Seong Geun Hong

Investigation of Toxin Gene Diversity, Molecular Epidemiology, and Antimicrobial Resistance of Clostridium difficile Isolated from 12 Hospitals in South Korea

BACKGROUND Clostridium difficile is a major cause of antibiotic-associated diarrhea. The objective of this study was to characterize clinical isolates of C. difficile obtained from various regions in Korea with regard to their toxin status, molecular type, and antimicrobial susceptibility. METHODS We analyzed a total of 408 C. difficile isolates obtained between 2006 and 2008 from 408 patients with diarrhea in 12 South Korean teaching hospitals. C. difficile toxin genes tcdA, tcdB, cdtA, and cdtB were detected by PCR. Molecular genotyping was performed by PCR ribotyping. Antimicrobial susceptibilities of the 120 C. difficile isolates were assessed by agar dilution methods. RESULTS Among 337 toxigenic isolates, 105 were toxin A-negative and toxin B-positive (A(-)B(+)) and 29 were binary toxin-producing strains. PCR ribotyping showed 50 different ribotype patterns. The 5 most frequently occurring ribotypes comprised 62.0% of all identified ribotypes. No isolate was susceptible to cefoxitin, and all except 1 were susceptible to piperacillin and piperacillin-tazobactam. The resistance rates of isolates to imipenem, cefotetan, moxifloxacin, ampicillin, and clindamycin were 25%, 34%, 42%, 51%, and 60%, respectively. The isolates showed no resistance to metronidazole or vancomycin. CONCLUSIONS This is the first nationwide study on the toxin status, including PCR ribotyping and antimicrobial resistance, of C. difficile isolates in Korea. The prevalence of A-B+ strains was 25.7%, much higher than that reported from other countries. Binary toxin-producing strains accounted for 7.1% of all strains, which was not rare in Korea. The most prevalent ribotype was ribotype 017, and all A-B+ strains showed this pattern. We did not isolate strains with decreased susceptibility to metronidazole or vancomycin.

First Outbreak of Klebsiella pneumoniae Clinical Isolates Producing GES-5 and SHV-12 Extended-Spectrum β-Lactamases in Korea

Klebsiella pneumoniae clinical isolates producing extended-spectrum β-lactamases (ESBLs; TEM and SHV types) are frequently implicated in hospital outbreaks. There are also reports of K. pneumoniae isolates producing various non-TEM, non-SHV ESBLs: CTX-M and GES/IBC types (1). Presently, the different GES-type ESBLs are designated by identical names (8). To clarify the misleading nomenclature of GES-type ESBLs, we propose maintaining the current denomination concerning the fully characterized GES-3 and GES-4 ESBLs reported by Wachino et al. (12, 13) and renaming the variants (GES-3 and GES-4) reported by Vourli et al. (11) as GES-5 and GES-6, respectively.

Comparison of Phoenix and VITEK 2 Extended-Spectrum-β-Lactamase Detection Tests for Analysis of Escherichia coli and Klebsiella Isolates with Well-Characterized β-Lactamases

ABSTRACT The VITEK 2 and Phoenix extended-spectrum β-lactamase (ESBL) detection systems, which comprise confirmatory tests and expert systems, were evaluated for their ability to discriminate between 102 well-characterized strains of ESBL-positive or -negative Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca. At least 38 distinct ESBLs were included. The strains were chosen to include some known to cause false-positive and false-negative CLSI ESBL confirmatory test results. Therefore, enzyme characterizations, rather than CLSI tests, were the reference methods for the Phoenix and VITEK 2 evaluations. A third arm of the study was conducted with the Phoenix test using two normally inactive expert rules intended to enhance ESBL detection, in addition to using the currently available software. The Phoenix ESBL confirmatory test and unmodified expert system exhibited 96% sensitivity and 81% specificity for ESBL detection. Activation of the two additional rules increased sensitivity to 99% but reduced the specificity to 58%. The VITEK 2 ESBL confirmatory test exhibited 91% sensitivity, which was reduced to 89% sensitivity by its expert system, while its specificity was 85%. Many of the expert system interpretations of both instruments were helpful, but some were suboptimal. The VITEK 2 expert system was potentially more frustrating because it provided more inconclusive interpretations of the results. Considering the high degree of diagnostic difficulty posed by the strains, both ESBL confirmatory tests were highly sensitive. The expert systems of both instruments require modification to update and enhance their utility.

Convenient Test Using a Combination of Chelating Agents for Detection of Metallo-β-Lactamases in the Clinical Laboratory

ABSTRACT Although transmissible metallo-β-lactamases (MBLs) are a serious threat to β-lactam antibiotic therapy, the CLSI currently does not recommend testing methods for the detection of MBLs. The aim of this study was to evaluate the capability of double-disk tests (DDTs) by using disks containing a combination of the chelators 2-mercaptopropionic acid (MPA) and Tris-EDTA (TE) to detect MBLs. Sixteen isolates (4 Acinetobacter baumannii isolates, 6 Pseudomonas aeruginosa isolates, 1 Serratia marcescens isolate, 1 Aeromonas hydrophila isolate, 1 Aeromonas veronii isolate, 2 Chryseobacterium meningosepticum isolates, and 1 Stenotrophomonas maltophilia isolate) producing IMP-1, IMP-1-like, IMP-18, GIM-1, SPM-1, VIM-2, VIM-2-like, and chromosomal MBLs and 20 isolates (7 Klebsiella pneumoniae isolates, 3 Escherichia coli isolates, 5 Enterobacter cloacae isolates, 2 S. marcescens isolates, 1 Proteus mirabilis isolate, and 2 A. baumannii isolates) producing non-MBL carbapenemases, AmpC β-lactamases, and extended-spectrum β-lactamases were tested. The DDT method was evaluated by using four types of chelator disks (TE, high-strength TE, MPA, and TE plus 20 μl of MPA [at various concentrations]) and the β-lactams imipenem (IPM), meropenem (MEM), ertapenem (ERT), and ceftazidime (CAZ). DDTs with IPM and a TE disk supplemented with 1:320 MPA detected all MBLs and yielded no false-positive results. Some, but not all, MBL producers were detected in IPM-based tests involving the single chelator TE or MPA alone or by ERT- or CAZ-based tests. IPM-based tests with MPA concentrations other than 1:320 and all MEM-based tests had suboptimal sensitivities or specificities. DDT with IPM and a TE disk supplemented with 20 μl of 1:320 MPA appears to be convenient for the detection of MBLs in the clinical laboratory.

Increasing Prevalence of Vancomycin-Resistant Enterococcus faecium, Expanded-Spectrum Cephalosporin-Resistant Klebsiella pneumoniae, and Imipenem-Resistant Pseudomonas aeruginosa in Korea: KONSAR Study in 2001

The 5th year KONSAR surveillance in 2001 was based on routine test data at 30 participating hospitals. It was of particular interest to find a trend in the resistances of enterococci to vancomycin, of Enterobacteriaceae to the 3rd generation cephalosporin and fluoroquinolone, and of Pseudomonas aeruginosa and acinetobacters to carbapenem. Resistance rates of Gram-positive cocci were: 70% of Staphylococcus aureus to oxacillin; 88% and 16% of Enterococcus faecium to ampicillin and vancomycin, respectively. Seventy-two percent of pneumococci were nonsusceptible to penicillin. The resistance rates of Enterobacteriaceae were: Escherichia coli, 28% to fluoroquinolone; Klebsiella pneumoniae, 27% to ceftazidime, and 20% to cefoxitin; and Enterobacter cloacae, ≥40% to cefotaxime and ceftazidime. The resistance rates of P. aeruginosa were 21% to ceftazidime, 17% to imipenem, and those of the acinetobacters were ≥61% to ceftazidime, aminoglycosides, fluoroquinolone and cotrimoxazole. Thirty-five percent of non-typhoidal salmonellae were ampicillin resistant, and 66% of Haemophilus influenzae were β-lactamase producers. Notable changes over the 1997-2001 period were: increases in vancomycin-resistant E. faecium, and amikacin- and fluoroquinolone-resistant acinetobacters. With the increasing prevalence of resistant bacteria, nationwide surveillance has become more important for optimal patient management, for the control of nosocomial infection, and for the conservation of the newer antimicrobial agents.

Multiplex PCR for Rapid Detection of Genes Encoding Class A Carbapenemases

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers.

In vivo selection of carbapenem-resistant Klebsiella pneumoniae by OmpK36 loss during meropenem treatment

We recovered a carbapenem-resistant Klebsiella pneumoniae isolate H224 under in vivo meropenem selection pressure. Insertional inactivation of a major porin gene, ompK36, by IS5 element might play a role in acquiring carbapenem resistance in this strain harboring plasmid-borne DHA-1 AmpC beta-lactamase.

Characteristics of clinical isolates of Acinetobacter genomospecies 10 carrying two different metallo-β-lactamases

Acquired metallo-beta-lactamase (MBL) production is an important mechanism of carbapenem resistance. To our knowledge, carriage of two different MBLs has not been described previously in Acinetobacter spp. We present the characteristics of five Acinetobacter isolates carrying two different MBL genes. The species of all five Acinetobacter isolates with two different MBL genes were Acinetobacter genomospecies 10, and bla(IMP-1), bla(VIM-2) and bla(SIM-1) may coexist in this species. Minimum inhibitory concentrations (MICs) of imipenem for all five isolates with two different MBLs were >or=32 microg/mL, whilst those for two segregants that lost both MBLs were 0.5 microg/mL. The presence of MBL gene-carrying integrons with identical structures suggested the easily transferable nature of the elements, whilst instability of the MBL genes indicated potentially erroneous phenotypic and genetic characterisation.

Trends in antimicrobial resistance of neisseria gonorrhoeae isolated from Korean patients from 2000 to 2006

Background: The recent emergence and spread of antimicrobial-resistant Neisseria gonorrhoeae has compromised treatment and control of gonorrhea. We determined recent trends in antimicrobial susceptibility of the isolates, analyzed recent use of antigonococcal agents, and investigated the relationship between fluoroquinolone nonsusceptibility and amino acid substitutions within the fluoroquinolone resistance-determining regions in Korea. Methods: The antimicrobial susceptibilities of 977 isolates of N. gonorrhoeae collected from 2000 to 2006 in Korea were determined with penicillin, ceftriaxone, spectinomycin, tetracycline, and ciprofloxacin disks. Some of the randomly selected isolates were tested by the Clinical and Laboratory Standards Institute agar dilution method, to determine subtle changes in susceptibility to the above antibiotics and cefixime. &bgr;-lactamase was detected using a cefinase disk. Results: All of the isolates exhibited plasmid- or chromosomally mediated resistance to penicillin; however, the proportions of penicillinase-producing N. gonorrhoeae decreased rapidly from 64% in 2000 to 21% in 2006. All isolates were susceptible to third-generation cephalosporins, except for 1 isolate that was not susceptible to cefixime. The proportion of ciprofloxacin-resistant isolates increased from 26% in 2000 to 83% in 2006. Of 7 substitution types, 5 (Ser-91-Phe in Gyrase A (GyrA), Ser-87-Arg in ParC subunit of topoisomerase IV (ParC); Ser-91-Phe and Asp-95-Ala in GyrA, and Ser-87-Asn in ParC; Ser-91-Phe and Asp-95-Gly in GyrA, and Asp-86-Asn in ParC; Ser-91-Tyr in GyrA; Ser-91-Phe in GyrA, and Asp-86-Asn in ParC) were new ones not identified in our 2004 study. All isolates were susceptible to spectinomycin. About half of the patients in our current study (52.6%–58.1%, depending on the year) received spectinomycin treatment. Majorities were resistant to tetracycline, and the rate of highly tetracycline-resistant N. gonorrhoeae increased from 3% in 2000 to 9% in 2006. Conclusions: The incidence of penicillinase-producing N. gonorrhoeae declined significantly, but none of the isolates were susceptible to penicillin G. All isolates were susceptible to spectinomycin, in contrast majority were resistant to tetracycline. Inappropriate use of fluoroquinolone was frequent. The minimum inhibitory concentrations of ceftriaxone were within the susceptible range for all isolates, but those of cefixime were slightly higher, and it was 0.5 &mgr;g/mL (nonsusceptible) for 1 isolate.

Acquisition of extensive drug-resistant Pseudomonas aeruginosa among hospitalized patients: risk factors and resistance mechanisms to carbapenems.

Extensive drug-resistant Pseudomonas aeruginosa (XDRPA) strains, defined as resistant to all available antipseudomonal antibiotics, have been reported recently. This study aimed to investigate the risk factors for XDRPA acquisition by patients and the resistance mechanisms to carbapenems. From June to November 2007, XDRPA isolates were collected from patients in eight tertiary care hospitals. A case-control study was performed to determine factors associated with XDRPA acquisition. EDTA-imipenem disc synergy tests, and polymerase chain reaction amplification and sequencing were performed to detect the presence of metallo-β-lactamases (MBLs). Risk factor analysis was performed for 33 patients. Mechanical ventilation [odds ratio (OR) 8.2, 95% confidence interval (CI) 1.3-52.2; P = 0.026] and APACHE II score (OR 1.2, 95% CI 1.0-1.3; P = 0.007) were identified as independent risk factors for XDRPA acquisition. Pulsed-field gel electrophoresis of XDRPA identified clonal epidemic isolates co-existing with sporadic isolates. Eight of 43 (19%) XDRPA isolates were shown to produce MBLs; four produced VIM-2 and four produced IMP-6. This study suggests a major role for mechanical ventilation in XDRPA acquisition. Moreover, pulsed-field gel electrophoresis identified a clonal epidemic within hospitals. Taken together, these results suggest that patient-to-patient transmission contributes to XDRPA acquisition in Korea.