In this study, we investigated the molecular characteristics of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae isolates that were recovered from an outbreak in a Korean hospital. A new multilocus sequence typing (MLST) scheme for K. pneumoniae based on five housekeeping genes was developed and was evaluated for 43 ESBL-producing isolates from an outbreak as well as 38 surveillance isolates from Korea and also a reference strain. Overall, a total of 37 sequence types (STs) and six clonal complexes (CCs) were identified among the 82 K. pneumoniae isolates. The result of MLST analysis was concordant with that of pulsedfield gel electrophoresis. Most of the outbreak isolates belonged to a certain clone (ST2), and they produced SHV-1 and CTX-M14 enzymes, which was a different feature from that of the K. pneumoniae isolates from other Korean hospitals (ST20 and SHV-12). We also found a different distribution of CCs between ESBL-producing and -nonproducing K. pneumoniae isolates. The MLST method we developed in this study could provide unambiguous and well-resolved data for the epidemiologic study of K. pneumoniae. The outbreak isolates showed different molecular characteristics from the other K. pneumoniae isolates from other Korean hospitals.
Electrophoresis, Gel, Pulsed-Field ; Hospitals ; Humans ; Klebsiella pneumoniae/*classification/enzymology/genetics/isolation & purification ; Sequence Analysis, DNA ; beta-Lactamases/*biosynthesis ; Mycobacteria, Atypical/*drug effects/genetics/isolation & purification

BACKGROUNDIn the present study, we characterized multidrug-resistant Pseudomonas aeruginosa (MDRP) clinical isolates from a paediatric facility and investigated the types and features of the metallo-beta-lactamases (MBLs) produced by carbapenem-resistant strains.

METHODSFour hundred and ninety-eight strains of Pseudomonas aeruginosa were isolated from patients at Beijing Children's Hospital between January 2005 and December 2006. The minimal inhibition concentrations (MICs) of the strains for 13 antibiotics were measured. A combination of the E test and PCR amplification/DNA sequencing was used to define the carbapenem-resistant strains.

RESULTSWe found that 24.1% (120/498) of the isolates were MDRP. The frequencies of resistance to imipenem and meropenem were 34.2% and 35.8%, respectively, and the MIC50 and MIC90 values for the two antibiotics were identical at 4 microg/ml and 32 microg/ml, respectively. The detection rate for carbapenem resistance was 49.2% (59/120). Among the 59 carbapenem-resistant Pseudomonas aeruginosa strains, 39 (66.1%) were positive for the MBL genotype; 35 (89.7%) strains carried the bla(IMP) gene and 4 (10.3%) strains carried the bla(VIM) gene. Neither bla(SPM) nor bla(GIM) was amplified from any of the 59 isolates. DNA sequencing revealed that IMP-1 was present in 35 IMP-producing isolates and VIM-2 was detected in four VIM-producing isolates.

CONCLUSIONSThese MDRP isolates exhibited high frequencies of resistance to carbapenems among clinical isolates from a paediatric facility in Beijing, China. The production of MBL appears to be an important mechanism for carbapenem resistance in Pseudomonas aeruginosa.

Carbapenems ; pharmacology ; Child ; Drug Resistance, Multiple, Bacterial ; Humans ; Microbial Sensitivity Tests ; Pseudomonas aeruginosa ; drug effects ; enzymology ; Sequence Analysis, DNA ; beta-Lactamases ; biosynthesis ; classification ; genetics

BACKGROUND: Concomitant quinolone resistance in extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is a crucial problem in the clinical management of infections. In foreign countries, the fluoroquinolone acetylating aminoglycoside-(6)-N-acetyltransferase (aac[6']-Ib-cr) gene, a novel plasmid-mediated quinolone resistance determinant has been reported to occur in conjunction with qnr. We aim to investigate the prevalence and characteristics of concomitant aac(6')-Ib-cr and qnr expression in ESBL-producing Escherichia coli and Klebsiella pneumoniae in Korea. METHODS: Between December 2007 and April 2008, we collected 60 and 69 clonally unrelated non-repetitive clinical isolates of ESBL-producing E. coli and K. pneumoniae, respectively. We studied the expressions of 11 types of ESBL-encoding genes, 4 types of 16s rRNA methylase genes; rmtA, rmtB, rmtC and armA, 3 types of qnr genes; qnrA, qnrB, qnrS and aac(6')-Ib. The presence of aac(6')-Ib-cr variants was detected by sequencing. The involvement of integrons was studied using multiplex PCR and sequencing of gene-cassette arrays. Conjugation experiments were performed to confirm plasmid-mediated resistance and the relationships among coharbored genes. RESULTS: We observed a high prevalence of the cr variant (61.1%) of aac(6')-Ib, and the prevalence of this variant in qnr and aac(6')-Ib-coharboring isolates (67.4%) was higher than in qnr-negative isolates (51.7%). The high prevalence of the cr variant was significantly related to the high minimum inhibitory concentrations (MICs) of ciprofloxacin, tobramycin, and amikacin and indicated the statistically significant roles of qnrB, qnrS, rmtA, and rmtB in quinolone and aminoglycoside resistance. CONCLUSIONS: The aac(6')-Ib-cr variants were widespread and showed significant relation to the high-level quinolone and aminoglycoside resistance in ESBL-producing E. coli and K. pneumoniae.
Acetyltransferases/*genetics ; Drug Resistance, Bacterial/*genetics ; Escherichia coli/enzymology/*genetics ; Genes, Bacterial/genetics ; Klebsiella pneumoniae/enzymology/*genetics ; Microbial Sensitivity Tests ; Phenotype ; Republic of Korea ; Sequence Analysis, DNA ; beta-Lactamases/*biosynthesis

BACKGROUND: Since metallo-beta-lactamase (MBL)-producing isolates can hydrolyze carbapenem and also easily transfer the resistance genes to other bacteria, a rapid and accurate detection of MBL has become very important. We evaluated the utility of Mueller Hinton agar (MHA) biplate containing dipicolinic acid (DPA) as a screening method to detect IMP-1 and VIM-2 type MBL-producing isolates. METHODS: Based on our preliminary tests using various concentrations of DPA, 200 and 300 microg/mL concentration of DPA were chosen for further study. Bacterial lawns were grown on MHA biplate, one half of which contained DPA while the other did not. The inhibition zone around the imipenem (IPM) disk on both sides of this plate was compared. The stability of DPA in the stored DPA-MHA biplate was also evaluated during three months using two MBL- and one non-MBL-producing isolates. RESULTS: When the criterion of a > or =7 mm increase of inhibition zone around the IPM disk on the MHA containing DPA compared to MHA without DPA was used, the sensitivities and specificities were 94.7% and 97.6% for 200 microg/mL DPA-MHA biplate, and 98.2% and 97.6% for 300 microg/mL DPA-MHA biplate, respectively. The activity of the DPA in this biplate was stable for three months. CONCLUSIONS: Assays using DPA 300-MHA biplate were highly sensitive and specific for the detection of IMP-1 and VIM-2 type MBL-producing bacteria. In addition, it is easy to perform; so, it may be useful to apply this method for detection of IMP-1 and VIM-2 type MBL in clinical laboratories.
Agar ; Anti-Bacterial Agents/*pharmacology ; Bacteriological Techniques ; Chelating Agents/chemistry/*pharmacology ; Drug Resistance, Bacterial ; Gram-Negative Bacteria/drug effects/enzymology/*isolation & purification ; Imipenem/*pharmacology ; Picolinic Acids/chemistry/*pharmacology ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; beta-Lactamases/*analysis/biosynthesis

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.
Acinetobacter Infections/microbiology ; Acinetobacter baumannii/*genetics/isolation & purification ; Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/genetics ; DNA Gyrase/genetics ; DNA, Bacterial/chemistry/genetics ; Drug Resistance, Bacterial/*genetics ; Humans ; Imipenem/*pharmacology ; Integrons/genetics ; Methyltransferases/genetics ; Microbial Sensitivity Tests ; Mutation ; Polymerase Chain Reaction ; Republic of Korea ; Sequence Analysis, DNA ; beta-Lactamases/biosynthesis/genetics

Carbapenem-resistant Klebsiella pneumoniae isolates producing K. pneumoniae carbapenemases (KPC) were first reported in the USA in 2001, and since then, this infection has been reported in Europe, Israel, South America, and China. In Korea, the first KPC-2-producing K. pneumoniae sequence type (ST) 11 strain was detected in 2010. We report the case of a patient with a urinary tract infection caused by KPC-2-producing K. pneumoniae. This is the second report of a KPC-2-producing K. pneumoniae infection in Korea, but the multilocus sequence type was ST258. The KPC-2-producing isolate was resistant to all tested beta-lactams (including imipenem and meropenem), amikacin, tobramycin, ciprofloxacin, levofloxacin, and trimethoprim-sulfamethoxazole, but was susceptible to gentamicin, colistin, polymyxin B, and tigecycline. The KPC-2-producing isolate was negative to phenotypic extended-spectrum beta-lactamase (ESBL) and AmpC detection tests and positive to modified Hodge test and carbapenemase inhibition test with aminophenylboronic acid.
Aged ; Bacterial Proteins/antagonists & inhibitors/metabolism ; Carbapenems/pharmacology ; Drug Resistance, Bacterial/genetics ; Female ; Humans ; Klebsiella pneumoniae/drug effects/genetics/*isolation & purification ; Microbial Sensitivity Tests ; Republic of Korea ; Sequence Analysis, DNA ; Urinary Tract Infections/*diagnosis/microbiology ; beta-Lactamases/antagonists & inhibitors/biosynthesis/*genetics/metabolism