Klebsiella pneumoniae ; enzymology ; isolation & purification ; Microbial Sensitivity Tests ; beta-Lactamases ; classification

No abstract available.
Aged ; Female ; Humans ; Klebsiella Infections/*diagnosis/microbiology ; Klebsiella pneumoniae/*enzymology/isolation & purification ; Pneumonia/*diagnosis/microbiology ; Turkey ; beta-Lactamases/*metabolism

BACKGROUND: The aim of this study was to compare the BD Phoenix (Beckton Dickinson Diagnostic Systems, USA) extended-spectrum beta-lactamase (ESBL) test with the Clinical and Laboratory Standards Institute (CLSI) ESBL phenotypic confirmatory test by disk diffusion (CLSI ESBL test) in Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca and Proteus mirabilis. METHODS: We tested 224 clinical isolates of E. coli, K. pneumoniae, K. oxytoca and P. mirabilis during May 2006 to March 2007. These isolates were examined by the Phoenix and the CLSI ESBL tests simultaneously. For the isolates showing discordant results between the two tests, boronic acid disk test was performed to differentiate AmpC beta-lactamase and ESBL. RESULTS: Among the 224 clinical isolates, 75 and 79 isolates were positive for ESBL by CLSI ESBL test and Phoenix test, respectively. Having detected 4 more isolates as ESBL-producers, Phoenix test showed a 98.2% agreement with a 100% sensitivity and 97.3% specificity compared with CLSI ESBL test. Among the four false positive isolates, three were AmpC-positive but ESBL-negative. CONCLUSIONS: The BD Phoenix ESBL test was sensitive and specific, and can be used as a rapid and reliable method to detect ESBL production in E. coli, Klebsiella species, and P. mirabilis.
Automation ; Bacterial Proteins/classification/*metabolism ; Disk Diffusion Antimicrobial Tests ; Escherichia coli/drug effects/*enzymology/isolation & purification ; Humans ; Klebsiella/*enzymology ; Klebsiella oxytoca/drug effects/enzymology/isolation & purification ; Klebsiella pneumoniae/drug effects/enzymology/isolation & purification ; *Microbial Sensitivity Tests ; Proteus mirabilis/drug effects/*enzymology/isolation & purification ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; beta-Lactamases/classification/*metabolism

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

Ethyl carbamate (EC) is a carcinogenic substance in many fermented foods. Enzymatic removal of ethyl carbamate from fermented foods is an important way to eliminate its potential health damage to consumers. To study the enzymatic properties of an ethyl carbamate hydrolase (urethanase) from Klebsiella pneumoniae, a strain isolated from murine somach, we purified the enzyme using ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The molecular mass of this enzyme was estimated to be 55 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its K(m) was 74 mmol/L when EC was used as the substrate. Moreover, its optimal reaction temperature was 55 degrees C, and the optimum pH was 7.0. The activity was enhanced by ethylene diamine tetraacetic acid (EDTA) and dithiothreitol (DTT), but strongly inhibited by Cu2+ and Zn2+. The enzyme was halophilic and tolerant to low concentration of ethanol. Therefore, it has the potential to remove EC from fermented foods.
Amidohydrolases ; chemistry ; isolation & purification ; Bacterial Proteins ; chemistry ; isolation & purification ; Chromatography, Gel ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Hydrogen-Ion Concentration ; Klebsiella pneumoniae ; enzymology ; Molecular Weight ; Substrate Specificity ; Temperature

The gldA gene coding glycerol dehydrogenase (GDH) was amplified by PCR with the genomic DNA of Klebsiella pneumoniae as the template. The gldA were inserted in pMD-18T to construct the recombinant cloning vector pMD-gldA. After the DNA sequence was determined, the gldA was subcloned into expression vector pET-32a (+) to construct the recombinant expression vector pET-32gldA. Upon lactose induction, soluble GDH was over-produced by E. coli BL21 (DE3) harboring the expression construct. Recombinant GDH purified by Ni-NTA affinity chromatography showed a single band about 54 kD on SDS-PAGE gel, and the specified activity was about 188 u/mg, the purification fold is 3 times and the activity recovery is 67.5%.
Chromatography, Affinity ; Cloning, Molecular ; DNA, Bacterial ; genetics ; Escherichia coli ; genetics ; isolation & purification ; metabolism ; Klebsiella pneumoniae ; enzymology ; genetics ; Sugar Alcohol Dehydrogenases ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo identify risk factors for Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) colonization in neonates hospitalized in the neonatal intensive care unit (NICU).

METHODSA case-control study was conducted. The case group included nine patients colonized with KPC-Kp between 1 August 2012 and 31 April 2013 and the controls were selected randomly from patients without KPC-Kp colonization during the same period. Univariable analysis and multivariable logistic regression analysis were conducted to identify risk factors for KPC-Kp colonization.

RESULTSThe univariable analysis showed 11 factors associated with KPC-Kp colonization: gestational age, birth weight, length of hospital stay, duration of mechanical ventilation, congenital heart disease, peripherally inserted central catheter, surgical operation, duration of intravenous nutrition, carbapenems use, duration of carbapenems use and glycopeptides use. The multivariable logistic regression analysis showed that exposure to more than 4 days of carbapenems use (OR=18.7, 95%CI: 1.98-175.5, P=0.01) was an independent risk factor for KPC-Kp colonization. The intervention to control KPC-Kp colonization included contact isolation, active surveillance, and rational use of antibiotics.

CONCLUSIONSExposure to prolonged use of carbapenems is an independent risk factor for the development of KPC-Kp colonization in neonates hospitalized in the NICU.

Bacterial Proteins ; biosynthesis ; Carbapenems ; adverse effects ; Female ; Humans ; Infant, Newborn ; Intensive Care Units, Neonatal ; Klebsiella pneumoniae ; enzymology ; isolation & purification ; Logistic Models ; Male ; Risk Factors ; beta-Lactamases ; biosynthesis

OBJECTIVETo study the prevalence and drug resistance of extended-spectrum-β-lactamases (ESBLs)-producing bacteria in blood culture isolated from children with hematological malignancy after chemotherapy.

METHODSBlood samples taken from 3264 children with hematological malignancy and severe infection following chemotherapy between 2002 and 2008 were cultured using the Bact/ALTER 3D blood culture system. VITEK 60 automicroscan was used to identify viral species and to conduct drug resistance tests. The results were indentified according to National Committee for Clinical Laboratory Standard guidelines.

RESULTSFifty-eight strains of Escherichia coli and fifty-one strains of Klebsiella pneumoniae were isolated. Thirty-eight strains of Escherichia coli and nineteen strains of Klebsiella pneumoniae were ESBLs-producing and these ESBLs-producing strains were less susceptible than those that were non-ESBLs-producing to most antibiotics. Both ESBL- and non-ESBL-producing strains were susceptible to imipenem, piperacillin/tazobactam and amikacin.

CONCLUSIONSThe prevalence of ESBLs-producing bacteria is high in childrn with hematological malignancy and infection following chemotherapy. ESBLs-producing bacteria are resistant to common antibiotics, suggesting that antibiotic treatment based on the result of antimicrobial susceptibility test is necessary in these children.

Adolescent ; Bacteremia ; microbiology ; Child ; Child, Preschool ; Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; enzymology ; isolation & purification ; Female ; Hematologic Neoplasms ; drug therapy ; microbiology ; Humans ; Klebsiella pneumoniae ; drug effects ; enzymology ; isolation & purification ; Male ; Microbial Sensitivity Tests ; beta-Lactamases ; biosynthesis

PURPOSE: Coexistence of different classes of beta-lactamases in a single bacterial isolate may pose diagnostic and therapeutic challenges. We investigated a spread of Klebsiella pneumoniae isolates co-producing an AmpC beta-lactamase and an extended-spectrum beta-lactamase (ESBL) in a university hospital. MATERIALS AND METHODS: Over a three-month period, a total of 11 K. pneumoniae isolates, which exhibited resistance to cefotaxime, aztreonam, and cefoxitin, were isolated. These isolates showed positive to ESBLs by double disk tests. Minimal inhibitory concentrations (MICs) were determined by broth microdilution testing. All isolates were examined by isoelectric focusing, PCR and sequence analysis to identify bla(SHV) and bla(DHA), and molecular typing by pulsed-field gel electrophoresis (PFGE). RESULTS: All 11 isolates were highly resistant (MIC, > or = 128microngram/ml) to ceftazidime, aztreonam, and cefoxitin, while they were susceptible (MIC, < or = 2microngram/ml) to imipenem. The bla(SHV-12) and bla(DHA-1) genes were detected by PCR and sequence analysis. PFGE revealed a similar pattern in 10 of the 11 strains tested. CONCLUSION: This is the first outbreak report of K. pneumoniae in Korea which co-produced SHV-12 and DHA-1 beta-lactamase, and we suggest a clonal spread of multidrug-resistant K. pneumoniae at a hospital.
Adult ; Aged ; Aged, 80 and over ; *Disease Outbreaks ; Disease Susceptibility ; *Drug Resistance, Multiple, Bacterial ; Female ; Genotype ; Hospitals ; Humans ; Klebsiella Infections/*epidemiology/*microbiology ; Klebsiella pneumoniae/classification/*enzymology/genetics/isolation & purification ; Korea ; Male ; Middle Aged ; Phenotype ; beta-Lactamases/*classification/genetics/*metabolism

BACKGROUND: Extended-spectrum beta-lactamases (ESBLs) are cephalosporinases that confer resistance to a wide variety of oxyimino cephalosporins and create serious therapeutic problems. Although ESBLs have been reported with increasing frequency in Korea, their prevalence and genotypic distribution in Daejeon remain unknown. This study was designed to evaluate the occurrence and genotypic distributions of ESBL-producing Escherichia coli and Klebsiella pneumoniae in Daejeon. METHODS: We tested a total of 427 isolates of E. coli and K. pneumoniae at Chungnam National University Hospital during the period from March to September 2006. ESBL production was determined by the Clinical and Laboratory Standards Institute ESBL confirmatory test; minimum inhibitory concentrations of beta-lactam antibiotics were determined by the broth dilution method. The ceftazidime or cefotaxime resistance of the ESBL-producers was transferred to azide-resistant E. coli J53 by conjugation. Searches for ESBL genes were performed by PCR amplification, and the genotypes of ESBLs were determined by direct nucleotide sequence analysis of the amplified products. The pIs of ESBL were determined by isoelectric focusing. RESULTS: The proportion of ESBL-producers was 10% of the E. coli and 28% of the K. pneumoniae isolates. The prevalence of ESBL-positive isolates was 60% in the intensive care units and 18.7% in the general wards. The most prevalent ESBL genotype in E. coli isolates was blaCTX-M and in K. pneumoniae was blaSHV-12. CONCLUSIONS: E. coli and K. pneumoniae isolates producing SHV-12 or CTX-M-type ESBLs are widespread in Daejeon.
Anti-Bacterial Agents/therapeutic use ; Disk Diffusion Antimicrobial Tests ; Drug Resistance, Bacterial ; Escherichia coli/*drug effects/enzymology/isolation & purification ; Escherichia coli Infections/drug therapy/microbiology ; Genotype ; Humans ; Klebsiella Infections/drug therapy/microbiology ; Klebsiella pneumoniae/*drug effects/enzymology/isolation & purification ; Korea ; *beta-Lactam Resistance ; beta-Lactamases/*analysis ; beta-Lactams/therapeutic use