Hye Hyun Cho

Genetic Basis of Multidrug-resistant Acinetobacter baumannii Clinical Isolates from Three University Hospitals in Chungcheong Province, Korea

BACKGROUND The emergence of multidrug-resistant (MDR) Acinetobacter baumannii as an important opportunistic pathogen has given rise to significant therapeutic challenges in the treatment of nosocomial infections. In the present study, we assess the antibiotic resistance mechanisms of MDR A. baumannii strains by estimating the prevalence of antibiotic resistance determinants, including integrons, β-lactamases, str genes, and gyrA and parC mutations. METHODS Thirty-five MDR A. baumannii clinical isolates were collected from 3 Korean university hospitals over a 2-yr period. A. baumannii was confirmed by rpoB gene analysis. For each isolate, the minimal inhibitory concentrations (MICs) of 9 antibiotics were determined by the agar dilution method. PCR and DNA sequencing were used to identify the genes that potentially contribute to each resistance phenotype. RESULTS Of the 35 MDR A. baumannii isolates examined, 7 antibiotic resistance gene determinants were detected. These resistance gene determinants included the gene bla(OXA-23), with an upstream element ISAba1 to promote increased gene expression and subsequent resistance to carbapenems, in 8 isolates (22.9%); aacA4, located within class 1 integrons, in 7 isolates (20.0%); and fluoroquinolone resistance conferred by gyrA and parC sense mutations in 31 isolates. CONCLUSIONS Of the 35 MDR A. baumannii isolates, 26 (74.3%) from both outbreak and sporadic cases possessed at least 4 of the 7 antibiotic resistance gene determinants that give rise to the MDR phenotype. The co-occurrence of several resistance determinants may present a significant threat.

Correlation between virulence genotype and fluoroquinolone resistance in carbapenem-resistant Pseudomonas aeruginosa.

Background Pseudomonas aeruginosa is a clinically important pathogen that causes opportunistic infections and nosocomial outbreaks. Recently, the type III secretion system (TTSS) has been shown to play an important role in the virulence of P. aeruginosa. ExoU, in particular, has the greatest impact on disease severity. We examined the relationship among the TTSS effector genotype (exoS and exoU), fluoroquinolone resistance, and target site mutations in 66 carbapenem-resistant P. aeruginosa strains. Methods Sixty-six carbapenem-resistant P. aeruginosa strains were collected from patients in a university hospital in Daejeon, Korea, from January 2008 to May 2012. Minimum inhibitory concentrations (MICs) of fluoroquinolones (ciprofloxacin and levofloxacin) were determined by using the agar dilution method. We used PCR and sequencing to determine the TTSS effector genotype and quinolone resistance-determining regions (QRDRs) of the respective target genes gyrA, gyrB, parC, and parE. Results A higher proportion of exoU+ strains were fluoroquinolone-resistant than exoS+ strains (93.2%, 41/44 vs. 45.0%, 9/20; P≤0.0001). Additionally, exoU+ strains were more likely to carry combined mutations than exoS+ strains (97.6%, 40/41 vs. 70%, 7/10; P=0.021), and MIC increased as the number of active mutations increased. Conclusions The recent overuse of fluoroquinolone has led to both increased resistance and enhanced virulence of carbapenem-resistant P. aeruginosa. These data indicate a specific relationship among exoU genotype, fluoroquinolone resistance, and resistance-conferring mutations.

Expression of Sme Efflux Pumps and Multilocus Sequence Typing in Clinical Isolates of Stenotrophomonas maltophilia

Background Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen, which causes infections that are often difficult to manage because of the inherent resistance of the pathogen to a variety of antimicrobial agents. In this study, we analyzed the expressions of smeABC and smeDEF and their correlation with antimicrobial susceptibility. We also evaluated the genetic relatedness and epidemiological links among 33 isolates of S. maltophilia. Methods In total, 33 S. maltophilia strains were isolated from patients in a tertiary hospital in Daejeon. Minimum inhibitory concentrations (MICs) of 11 antimicrobial agents were determined by using agar dilution method and E-test (BioMérieux, France). Real-time PCR analysis was performed to evaluate the expression of the Sme efflux systems in the S. maltophilia isolates. Additionally, an epidemiological investigation was performed using multilocus sequence typing (MLST) assays. Results The findings of susceptibility testing showed that the majority of the S. maltophilia isolates were resistant to β-lactams and aminoglycosides. Twenty-one clinical isolates overexpressed smeABC and showed high resistance to ciprofloxacin. Moreover, a high degree of genetic diversity was observed among the S. maltophilia isolates; 3 sequence types (STs) and 23 allelic profiles were observed. Conclusions The smeABC efflux pump was associated with multidrug resistance in clinical isolates of S. maltophilia. In particular, smeABC efflux pumps appear to perform an important role in ciprofloxacin resistance of S. maltophilia. The MLST scheme for S. maltophilia represents a discriminatory typing method with stable markers and is appropriate for studying population structures.

Antimicrobial Resistance and Multilocus Sequence Typing of Vancomycin-Resistant Enterococcus faecium Isolated from the Chungcheong Area

Background: Enterococcus faecium has emerged as an important nosocomial pathogen worldwide, and this trend has been associated with the dissemination of a genetic lineage designated clonal complex 17 (CC17). In the present study, characterization of the glycopeptide resistance mechanism, genetic relatedness, and pathogenicity in isolates of vancomycin-resistant E. faecium in the Chungcheong area were investigated. Methods: A total of 37 consecutive, non-duplicate, vancomycin-resistant E. faecium were isolated at three university hospitals in the Chungcheong area. The mechanism of glycopeptide resistance and pathogenicity factors were studied using PCR, and the genetic relatedness was determined via multilocus sequence type and esp repeat profile analysis. Additionally, the quinolone resistance-determining regions of parC and gyrA were sequenced to identify mutations involved in ciprofloxacin resistance. Results: Two genotypes of VRE were confirmed: VanAphenotype vanA genotype VRE (25 isolates) and VanB-phenotype vanA genotype VRE (12 isolates). MLST analysis revealed five sequence types. A significant result was that ST414 and CNS4 (4-11-1-1-1-1) were considered as belonging to CC17. The esp and hyl genes were found in 100% and 86.4% of the isolates, respectively. A total of 37 isolates showed genetic mutations in parC and gyrA. Conclusion: All isolated strains in the present study belonged to one of the CC17 genotypes including ST414 and CNS4 (4-1-1-1-1-1-1), which were not previously detected in Korea. The combination of MLST and the esp gene repeat profiles can be useful for genetic characterization of VREF isolates with regard to the evolutionary process and epidemiology of the clones. (Korean J Clin Microbiol 2011;14:6066)

Distribution of Pseudomonas-Derived Cephalosporinase and Metallo-β-Lactamases in Carbapenem-Resistant Pseudomonas aeruginosa Isolates from Korea.

The emergence of carbapenem resistance among Pseudomonas aeruginosa is an increasing problem in many parts of the world. In particular, metallo-β-lactamases (MBLs) and AmpC β- lactamases are responsible for high-level resistance to carbapenem and cephalosporin. We studied the diversity and frequency of β-lactamases and characterized chromosomal AmpC β- lactamase from carbapenem-resistant P. aeruginosa isolates. Sixty-one carbapenem-resistant P. aeruginosa isolates were collected from patients in a tertiary hospital in Daejeon, Korea, from January 2011 to June 2014. Minimum inhibitory concentrations (MICs) of four antimicrobial agents were determined using the agar-dilution method. Polymerase chain reaction and sequencing were used to identify the various β-lactamase genes, class 1 integrons, and chromosomally encoded and plasmid-mediated ampC genes. In addition, the epidemiological relationship was investigated by multilocus sequence typing. Among 61 carbapenem-resistant P. aeruginosa isolates, 25 isolates (41.0%) were MBL producers. Additionally, 30 isolates producing PDC (Pseudomonas-derived cephalosporinase)-2 were highly resistant to ceftazidime (MIC50 = 256 μg/ml) and cefepime (MIC50 = 256 μg/ml). Of all the PDC variants, 25 isolates harboring MBL genes showed high levels of cephalosporin and carbapenem resistance, whereas 36 isolates that did not harbor MBL genes revealed relatively low-level resistance (ceftazidime, p < 0.001; cefepime, p < 0.001; imipenem, p = 0.003; meropenem, p < 0.001). The coexistence of MBLs and AmpC β-lactamases suggests that these may be important contributing factors for cephalosporin and carbapenem resistance. Therefore, efficient detection and intervention to control drug resistance are necessary to prevent the emergence of P. aeruginosa possessing this combination of β-lactamases.

AbaR7, a genomic resistance island found in multidrug-resistant Acinetobacter baumannii isolates in Daejeon, Korea.

Background Acinetobacter baumannii resistance islands (AbaRs) have been recently recognized as mobile genetic elements that harbor multiple resistance determinants and are associated with multidrug resistance (MDR). In the present study, we aimed to determine the AbaRs conferring multiple antimicrobial resistance and their clonal relatedness to MDR A. baumannii clinical isolates obtained from a university hospital in Daejeon, Korea. Methods This study included 29 MDR A. baumannii strains isolated in Daejeon, Korea. The minimal inhibitory concentrations (MICs) were determined by Etest. A. baumannii isolates were characterized using the 2 multiplex PCR assays and multilocus sequence typing (MLST) scheme. To detect and characterize AbaRs, PCR and PCR mapping experiments were performed. Results Twenty-seven of the 29 isolates belonged to the European (EU) clone II lineage and contained 5 sequence types (STs) (75, 92, 137, 138, and 357). In this study, ST357 was confirmed for the first time in Korea. Only 2 of the 29 isolates belonged to the EU clone I lineage, and were confirmed as ST109. These 2 isolates harbored the 22-kb AbaR7 aacC1-orfP-orfQ-aadA1 gene cassette array. In contrast, AbaR was not found in EU clone II isolates. Conclusions This is the first study that attempted to determine the AbaRs in MDR A. baumannii isolates in Korea. We found 2 EU clone I isolates (ST109) that harbored AbaR7.

Characteristics of the Molecular Epidemiology of CTX-M-Producing Escherichia coli Isolated from a Tertiary Hospital in Daejeon, Korea.

The aims of this study were to characterize the molecular epidemiological profiles of CTX-M-producing uropathogenic Escherichia coli isolates from a tertiary hospital in Daejeon, Korea, and to investigate the genetic diversity and compare the prevalence of sequence types (STs) in different areas. Extended spectrum β-lactamase-producing E. coli strains isolated from urine were analyzed for CTX-M, integrons, and insertion sequence common regions (ISCRs) by PCR and sequencing. Multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), phylogenetic analysis, and rep-PCR were also used for molecular typing of the isolates. Of 80 CTX-M producers, 31 and 46 expressed CTX-M-15 and CTX-M-14, respectively. MLST analysis indicated that the most prevalent ST was ST131 (n = 34, 42.5%), followed by ST38 (n = 22, 27.5%), ST405 (n = 8, 10.0%), and ST69 (n = 6, 7.5%). Most CTX-M producers harbored class 1 integrons. ST131 strains belonged to phylogenetic group B2 and showed identical rep-PCR patterns, whereas ST69, ST38, and ST405 strains belonged to phylogenetic group D; the ST38 and ST405 strains displayed the same rep-PCR pattern, respectively. ST131 and ST38 isolates showed 21 and 19 distinct types, respectively, by PFGE. In Daejeon, D-ST38 CTX-M-14 producers were relatively more prevalent than in other countries and Korean cities. Our results indicate that CTX-M-producing E. coli isolates belonged mostly to ST131 or ST38 and were more related to hospital-onset than to community-onset infections and that the blaCTX-M gene may vary according to the ST.

Nosocomial infection by sequence type 357 multidrug-resistant Acinetobacter baumannii isolates in a neonatal intensive care unit in Daejeon, Korea.

Acinetobacter baumannii is an important microorganism responsible for a number of nosocomial outbreaks, in particular, in intensive care units (ICUs). We investigated a nosocomial infection caused by multidrug-resistant (MDR) A. baumannii in a neonatal intensive care unit (NICU) in Korea. A. baumannii isolates were characterized using Etest (AB Biodisk, Sweden), two multiplex PCR assays, and multilocus sequence typing (MLST) scheme. PCR and PCR mapping experiments were performed for detecting and characterizing the determinants of antimicrobial resistance. Eight strains isolated from an NICU belonged to European (EU) clone II and revealed only one sequence type (ST), namely, ST357. All the isolates were susceptible to imipenem but were resistant to amikacin, gentamicin, ceftazidime, cefepime, and ciprofloxacin. To the best of our knowledge, this is the first report of a nosocomial infection in an NICU in Korea caused by ST357 MDR/carbapenem-susceptible A. baumannii strains. This result demonstrates that nosocomial outbreaks of MDR/carbapenem-susceptible strains as well as MDR/carbapenem-resistant isolates may occur in NICUs.

Dissemination of an AbaR-type Resistance Island in Multidrug-resistant Acinetobacter baumannii Global Clone 2 in Daejeon of Korea

Background: Acinetobacter baumannii resistance islands (AbaRs) are transposons that have the role of important vehicles for the acquisition of antimicrobial resistance genes, and are associated with multidrug resistance (MDR). In this study, we aimed to determine the AbaRs in MDR A. baumannii global clone 2 (GC2) clinical isolates obtained from a university hospital in Daejeon, Korea. Methods: This study included 17 MDR A. baumannii strains isolated in Daejeon, Korea. The minimal inhibitory concentrations (MICs) were determined by Etest. A. baumannii isolates were characterized using 2 multiplex PCR assays and a multilocus sequence typing (MLST) scheme. To detect and characterize AbaRs, PCR and PCR mapping experiments were performed. Results: All 17 MDR A. baumannii isolates tested in this study belonged to GC2 and contained 5 sequence types (STs): 75, 92, 137, 138, and 357. Tn6166 that contains antimicrobial resistance genes and is also known as AbaR4a was found in all 17 GC2 strains. This is the first report of Tn6166 in MDR A. baumannii GC2 isolates in Korea. In contrast, AbaR4 was not found in the GC2 isolates. Conclusion: Tn6166 has been disseminated among MDR A. baumannii GC2 isolates in Korea. Further investigation is needed to recover the various types of AbaRs in MDR A. baumannii GC2 isolates in Korea are responsible for the multiple antimicrobial resistance mechanisms. (Ann Clin Microbiol 2013;16: 75-80)

Chromosomal Mutations in oprD, gyrA, and parC in Carbapenem Resistant Pseudomonas aeruginosa

Background: Outbreaks of carbapenem resistant P. aeruginosa give rise to significant therapeutic challenges for treating nosocomial infections. In this study, we analyzed carbapenem resistance mechanisms in carbapenem resistant and clonally different P. aeruginosa strains. We analyzed chromosomal alterations in the genes of OprD and efflux system regulatory proteins (MexR, NalC, NalD, MexT, and MexZ). We also investigated chromosomal alterations in the quinolone resistance-determining region (QRDR) for quinolone resistance mechanisms. Methods: Twenty-one clonally different P. aeruginosa strains were isolated by repetitive extragenic palindromic sequence-based PCR (rep-PCR). PCR and DNA sequencing were conducted for the detection of β-lactamase genes and chromosomal alterations of efflux pump regulatory genes, oprD, and QRDR in gyrA, gyrB, parC, and parE. Results: Only one (P28) of the 21 strains harbored blaVIM-2. Two isolates had mutations in nalD or mexZ that were associated with efflux pump overexpression. Chromosomal alterations causing loss of OprD were found in 4 out of 21 carbapenem resistant P. aeruginosa strains. Nine of 10 imipenem and ciprofloxacin resistant strains had alterations in gyrA and/or parC. Conclusion: Carbapenem resistance in P. aeruginosa was mediated by several mechanisms, including loss of the OprD, overexpression of efflux systems, and production of carbapenemase. Resistance to quinolone is frequently caused by point mutations in gyrA and/or parC. (Korean J Clin Microbiol 2011;14: 131-137)