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

BACKGROUND: The emergence of carbapenem-resistant Klebsiella pneumoniae poses a serious problem to antibiotic management. We investigated the beta-lactamases in a group of carbapenem-resistant K. pneumoniae clinical isolates from Turkey. METHODS: Thirty-seven strains of K. pneumoniae isolated from various clinical specimens were analyzed by antimicrobial susceptibility testing, PCR for the detection of beta-lactamase genes, DNA sequencing, and repetitive extragenic palindronic (REP)-PCR analysis. RESULTS: All 37 isolates were resistant to ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, ceftazidime, cefoperazone/sulbactam, cefepime, imipenem, and meropenem. The lowest resistance rates were observed for colistin (2.7%), tigecycline (11%), and amikacin (19%). According to PCR and sequencing results, 98% (36/37) of strains carried at least one carbapenemase gene, with 32 (86%) carrying OXA-48 and 7 (19%) carrying NDM-1. No other carbapenemase genes were identified. All strains carried a CTX-M-2-like beta-lactamase, and some carried SHV- (97%), TEM- (9%), and CTX-M-1-like (62%) beta-lactamases. Sequence analysis of bla(TEM) genes identified a bla(TEM-166) with an amino acid change at position 53 (Arg53Gly) from bla(TEM-1b), the first report of a mutation in this region. REP-PCR analysis revealed that there were seven different clonal groups, and temporo-spatial links were identified within these groups. CONCLUSIONS: Combinations of beta-lactamases were found in all strains, with the most common being OXA-48, SHV, TEM, and CTX-M-type (76% of strains). We have reported, for the first time, a high prevalence of the NDM-1 (19%) carbapenemase in carbapenem-resistant K. pneumoniae from Turkey. These enzymes often co-exist with other beta-lactamases, such as TEM, SHV, and CTX-M beta-lactamases.
Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/*genetics/metabolism ; Carbapenems/*pharmacology ; DNA, Bacterial/chemistry/genetics/metabolism ; Drug Resistance, Bacterial ; Genotype ; Humans ; Klebsiella Infections/diagnosis/microbiology ; Klebsiella pneumoniae/*drug effects/enzymology/isolation & purification ; Microbial Sensitivity Tests ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Turkey ; beta-Lactamases/*genetics/metabolism

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

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

The prevalence of antibiotic resistance is higher in patients undergoing renal replacement therapy (RRT) than in patients who did not undergo RRT. We investigated the presence of KP (Klebsiella pneumoniae) in patients who underwent RRT. All data were collected retrospectively by accessing patient medical records from 2004 to 2011 for the culture results of all patients who were positive for KP. We grouped the patients by the presence of extended-spectrum beta-lactamase (ESBL) into a KP ESBL(-) group (KP[-]) and a KP ESBL(+) group (KP[+]). In total, 292 patients (23.1%) were in the KP(+) group, and 974 patients (76.9%) were in the KP(-) group. A greater percentage of KP(+) was found in patients who underwent RRT (7.5%) than in patients who did not undergo RRT (3.2%) (OR, 2.479; 95% CI,1.412-4.352). A Cox's hazard proportional model analysis was performed, and for patients with pneumonia, the risk of KP(+) was 0.663 times higher in patients who had lower albumin levels, 2.796 times higher in patients who had an inserted Levin tube, and 4.551 times higher in patients who underwent RRT. In conclusion, RRT can be a risk factor for KP(+) in patients with pneumonia.
Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Kaplan-Meier Estimate ; Kidney Failure, Chronic/*microbiology/therapy ; Klebsiella Infections/*epidemiology/microbiology/mortality ; Klebsiella pneumoniae/*enzymology/isolation & purification ; Male ; Middle Aged ; Odds Ratio ; Pneumonia/diagnosis/*epidemiology/microbiology ; Prevalence ; Proportional Hazards Models ; Renal Replacement Therapy ; Retrospective Studies ; Risk Factors ; Serum Albumin/analysis ; beta-Lactamases/*metabolism

We investigated the occurrence and genetic basis of AmpC beta-lactamase (AmpC)-mediated antibiotic resistance, by examining Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a university hospital, from 2007 to 2010. The ampC genes were detected by multiplex AmpC PCR, and AmpC-positive strains were subjected to DNA sequencing. Extended-spectrum beta-lactamase (ESBL) production was assessed using the ESBL disk test based on the utilization of boronic acid. Carbapenem-resistant isolates were further investigated by the modified Hodge test, a carbapenemase inhibition test and SDS-PAGE experiments. AmpC expression was detected in 1.6% of E. coli (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, 7.2% of K. pneumoniae (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, and 2.5% of P. mirabilis (8 CMY-2 and 1 CMY-1) isolates. Of the 198 acquired AmpC producers, 58 isolates (29.3%) also produced an ESBL enzyme. Among the acquired AmpC-producing K. pneumoniae isolates, the minimum inhibitory concentration (MIC) MIC50/MIC90 values for cefoxitin, cefotaxime, cefepime, imipenem, and meropenem were >32/>32, 16/>32, 1/16, 0.25/0.5, and <0.125/0.125 microg/mL, respectively. The MIC values for carbapenem were > or =2 microg/mL for 2 K. pneumoniae isolates, both of which carried the blaDHA-1 gene with a loss of OmpK36 expression, but were negative for carbapenemase production. The acquisition of AmpC-mediated resistance in K. pneumoniae isolates increased, as did the proportion of AmpC and ESBL co-producers among the hospital isolates. The accurate identification of isolates producing AmpCs and ESBLs may aid in infection control and will assist physicians in selecting an appropriate antibiotic regimen.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*genetics ; DNA, Bacterial/genetics ; Enterobacteriaceae Infections/*epidemiology/*microbiology ; Escherichia coli/drug effects/enzymology/isolation & purification ; Hospitals, University/statistics & numerical data ; Humans ; Klebsiella pneumoniae/drug effects/enzymology/isolation & purification/*physiology ; Microbial Sensitivity Tests ; Multiplex Polymerase Chain Reaction ; Proteus mirabilis/drug effects/enzymology/isolation & purification ; Republic of Korea/epidemiology ; beta-Lactamases/*genetics

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

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

BACKGROUND: Accurate and rapid detection of extended-spectrum beta-lactamases (ESBLs) is important in guiding proper antimicrobial therapy for infected patients. We evaluated the performance of MicroScan NegCombo Type 44 panel (Dade Behring, USA), which was developed to confirm ESBL-producing Enterobacteriaceae using ceftazidime/clavulanate and cefotaxime/clavulanate. METHODS: From August 30 to September 20, 2007, 206 non-duplicate clinical isolates, including 106 Escherichia coli, 81 Klebsiella pneumoniae, 11 Klebsiella oxytoca, and 8 Proteus mirabilis were subcultured and tested with Type 32 and Type 44 panels. The results were compared with those of the CLSI phenotypic confirmatory test (CLSI-PCT) and disk approximation test (DAT). Isolates not susceptible to cefotetan or flagged as "Possible ESBL, unable to interpret confirm test (Possible ESBL)" on Type 44 panel were tested with boronic acid disks to confirm AmpC beta-lactamases (AmpC) production. RESULTS: Of the 206 isolates tested, 44 (21.4%) produced ESBL by CLSI-PCT or DAT, including 27 E. coli, 14 K. pneumoniae, 2 K. oxytoca, and 1 P. mirabilis. Thirty-eight isolates flagged as "Confirmed ESBL" on Type 44 panel were all confirmed as ESBL-producers. Of 14 K. pneumoniae flagged as "Possible ESBL", 6 were confirmed as ESBL and AmpC co-producers and 8 as AmpC-producers. CONCLUSIONS: Type 44 panel showed an excellent performance in detecting ESBL-producing E. coli, Klebsiella spp., and P. mirabilis. When flagged as "Confirmed ESBL", no other confirmatory test was necessary to report as ESBL; however, "Possible ESBL" required a differential test for AmpC production.
Bacterial Proteins/*biosynthesis ; Cefotetan/pharmacology ; Disk Diffusion Antimicrobial Tests ; Drug Resistance, Bacterial ; Escherichia coli/*enzymology/isolation & purification ; Humans ; Klebsiella/*enzymology/isolation & purification ; Proteus mirabilis/*enzymology/isolation & purification ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; 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