Development and Evaluation of Multiplex PCR for the Detection of Carbapenemase-Producing Enterobacteriaceae / 대한임상미생물학회지

BACKGROUND: The isolation of carbapenemase-producing Enterobacteriaceae (CPE) has become increasingly common. Continuous surveillance for these organisms is essential because their infections are closely related to outbreaks of illness and are associated with high mortality rates. The aim of this study was to develop and evaluate multiplex PCR as a means of detecting several important CPE genes simultaneously. METHODS: We aimed to develop a multiplex PCR that could detect seven CPE genes simultaneously. The multiplex PCR was composed of seven primer sets for the detection of KPC, IMP, VIM, NDM-1, GES, OXA-23, and OXA-48. We designed different PCR product sizes of at least 100 bp. We evaluated the performance of this new test using 69 CPE-positive clinical isolates. Also, we confirmed the specificity to rule out false-positive reactions by using 71 carbapenem-susceptible clinical strains. RESULTS: A total of 69 CPE clinical isolates showed positive results and were correctly identified as KPC (N=14), IMP (N=13), OXA-23 (N=12), OXA-48 (N=11), VIM (N=9), GES (N=5), and NDM (N=5) by the multiplex PCR. All 71 carbapenem-susceptible clinical isolates, including Enterococcus faecalis , Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, showed negative results. CONCLUSION: This multiplex PCR can detect seven CPE genes at a time and will be useful in clinical laboratories.

Recurrent Familial Furunculosis Associated with Panton-Valentine Leukocidin-Positive Methicillin-Susceptible Staphylococcus aureus ST1

Staphylococcus aureus is now a major community-acquired pathogen worldwide, notably associated with skin and soft tissue infections. Staphylococci are present in the form of colonizers or environmental contaminants at home and increase the risk of recurrent infection. We are describing recurrent familial furunculosis caused by Panton-Valentine Leukocidin-positive methicillin susceptible S. aureus ST1 in Korea. An infant, his father and mother had furunculosis due to methicillin-sensitive S. aureus (MSSA) infection with identical susceptibility patterns. ST1 accounted for all 3 isolates and they were confirmed of having agr group I. Both sec and seh were detected in all isolates using polymerase chain reaction (PCR) assays, and all isolates contained Panton-Valentine leukocidin (PVL) genes. Risk factors for the household spread of S. aureus include skin conditions and close physical contact among household members. The relationship between S. aureus colonization of household contacts and the occurrence of S. aureus infection should be studied into more detail.

Identification of Acinetobacter Species Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

BACKGROUND: Acinetobacter baumannii has a greater clinical impact and exhibits higher antimicrobial resistance rates than the non-baumannii Acinetobacter species. Therefore, the correct identification of Acinetobacter species is clinically important. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has recently become the method of choice for identifying bacterial species. The purpose of this study was to evaluate the ability of MALDI-TOF MS (Bruker Daltonics GmbH, Germany) in combination with an improved database to identify various Acinetobacter species. METHODS: A total of 729 Acinetobacter clinical isolates were investigated, including 447 A. baumannii, 146 A. nosocomialis, 78 A. pittii, 18 A. ursingii, 9 A. bereziniae, 9 A. soli, 4 A. johnsonii, 4 A. radioresistens, 3 A. gyllenbergii, 3 A. haemolyticus, 2 A. lwoffii, 2 A. junii, 2 A. venetianus, and 2 A. genomospecies 14TU. After 212 isolates were tested with the default Bruker database, the profiles of 63 additional Acinetobacter strains were added to the default database, and 517 isolates from 32 hospitals were assayed for validation. All strains in this study were confirmed by rpoB sequencing. RESULTS: The addition of the 63 Acinetobacter strains' profiles to the default Bruker database increased the overall concordance rate between MALDI-TOF MS and rpoB sequencing from 69.8% (148/212) to 100.0% (517/517). Moreover, after library modification, all previously mismatched 64 Acinetobacter strains were correctly identified. CONCLUSIONS: MALDI-TOF MS enables the prompt and accurate identification of clinically significant Acinetobacter species when used with the improved database.

Combined Use of the Modified Hodge Test and Carbapenemase Inhibition Test for Detection of Carbapenemase-Producing Enterobacteriaceae and Metallo-beta-Lactamase-Producing Pseudomonas spp

BACKGROUND: We evaluated the combined use of the modified Hodge test (MHT) and carbapenemase inhibition test (CIT) using phenylboronic acid (PBA) and EDTA to detect carbapenemase-producing Enterobacteriaceae (CPE) and metallo-beta-lactamase (MBL)-producing Pseudomonas spp. METHODS: A total of 49 isolates of CPE (15 Klebsiella pneumoniae carbapenemase [KPC], 5 Guiana extended-spectrum beta-lactamase [GES]-5, 9 New Delhi metallo-beta-lactamase [NDM]-1, 5 Verona integron-encoded metallo-beta-lactamase [VIM]-2, 3 imipenem-hydrolyzing beta-lactamase [IMP], and 12 oxacillinase [OXA]-48-like), 25 isolates of MBL-producing Pseudomonas spp. (14 VIM-2 and 11 IMP), and 35 carbapenemase-negative controls were included. The MHT was performed for all isolates as recommended by the Clinical and Laboratory Standards Institute. Enhanced growth of the indicator strain was measured in mm with a ruler. The CIT was performed by directly dripping PBA and EDTA solutions onto carbapenem disks that were placed on Mueller-Hinton agar plates seeded with the test strain. RESULTS: Considering the results of the MHT with the ertapenem disk in Enterobacteriaceae and Pseudomonas spp., the CIT with the meropenem disk in Enterobacteriaceae, and the imipenem disk in Pseudomonas spp., three combined disk tests, namely MHT-positive plus PBA-positive, EDTA-positive, and MHT-positive plus PBA-negative plus EDTA-negative, had excellent sensitivity and specificity for the detection of KPC- (100% sensitivity and 100% specificity), MBL- (94% sensitivity and 100% specificity), and OXA-48-like-producing isolates (100% sensitivity and 100% specificity), respectively. CONCLUSIONS: Combined use of the MHT and CIT with PBA and EDTA, for the detection of CPE and MBL-producing Pseudomonas spp., is effective in detecting and characterizing carbapenemases in routine laboratories.

Detection of Carbapenemases in Clinical Enterobacteriaceae Isolates Using the VITEK AST-N202 Card / 감염과화학요법

BACKGROUND: The rapid and accurate detection of carbapenemase-producing Enterobacteriaceae (CPE) in clinical microbiology laboratories is essential for the treatment and control of infections caused by these microorganisms. This study was performed to evaluate the ability of the VITEK AST-N202 card to detect CPE isolates. MATERIALS AND METHODS: A total of 43 (Klebsiella pneumoniae, n = 37; Escherichia coli, n = 3; and Enterobacter cloacae, n = 3) CPE isolates and 79 carbapenemase-non-producing Enterobacteriaceae (CNE) isolates were included in this study. The CPE isolates harbored KPC-2 (n = 11), KPC-3 (n = 20), GES-5 (n = 5), VIM-2 (n = 2), IMP-1 (n = 1), NDM-1 (n = 2), or OXA-232 (n = 2). Of the 79 CNE isolates, eight K. pneumoniae isolates were resistant to ertapenem, imipenem, and meropenem, while the remaining 71 isolates were susceptible to the carbapenems. Antimicrobial susceptibilities were tested using the VITEK AST-N202 card, and the results were interpreted as positive when the isolates showed resistant or intermediate results. Modified-Hodge tests (MHTs) were performed using ertapenem or meropenem disks for the screening of carbapenemase production. Polymerase chain reaction (PCR) and direct sequencing were used to identify beta-lactamase genes. RESULTS: Sensitivity of MHT with ertapenem and meropenem disks for the detection of carbapenemase was 81.4% (35/43) and 81.4% (35/43), respectively, and a combination with both antibiotic disks increased the sensitivity to 88.4% (38/43). Specificity of the MHT was 100% (79/79) for the CNE isolates. Sensitivity of ertapenem, imipenem, and meropenem as assessed by the VITEK AST-N202 card was 100% (43/43), 93% (40/43), and 95.3% (41/43), respectively. Specificity (89.8%, 71/79) of the test with each carbapenem was improved to 100% (71/71) when eight carbapenem-resistant CNE isolates were excluded from the testing. CONCLUSION: The VITEK AST-N202 card showed high sensitivity for the detection of carbapenemases in Enterobacteriaceae strains. PCR and sequencing experiments for the detection of carbapenemases are recommended when clinical Enterobacteriaceae isolates show non-susceptibility to carbapenems.

Epidemiology and Characteristics of Metallo-beta-Lactamase-Producing Pseudomonas aeruginosa / 감염과화학요법

Metallo-beta-lactamase-producing Pseudomonas aeruginosa (MPPA) is an important nosocomial pathogen that shows resistance to all beta-lactam antibiotics except monobactams. There are various types of metallo-beta-lactamases (MBLs) in carbapenem-resistant P. aeruginosa including Imipenemase (IMP), Verona integron-encoded metallo-beta-lactamase (VIM), Sao Paulo metallo-beta-lactamase (SPM), Germany imipenemase (GIM), New Delhi metallo-beta-lactamase (NDM), Florence imipenemase (FIM). Each MBL gene is located on specific genetic elements including integrons, transposons, plasmids, or on the chromosome, in which they carry genes encoding determinants of resistance to carbapenems and other antibiotics, conferring multidrug resistance to P. aeruginosa. In addition, these genetic elements are transferable to other Gram-negative species, increasing the antimicrobial resistance rate and complicating the treatment of infected patients. Therefore, it is essential to understand the epidemiology, resistance mechanism, and molecular characteristics of MPPA for infection control and prevention of a possible global health crisis. Here, we highlight the characteristics of MPPA.

Characteristics of Metallo-beta-Lactamase-Producing Pseudomonas aeruginosa in Korea / 감염과화학요법

BACKGROUND: The aim of this study was to investigate the molecular epidemiological characteristics of metallo-beta-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.

In Vivo Selection of Pan-Drug Resistant Acinetobacter baumannii during Antibiotic Treatment

PURPOSE: Colistin resistance in Acinetobacter baumannii (A. baumannii) is mediated by a complete loss of lipopolysaccharide production via mutations in lpxA, lpxC, and lpxD gene or lipid A modifications via mutations in the pmrA and pmrB genes. However, the exact mechanism of therapy-induced colistin resistance in A. baumannii is not well understood. MATERIALS AND METHODS: We investigated the genotypic and phenotypic changes that underlie pan-drug resistance mechanisms by determining differences between the alterations in extensively drug-resistant (XDR) A. baumannii (AB001 and AB002) isolates and a pan-drug resistant (PDR) counterpart (AB003) recovered from one patient before and after antibiotic treatment, respectively. RESULTS: All three clinical isolates shared an identical sequence type (ST138), belonging to the global epidemic clone, clonal complex 92, and all produced OXA-23 carbapenemase. The PDR AB003 showed two genetic differences, acquisition of armA gene and an amino acid substitution (Glu229Asp) in pmrB gene, relative to XDR isolates. No mutations were detected in the pmrA, pmrC, lpxA, lpxC, or lpxD genes in all three isolates. In matrix-assisted laser desorption ionization-time of flight analysis, the three isolates commonly showed two major peaks at 1728 m/z and 1912 m/z, but peaks at 2034 m/z, 2157 m/z, 2261 m/z, and 2384 m/z were detected only in the PDR A. baumannii AB003 isolate. CONCLUSION: Our results show that changes in lipid A structure via a mutation in the pmrB gene and acquisition of armA gene might confer resistance to colistin and aminoglycosides to XDR A. baumannii strains, resulting in appearance of a PDR A. baumannii strain of ST138.

Postsurgical Wound Infection Caused by Mycobacterium conceptionense Identified by Sequencing of 16S rRNA, hsp65, and rpoB Genes in an Immunocompetent Patient / 대한임상미생물학회지

Rapidly growing mycobacteria are ubiquitous in the environment and are increasingly being recognized as opportunistic pathogens. Recently, a new species, Mycobacteium conceptionense, has been validated from the Mycobacterium fortuitum third biovariant complex by molecular analysis. However, there are few reports, and postsurgical wound infection by this species is rare. We report a case of postsurgical wound infection caused by M. conceptionense in an immunocompetent patient that was identified by a sequencing analysis of 16S rRNA, hps65, and rpoB genes.

Risk Factors and Molecular Epidemiology of Community-Onset Extended-Spectrum beta-Lactamase-Producing Escherichia coli Bacteremia

PURPOSE: Inadequate empirical therapy for severe infections caused by extended-spectrum beta-lactamase-producing Escherichia coli (ESBLEC) is associated with poor outcomes. This study was designed to investigate risk factors for community-onset ESBLEC bacteremia at admission to a tertiary care hospital. MATERIALS AND METHODS: A case-control study was performed that included all episodes of ESBLEC bacteremia in the outpatient department or within 48 hours of admission from January 2005 to March 2009. Data on predisposing factors were collected. The molecular epidemiology of ESBLEC clinical isolates was also determined. RESULTS: Among 25281 blood cultures, 60 episodes of ESBLEC bacteremia were studied, which accounted for 7% of all E. coli bacteremia at admission. Healthcare-associated infection [odds ratio (OR), 8.3; 95% confidence interval (CI), 2.4-28.7; p=0.001], malignancy (OR, 4.6; 95% CI, 1.3-16.3; p=0.018), urinary tract infection (OR, 139.1; 95% CI, 24.6-788.2; p<0.001), hepatobiliary infection (OR, 79.1; 95% CI, 13.5-463.8; p<0.001), third generation cephalosporin usage during preceding 3 months (OR, 16.4; 95% CI, 2.0-131.8; p=0.008), and severe sepsis/septic shock (OR, 73.7; 95% CI, 12.4-438.5; p<0.001) were determined as independent risk factors for community-onset ESBLEC bacteremia. The most common extended-spectrum beta-lactamase (ESBL) gene identified was bla(CTX-M-15) (n=31) followed by bla(CTX-M-14) (n=23). CONCLUSION: The most common types of ESBLs in E. coli causing community-onset bacteremia were CTX-M-15 and CTX-M-14 in Korea. By result of decision tree analysis, the empirical use of carbapenems is suggested only for patients with severe sepsis/septic shock, hepatobiliary infection, or healthcare-associated urinary tract infection.

Comparison Cytomegalovirus Qualitative Assay Using Real-Time PCR and Conventional PCR / 대한임상미생물학회지

BACKGROUND: Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality in immunocompromised patients. We compared the abilities of the recently developed Real-Q Cytomegalovirus Kit (Biosewoom Inc., Korea) and the previously used PANA mPCR CMV Detection Kit (Panagene Inc., Korea) to detect CMV. METHODS: We analyzed 300 samples (whole blood: 262, urine: 37, CSF: 1) submitted for qualitative CMV PCR testing during October 2011 at Yonsei University College of Medicine Severance Hospital. Real-time PCR was performed with a Real-Q Cytomegalovirus Kit and conventional PCR was conducted with a PANAmPCR CMV Detection Kit. RESULTS: The positive rates of both Real-time PCR and conventional PCR were 25.3% (76/300), and the kappa coefficient (K) was 0.96 (95% confidence interval (CI), 0.93-1.00). The concordance rate of the Real-Q Cytomegalovirus Kit and the PANAmPCR CMV Detection Kit was 98.7% (296/300), and four out of 300 samples showed discordant results. If the concordant results of 296 samples and the four results confirmed by direct sequencing were assumed to be true, the sensitivity and specificity of the Real-Q Cytomegalovirus Kit were 97.4% (95% CI, 93.8-100.0%) and 99.1% (95% CI, 97.9-100.0%), respectively. CONCLUSION: The recently developed Real-Q Cytomegalovirus Kit showed excellent sensitivity and specificity, and had a high concordance rate with the previously established PANAmPCR CMV Detection Kit, which uses conventional PCR. Furthermore, Real-time PCR could decrease the test time, as the electrophoresis step required for conventional PCR is not required for Real-time PCR.

Carbapenem-Resistant Acinetobacter baumannii / 대한임상미생물학회지

Clinical isolates of Acinetobacter spp. in Korea exhibit higher antimicrobial resistance rates than in foreign countries and frequently show multi-drug resistance. Approximately 67% (272/405) of Acinetobacter baumannii isolates collected from 19 hospitals in Korea in 2008 exhibited intermediate susceptibility or resistance to imipenem and/or meropenem. The most important mechanisms in acquiring carbapenem resistance in A. baumannii in Korea are production of OXA-23 and overproduction of OXA-51, while that in non-baumannii Acinetobacter is the production of metallo-beta-lactamases. All the carbapenem-resistant A. baumannii isolates were identified as clonal complex 92 and belonged to worldwide clone 2.

Risk Factors Associated with Gastrointestinal Tract Colonization by Enterobacteriaceae in Neonatal Intensive Care Unit Patients

PURPOSE: The incidence of nosocomial infection caused by Gram-negative bacilli (GNB) has increased in neonatal intensive care units (NICU). This study identified the progression of sepsis caused by GNB colonization and analyzed the risk factors associated with using periodic stool culture surveillance. METHODS: We included 86 newborns admitted to the NICU, Kosin University Gospel Hospital from October 2007 to May 2008. Three stool specimens were collected right after birth and two more were collected at 2 week intervals. The risk factors related to GNB colonization were established from each medical record and related references. RESULTS: The incidence of colonization by GNB was 22 (25.6%) per 86 neonates but none had culture-proven sepsis. The three most commonly isolated GNB were Pseudomonas aeruginosa, Enterobacter cloacae, and Citrobacter freundii. Approximately 89% (32/36) of isolated GNB were susceptible to amikacin. The probability of GNB colonization increased in infants who were fed a small volume during enteral feeding. In contrast, delayed enteral feeding resulted in a decreased probability for GNB colonization. CONCLUSION: Colonized GNB in the intestine was confirmed by enteric surveillance culture of newborns admitted to the NICU. However, we found no evidence of culture-proven GNB sepsis. As lower feeding volume on the colonization day is a risk factor for GNB colonization, the chance for GNB colonization should be considered when feeding intolerance is present.

Prevalence of Plasmid-mediated Quinolone Resistance and Its Association with Extended-spectrum Beta-lactamase and AmpC Beta-lactamase in Enterobacteriaceae / 대한진단검사의학회지

BACKGROUND: We investigated the prevalence of plasmid-mediated quinolone resistance and its association with extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase in Enterobacteriaceae. METHODS: A total of 347 non-duplicated isolates of Enterobacteriaceae were collected between August and October 2006 from 2 hospitals. Qnr determinant screening was conducted using PCR amplification, and all positive results were confirmed by direct sequencing. Qnr-positive strains were determined on the basis of the presence of ESBL and AmpC beta-lactamase genes. RESULTS: The qnr gene was detected in 47 of 347 clinical Enterobacteriaceae isolates. Among the 47 qnr-positive strains, Klebsiella pneumoniae (N=29) was the most common, followed by Escherichia coli (N=6), Enterobacter cloacae (N=6), Citrobacter freundii (N=5), and Enterobacter aerogenes (N=1). These isolates were identified as qnrA1 (N=6), 8 qnrB subtypes (N=40), and qnrS1 (N=1). At least 1 ESBL was detected in 38 of the 47 qnr-positive strains. Qnr-positive strains also showed high positive rates of ESBL or AmpC beta-lactamase, such as TEM, SHV, CTX-M, and DHA. DHA-1 was detected in 23 of 47 qnr-positive strains, and this was co-produced with 1 qnrA1 and 22 qnrB4. Strains harboring MIR-1T and CMY were also detected among the qnr-positive strains. Antimicrobial-resistance rates of qnr-positive strains to ciprofloxacin, levofloxacin, norfloxacin, nalidixic acid, and moxifloxacin were 51.1%, 46.8%, 46.8%, 74.5%, and 53.2%, respectively. CONCLUSIONS: The qnr genes were highly prevalent in Enterobacteriaceae, primarily the qnrB subtypes. They were closely associated with EBSL and AmpC beta-lactamase.

Frequency of Extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase Genes in Escherichia coli and Klebsiella pneumoniae over a Three-year Period in a University Hospital in Korea / 대한진단검사의학회지

BACKGROUND: The aim of this study was to determine the yearly prevalence and genotype distribution of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae collected over a 3-yr period in Gwangju, Korea. METHODS: Clinical isolates of E. coli and K. pneumoniae collected at Chosun University Hospital from September 15, 2005 to September 14, 2008 were evaluated. Antimicrobial susceptibility testing was performed using the Vitek II system (bioMerieux, USA) and agar dilution methods. Screening for ESBL and AmpC beta-lactamase genes was performed using PCR amplification of plasmid DNA followed by direct sequencing of the PCR products. RESULTS: The percentage of ESBL-producing isolates was 12.6% (196/1,550) for E. coli and 26.2% (294/1,121) for K. pneumoniae. The ESBL gene sequencing results showed that the most prevalent ESBL types were CTX-M (93.5%) and SHV (12.9%) in E. coli, and SHV (73.2%) and CTX-M (46.3%) in K. pneumoniae. The most common ESBL in E. coli was CTX-M-15-like, followed by CTX-M-14-like, SHV-2a-like, and SHV-12-like. The most prevalent ESBL type in K. pneumoniae was SHV-12, followed by CTX-M-14-like and CTX-M-15-like. Fifty-one percent (21/41) of ESBL-producing K. pneumoniae with ESBL types verified by sequencing also had DHA-1-like AmpC beta-lactamases. However, none of the ESBL-producing E. coli was positive in the AmpC beta-lactamase PCR analysis. CONCLUSIONS: In this study, the most common types of class A ESBLs identified were CTX-M-15-like in E. coli and SHV-12-like in K. pneumoniae.

Prevalence of the Extended-Spectrum beta-Lactamase and qnr Genes in Clinical Isolates of Escherichia coli / 대한진단검사의학회지

BACKGROUND: This study was performed to investigate the prevalence of qnr genes in clinical isolates of Escherichia coli from Korea that produce extended-spectrum beta-lactamases (ESBLs). METHODS: During the period of May to June 2005, we collected clinical isolates of E. coli that were intermediate or resistant to ceftazidime and/or cefotaxime from 11 Korean hospitals. Antimicrobial susceptibility was determined by the disk diffusion and agar dilution methods. ESBL production was confirmed phenotypically by the double-disk synergy test. ESBL and qnr genes were searched for by PCR amplification, and the PCR products were then subjected to direct sequencing. RESULTS: Double-disk synergy tests were positive in 84.3% (118/140) of ceftazidime- and/or cefotaxime-nonsusceptible E. coli isolates. The most prevalent types of ESBL in E. coli isolates were CTX-M-14 (N=41) and CTX-M-15 (N=58). Other ESBLs were also identified, including CTX-M-3 (N=7), CTX-M-9 (N=8), CTX-M-12 (N=1), CTX-M-57 (N=1), SHV-2a (N=2), SHV-12 (N=17) and TEM-52 (N=4). The qnrA1 and qnrB4 genes were identified in 4 and 7 ESBL-producing isolates, respectively. CONCLUSIONS: CTX-M-type enzymes were the most common type of ESBL in E. coli isolates from Korea, and the qnr genes were not uncommon in ESBL-producing E. coli isolates. Dissemination of E. coli containing both ESBL and qnr genes could compromise the future usefulness of the expanded-spectrum antibiotics for the treatment of infections.

Antibiotic Resistance Mechanisms of Escherichia coli Isolates from Urinary Specimens / 대한진단검사의학회지

BACKGROUND: This study was designed to characterize urinary isolates of Escherichia coli that produce extended-spectrum beta-lactamases (ESBLs) and to determine the prevalence of other antimicrobial resistance genes. METHODS: A total of 264 non-duplicate clinical isolates of E. coli were recovered from urine specimens in a tertiary-care hospital in Busan in 2005. Antimicrobial susceptibility was determined by disk diffusion and agar dilution methods, ESBL production was confirmed using the double-disk synergy (DDS) test, and antimicrobial resistance genes were detected by direct sequencing of PCR amplification products. E. coli isolates were classified into four phylogenetic biotypes according to the presence of chuA, yjaA, and TSPE4. RESULTS: DDS testing detected ESBLs in 27 (10.2%) of the 264 isolates. The most common type of ESBL was CTX-M-15 (N=14), followed by CTX-M-3 (N=8) and CTX-M-14 (N=6). All of the ESBL-producing isolates were resistant to ciprofloxacin. PCR experiments detected genes encoding DHA-1 and CMY-10 AmpC beta-lactamases in one and two isolates, respectively. Also isolated were 5 isolates harboring 16S rRNA methylases, 2 isolates harboring Qnr, and 19 isolates harboring AAC(6')-Ib-cr. Most ESBL-producing isolates clustered within phylogenetic groups B2 (N=14) and D (N=7). CONCLUSION: CTX-M enzymes were the dominant type of ESBLs in urinary isolates of E. coli, and ESBL-producing isolates frequently contained other antimicrobial resistance genes. More than half of the urinary E. coli isolates harboring CTX-M enzymes were within the phylogenetic group B2.

The Emerging Strong Among Extended-Spectrum beta-Lactamases: CTX-M Enzymes / 병원감염관리

CTX-M-type extended-spectrum beta-lactamases (ESBLs) are the most wide spread enzymes among non-TEM and non-SHV plamid-mediated ESBLs, and have been found predominantly in Escherichia coli. CTX-M ESBLs have a wide substrate range, including penicillins and narrow- and expanded-spectrum cephalosporins, and as the designation "CTX" refers, these enzymes preferentially hydrolyze cefotaxime but not ceftazidime. At present, the CTX-M family comprises more than 60 enzymes that can be subclassified into 5 clusters by amino acid sequence similarities. In Korea, members of CTX-M-1 (CTX-M-3, CTX-M-12, CTX-M-15, and CTX-M-54) and CTX-M-9 (CTX-M-9 and CTX-M-14) clusters have been found. The rapid dissemination of CTX-M ESBLs involves strain or plasmid epidemics, but it also involves mobile elements including ISEcp1-like insertion sequences and ISCR1 element. A recent report shows that the blaCTX-M-14 gene from Korea is associated with not only ISEcp1-like insertion sequences but also ISCR1 element. ISCR1 element is a powerful genetic tool that can mobilize antibiotic resistance genes; therefore, further spread of the blaCTX-M-14 gene can be anticipated.

The Emerging Strong Among Extended-Spectrum beta-Lactamases: CTX-M Enzymes / 병원감염관리

CTX-M-type extended-spectrum beta-lactamases (ESBLs) are the most wide spread enzymes among non-TEM and non-SHV plamid-mediated ESBLs, and have been found predominantly in Escherichia coli. CTX-M ESBLs have a wide substrate range, including penicillins and narrow- and expanded-spectrum cephalosporins, and as the designation "CTX" refers, these enzymes preferentially hydrolyze cefotaxime but not ceftazidime. At present, the CTX-M family comprises more than 60 enzymes that can be subclassified into 5 clusters by amino acid sequence similarities. In Korea, members of CTX-M-1 (CTX-M-3, CTX-M-12, CTX-M-15, and CTX-M-54) and CTX-M-9 (CTX-M-9 and CTX-M-14) clusters have been found. The rapid dissemination of CTX-M ESBLs involves strain or plasmid epidemics, but it also involves mobile elements including ISEcp1-like insertion sequences and ISCR1 element. A recent report shows that the blaCTX-M-14 gene from Korea is associated with not only ISEcp1-like insertion sequences but also ISCR1 element. ISCR1 element is a powerful genetic tool that can mobilize antibiotic resistance genes; therefore, further spread of the blaCTX-M-14 gene can be anticipated.

Emergence of CTX-M-12 and A Novel CTX-M Type Extended-Spectrum beta-Lactamaseproducing Klebsiella pneumoniae / 대한진단검사의학회지

BACKGROUND: The aims of this study were to survey the nation-wide susceptibilities of Klebsiella pneumoniae isolates against ceftazidime and cefotaxime and to determine the prevalence of class A extended-spectrum beta-lactamases (ESBLs). METHODS: During the period of February to July 2004, K. pneumoniae isolates intermediate or resistant to ceftazidime and/or cefotaxime were collected from 12 hospitals in Korea. Antimicrobial susceptibilities were determined by the disk diffusion and the agar dilution methods and ESBL-production was by double-disk synergy test. Ceftazidime or cefotaxime-resistance determinants of the ESBLproducers were transfered to Escherichia coli J53 by transconjugation. Searches for class A ESBL genes were performed by PCR amplication. RESULTS: Among 212 clinical K. pneumoniae isolates, 172 (81%) isolates showed positive results in double-disk synergy test; the most prevalent ESBL was SHV-12 (n=104). Genes encoding ESBLs including SHV-2 (n=6), SHV-2a (n=17), CTX-M-3 (n=18), CTX-M-9 (n=6), CTX-M-12 (n=1), CTX-M- 14 (n=27), CTX-M-15 (n=3), and a novel CTX-M-type beta-lactamases were also detected. CONCLUSIONS: It is concluded that diversity of ESBLs in K. pneumoniae isolates are increasing in Korea. CTX-M-12 has never been reported in Asia, and a novel CTX-M-type ESBL has emerged.