. RESULTS: The complete genome of KBN10P05679 comprises a circular chromosome of 3 990 428 bp and two plasmids (74 294 and 8731 bp) and was assigned to the ST451 sequence type. Clusters of Orthologous Gene annotation identified 3810 genes, including those involved in amino acid transport and metabolism, transcription, inorganic ion transport, energy production and conversion, replication, recombination and repair, and carbohydrate and protein metabolism. The antibiotic resistance genes were investigated by searching the Comprehensive Antibiotic Resistance Database, and the genome was found to harbour 30 different antibiotic resistance genes. Analysis of the Virulence Factor Database revealed 86 virulence factor genes in the KBN1005679 genome. The KBN10P05679 strain was found to have a higher capacity for biofilm formation and expressed biofilm-related genes at a higher level than the other tested strains. CONCLUSIONS: The antibiotic resistance genotype and potential virulence factor-related data obtained in this study would help direct future studies for developing the control measures for this multidrug-resistant pathogen.

Complete Genome Sequence of Acinetobacter baumannii Strain KBN10P04593, a Clinical Isolate from South Korea.

Acinetobacter baumannii is an opportunistic nosocomial pathogen that is responsible for various life-threating infections in immunocompromised hosts. We present the complete 3.93-Mb genome sequence of A. baumannii KBN10P04593, generated by combining PacBio and Illumina technologies.

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.

Complementary Regulation of BfmRS Two-Component and AbaIR Quorum Sensing Systems to Express Virulence-Associated Genes in Acinetobacter baumannii.

Acinetobacter baumannii expresses various virulence factors to adapt to hostile environments and infect susceptible hosts. This study investigated the regulatory network of the BfmRS two-component and AbaIR quorum sensing (QS) systems in the expression of virulence-associated genes in A. baumannii ATCC 17978. The ΔbfmS mutant exhibited a significant decrease in surface motility, which presumably resulted from the low expression of pilT and A1S_0112-A1S_0119 gene cluster. The ΔbfmR mutant displayed a significant reduction in biofilm and pellicle formation due to the low expression of csu operon. The deletion of abaR did not affect the expression of bfmR or bfmS. However, the expression of abaR and abaI was upregulated in the ΔbfmR mutant. The ΔbfmR mutant also produced more autoinducers than did the wild-type strain, suggesting that BfmR negatively regulates the AbaIR QS system. The ΔbfmS mutant exhibited no autoinducer production in the bioassay system. The expression of the A1S_0112-A1S_0119 gene cluster was downregulated in the ΔabaR mutant, whereas the expression of csu operon was upregulated in this mutant with a high cell density. In conclusion, for the first time, we demonstrated that the BfmRS-AbaIR QS system axis regulated the expression of virulence-associated genes in A. baumannii. This study provides new insights into the complex network system involved in the regulation of virulence-associated genes underlying the pathogenicity of A. baumannii.

Engineering of lysin by fusion of antimicrobial peptide (cecropin A) enhances its antibacterial properties against multidrug-resistant Acinetobacter baumannii.

Most clinical isolates of Acinetobacter baumannii, a nosocomial pathogen, are multidrug-resistant (MDR), fueling the search for alternative therapies. Bacteriophage-derived endolysins have potent antibacterial activities and are considered as alternatives to antibiotics against A. baumannii infection. Gram-negative bacteria possess outer lipid membrane that prevents direct contact between the endolysins and the cell wall. We hypothesized that the fusion of antimicrobial peptide (AMP) with endolysin could help to reduce bacterial endolysin resistance and increase antimicrobial activity by membrane permeability action. Accordingly, we fused cecropin A, a commonly used AMP, with the N-terminus of AbEndolysin, which enhances the bactericidal activity of the chimeric endolysin. The bactericidal activity of cecropin A-fused AbEndolysin increased by at least 2-8 fold for various MDR A. baumannii clinical isolates. The in vitro bactericidal activity results also showed higher bacterial lysis by the chimeric endolysin than that by the parental lysin. The engineered AbEndolysin (eAbEndolysin) showed synergistic effects with the beta-lactam antibiotics cefotaxime, ceftazidime, and aztreonam, and an additive effect with meropenem and imipenem. eAbEndolysin had no cytotoxic effect on A549 cell line and rescued mice (40% survival rate) from systemic A. baumannii infection. Together, these findings suggest the potential of lysin therapy and may prompt its use as an alternative to antibiotics.

DksA Modulates Antimicrobial Susceptibility of Acinetobacter baumannii.

The stringent response regulators, (p)ppGpp and DksA, modulate various genes involved in physiological processes, virulence, and antimicrobial resistance in pathogenic bacteria. This study investigated the role of DksA in the antimicrobial susceptibility of Acinetobacter baumannii. The ∆dksA mutant (KM0248D) of A. baumannii ATCC 17978 and its complemented strain (KM0248C) were used, in addition to the ∆dksA mutant strain (NY0298D) of clinical 1656-2 strain. The microdilution assay was used to determine the minimum inhibitory concentrations (MICs) of antimicrobial agents. Quantitative real-time PCR was performed to analyze the expression of genes associated with efflux pumps. The KM0248D strain exhibited an increase of MICs to quinolones and tetracyclines, whereas KM0248D and NY0298D strains exhibited a decrease of MICs to aminoglycosides. The expression of genes associated with efflux pumps, including adeB, adeI/J, abeM, and/or tetA, was upregulated in both ∆dksA mutant strains. The deletion of dksA altered bacterial morphology in the clinical 1656-2 strain. In conclusion, DksA modulates the antimicrobial susceptibility of A. baumannii. The ∆dksA mutant strains of A. baumannii upregulate efflux pump gene expression, whereas (p)ppGpp-deficient mutants downregulate efflux pump gene expression. (p)ppGpp and DksA conduct opposite roles in the antimicrobial susceptibility of A. baumannii via efflux pump gene regulation.

Therapeutic Effects of Inhibitor of ompA Expression against Carbapenem-Resistant Acinetobacter baumannii Strains.

The widespread of carbapenem-resistant Acinetobacter baumannii (CRAB) is of great concern in clinical settings worldwide. It is urgent to develop new therapeutic agents against this pathogen. This study aimed to evaluate the therapeutic potentials of compound 62520, which has been previously identified as an inhibitor of the ompA promoter activity of A. baumannii, against CRAB isolates, both in vitro and in vivo. Compound 62520 was found to inhibit the ompA expression and biofilm formation in A. baumannii ATCC 17978 at sub-inhibitory concentrations in a dose-dependent manner. These inhibitory properties were also observed in clinical CRAB isolates belonging to sequence type (ST) 191. Additionally, compound 62520 exhibited a bacteriostatic activity against clinical clonal complex (CC) 208 CRAB isolates, including ST191, and ESKAPE pathogens. This bacteriostatic activity was not different between STs of CRAB isolates. Bacterial clearance was observed in mice infected with bioimaging A. baumannii strain 24 h after treatment with compound 62520. Compound 62520 was shown to significantly increase the survival rates of both immunocompetent and neutropenic mice infected with A. baumannii ATCC 17978. This compound also increased the survival rates of mice infected with clinical CRAB isolate. These results suggest that compound 62520 is a promising scaffold to develop a novel therapeutic agent against CRAB infections.

LeuO, a LysR-Type Transcriptional Regulator, Is Involved in Biofilm Formation and Virulence of Acinetobacter baumannii.

Acinetobacter baumannii is an important nosocomial pathogen that can survive in different environmental conditions and poses a severe threat to public health due to its multidrug resistance properties. Research on transcriptional regulators, which play an essential role in adjusting to new environments, could provide new insights into A. baumannii pathogenesis. LysR-type transcriptional regulators (LTTRs) are structurally conserved among bacterial species and regulate virulence in many pathogens. We identified a novel LTTR, designated as LeuO encoded in the A. baumannii genome. After construction of LeuO mutant strain, transcriptome analysis showed that LeuO regulates the expression of 194 upregulated genes and 108 downregulated genes responsible for various functions and our qPCR validation of several differentially expressed genes support transcriptome data. Our results demonstrated that disruption of LeuO led to increased biofilm formation and increased pathogenicity in an animal model. However, the adherence and surface motility of the LeuO mutant were reduced compared with those of the wild-type strain. We observed some mutations on amino acids sequence of LeuO in clinical isolates. These mutations in the A. baumannii biofilm regulator LeuO may cause hyper-biofilm in the tested clinical isolates. This study is the first to demonstrate the association between the LTTR member LeuO and virulence traits of A. baumannii.

Global regulator DksA modulates virulence of Acinetobacter baumannii.

DksA with (p)ppGpp regulates a wide range of gene transcriptions during the stringent response. The aim of this study was to identify a DksA ortholog in Acinetobacter baumannii and clarify the roles of DksA in bacterial physiology and virulence. The ∆dksA mutant and its complemented strains were constructed using A. baumannii ATCC 17978. The AlS_0248 in A. baumannii ATCC 17978 was identified to dksA using sequence homology, protein structure prediction, and gene expression patterns under different culture conditions. The ∆dksA mutant strain showed a filamentous morphology compared with the wild-type (WT) strain. Bacterial growth was decreased in the ∆dksA mutant strain under static conditions. Surface motility was decreased in the ∆dksA mutant strain compared with the WT strain. In contrast, biofilm formation was increased and biofilm-associated genes, such as bfmR/S and csuC/D/E, were upregulated in the ∆dksA mutant strain. The ∆dksA mutant strain produced less autoinducers than the WT strain. The expression of abaI and abaR was significantly decreased in the ∆dksA mutant strain. Furthermore, the ∆dksA mutant strain showed less bacterial burden and milder histopathological changes in the lungs of mice than the WT strain. Mice survival was also significantly different between the ∆dksA mutant and WT strains. Conclusively, DksA is directly or indirectly involved in regulating a wide range of genes associated with bacterial physiology and virulence, which contributes to the pathogenesis of A. baumannii. Thus, DksA is a potential anti-virulence target for A. baumannii infection.

Characterization of a Novel Phage ΦAb1656-2 and Its Endolysin with Higher Antimicrobial Activity against Multidrug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial pathogen, which is a problem worldwide due to the emergence of a difficult-to-treat multidrug-resistant A. baumannii (MDRAB). Endolysins are hydrolytic enzymes produced by a bacteriophage that can be used as a potential therapeutic agent for multidrug-resistant bacterial infection in replacing antibiotics. Here, we isolated a novel bacteriophage through prophage induction using mitomycin C from clinical A. baumannii 1656-2. Morphologically, ΦAb1656-2 was identified as a Siphoviridae family bacteriophage, which can infect MDRAB. The whole genome of ΦAb1656-2 was sequenced, and it showed that it is 50.9 kb with a G + C content of 38.6% and 68 putative open reading frames (ORFs). A novel endolysin named AbEndolysin with an N-acetylmuramidase-containing catalytic domain was identified, expressed, and purified from ΦAb1656-2. Recombinant AbEndolysin showed significant antibacterial activity against MDRAB clinical strains without any outer membrane permeabilizer. These results suggest that AbEndolysin could represent a potential antimicrobial agent for treating MDRAB clinical isolates.

ppGpp signaling plays a critical role in virulence of Acinetobacter baumannii.

Acinetobacter baumannii, a major nosocomial pathogen, survives in diverse hospital environments, and its multidrug resistance is a major concern. The ppGpp-dependent stringent response mediates the reprogramming of genes with diverse functions in several bacteria. We investigated whether ppGpp is involved in A. baumannii's pathogenesis by examining biofilm formation, surface motility, adhesion, invasion, and mouse infection studies. Transcriptome analysis of early stationary phase cultures revealed 498 differentially-expressed genes (≥ 2-fold change) in a ppGpp-deficient A. baumannii strain; 220 and 278 genes were up and downregulated, respectively. Csu operon expression, important in pilus biosynthesis during early biofilm formation, was significantly reduced in the ppGpp-deficient strain. Our findings suggest that ppGpp signaling influences A. baumannii biofilm formation, surface motility, adherence, and virulence. We showed the association between ppGpp and pathogenicity in A. baumannii for the first time; ppGpp may be a novel antivirulence target in A. baumannii.

Screening of small molecules attenuating biofilm formation of Acinetobacter baumannii by inhibition of ompA promoter activity.

Anti-virulence therapeutic strategies are promising alternatives against drug-resistant pathogens. Outer membrane protein A (OmpA) plays a versatile role in the pathogenesis and antimicrobial resistance of Acinetobacter baumannii. Therefore, OmpA is an innovative target for anti-virulence therapy against A. baumannii. This study aimed to develop a high-throughput screening (HTS) system to discover small molecules inhibiting the ompA promoter activity of A. baumannii and screen chemical compounds using the bacterial growth-based HTS system. The ompA promoter and open reading frame of nptI fusion plasmids that controlled the expression of nptI encoding resistance to kanamycin by the ompA promoter were constructed and then transformed into A. baumannii ATCC 17978. This reporter strain was applied to screen small molecules inhibiting the ompA promoter activity in a chemical library. Of the 7,520 chemical compounds, 15 exhibited ≥ 70% growth inhibition of the report strain cultured in media containing kanamycin. Three compounds inhibited the expression of ompA and OmpA in the outer membrane of A. baumannii ATCC 17978, which subsequently reduced biofilm formation. In conclusion, our reporter strain is useful for large-scale screening of small molecules inhibiting the ompA expression in A. baumannii. Hit compounds identified by the HTS system are promising scaffolds to develop novel therapeutics against 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.

Production of Membrane Vesicles in Listeria monocytogenes Cultured with or without Sub-Inhibitory Concentrations of Antibiotics and Their Innate Immune Responses In Vitro.

Listeriosis is a food-borne illness caused by Listeria monocytogenes. Ampicillin (AMP) alone or in combination with gentamicin (GEN) is the first-line treatment option. Membrane vesicle (MV) production in L. monocytogenes under antibiotic stress conditions and pathologic roles of these MVs in hosts have not been reported yet. Thus, the aim of this study was to investigate the production of MVs in L. monocytogenes cultured with sub-minimum inhibitory concentrations (MICs) of AMP, GEN, or trimethoprim/sulfamethoxazole (SXT) and determine pathologic effects of these MVs in colon epithelial Caco-2 cells. L. monocytogenes cultured in tryptic soy broth with 1/2 MIC of AMP, GEN, or SXT produced 6.0, 2.9, or 1.5 times more MV particles, respectively, than bacteria cultured without antibiotics. MVs from L. monocytogenes cultured with AMP (MVAMP), GEN (MVGEN), or SXT (MVSXT) were more cytotoxic to Caco-2 cell than MVs obtained from cultivation without antibiotics (MVTSB). MVAMP induced more expression of tumor necrosis factor (TNF)-α gene than MVTSB, MVGEN and MVSXT, whereas MVTSB induced more expression of interleukin (IL)-1ß and IL-8 genes than other MVs. Expression of pro-inflammatory cytokine genes by L. monocytogenes MVs was significantly inhibited by proteinase K treatment of MVs. In conclusion, antibiotic stress can trigger the biogenesis of MVs in L. monocytogenes and MVs produced by L. monocytogenes exposed to sub-MIC of AMP can induce strong pro-inflammatory responses by expressing TNF-α gene in host cells, which may contribute to the pathology of listeriosis.