Wonkeun Song

Detection of Extended-Spectrum β-Lactamases by Using Boronic Acid as an AmpC β-Lactamase Inhibitor in Clinical Isolates of Klebsiella spp. and Escherichia coli

ABSTRACT We evaluated highly sensitive methods using boronic acid (BA) to detect extended-spectrum β-lactamase (ESBL) production. A total of 182 clinical isolates of Klebsiella spp. (n = 118) and Escherichia coli (n = 64) were analyzed: 62 harbored only ESBLs, 80 harbored both ESBLs and plasmid-mediated AmpC β-lactamases (pAmpCs), and 40 harbored only pAmpCs. The CLSI confirmatory test detected all isolates that produce only ESBLs but detected 85% of isolates that produce both enzymes. When a ≥5-mm increase in the zone diameter of either the cefotaxime (CTX) or the ceftazidime (CAZ) disk in the presence of both clavulanic acid (CA) and BA was considered to be a positive result, the test detected all isolates that harbor ESBLs (± pAmpCs) but showed frequent false-positive results (50%) for isolates that produce only pAmpCs. Meanwhile, when a ≥3-mm increase in the zone diameter of either the CTX/BA or the CAZ/BA disk in the presence of CA was considered to be a positive result, the test also detected all isolates that harbor ESBLs (± pAmpCs) and showed less frequent false-positive results (5%) in isolates that produce only pAmpCs. The latter new interpretive guideline has enhanced detection of ESBLs in clinical isolates of Klebsiella spp. and Escherichia coli and allowed detection of an ESBL even when potentially masked by a pAmpC.

Subcellular localization of hepatitis C viral proteins in mammalian cells

SummaryWe determined the subcellular localization of hepatitis C viral (HCV) proteins as a first step towards the understanding of the functions of these proteins in the mammalian cell (CHO-K1). We used fluorescence emitted from green fluorescent protein (GFP)-fused to the viral proteins to determine the subcellular localization of the viral proteins. We found that most of the viral proteins were excluded from the nucleus. Core exhibited a globular pattern near the nucleus. NS2 was concentrated in the perinuclear space. NS4A accumulated in the ER and the Golgi regions. NS3 was detected in the nucleus as well as the cytoplasm, when it was expressed by itself. However, NS3 became restricted to the cytoplasm, when it was produced together with NS4A. NS4B showed a spot-like pattern throughout the cytoplasm. NS5A and NS5B were distributed throughout the cytoplasm in a mesh-like pattern. These results can provide a basis for further investigations into the functions of the HCV proteins.

CTX-M-14 and CTX-M-15 enzymes are the dominant type of extended-spectrum β-lactamase in clinical isolates of Escherichia coli from Korea

This study was performed to assess the prevalence and genotypes of plasmid-borne extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases in Escherichia coli in Korea. A total of 576 isolates of E. coli was collected from 12 Korean hospitals during May and July 2007. A phenotypic confirmatory test detected ESBLs in 82 (14.2 %) of the 576 E. coli isolates. The most common types of ESBLs identified were CTX-M-14 (n=32) and CTX-M-15 (n=27). The prevalence and diversity of the CTX-M mutants, including CTX-M-15, CTX-M-27 and CTX-M-57, with significant hydrolytic activity against ceftazidime were increased. PCR experiments detected genes encoding plasmid-borne AmpC β-lactamases in 15/56 cefoxitin-intermediate or cefoxitin-resistant isolates, and the most common type of AmpC β-lactamase identified was DHA-1 (n=10). These data suggest that the incidence of ESBLs in E. coli has increased as a result of the dissemination of CTX-M enzymes in Korea. In addition, CTX-M-22, CTX-M-27 and CTX-M-57 have appeared in Korea.

Microbiologic aspects of predominant bacteria isolated from the burn patients in Korea

The risk of infection in burns is well-known. In recent decades, the antimicrobial resistance of bacteria isolated from burn patients has increased. For this reason, we have carried out a study of the predominant bacterial profiles and antimicrobial resistance patterns of isolates from a burn center in Korea. A retrospective study was undertaken at Hallym University, Hangang Sacred Heart Hospital to examine the bacterial isolates from the burn patients and to compare the antibiograms of the predominant bacteria isolated from these patients with those of the other wards over a period of 3 years. Pseudomonas aeruginosa was the most common (n=2997, 45.7%) isolate from the burn patients followed by Staphylococcus aureus (n=21261, 19.2%) and Acinetobacter baumannii (n=878, 13.4%). These bacteria, isolated from the burn patients, were almost all higher in antimicrobial resistance rate than those in the non-burn patients (P<0.05). Because these bacteria showed very high resistant rates, they must be avoided in order to control a hospital-acquired infection. Our results seem helpful in providing useful guidelines for choosing effective empiric antimicrobial therapy against bacteria isolated from the burn patients in Korea.

In vitro activity of β-lactams in combination with other antimicrobial agents against resistant strains of Pseudomonas aeruginosa

Abstract Using the chequerboard titration method, the activity in combination of β-lactams, fluoroquinolones and aminoglycosides was investigated against 24 Pseudomonas aeruginosa isolates resistant to these antibiotics. Synergy was detected with one or more antimicrobial combinations against 15 of 24 (63%) isolates and partial synergy was detected with one or more combinations against all 24 isolates. No antagonism was seen with any combination. Ceftazidime and cefepime with aztreonam, amikacin and isepamicin showed synergy or partial synergy against 12–20 (50–80%) isolates. Imipenem and meropenem with amikacin and isepamicin showed synergy or partial synergy against eight to 12 (33–50%) isolates. The results of this study indicate that against P. aeruginosa , synergy may occur between β-lactams, fluoroquinolones and aminoglycosides although the strains are resistant to the individual antibiotics.

Prevalence and diversity of carbapenemases among imipenem-nonsusceptible Acinetobacter isolates in Korea: emergence of a novel OXA-182

Increase in multidrug-resistant Acinetobacter poses a serious problem in Korea. In this study, 190 imipenem (IPM)-nonsusceptible (NS) Acinetobacter isolates from 12 Korean hospitals in 2007 were used to determine species, prevalence, and antimicrobial susceptibility of OXA carbapenemase- and metallo-β-lactamase (MBL)-producing isolates. bla(OXA)-₂₃-like and ISAba1-asssociated bla(OXA)-₅₁-like genes were detected in 80% and 12% of 178 IPM-NS Acinetobacter baumannii isolates, respectively. A novel bla(OXA)-₁₈₂ was detected in 12 IPM-NS A. baumannii isolates. Twelve out of 14 MBL-producing isolates were non-baumanniiAcinetobacter. A. baumannii isolates with OXA carbapenemase were more often resistant to aminoglycosides, ciprofloxacin, and tigecycline than non-baumannii Acinetobacter isolates with MBL. Identical pulsed- field gel electrophoresis patterns were observed in 89% of A. baumannii isolates with bla(OXA)-₂₃-like gene. In conclusion, extremely rapid increase of IPM-NS A. baumannii in previous Korean studies was mainly due to clonal spread of OXA-23-producing A. baumannii isolates. A novel OXA-182 emerged in Korea.

Chromosome-encoded AmpC and CTX-M extended-spectrum β-lactamases in clinical isolates of Proteus mirabilis from Korea

ABSTRACT Among 222 Proteus mirabilis clinical isolates collected from 17 hospitals in Korea in 2008, 28 (12.6%) and 8 (3.6%) isolates exhibited extended-spectrum β-lactamase (ESBL) and AmpC phenotypes, respectively. The most common type of ESBL gene identified by PCR and sequencing experiments was bla CTX-M-14a (n = 12). The bla CTX-M-90 (n = 4), bla CTX-M-15 (n = 3), bla CTX-M-12 (n = 3), bla CTX-M-2 (n = 2), bla CTX-M-14b (n = 1), bla TEM-52 (n = 5), and bla SHV-12 (n = 1) genes were also detected. Eight isolates carried an AmpC β-lactamase gene, such as bla CMY-2 (n = 6) or bla DHA-1 (n = 2). All bla genes encoding CTX-M-1- and CTX-M-9-type enzymes and all bla CMY-2 genes were preceded by ISEcp1-like elements. The bla CTX-M-2 gene found in two isolates was located on a complex class 1 integron. The bla DHA-1 gene was preceded by a transcriptional regulator gene and was followed by phage shock protein genes. The bla CTX-M genes were located on the chromosome in 21 isolates. A plasmid location for the bla CTX-M gene was found in only four isolates: the bla CTX-M-14a gene was located on ∼150-kbp IncA/C plasmids in three isolates and on a ∼50-kbp IncN plasmid in one isolate. The bla TEM-52 gene was located on ∼50-kbp IncN plasmids in all five isolates. The AmpC β-lactamase genes were located on the chromosome in seven of eight isolates; one isolate carried the bla CMY-2 gene on a ∼150-kbp IncA/C plasmid. Our results show that a chromosomal location of CTX-M ESBL and AmpC β-lactamase genes in P. mirabilis is no longer an unusual phenomenon in hospital environments.

Boronic acid disk tests for identification of extended-spectrum β-lactamase production in clinical isolates of Enterobacteriaceae producing chromosomal AmpC β-lactamases

A study using boronic acid (BA) was designed to detect the extended-spectrum beta-lactamases (ESBLs) in Enterobacteriaceae producing chromosomal AmpC beta-lactamases. A total of 197 clinical isolates of Enterobacter spp. (n=100), Serratia marcescens (n=62) and Citrobacter freundii (n=35) were analysed. Genes encoding ESBLs were detected by polymerase chain reaction (PCR) amplification followed by direct sequencing of PCR products. The Clinical and Laboratory Standards Institute confirmatory test detected only 72.1% of the ESBL-producing isolates. When a > or =5mm increase in the zone diameter of either the cefotaxime/clavulanic acid and/or the ceftazidime/clavulanic acid disks tested in combination with BA versus cefotaxime and/or ceftazidime containing BA was considered to be a positive for ESBL, the method detected 60 (98.4%) of the 61 isolates that harboured ESBLs and showed no false-positive results for ESBL-non-producing isolates. In conclusion, the BA disk test is a highly sensitive and specific method for the detection of ESBLs in Enterobacteriaceae producing chromosomal AmpC beta-lactamases.

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.

Rates of fecal transmission of extended-spectrum β-lactamase-producing and carbapenem-resistant Enterobacteriaceae among patients in intensive care units in Korea.

Background We investigated the rates of fecal transmission of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) and carbapenem-resistant Enterobacteriaceae (CRE) among patients admitted to intensive care units (ICUs). Methods From June to August 2012, rectal cultures were acquired from all patients at ICU admission. For patients not carrying ESBL-E or CRE at admission, follow-up cultures were performed to detect acquisition. A chromogenic assay was used to screen for ESBL-E and CRE. Bacterial species identification and antibiotic susceptibility tests were performed using the Vitek 2 system (bioMérieux, France). ESBL genotypes were determined by PCR, and clonal relatedness of the isolates was assessed by pulsed-field gel electrophoresis. Results Out of 347 ICU admissions, 98 patients were found to be carriers of ESBL-E (28.2%, 98/347). Follow-up cultures were acquired from 91 of the patients who tested negative for ESBL-E at admission; the acquisition rate in this group was 12.1% (11/91), although none was a nosocomial transmission. For CRE, the prevalence of fecal carriage was 0.3% (1/347), and the acquisition rate was 2.9% (4/140). None of the CRE isolates were carbapenemase-producers. Conclusions The high prevalence of ESBL-E carriage on admission (28.2%), coupled with rare nosocomial transmission and the very low carriage rate of CRE (0.3%), challenge the routine use of active surveillance in non-epidemic settings. Nevertheless, passive surveillance measures, such as rapid and accurate screening of clinical specimens, will be critical for controlling the spread of CRE.