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Studies on plant-derived natural products that have no side-effects have attracted great attention from the cosmetics industry. Plant-derived exosomes are nanovesicles (30–150 nm in size) that contain biomolecules, which protect plants from stress and pathogens. In this study, we determined the availability of physiologically active Centella asiatica (Cica) extract and Cica exosome for cosmetic use with human keratinocytes using an in vitro efficacy assay, transcriptome analysis, and miRNA profiling. In vitro efficacy analysis revealed that Cica extract and Cica exosome have regenerative and wrinkle-improving effects. Comparative transcriptome analysis between Cica exosome- and Cica extract-treated-human keratinocytes revealed 46% more gene expression in Cica exosome-treated cells than Cica extract-treated cells; moreover, the expression of marker genes involved in skin aging and regeneration was upregulated. miRNA analysis of Cica exosomes identified 11 novel miRNAs. Prediction of miRNA targets revealed that Cica exosomal miRNA can inhibit genes involved in melanin biosynthesis and dermatitis. Transcriptome analysis confirmed that the predicted human target genes were downregulated by Cica exosome miRNA compared with the control. Thus, Cica exosomes and Cica extracts can have a positive effect on skin regeneration, wrinkle improvement, and skin barrier improvement. Cica exosomal miRNA has potential whitening and anti-dermatitis effects.

Background As carbapenem-resistant Acinetobacter baumannii is dominant in clinical settings, the old polymyxin antibiotic colistin has been revived as a therapeutic option. The development of colistin resistance during treatment is becoming a growing concern. Objectives To access low- to mid-level colistin-resistant A. baumannii blood isolates recovered from an outbreak in a tertiary care hospital from a national antimicrobial surveillance study. Methods The entire bacterial genome was sequenced through long-read sequencing methodology. Quantitative RT–PCR was carried out to determine the level of gene expression. Relative growth rates were determined to estimate fitness costs of each isolate caused by the genetic alterations. Results The A. baumannii isolates belonged to global clone 2 harbouring two intrinsic phosphoethanolamine transferases. Cumulative alterations continuing the colistin resistance were observed. PmrC overproduction caused by the PmrBA226T alteration was identified in A. baumannii isolates with low-level colistin resistance and an additional PmrCR109H substitution led to mid-level colistin resistance. Truncation of the PmrC enzyme by insertion of ISAba59 was compensated by ISAba10-mediated overproduction of EptA and, in the last isolate, the complete PmrAB two-component regulatory system was eliminated to restore the biological cost of the bacterial host. Conclusions During the in-hospital outbreak, a trajectory of genetic modification in colistin-resistant A. baumannii isolates was observed for survival in the harsh conditions imposed by life-threatening drugs with the clear purpose of maintaining drug resistance above a certain level with a reasonable fitness cost.

Approximately half of the BSIs caused by E. coli are secondary to E. coli UTIs. Since the uropathogenic E. coli causing most of the UTIs is genetically diverse, understanding the risk factors in the E. coli urine isolates causing the BSI is important for pathophysiology.

Increasing prevalence of E. coli producing CTX-M ESBL is a major concern in clinical settings because it significantly limits treatment options. Thus, it is important to keep watching current molecular mechanisms of resistance and the scheme for dissemination.

Class D β-lactamases are composed of 14 families and the majority of the member enzymes are included in the OXA family. The genes for class D β-lactamases are frequently identified in the chromosome as an intrinsic resistance determinant in environmental bacteria and a few of these are found in mobile genetic elements carried by clinically significant pathogens. The most dominant OXA family among class D β-lactamases is superheterogeneous and the family needs to have an updated scheme for grouping OXA subfamilies through phylogenetic analysis. The OXA enzymes, even the members within a subfamily, have a diverse spectrum of resistance. Such varied activity could be derived from their active sites, which are distinct from those of the other serine β-lactamases. Their substrate profile is determined according to the size and position of the P-, Ω- and β5–β6 loops, assembling the active-site channel, which is very hydrophobic. Also, amino acid substitutions occurring in critical structures may alter the range of hydrolysed substrates and one subfamily could include members belonging to several functional groups. This review aims to describe the current class D β-lactamases including the functional groups, occurrence types (intrinsic or acquired) and substrate spectra and, focusing on the major OXA family, a new model for subfamily grouping will be presented.

Various forms of adaptive evolution occur in clinical isolates in response to the presence of antimicrobial drugs. Among a total of 171 CTX-M-9 group/family extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli blood isolates recovered between 2016 and 2017 in six general hospitals, 50.3% of the isolates possessed the

Objectives A MALDI-TOF MS-based identification method for KPC-producing Enterobacterales was developed. Methods The molecular mass of the intact KPC-2 polypeptide was estimated for blaKPC-2 transformants using MALDI Microflex and the exact mass was confirmed by LC and a high-resolution MS/MS system. A total of 1181 clinical Enterobacterales strains, including 369 KPC producers and 812 KPC non-producers, were used to set up the methodology and the results were compared with those from PCR analyses. For external validation, a total of 458 Enterobacterales clinical isolates from a general hospital between December 2018 and April 2019 were used. Results The exact molecular mass of the intact KPC-2 protein was 28 718.13 Da and KPC peaks were observed at m/z 28 708.87–28 728.34 using MALDI Microflex. Most of the KPC-2 (99.1%, 335/338) and KPC-3 (100%, 6/6) producers presented a clear peak via this method, while 12.0% (3/25) of the KPC-4 producers had a peak of weak intensity associated with low levels of gene expression. It took less than 20 min for the entire assay to be performed with colonies on an agar plate. External validation showed that the analytical sensitivity and specificity of the method compared with PCR were 100% (59/59) and 99.50% (397/399), respectively. Conclusions The MALDI-TOF MS-based method for directly detecting the intact KPC protein is applicable to routine tests in clinical microbiology laboratories, supported by its speed, low cost and excellent sensitivity and specificity.

Background Carbapenem-resistant K. pneumoniae 2297, isolated from a patient treated with tigecycline for pneumonia, developed tigecycline resistance, in contrast to carbapenem-resistant isolate 1215, which was collected four months prior to the 2297 isolate. Mechanisms underlying tigecycline resistance were elucidated for the clinical isolates. Methods The tigecycline minimum inhibitory concentration (MIC) was determined using the broth microdilution method, with or without phenylalanine-arginine β-naphthylamide (PABN), and whole-genome sequencing was carried out by single-molecule real-time sequencing. The expression levels of the genes acrA, oqxA, ramA, rarA, and rpoB were determined by reverse-transcription quantitative PCR. Results Both isolates presented identical antibiograms, except for tigecycline, which showed an MIC of 0.5 mg/L in 1215 and 2 mg/L in 2297. The addition of PABN to tigecycline-resistant 2297 caused a four-fold decrease in the tigecycline MIC to 0.5 mg/L, although acrA expression (encoding the AcrAB efflux pump) was upregulated by 2.5 fold and ramA expression (encoding the pump activator RamA) was upregulated by 1.4 fold. We identified a 6,096-bp fragment insertion flanking direct TATAT repeats that disrupted the romA gene located upstream of ramA in the chromosome of K. pneumoniae 2297; the insertion led the ramA gene promoter replacement resulting in stronger activation of the gene. Conclusions The K. pneumoniae isolate developed tigecycline resistance during tigecycline treatment. It was related to the overexpression of the AcrAB resistance-nodulation-cell division efflux system due to promoter replacement.

Plant exosomes are nanosized (30–150 nm) membrane vesicles that contain biomolecules and influence the development of a plant and protect the plant from pathogens. Recently, plant exosomes are in the spotlight as a new biologically active substance. However, whether plant exosomes have similar efficacy to conventional secondary metabolites of plants is unknown. In this study, the difference in efficacy between plant exosomes and conventional secondary metabolites was analyzed with three or four types of plant extracts, including ginseng (Panax ginseng) and green tea (Camellia sinensis). After 6 h of treatment, the analysis of gene expression pattern of each sample showed that the exosome treatment group and the extract treatment group were clearly distinguished. After selecting the genes that showed differential expression of > twofold change, the number of genes that were up- or downregulated appeared to be 398 or 438 for the extract and 861 or 648 for the exosome, on average. This suggests that the change in transcriptome is more expressed in the exosome treatment group than in the extract treatment group. In addition, in the comparative analysis of expression of genes that are known to affect aging, regeneration, skin barrier, and moisturization—MMP12, MMP13, NOTCH3, FGF12, HS3ST3A1, LOX, VIM, ELOVL3, and KRTI—the exosome treatment group was predicted to more effectively contribute to maintaining a healthy skin when compared to the extract treatment group. The number of genes that were identified to specifically react to the Panax ginseng or Camellia sinensis treatment group during the transcriptome change phase was 11 and 8, respectively. This suggests that exosomes bear its specific effect according to the plant it is derived from. In conclusion, the results of this study indicate that plant exosomes, as natural biologically active substances, have different effects from conventional plant extracts, and have the potential to be commercialized as a cosmeceutical product.

To monitor national antimicrobial resistance (AMR), the Korea Global AMR Surveillance System (Kor-GLASS) was established. This study analyzed bloodstream infection (BSI) cases from Kor-GLASS phase I from January 2017 to December 2019. Nine non-duplicated Kor-GLASS target pathogens, including Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter spp., and Salmonella spp., were isolated from blood specimens from eight sentinel hospitals. Antimicrobial susceptibility testing, AMR genotyping, and strain typing were carried out. Among the 20,041 BSI cases, 15,171 cases were caused by one of the target pathogens, and 12,578 blood isolates were collected for the study. Half (1,059/2,134) of S. aureus isolates were resistant to cefoxitin, and 38.1% (333/873) of E. faecium isolates were resistant to vancomycin. Beta-lactamase-non-producing ampicillin-resistant and penicillin-resistant E. faecalis isolates by disk diffusion method were identified, but the isolates were confirmed as ampicillin-susceptible by broth microdilution method. Among E. coli, an increasing number of isolates carried the blaCTX–M–27 gene, and the ertapenem resistance in 1.4% (30/2,110) of K. pneumoniae isolates was mostly (23/30) conferred by K. pneumoniae carbapenemases. A quarter (108/488) of P. aeruginosa isolates were resistant to meropenem, and 30.5% (33/108) of those carried acquired carbapenemase genes. Over 90% (542/599) of A. baumannii isolates were imipenem-resistant, and all except one harbored the blaOXA–23 gene. Kor-GLASS provided comprehensive AMR surveillance data, and the defined molecular mechanisms of resistance helped us to better understand AMR epidemiology. Comparative analysis with other GLASS-enrolled countries is possible owing to the harmonized system provided by GLASS.

Here, we aimed to elucidate the differences in microbiota composition between patients with gout and those with asymptomatic hyperuricemia (asHU) and determine the effect of uric acid-lowering therapy (ULT) on the gut microbiome.Stool samples from patients with asHU (n=8) and three groups of gout patients, i.e., acute gout patients before ULT (0ULT, n=14), the same acute gout patients after 30-day ULT (30ULT, n=9), and chronic gout patients after ≥6-month ULT (cULT, n=18) were collected and analyzed using 16S rRNA gene-based pyrosequencing. The composition of microbial taxonomy and communities, species diversity, and relationships among microbial communities were elucidated by bioinformatic analysis.Gout patients showed less diverse gut microbiota than asHU patients. The microbiota of the asHU group exhibited a higherWe found that microbial composition differs between asHU and gout patients. The differential gut microbiota in asHU patients may protect against gout development, whereas that in gout patients may have a role in gout provocation. ULT in gout patients altered the gut microbiota, and may help alleviate gout pathology and mitigate gout progression.

Species identification by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a routine diagnostic process for infectious diseases in current clinical settings. The rapid, low-cost, and simple to conduct methodology is expanding its application in clinical microbiology laboratories to diagnose the antimicrobial resistance (AMR) in microorganisms. Primarily, antimicrobial susceptibility testing is able to be carried out either by comparing the area under curve of MALDI spectra of bacteria grown in media with antimicrobial drugs or by identifying the shift peaks of bacteria grown in media including 13C isotope with antimicrobial drugs. Secondly, the antimicrobial resistance is able to be determined through identifying (i) the antimicrobial-resistant clonal groups based on the fingerprints of the clone, (ii) the shift peak of the modified antimicrobial drug, which is inactivated by the resistance determinant, (iii) the shift peak of the modified antimicrobial target, (iv) the peak specific for the antimicrobial determinant, and (v) the biomarkers that are coproduced proteins with AMR determinants. This review aims to present the current usage of the MALDI-TOF MS technique for diagnosing antimicrobial resistance in bacteria, varied approaches for AMR diagnostics using the methodology, and the future applications of the methods for the accurate and rapid identification of AMR in infection-causing bacterial pathogens.

Background The emergence of carbapenem resistance among gram-negative bacilli (GNB), mediated by carbapenemase production, has necessitated the development of a simple and accurate device for detecting minimum inhibitory concentrations (MICs) and resistance mechanisms, especially carbapenemase production. We evaluated the performance of the BD Phoenix NMIC-500 panel (BD Diagnostic Systems, Sparks, MD, USA) for antimicrobial susceptibility testing (AST) and carbapenemase-producing organism (CPO) detection. Methods We used 450 non-duplicate clinical GNB isolates from six general hospitals in Korea (409 Enterobacteriaceae and 41 glucose non-fermenting bacilli [GNFB] isolates). AST for meropenem, imipenem, ertapenem, ceftazidime, and ceftazidime/avibactam, and CPO detection were performed using the Phoenix NMIC-500 panel. Broth microdilution was used as the reference method for AST. The rates of categorical agreement (CA), essential agreement (EA), minor error (mE), major error (ME), and very major error (VME) were calculated in each antimicrobial. In addition, PCR and sequencing were performed to evaluate the accuracy of CPO detection by the BD Phoenix NMIC-500 panel, and the rate of correct identification was calculated. Results The CA rates were >90% for all antimicrobials tested with the Enterobacteriaceae isolates, except for imipenem (87.2%). The GNFB CA rates ranged from 92.7% to 100% for all antimicrobials. The ME rates were 1.7% for Enterobacteriaceae and 0% for GNFB. The panel identified 97.2% (243/250) of the carbapenemase-producing isolates. Conclusions The BD Phoenix NMIC-500 panel shows promise for AST and CPO detection.

BackgroundTo assess the mortality dynamics of patients with Pseudomonas aeruginosa bloodstream infections (BSIs) and the influence of OprD deficiencies of the microorganism on early mortality.MethodsA prospective multicentre observational study was conducted with 120 patients with P. aeruginosa BSIs occurring between May 2016 and April 2017 in six general hospitals in South Korea. PCR and sequencing were carried out to identify the alterations in oprD and the presence of virulence factors. Cox regression was used to estimate the risk factors for mortality at each timepoint and Kaplan–Meier survival analyses were performed to determine the mortality dynamics.ResultsDuring the 6 week follow-up, 10.8% (13/120) of the patients with P. aeruginosa BSIs died in 2 weeks, 14.2% (17/120) in 4 weeks and 20.0% (24/120) in 6 weeks, revealing a steep decrease in cumulative survival between the fourth and sixth weeks. ICU admission and SOFA score were risk factors for mortality in any weeks after BSI onset and causative OprD-defective P. aeruginosa had a risk tendency for mortality within 6 weeks. Among the 120 P. aeruginosa blood isolates, 14 were XDR, nine produced either IMP-6 or VIM-2 MBL, and 21 had OprD deficiency.ConclusionsBSIs caused by OprD-defective P. aeruginosa resulted in a 2-fold higher 6 week mortality rate (33.3%) than that of BSIs caused by OprD-intact P. aeruginosa (17.2%), likely due to the decreased susceptibility to carbapenems and bacterial persistence in clinical settings.

Carbapenem-resistantPseudomonas aeruginosais one of the major concerns in clinical settings impelling a great challenge to antimicrobial therapy for patients with infections caused by the pathogen. While membrane permeability, together with derepression of the intrinsic beta-lactamase gene, is the global prevailing mechanism of carbapenem resistance inP. aeruginosa, the acquired genes for carbapenemases need special attention because horizontal gene transfer through mobile genetic elements, such as integrons, transposons, plasmids, and integrative and conjugative elements, could accelerate the dissemination of the carbapenem-resistantP. aeruginosa. This review aimed to illustrate epidemiologically the carbapenem resistance inP. aeruginosa, including the resistance rates worldwide and the carbapenemase-encoding genes along with the mobile genetic elements responsible for the horizontal dissemination of the drug resistance determinants. Moreover, the modular mobile elements including the carbapenemase-encoding gene, also known as theP. aeruginosaresistance islands, are scrutinized mostly for their structures.

A novel Klebsiella pneumoniae carbapenemase (KPC) variant, KPC-55, produced by a K. pneumoniae ST307 strain was characterized. K. pneumoniae strain BS407 was recovered from an active surveillance rectal swab of a patient newly admitted to a general hospital in Busan, South Korea. Carbapenemase production was confirmed by the modified Hodge test, and the MICs of β-lactams were determined by the broth microdilution method. The whole genome was sequenced. Cloning and expression of the bla KPC-55 gene in Escherichia coli and MIC determination were performed. The enzyme KPC-55 was used for kinetic assays against β-lactams and compared with the KPC-2 enzyme. The new allele of the bla KPC gene had a T794A alteration compared to the bla KPC-2 gene, resulting in the amino acid substitution Y264N in the middle of the β9-sheet. Compared to the KPC-2-producing strain, the KPC-55-producing strain exhibited a lower level of resistance to most β-lactam drugs tested, however, the KPC-55 enzyme catalyzed aztreonam and meropenem at an increased efficiency compared to the catalytic activity of KPC-2. KPC subtypes could have varied phenotypes due to alterations in amino acid sequences, and such an unexpected resistance phenotype emphasizes the importance of detailed characterizations for the carbapenemase-producing Enterobacterales.

Carbapenem-resistant Pseudomonas aeruginosa is one of the major concerns in clinical settings impelling a great challenge to antimicrobial therapy for patients with infections caused by the pathogen. While membrane permeability, together with derepression of the intrinsic beta-lactamase gene, is the global prevailing mechanism of carbapenem resistance in P. aeruginosa, the acquired genes for carbapenemases need special attention because horizontal gene transfer through mobile genetic elements, such as integrons, transposons, plasmids, and integrative and conjugative elements, could accelerate the dissemination of the carbapenem-resistant P. aeruginosa. This review aimed to illustrate epidemiologically the carbapenem resistance in P. aeruginosa, including the resistance rates worldwide and the carbapenemase-encoding genes along with the mobile genetic elements responsible for the horizontal dissemination of the drug resistance determinants. Moreover, the modular mobile elements including the carbapenemase-encoding gene, also known as the P. aeruginosa resistance islands, are scrutinized mostly for their structures.

Dominant F-type plasmids harboring the gene have been pointed out to be responsible for the dissemination of the CTX-M extended-spectrum-β-lactamase (ESBL)-producing K. pneumoniae. Recently, the emergence of K. pneumoniae isolates with the blaCTX-M gene in their chromosomes has been reported occasionally worldwide. Such a chromosomal location of the resistance gene could be beneficial for stable propagation, as was the Acinetobacter baumannii ST191 harboring chromosomal blaOXA-23 that is endemic to South Korea. Through the present study, particular clones were identified as having built-in resistance genes in their chromosomes, and the chromosomal integration events were tracked by assessing their genomes. The cefotaxime-resistant K. pneumoniae clones of this study were particularized as results of the fastidiousness for plasmids to acquire the blaCTX-M gene for securing the diversity and of the chromosomal addiction of the blaCTX-M gene for ensuring propagation., The acquired CTX-M-type extended-spectrum-β-lactamase (ESBL)-producing Enterobacterales are of great concern in clinical settings because they limit therapeutic options for patients infected by the pathogens. An intriguing clonality of CTX-M ESBL-producing Klebsiella pneumoniae blood isolates was observed from a national cohort study, and comparative genomics were assessed for the 115 K. pneumoniae blood isolates carrying the blaCTX-M gene. The plasmid preference of particular clones of a sequence type (ST) was assessed by liquid mating. A quarter of the blaCTX-M gene-carrying K. pneumoniae blood isolates harbor the gene in their chromosome, and most of those with the built-in blaCTX-M gene belonged either to ST307 or ST48. Notably, all 16 K. pneumoniae ST48 isolates harbored two copies of the blaCTX-M-15 gene in the chromosome. The chromosomal integration of the blaCTX-M-15 gene was mostly derived from the ISEcp1-targeting 5-bp AT-rich locus in the chromosome. The IS26-mediated chromosomal integration occurred when the upstream ISEcp1 from the blaCTX-M gene was truncated, targeting the anchor IS26 copy in the chromosome. Higher transfer efficiency of the blaCTX-M-15 gene-carrying FIA:R plasmid was observed in ST17 than that of the blaCTX-M-14 gene-carrying FIB:FII plasmid. The transfer efficiency of the plasmid differed by isolate among the ST307 members. The K. pneumoniae clones ST307 and ST48 harboring the blaCTX-M-15 gene in the chromosome were able to disseminate stably in clinical settings regardless of the environmental pressure, and the current population of K. pneumoniae blood isolates was constructed. Further follow-up is needed for the epidemiology of this antimicrobial resistance. IMPORTANCE Dominant F-type plasmids harboring the gene have been pointed out to be responsible for the dissemination of the CTX-M extended-spectrum-β-lactamase (ESBL)-producing K. pneumoniae. Recently, the emergence of K. pneumoniae isolates with the blaCTX-M gene in their chromosomes has been reported occasionally worldwide. Such a chromosomal location of the resistance gene could be beneficial for stable propagation, as was the Acinetobacter baumannii ST191 harboring chromosomal blaOXA-23 that is endemic to South Korea. Through the present study, particular clones were identified as having built-in resistance genes in their chromosomes, and the chromosomal integration events were tracked by assessing their genomes. The cefotaxime-resistant K. pneumoniae clones of this study were particularized as results of the fastidiousness for plasmids to acquire the blaCTX-M gene for securing the diversity and of the chromosomal addiction of the blaCTX-M gene for ensuring propagation.

This study was performed to evaluate the impacts of vanA positivity of Enterococcus faecium exhibiting diverse susceptibility phenotypes to glycopeptides on clinical outcomes in patients with a bloodstream infection (BSI) through a prospective, multicenter, observational study. A total of 509 patients with E. faecium BSI from eight sentinel hospitals in South Korea during a 2-year period were enrolled in this study. Risk factors of the hosts and causative E. faecium isolates were assessed to determine associations with the 30-day mortality of E. faecium BSI patients via multivariable logistic regression analyses. The vanA gene was detected in 35.2% (179/509) of E. faecium isolates; 131 E. faecium isolates exhibited typical VanA phenotypes (group vanA-VanA), while the remaining 48 E. faecium isolates exhibited atypical phenotypes (group vanA-atypical), which included VanD (n = 43) and vancomycin-variable phenotypes (n = 5). A multivariable logistic regression indicated that vanA positivity of causative pathogens was independently associated with the increased 30-day mortality rate in the patients with E. faecium BSI; however, there was no significant difference in survival rates between the patients of the vanA-VanA and vanA-atypical groups (log rank test, P = 0.904). A high 30-day mortality rate was observed in patients with vanA-positive E. faecium BSIs, and vanA positivity of causative E. faecium isolates was an independent risk factor for early mortality irrespective of the susceptibility phenotypes to glycopeptides; thus, intensified antimicrobial stewardship is needed to improve the clinical outcomes of patients with vanA-positive E. faecium BSI.

A novel Klebsiella pneumoniae carbapenemase (KPC) variant, KPC-55, produced by a K. pneumoniae ST307 strain was characterized. K. pneumoniae strain BS407 was recovered from an active surveillance rectal swab of a patient newly admitted to a general hospital in Busan, South Korea. Carbapenemase production was confirmed by the modified Hodge test, and the MICs of β-lactams were determined by the broth microdilution method. The whole genome was sequenced. Cloning and expression of the blaKPC–55 gene in Escherichia coli and MIC determination were performed. The enzyme KPC-55 was used for kinetic assays against β-lactams and compared with the KPC-2 enzyme. The new allele of the blaKPC gene had a T794A alteration compared to the blaKPC–2 gene, resulting in the amino acid substitution Y264N in the middle of the β9-sheet. Compared to the KPC-2-producing strain, the KPC-55-producing strain exhibited a lower level of resistance to most β-lactam drugs tested, however, the KPC-55 enzyme catalyzed aztreonam and meropenem at an increased efficiency compared to the catalytic activity of KPC-2. KPC subtypes could have varied phenotypes due to alterations in amino acid sequences, and such an unexpected resistance phenotype emphasizes the importance of detailed characterizations for the carbapenemase-producing Enterobacterales.

Background: Ideal dose of the antimicrobials should be decided by considering their killing dynamics since sufficient elimination of the causative microorganisms is critical for proper antimicrobial treatment. In this study, the bactericidal activities of carbapenems by resistance mechanisms were assessed for carbapenem-resistant Pseudomonas aeruginosa. Methods: Minimal inhibition concentrations (MICs) of carbapenems were determined by broth dilution method and the resistance mechanisms were identified by PCR and DNA sequencing. The expression levels of efflux pumps were determined by reverse transcriptase real-time PCR. Time-kill curves were plotted by time-course numeration of the viable cells grown under imipenem and meropenem at 1× and 4× MICs, respectively. Results: One P. aeruginosa strain was susceptible, whereas three were resistant to carbapenems by defective OprD, efflux pump overproduction, and/or IMP-6 production. The susceptible strain had imipenem and meropenem MICs of 2 and 1 mg/L, respectively. The MICs were elevated by eight-fold by defective OprD, 16- and 32-fold by the pump overproduction, and four- and

Background The emerging mobile colistin resistance gene, mcr-1, is an ongoing worldwide concern and an evaluation of clinical isolates harboring this gene is required in Korea. We investigated mcr-1-possessing Enterobacteriaceae among Enterobacteriaceae strains isolated in Korea, and compared the genetic details of the plasmids with those in Escherichia coli isolates from livestock. Methods Among 9,396 Enterobacteriaceae clinical isolates collected between 2010 and 2015, 1,347 (14.3%) strains were resistant to colistin and those were screened for mcr-1 by PCR. Colistin minimum inhibitory concentrations (MICs) were determined by microdilution, and conjugal transfer of the mcr-1-harboring plasmids was assessed by direct mating. Whole genomes of three mcr-1-positive Enterobacteriaceae clinical isolates and 11 livestock-origin mcr-1-positive E. coli isolates were sequenced. Results Two E. coli and one Enterobacter aerogenes clinical isolates carried carried IncI2 plasmids harboring mcr-1, which conferred colistin resistance (E. coli MIC, 4 mg/L; E. aerogenes MIC, 32 mg/L). The strains possessed the complete conjugal machinery except for E. aerogenes harboring a truncated prepilin peptidase. The E. coli plasmid transferred more efficiently to E. coli than to Klebsiella pneumoniae or Enterobacter cloacae recipients. Among the three bacterial hosts, the colistin MIC was the highest for E. coli owing to the higher mcr-1-plasmid copy number and mcr-1 expression levels. Ten mcr-1-positive chicken-origin E. coli strains also possessed mcr-1-harboring IncI2 plasmids closely related to that in the clinical E. aerogenes isolate, and the remaining one porcine-origin E. coli possessed an mcr-1-harboring IncX4 plasmid. Conclusions mcr-1-harboring IncI2 plasmids were identified in clinical Enterobacteriaceae isolates. These plasmids were closely associated with those in chicken-origin E. coli strains in Korea, supporting the concept of mcr-1 dissemination between humans and livestock.

Background The increasing morbidity and mortality rates associated with Acinetobacter baumannii are due to the emergence of drug resistance and the limited treatment options. We compared characteristics of colistin-resistant Acinetobacter baumannii (CR-AB) clinical isolates recovered from patients with and without prior colistin treatment. We assessed whether prior colistin treatment affects the resistance mechanism of CR-AB isolates, mortality rates, and clinical characteristics. Additionally, a proper method for identifying CR-AB was determined. Methods We collected 36 non-duplicate CR-AB clinical isolates resistant to colistin. Antimicrobial susceptibility testing, Sanger sequencing analysis, molecular typing, lipid A structure analysis, and in vitro synergy testing were performed. Eleven colistin-susceptible AB isolates were used as controls. Results Despite no differences in clinical characteristics between patients with and without prior colistin treatment, resistance-causing genetic mutations were more frequent in isolates from colistin-treated patients. Distinct mutations were overlooked via the Sanger sequencing method, perhaps because of a masking effect by the colistin-susceptible AB subpopulation of CR-AB isolates lacking genetic mutations. However, modified lipid A analysis revealed colistin resistance peaks, despite the population heterogeneity, and peak levels were significantly different between the groups. Conclusions Although prior colistin use did not induce clinical or susceptibility differences, we demonstrated that identification of CR-AB by sequencing is insufficient. We propose that population heterogeneity has a masking effect, especially in colistin non-treated patients; therefore, accurate testing methods reflecting physiological alterations of the bacteria, such as phosphoethanolamine-modified lipid A identification by matrix-assisted laser desorption ionization-time of flight, should be employed.

Aim: To assess the epidemiology of carbapenemase-producing Enterobacteriaceae (CPE) in South Korea. Materials & methods: From 2011 to 2015, 2487 carbapenem-nonsusceptible Enterobacteriaceae were collected through the Korean National Laboratory Surveillance System. Disk-diffusion for antimicrobial susceptibility, PCR/sequencing to detect carbapenemase genes and multilocus sequence typing for molecular epidemiology were carried out. Results: The number of carbapenem-nonsusceptible Enterobacteriaceae was increasing approximately 1.5-fold per year and the proportion of CPEs was exponentially confirmed from 2014. KPC was the most dominant, mostly associated with Klebsiella pneumoniae ST11 and ST307, NDM was the second and OXA-48-like was the third dominant carbapenemases. The IMP, VIM and GES-5 CPEs were identified sporadically. Conclusion: The nation-wide spreads of KPC, NDM and OXA-48-like CPEs were in an alarming epidemiological stage.

Pseudomonas putida group are Gram-negative bacilli with polar flagellation, which are ubiquitous in the environment, although they are rarely involved in human infections. The aim of this study was to identify the dissemination of VIM-2–producing P. putida group in clinical isolates from a hospital in Korea. Thirteen strains were collected from 2014 to 2015 for the study. The isolates were recovered from urine cultures of both inpatients and outpatients at the hospital. Minimum inhibitory concentrations of antibiotics were determined by Etest. Carbapenemase genes were amplified by polymerase chain reaction and sequenced. Pulsed-field gel electrophoresis was performed for strain typing. Whole-genome sequencing was carried out randomly for two strains chosen from each year of the study to analyze the plasmid structure carrying the blaVIM-2 genes. The 13 isolates carried nine different class I integrons harboring VIM-2 and were resistant to meropenem and imipenem (minimum inhibitory concentrations, ≥32 μg/ml...

BACKGROUND Extensively drug-resistant (XDR) Enterobacteriaceae carrying the bla(KPC) gene have emerged as a major global therapeutic concern. The purpose of this study was to analyze the complete sequences of plasmids from KPC-2 carbapenemase-producing XDR Escherichia coli sequence type (ST) 1642 isolates. METHODS We performed antimicrobial susceptibility testing, PCR, multilocus sequence typing (MLST), and whole-genome sequencing to characterize the plasmid-mediated KPC-2-producing E. coli clinical isolates. RESULTS The isolates were resistant to most available antibiotics, including meropenem, ampicillin, ceftriaxone, gentamicin, and ciprofloxacin, but susceptible to tigecycline and colistin. The isolates were identified as the rare ST1642 by MLST. The isolates carried four plasmids: the first 69-kb conjugative IncX3 plasmid harbors bla(KPC-2) within a truncated Tn4401a transposon and bla(SHV-11) with duplicated conjugative elements. The second 142-kb plasmid with a multireplicon consisting of IncQ, IncFIA, and IncIB carries bla(TEM-1b) and two class 1 integrons. This plasmid also harbors a wide variety of additional antimicrobial resistance genes including aadA5, dfrA17, mph(A), sul1, tet(B), aac(3')-IId, strA, strB, and sul2. CONCLUSIONS The complete sequence analysis of plasmids from an XDR E. coli strain related to persistent infection showed the coexistence of a bla(KPC-2)-carrying IncX3-type plasmid and a class 1 integron-harboring multireplicon, suggesting its potential to cause outbreaks. Of additional clinical significance, the rare ST1642, identified in a cat, could constitute the source of human infection.

A total of 431 Pseudomonas aeruginosa clinical isolates were collected from 29 general hospitals in South Korea in 2015. Antimicrobial susceptibility was tested by the disk diffusion method, and MICs of carbapenems were determined by the agar dilution method. Carbapenemase genes were amplified by PCR and sequenced, and the structures of class 1 integrons surrounding the carbapenemase gene cassettes were analyzed by PCR mapping. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed for strain typing. Whole-genome sequencing was carried out to analyze P. aeruginosa genomic islands (PAGIs) carrying the bla IMP-6 , bla IMP-10 , and bla GES-24 genes. The rates of carbapenem-nonsusceptible and carbapenemase-producing P. aeruginosa isolates were 34.3% (148/431) and 9.5% (41/431), respectively. IMP-6 was the most prevalent carbapenemase type, followed by VIM-2, IMP-10, and GES-24. All carbapenemase genes were located on class 1 integrons of 6 different types on the chromosome. All isolates harboring carbapenemase genes exhibited genetic relatedness by PFGE (similarity > 80%); moreover, all isolates were identified as sequence type 235 (ST235), with the exception of two ST244 isolates by MLST. The bla IMP-6 , bla IMP-10 , and bla GES-24 genes were found to be located on two novel PAGIs, designated PAGI-15 and PAGI-16. Our data support the clonal spread of an IMP-6-producing P. aeruginosa ST235 strain, and the emergence of IMP-10 and GES-24 demonstrates the diversification of carbapenemases in P. aeruginosa in Korea.

This study was performed to evaluate the impacts of vanA positivity of Enterococcus faecium exhibiting diverse susceptibility phenotypes to glycopeptides on clinical outcomes in patients with a bloodstream infection (BSI) through a prospective, multicenter, observational study. A total of 509 patients with E. faecium BSI from eight sentinel hospitals in South Korea during a 2-year period were enrolled in this study. Risk factors of the hosts and causative E. faecium isolates were assessed to determine associations with the 30-day mortality of E. faecium BSI patients via multivariable logistic regression analyses. The vanA gene was detected in 35.2% (179/509) of E. faecium isolates; 131 E. faecium isolates exhibited typical VanA phenotypes (group vanA-VanA), while the remaining 48 E. faecium isolates exhibited atypical phenotypes (group vanA-atypical), which included VanD (n = 43) and vancomycin-variable phenotypes (n = 5). A multivariable logistic regression indicated that vanA positivity of causative pathogens was independently associated with the increased 30-day mortality rate in the patients with E. faecium BSI; however, there was no significant difference in survival rates between the patients of the vanA-VanA and vanA-atypical groups (log rank test, P = 0.904). A high 30-day mortality rate was observed in patients with vanA-positive E. faecium BSIs, and vanA positivity of causative E. faecium isolates was an independent risk factor for early mortality irrespective of the susceptibility phenotypes to glycopeptides; thus, intensified antimicrobial stewardship is needed to improve the clinical outcomes of patients with vanA-positive E. faecium BSI.

This study was performed to evaluate the clinical impacts of putative risk factors in patients with Staphylococcus aureus bloodstream infections (BSIs) through a prospective, multicenter, observational study. All 567 patients with S. aureus BSIs that occurred during a 1-year period in six general hospitals were included in this study. Host- and pathogen-related variables were investigated to determine risk factors for the early mortality of patients with S. aureus BSIs. The all-cause mortality rate was 15.0% (85/567) during the 4-week follow-up period from the initial blood culture, and 76.5% (65/85) of the mortality cases occurred within the first 2 weeks. One-quarter (26.8%, 152/567) of the S. aureus blood isolates carried the tst-1 gene, and most (86.2%, 131/152) of them were identified to be clonal complex 5 agr type 2 methicillin-resistant S. aureus (MRSA) strains harboring staphylococcal cassette chromosome mec type II, belonging to the New York/Japan epidemic clone. A multivariable logistic regression showed that the tst-1 positivity of the causative S. aureus isolates was associated with an increased 2-week mortality rate both in patients with S. aureus BSIs (adjusted odds ratio [aOR], 1.62; 95% confidence interval [CI], 0.90 to 2.88) and in patients with MRSA BSIs (aOR, 2.61; 95% CI, 1.19 to 6.03). Two host-related factors, an increased Pitt bacteremia score and advanced age, as well as a pathogen-related factor, carriage of tst-1 by causative MRSA isolates, were risk factors for 2-week mortality in patients with BSIs. Careful management of patients with BSIs caused by the New York/Japan epidemic clone is needed to improve clinical outcomes.

At the end of 2015, a global action plan on antimicrobial resistance (AMR) was proposed by the World Health Organization, and the Global AMR Surveillance System (GLASS) was subsequently initiated. The Centers for Disease Control and Prevention of South Korea established a customized AMR surveillance system for South Korea, called Kor-GLASS, in early 2016. A pilot phase of Kor-GLASS was operated from May to December 2016 with six sentinel hospitals, and phase I of Kor-GLASS started in January 2017 with eight sentinel hospitals. Previous surveillance data for overestimated AMR due to duplicate isolation of drug-resistant pathogens were corrected and error-free AMR data were compared with those from other countries. One-half (53.2%, 377/708) of Staphylococcus aureus blood strains exhibited resistance to cefoxitin, indicating methicillin-resistant S. aureus. Resistance to ampicillin in Enterococcus faecalis blood strains was rare (0.6%, 1/175), while the resistance rate to penicillin was 26.3% (46/175). Resistance to vancomycin (34.0%, 98/288) and teicoplanin (18.8%, 98/288) was frequently observed in Enterococcus faecium strains. The resistance rate of Escherichia coli strains to cefotaxime was 32.4% (574/1772), and that of Klebsiella pneumoniae strains was 26.1% (181/693). The resistance rates of Pseudomonas aeruginosa strains to imipenem and meropenem were 19.5% (29/149) and 18.1% (27/149), respectively. And 92.1% (187/203) of Acinetobacter baumannii strains were resistant to both imipenem and meropenem. The high incidence of bacteremia caused by major AMR pathogens among hospitalized patients especially in intensive care units emphasized the importance of hospital infection control and the need to improve the crowded hospitalization system in South Korea. The isolation rate of the Salmonella spp. is decreasing, reflecting the current socio-economic status of South Korea. The proportions of bacterial species in the blood strains were similar to those in other Asian countries with similar lifestyles.

Background Carbapenem-resistant K. pneumoniae 2297, isolated from a patient treated with tigecycline for pneumonia, developed tigecycline resistance, in contrast to carbapenem-resistant isolate 1215, which was collected four months prior to the 2297 isolate. Mechanisms underlying tigecycline resistance were elucidated for the clinical isolates. Methods The tigecycline minimum inhibitory concentration (MIC) was determined using the broth microdilution method, with or without phenylalanine-arginine β-naphthylamide (PABN), and whole-genome sequencing was carried out by single-molecule real-time sequencing. The expression levels of the genes acrA, oqxA, ramA, rarA, and rpoB were determined by reverse-transcription quantitative PCR. Results Both isolates presented identical antibiograms, except for tigecycline, which showed an MIC of 0.5 mg/L in 1215 and 2 mg/L in 2297. The addition of PABN to tigecycline-resistant 2297 caused a four-fold decrease in the tigecycline MIC to 0.5 mg/L, although acrA expression (encoding the AcrAB efflux pump) was upregulated by 2.5 fold and ramA expression (encoding the pump activator RamA) was upregulated by 1.4 fold. We identified a 6,096-bp fragment insertion flanking direct TATAT repeats that disrupted the romA gene located upstream of ramA in the chromosome of K. pneumoniae 2297; the insertion led the ramA gene promoter replacement resulting in stronger activation of the gene. Conclusions The K. pneumoniae isolate developed tigecycline resistance during tigecycline treatment. It was related to the overexpression of the AcrAB resistance-nodulation-cell division efflux system due to promoter replacement.

Objectives The spread of carbapenemase-producing organisms has been continuously reported over the past decade. Rapid and accurate detection of carbapenemase production is essential for adequate infection control and appropriate antimicrobial treatment in the clinical setting. In this study, the performance of the newly developed PANA RealTyper™ CRE Kit (PANAGENE, Daejeon, South Korea) for the detection of six common carbapenemase genes (blaKPC, blaGES, blaIMP, blaNDM, blaVIM and blaOXA-48-like) was evaluated. Methods A total of 479 non-duplicate clinical isolates of Gram-negative bacilli, including 391 carbapenemase-producers and 88 non-producers, were tested. Conventional PCR and sequencing were performed as reference methods for performance evaluation of the PANA RealTyper™ CRE Kit. Results The PANA RealTyper™ CRE Kit showed a reliable performance for the detection of carbapenemase-producing organisms compared with the monoplex PCR system, with sensitivity and specificity values both >95%, with only a few discrepant results for six types of carbapenemase genes. Conclusion This kit is rapid and accurate for simultaneously detecting various carbapenemase genes in a single reaction and could contribute to early decisions for appropriate antimicrobial treatment in the clinical setting.

Administration of an efficient alginate lyase (AlgL) or AlgL mutant may be a promising therapeutic strategy for treatment of cystic fibrosis patients with Pseudomonas aeruginosa infections. Nevertheless, the catalytic activity of wild-type AlgL is not sufficiently high. It is highly desired to design and discover an AlgL mutant with significantly improved catalytic efficiency against alginate substrates. For the purpose of identifying an AlgL mutant with significantly improved catalytic activity, in this study, we first constructed and validated a structural model of AlgL interacting with substrate, providing a better understanding of the interactions between AlgL and its substrate. Based on the modeling insights, further enzyme redesign and experimental testing led to discovery of AlgL mutants, including the K197D/K321A mutant, with significantly improved catalytic activities against alginate and acetylated alginate in ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA) biofilms. Further anti-biofilm activity assays have confirmed that the K197D/K321A mutant with piperacillin/tazobactam (PT) is indeed effective in degrading the CRPA biofilms. Co-administration of the potent mutant AlgL and an antibiotic (such as a nebulizer) could be effective for therapeutic treatment of CRPA-infected patients with cystic fibrosis. This article is protected by copyright. All rights reserved.

OBJECTIVES The aac(6')-Ih gene encoding aminoglycoside 6'-N-acetyltransferase type I subtype h [AAC(6')-Ih] is plasmid-borne in Acinetobacter baumannii where it confers high-level amikacin resistance, but its origin remains unknown. We searched for the gene in the genomes of a collection of 133 Acinetobacter spp. and studied its species specificity, expression and dissemination. METHODS Gene copy number was determined by quantitative PCR, expression by quantitative RT-PCR, MIC by microdilution and transfer by plasmid mobilization. RESULTS The aac(6')-Ih gene was present in the chromosome of the two Acinetobacter gyllenbergii of the collection and was detected in all seven A. gyllenbergii clinical isolates. They had indistinguishable flanking regions indicating that the gene was intrinsic to this species. A. baumannii PIS Aba23 promoters were provided by insertion of ISAba23, which disrupted the Pnative promoter in A. gyllenbergii. Both types of promoters were similarly potent in Escherichia coli and A. baumannii. Aminoglycoside MICs for A. baumannii harbouring pIP1858 were higher than for A. gyllenbergii due to gene dosage. The non-self-transferable plasmid could be mobilized to other A. baumannii cells by the broad host range plasmid RP4. CONCLUSIONS We have found the origin of aac(6')-Ih in A. gyllenbergii, a species isolated, although rarely, in humans, and documented that dissemination of this gene is restricted to the Acinetobacter genus.

Objectives To investigate the risk factors of patients with Klebsiella pneumoniae (KP) bloodstream infection (BSI) with a focus on antimicrobial resistance and virulence factors. Methods All KP BSI patients (n = 579) from six general hospitals during a 1 year period were included in this study. The risk factors of hosts and causative KP isolates were assessed to determine associations with the 30 day mortality of KP BSI patients by multivariate Cox hazards modelling. Results The 30 day mortality rate of KP BSI patients was 16.9% (98/579). Among the host-associated factors, increased SOFA score and leucopenia status exhibited strong associations with increased 30 day mortality. Among the pathogenic factors, carriage of the pks gene cluster (adjusted HR 1.80; 95% CI 1.16-2.79) was a risk factor, especially when accompanied by MDR. In this regard, KP isolates of the wzi50 capsular type (n = 22) frequently harboured pks (63.6%, 14/22) and ybtA (68.2%, n = 15) and mostly exhibited MDR (63.6%, n = 14), resulting in increased 30 day mortality. In contrast, hypermucoviscous KP isolates showed an inverse association with 30 day mortality (adjusted HR 0.55; 95% CI 0.33-0.90). Conclusions Despite the reported virulence of hypermucoviscous KP strains, they were associated with good prognoses in KP BSI patients. Importantly, carriage of the pks gene cluster, which is responsible for the synthesis of colibactin, was a relevant marker of early mortality.

A total of 281 nonduplicated Staphylococcus aureus blood isolates were collected from January to May 2017 from eight hospitals in South Korea to investigate the epidemiological traits of ceftaroline resistance in methicillin-resistant S. aureus (MRSA). Cefoxitin-disk diffusion tests and the mecA gene PCR revealed that 56.6% (159/281) of the S. aureus isolates were MRSA, and most belonged to ST5 (50.3%, 80/281) and ST72 (41.5%, 66/281). Of the MRSA isolates, 44.0% (70/159) were nonsusceptible to ceftaroline (MIC ≥ 2 mg/liter), whereas all of the methicillin-susceptible S. aureus isolates were susceptible to the drug. Eight amino acid substitutions in penicillin-binding protein 2a (PBP2a), including four (L357I, E447K, I563T, and S649A) in the penicillin-binding domain (PBD) and four (N104K, V117I, N146K, and A228V) in the non-PBD (nPBD) of PBP2a, were associated with ceftaroline resistance. The accumulation of substitutions in PBP2a resulted in the elevation of ceftaroline MICs: one substitution at 1 to 2 mg/liter, two or three substitutions at 2 to 4 mg/liter, and five substitutions at 4 or 16 mg/liter. Ceftaroline resistance in MRSA might be the result of clone-specific PBP2a polymorphism, along with substitutions both in PBD and nPBD, and the elevated ceftaroline MICs were associated with the substitution sites and accumulation of substitutions.

Pseudomonas putida group are Gram-negative bacilli with polar flagellation, which are ubiquitous in the environment, although they are rarely involved in human infections. The aim of this study was to identify the dissemination of VIM-2-producing P. putida group in clinical isolates from a hospital in Korea. Thirteen strains were collected from 2014 to 2015 for the study. The isolates were recovered from urine cultures of both inpatients and outpatients at the hospital. Minimum inhibitory concentrations of antibiotics were determined by Etest. Carbapenemase genes were amplified by polymerase chain reaction and sequenced. Pulsed-field gel electrophoresis was performed for strain typing. Whole-genome sequencing was carried out randomly for two strains chosen from each year of the study to analyze the plasmid structure carrying the bla

Zn x Cd1−x S (x = 0.15, 0.44, 0.62, 0.80, and 0.95) thin films of about 340 nm in thickness were deposited on indium-tin-oxide (ITO)-coated glass substrates by using thermal evaporation of high-purity ZnS and CdS mixed tablets in high vacuum. X-ray diffraction spectra showed that the Zn x Cd1−x S thin films were preferentially grown along the (111) orientation. The Zn x Cd1−x S crystal structure was a mixture structure of the ZnS and the CdS cubic zincblende structures with lattice constants a = 5.670 A to a = 5.734 A for CdS and a = 5.437 A for ZnS. The (αhν)2 vs. hν plots for the Zn x Cd1−x S thin films showed that all samples had direct transition band gaps. The energy band gaps of the Zn x Cd1−x S thin films increased monotonically from 2.45 eV for x = 0.15 to 3.37 eV for x = 0.95. The dynamical behavior of the charge carriers in the Zn x Cd1−x S thin films was investigated by using the photoinduced discharge characteristics (PIDC) technique.

Resistance to β-lactams in Acinetobacter baumannii involves various mechanisms. To decipher them, whole genome sequencing (WGS) and real-time quantitative polymerase chain reaction (RT-qPCR) were complemented by mass spectrometry (MS) in selected reaction monitoring mode (SRM) in 39 clinical isolates. The targeted label-free proteomic approach enabled, in one hour and using a single method, the quantitative detection of 16 proteins associated with antibiotic resistance: eight acquired β-lactamases (i.e. GES, NDM-1, OXA-23, OXA-24, OXA-58, PER, TEM-1, and VEB), two resident β-lactamases (i.e. ADC and OXA-51-like) and six components of the two major efflux systems (i.e. AdeABC and AdeIJK). Results were normalized using “bacterial quantotypic peptides,” i.e. peptide markers of the bacterial quantity, to obtain precise protein quantitation (on average 8.93% coefficient of variation for three biological replicates). This allowed to correlate the levels of resistance to β-lactam with those of the production of acquired as well as resident β-lactamases or of efflux systems. SRM detected enhanced ADC or OXA-51-like production and absence or increased efflux pump production. Precise protein quantitation was particularly valuable to detect resistance mechanisms mediated by regulated genes or by overexpression of chromosomal genes. Combination of WGS and MS, two orthogonal and complementary techniques, allows thereby interpretation of the resistance phenotypes at the molecular level.

Objectives High rates of carbapenem resistance in the human pathogen Acinetobacter baumannii threaten public health and need to be scrutinized. Methods A total of 356 A. baumannii and 50 non-baumannii Acinetobacter spp. (NBA) strains collected in 2013 throughout South Korea were studied. The type of blaOXA-23 transposon was determined by PCR mapping and molecular epidemiology was assessed by MLST. Twelve representative strains and two comparative A. baumannii were entirely sequenced by single-molecule real-time sequencing. Results The carbapenem resistance rate was 88% in A. baumannii, mainly due to blaOXA-23, with five exceptional cases associated with ISAba1-blaOXA-51-like. The blaOXA-23 gene in A. baumannii was carried either by Tn2006 (44%) or Tn2009 (54%), with a few exceptions carried by Tn2008 (1.6%). Of the NBA strains, 14% were resistant to carbapenems, two with blaOXA-58 and five with blaOXA-23 associated with Tn2006. The Tn2006-possessing strains belonged to various STs, whereas Tn2008- and Tn2009-possessing strains were limited to ST208 and ST191, respectively. The three transposons were often multiplied in the chromosome, and the gene copy number and the carbapenem MICs presented linear relationships either very strongly for Tn2008 or moderately for Tn2006 and Tn2009. Conclusions The dissemination of Tn2006 was facilitated by its capability for intercellular transfer and that of Tn2009 was attributable to successful dissemination of the ST191 bacterial host carrying the transposon. Tn2008 was infrequent because of its insufficient ability to undergo intercellular transfer and the scarce bacterial host A. baumannii ST208. Gene amplification is an adaptive mechanism for bacteria that encounter antimicrobial drugs.

Pullorum disease and fowl typhoid are septicemic avian diseases transmitting through egg by trans-ovarian infections. A series of tests has been performing in breeding flocks of chickens and test plans for proper inspection have been modified by government veterinary institute to control of such diseases. To improve inspection plans, different test methods were compared using fowl typhoid positive samples from a poultry farm located in Jeonbuk state in 2012. Based on first inspection, 11 samples among total 200 samples were positive by rapid slide agglutination (RSA) test and 7 samples among RSA positive samples were finally diagnosed as Salmonella Gallinarum infection by ELISA, bacterial isolation, PCR, and histopathologic examination. In the second inspection, 20 samples among total 100 samples were positive by RSA test. Among RSA positive ones, 19 samples were positive by ELISA, S. Gallinarm were successfully isolated in 3 samples, and 16 samples were positive by PCR in the cecal tonsils where were not successful for bacterial isolation. Based on histopathologic examination, severe inflammation in the 13 cecal tonsils and infiltration of lymphocytes and heterophils in the 11 livers were observed. Therefore, we suggest that bacterial isolation, PCR, and histopathologic examination methods in the third inspection need to be further used in various tissues for correct diagnosis and for final eradication of pullorum disease and fowl typhoid in breeding flocks of chickens.