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< or =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.

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

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.

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.

The Genetic Characteristics of Multidrug-resistant Acinetobacter baumannii Coproducing 16S rRNA Methylase armA and Carbapenemase OXA-23

Acinetobacter baumannii is a gram-negative organism reported worldwide as a cause of health-care associated infections. Due to its increasing drug resistance, several studies on coproduction of armA and carbapenemase in South Korea and other parts of the world were reported, which can pose significant therapeutic threat. The aim of this study was to investigate genetic characteristics of multidrug-resistant A. baumannii coproducing armA and carbapenemase and its epidemiological relatedness. Forty-five multidrug resistant (MDR) A. baumannii clinical isolates were collected. Antimicrobial susceptibility was determined by agar dilution, Etest and VITEK 2 system. The presence of 16S rRNA methylase and carbapenemase were analyzed by polymerase chain reaction (PCR) and sequencing. Repetitive element palindromic (REP)-PCR was also performed for epidemiologic investigation. All of A. baumannii isolates harbored blaOXA-51 -like gene and 10 isolates showed an upstream ISAba1. 36 isolates (80%) showed amplification of OXA-23, all of which except one had an upstream ISAba1. 16S rRNA methylase armA was found in 44 isolates with high level resistance to aminoglycosides. The rate of coproduction was found in 36 isolates (80%). All isolates showed dominant two patterns in REP-PCR profile. The prevalence of MDR A. baumannii coproducing OXA-23 and armA was high, which the rate of blaOXA-23 coproduction was also high.

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.

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.

Working Memory Deficits in Ultra-High Risk for Psychosis and Schizophrenia / 대한정신분열병학회지

OBJECTIVES: The aim of this study was to investigate whether verbal and spatial working memory functions were impaired not only in patients with schizophrenia but also in people at ultra-high risk for first-episode psychosis. METHODS: Twenty-five patients (M 13, F 12) with schizophrenia (SPR), 21 people at ultra-high risk for psychosis (UHR)(M 10, F 11) and 19 normal controls (NC)(M 10, F 9) were recruited. The working memory was assessed by using the verbal and spatial n-back test. The working memory load increased incrementally from the 0-back to the 3-back condition. RESULTS: SPR performed significantly lower than NC and UHR in terms of hit rates of verbal and spatial n-back test. UHR subjects conducted significantly lower than NC and higher in trend-level than SPR in terms of hit rates of verbal and spatial n-back test. These differences were derived from the high working memory load (2-back and 3-back), not from the low working memory load (0-back and 1-back). There was no significant difference between the verbal and spatial n-back test across the three groups. CONCLUSION: These findings suggest that verbal and spatial working memory dysfunction may be general rather than differential in terms of stimuli modality, and this working memory deficit may be an important trait factor in schizophrenia.

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 (BioMerieux, 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 beta-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 / 대한임상미생물학회지

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: VanA-phenotype 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 (4-1-1-1-1-1-1) 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.

Antimicrobial Resistance Determinants in Imipenem-nonsusceptible Acinetobacter calcoaceticus-baumannii Complex Isolated in Daejeon, Korea / 대한진단검사의학회지

BACKGROUND: Members of the Acinetobacter calcoaceticus-baumannii (Acb) complex are important opportunistic bacterial pathogens and present significant therapeutic challenges in the treatment of nosocomial infections. In the present study, we investigated the integrons and various genes involved in resistance to carbapenems, aminoglycosides, and fluoroquinolones in 56 imipenem-nonsusceptible Acb complex isolates. METHODS: This study included 44 imipenem-nonsusceptible A. baumannii, 10 Acinetobacter genomic species 3, and 2 Acinetobacter genomic species 13TU strains isolated in Daejeon, Korea. The minimum inhibitory concentrations (MICs) were determined by Etest. PCR and DNA sequencing were used to identify the genes that potentially contribute to each resistance phenotype. RESULTS: All A. baumannii isolates harbored the blaOXA-51-like gene, and 21 isolates (47.7%) co-produced OXA-23. However, isolates of Acinetobacter genomic species 3 and 13TU only contained blaIMP-1 or blaVIM-2. Most Acb complex isolates (94.6%) harbored class 1 integrons, armA, and/or aminoglycoside-modifying enzymes (AMEs). Of particular note was the fact that armA and aph(3')-Ia were only detected in A. baumannii isolates, which were highly resistant to amikacin (MIC50> or =256) and gentamicin (MIC50> or =1,024). In all 44 A. baumannii isolates, resistance to fluoroquinolones was conferred by sense mutations in the gyrA and parC. However, sense mutations in parC were not found in Acinetobacter genomic species 3 or 13TU isolates. CONCLUSIONS: Several differences in carbapenem, aminoglycoside, and fluoroquinolone resistance gene content were detected among Acb complex isolates. However, most Acb complex isolates (87.5%) possessed integrons, carbapenemases, AMEs, and mutations in gyrA. The co-occurrence of several resistance determinants may present a significant threat.

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

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 beta-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 bla VIM-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.

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

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, beta-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.

Analysis of Acquired Resistance Genes in Stenotrophomonas maltophilia / 대한진단검사의학회지

BACKGROUND: Stenotrophomonas maltophilia is a gram-negative bacillus and a nosocomial pathogen in immunocompromised patients. Trimethoprim/sulfamethoxazole (TMP/SMX) is the drug of choice for treating S. maltophilia infection; however, resistance to TMP/SMX is increasing. In this study, we investigated the relationship between the incidence of TMP/SMX resistance and the presence of sul genes and mobile elements. METHODS: A total of 120 S. maltophilia isolates were collected from 3 university hospitals between April 2007 and April 2009. Antimicrobial susceptibilities were determined using the disk diffusion method. PCR and DNA sequencing were conducted for the detection of sul1, sul2, class 1 integron, and ISCR2 element. Repetitive extragenic palindromic sequence-based PCR (REP-PCR) was carried out to evaluate the genetic relatedness. RESULTS: The TMP/SMX-resistant (R) isolates harbored a significantly higher proportion of sul1 gene and class 1 integron than TMP/SMX-susceptible (S) isolates (P<0.001). Seventeen of 28 isolates with sul1 also had a class 1 integron, but none of the isolates without sul1 had a class 1 integron. The identified gene cassettes within class 1 integrons include aacA4, aadA1, aac6'-II, and qac. None of the 120 isolates carried sul2, glmM, or ISCR2 element. REP-PCR did not show any genetic relatedness among the isolates. CONCLUSIONS: In Korea, the resistance of S. maltophilia isolates to TMP/SMX is due to sul1 within a class 1 integron rather than to sul2. The class 1 integron also harbors multiple antibiotic resistance genes in addition to sul1, and therefore it could mediate multidrug resistance in S. maltophilia.