Jun Sung Hong

Characteristics of Metallo-β-Lactamase-Producing Pseudomonas aeruginosa in Korea

Background The aim of this study was to investigate the molecular epidemiological characteristics of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa clinical isolates in Korea. Materials and Methods Three hundred and twenty nine P. aeruginosa clinical isolates were collected from 23 general hospitals in Korea from March to June 2014. Species were identified by matrix-assited laser desorption/ionization-time of flight and 16S rRNA sequencing. Antimicrobial susceptibility was determined by disk diffusion methods. Further, minimum inhibitory concentrations of carbapenems were determined by Etest. Polymerase chain reaction and sequencing were performed to identify genes encoding MBLs. Multi-locus sequence typing and pulsed-field gel electrophoresis were performed to determine epidemiological characteristics of MBL-producing P. aeruginosa isolates. Results Of the 329 isolates, 229 (69.6%) were susceptible to the carbapenems tested, including imipenem and meropenem; while 100 (30.4%) were non-susceptible to more than one of the carbapenems. Genes encoding imipenemase-6 (IMP-6) and Verona imipenemase-2 (VIM-2) MBLs were identified in 21 (6.4%) isolates (n = 17 and 4, respectively). All MBL-producing isolates showed multi-drug resistant phenotype, and a majority (n = 19) of the isolates were identified as sequence type 235 (ST235). The remaining isolates (n = 2) were identified as ST309 and ST463. Conclusion P. aeruginosa ST235 might play an important role in dissemination of MBL genes in Korea.

Clonal Dissemination of Pseudomonas aeruginosa Sequence Type 235 Isolates Carrying blaIMP-6 and Emergence of blaGES-24 and blaIMP-10 on Novel Genomic Islands PAGI-15 and -16 in South Korea

ABSTRACT 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 blaIMP-6, blaIMP-10, and blaGES-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 blaIMP-6, blaIMP-10, and blaGES-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.

Prevalence and Molecular Characteristics of Carbapenemase-Producing Enterobacteriaceae From Five Hospitals in Korea

Background The emergence of carbapenemase-producing Enterobacteriaceae (CPE) represents a major clinical problem because these bacteria are resistant to most antibiotics. CPE remain relatively uncommon in Korea. We report the prevalence, clinical characteristics, and molecular epidemiology of CPE isolates collected from five university hospitals in Korea. Methods Between January and December 2015, 393 non-duplicated isolates that were nonsusceptible to ertapenem were analyzed. Production of carbapenemase, extended-spectrum β-lactamase, and AmpC β-lactamase was determined by genotypic tests. Antimicrobial susceptibility profiles were determined by using an Etest. Clonality of Klebsiella pneumoniae carbapenemase (KPC)-2-producing and oxacillinase (OXA)-232-producing Klebsiella pneumoniae isolates was determined by pulsed-field gel electrophoresis (PFGE). Results Of the 393 isolates tested, 79 (20.1%) were CPE. Of these 79 isolates, 47 (59.5%) harbored the blaOXA-232 gene while the remaining isolates carried genes blaKPC-2 (n=27), blaIMP-1 (n=4), and blaNDM-1 (n=1). Among the 24 KPC-2 K. pneumoniae isolates from hospital B, 100% were resistant to carbapenems, 8% to colistin, and 0% to tigecycline. Among the 45 OXA-232 K. pneumoniae at hospital C, 95% were resistant to ertapenem, 68% to imipenem, 95% to meropenem, 10% to colistin, and 24% to tigecycline. PFGE analysis revealed a unique pattern for KPC-2 K. pneumoniae and identified 30 isolates belonging to the dominant pulsotypes (PT)1 and PT2 among 41 OXA-232 K. pneumoniae isolates. Conclusions CPE strains are present in Korea, with the majority of K. pneumoniae isolates producing OXA-232 and KPC-2. The prevalence and predominant genotypes of CPE show hospital-specific differences.

First Case Report of Human Infection With Ochrobactrum tritici Causing Bacteremia and Cholecystitis.

Dear Editor, Herein, we report the first case of human infection with Ochrobactrum tritici, primarily isolated from environmental sources and not previously known to infect humans. A 70-yr-old male patient was admitted to a tertiary-care hospital for jaundice. He had a medical history of cholangiocellular carcinoma (CCC). On the first day of hospitalization, he presented a 38.1℃ body temperature, 98/81 mmHg blood pressure, 87 beats per minute heart rate, and abdominal distention. Laboratory findings indicated a white blood cell count of 5.63×109/L with a differential of 71% neutrophils, a hemoglobin level of 10 g/dL, a platelet count of 25×109/L, and a C-reactive protein level of 48.5 mg/L (reference range, 0.1–6.0 mg/L). Two sets of blood cultures were performed at the time of high fever on admission. Among these four cultures, only one aerobic culture was positive for gram-negative short rods after 4 days of incubation. Intravenous antibiotics with metronidazole (500 mg every 8 hr) and cefoperazone/ sulbactam (2,000 mg every 12 hr) were started on the first day of hospitalization. His condition improved after antibiotic therapy and he was discharged from the hospital on day five. The isolate (named GNOT01) was identified as Ochrobactrum anthropi by matrix-assisted laser desorption ionization–time of flight mass spectrometer (MALDI-TOF MS; MicroFlex LT, Bruker Daltonics, Bremen, Germany). Two weeks later, the patient presented with exacerbated jaundice and abdominal pain. Physical examination revealed abdominal distention and ascites. Laboratory analysis indicated a leukocyte count of 6.5×109/L with a differential of 71% neutrophils, a platelet count of 83×109/L, a C-reactive protein level of 77.4 mg/L, and a total bilirubin of 6.7 mg/dL (reference range, 0.3–1.8 mg/dL). He received intravenous cefoperazone/sulbactam (2,000 mg every 12 hr) from the first day of hospitalization. Culture of the bile specimen collected during percutaneous transhepatic biliary drainage procedure yielded gram-negative rods (isolate GNOT02) and gram-positive cocci, identified as Ochrobactrum species and Enterococcus faecalis, respectively, by MALDI-TOF MS. The patient fully recovered from cholecystitis without any evidence of infection and was discharged on day 28 without specific complications. The clinical isolates, GNOT01 and GNOT02, were biochemically identified as O. anthropi with >99% probability. However, PCR and sequencing indicated that the 16S rRNA partial sequences of both isolates were 100% identical to those of strain CCUG 47104T, the O. tritici type strain. Scanning electron microscopy (SEM) images of the isolates revealed that they had a single polar flagellum, which was not observed in the type strain CCUG 47104T (Fig. 1). On bacterial motility test using motility indole ornithine (MIO) agar, GNOT01 and GNOT02 showed upper stab lines that diffused out just beneath the surface of the MIO agar creating an umbrella-shape, which suggests that they are strictly aerobic and motile. No finding related to motility was obtained with CCUG 47104T. Fig. 1 Scanning electron micrographs of Ochrobactrum tritici clinical isolate GNOT01 (A) and the type strain CCUG 47104T (B). GNOT01 isolate presented a single polar flagellum, while CCUG 47104T strain did not (Magnification, ×20,000). Pulsed-field gel electrophoresis (PFGE) presented identical XbaI macro-restriction banding patterns between the two isolates, which were different from that of CCUG 47104T (similarity, 84.2%) indicating that the bacteremia of the patient originated from cholecystitis. Antimicrobial susceptibility testing showed that CCUG 47104T was susceptible to ticarcillin, ceftriaxone, and cefepime, while GNOT01 and GNOT02 were resistant to these antimicrobials. Extended-spectrum β-lactamase (ESBL) genes and plasmid-encoded AmpC β-lactamase genes were not detected in GNOT01 and GNOT02 isolates by double disk synergy test and PCR, suggesting that they acquired resistance to β-lactams not by production of plasmid-mediated ESBLs or AmpC β-lactamases, but by overexpression of species-specific intrinsic AmpC β-lactamases. To date, only four species, O. anthropi, O. intermedium, O. haematophilum, and O. pseudogrignonense, have been reported to cause human infections [1,2,3,4]. Among these, O. anthropi is the most frequently reported human pathogen, especially in immunocompromised patients. Phylogenetic analysis of 16S rRNA gene sequences revealed a high similarity between O. tritici and O. anthropi with 98% identity [5]. However, O. tritici strains exhibit low DNA-DNA re-association values (< 60%) with O. anthropi probes and showed different phenotypic characteristics. These genotypic and phenotypic differences resulted in the classification of O. tritici as a distinct species. In summary, we reported the first case of human infection with an O. tritici motile strain isolated from the blood and bile. O. tritici should be considered as a potential pathogen that can cause bloodstream and biliary tract infections in humans. In addition, both commercial bacterial identification kits and MALDI-TOF MS misidentified O. tritici clinical isolates as O. anthropic. Thus, 16S rRNA sequencing should be performed when a clinical isolate is identified as O. anthropi by routine bacterial identification processes.

Differences in Colistin-resistant Acinetobacter baumannii Clinical Isolates Between Patients With and Without Prior Colistin Treatment

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.

Detection of mcr-1 Plasmids in Enterobacteriaceae Isolates From Human Specimens: Comparison With Those in Escherichia coli Isolates From Livestock in Korea

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.

Emergence of multidrug-resistant Providencia rettgeri isolates co-producing NDM-1 carbapenemase and PER-1 extended-spectrum β-lactamase causing a first outbreak in Korea

BackgroundNosocomial outbreak due to carbapenem-resistant Enterobacteriaceae has become serious challenge to patient treatment and infection control. We describe an outbreak due to a multidrug-resistant Providencia rettgeri from January 2016 to January 2017 at a University Hospital in Seoul, Korea.MethodsA total of eight non-duplicate P. rettgeri isolates were discovered from urine samples from eight patients having a urinary catheter and admitted in a surgical intensive care unit. The β-lactamase genes were identified using polymerase chain reaction and direct sequencing, and strain typing was done with pulsed-field gel electrophoresis (PFGE).ResultsAll isolates showed high-level resistance to extended-spectrum cephalosporins, aztreonam, meropenem, ertapenem, ciprofloxacin, and amikacin. They harbored the blaNDM-1 carbapenemase and the blaPER-1 type extended-spectrum β-lactamases genes. PFGE revealed that all isolates from eight patients were closely related strains.ConclusionsThe 13-month outbreak ended following reinforcement of infection control measures, including contact isolation precautions and environmental disinfection. This is the first report of an outbreak of a P. rettgeri clinical isolates co-producing NDM-1 and PER-1 β-lactamase.

Massilia varians Isolated from a Clinical Specimen

We report a case of Massilia varians isolated from a deep finger wound following orthopedic surgery on an immunocompetent patient. The bacterium was identified by 16S rDNA sequence analysis. This is the first case of M. varians isolated from a clinical specimen since the first report in 2008.