OBJECTIVETo investigate the possible differences in antimicrobial resistance of Escherichia coli isolated from different samples in children.

METHODSSix hundred and twenty-nine samples from urine, sputum, blood and secretion were collected from June 2004 to May 2009 for bacterial identification by VITEK-32 automatic system and antimicrobial susceptibility tests by Kirby-Bauer method. The drug resistance rate of Escherichia coli isolated from different samples was compared.

RESULTSTwo hundred and sixty strains of Escherichia coli were isolated , and 108 of which were from urine , 64 from sputum, 54 from secretion and 23 from blood. ESBLs were detected in 96 (36.9%) of the 260 isolates, AmpC enzymes in 32 (12.3%), and ESBLs+AmpC in 8 (3.1%). The ESBLs positive rate of Escherichia coli isolates from sputum was significantly higher than that from other samples (P<0.05). The antimicrobial resistance rate of Escherichia coli strains from different samples to amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, piperacillin, cefotaxime, cefuroxime, cefepime, gentamicin, cotrimoxazole, and nitrofurantoin was different. The resistance rate of the strains from sputum samples was higher than that from the other samples (P<0.05).

CONCLUSIONSEscherichia coli isolated from different samples have different antimicrobial resistance rates in children, so the selection of antibiotics for infections confirmed by bacterial cultures from different samples should based on drug sensitivity results.

Adolescent ; Child ; Child, Preschool ; Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; enzymology ; isolation & purification ; Female ; Humans ; Male ; beta-Lactamases ; analysis

OBJECTIVETo study the genotype distribution of extended-spectrum beta-lactamases (ESBLs) in ESBLs-producing Escherichia coli (E. coli) isolates from posthepatitic cirrhosis' patients with bloodstream infection.

METHODSE. coli were isolated in bloodstream from patients with posthepatitic cirrhosis between January and December in 2011. The strains were identified by VITEK-II. The antibiol susceptibility tests were performed with K-B method. beta-lactamases genes were detected multi-PCR, PCR, sequence and blast.

RESULTSA total of 79 non-duplicate clinical isolates of E coli were consecutively collected from liver cirrhosis' patients with bloodstream infection. There were 20 isolates produced TEM-1 type beta-lactamases and 1 isolate produced SHV-1 typebeta-lactamases. 40 clinical isolates were detected to produce CTX-M type ESBLs, there were 20 CTX-M-1 group and 26 CTX-M-9 group, including 6 stains habouring both CTX-M-1 and CTX-M-9 group. Eight CTX-M genotypes were confirmed by sequencing of the PCR products, including CTX-M-3, CTX-M-14, CTX-M-15, CTX-M-24, CTX-M-28, CTX-M-31, CTX-M-65 and CTX-M-79.

CONCLUSIONCTX-M genotype ESBLs was the most popular extended-spectrum beta-lactamases in E. coli isolated from liver cirrhosis' patients with bloodstream infection. The CTX-M-14 is the dominant epidemic type.

Bacteremia ; microbiology ; Cross Infection ; microbiology ; Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; enzymology ; genetics ; isolation & purification ; Escherichia coli Infections ; microbiology ; Escherichia coli Proteins ; genetics ; Genotype ; Hospitalization ; statistics & numerical data ; Humans ; Liver Cirrhosis ; therapy ; Microbial Sensitivity Tests ; beta-Lactamases ; genetics ; 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

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

The cDNA coding spinach glycolate oxidase (GO) was amplified by RT-PCR using the total RNA of spinach leaves as the template, and was cloned into cloning vector pMD18-T. After the DNA sequence was determined, the go gene was subcloned into E. coli expression vector pBV220, pET-22b(+), pTIG-Trx and pThioHisC. SDS-PAGE analysis revealed that the recombinant E. coli BL21 (DE3) (pTIG-Trx-GO) and E. coli BL21 (DE3) (pET-22b(+)-GO) expressed the predicted 38 kD glycolate oxidase, and the enzyme activity was also detected.
Alcohol Oxidoreductases ; genetics ; isolation & purification ; metabolism ; Cloning, Molecular ; Escherichia coli ; Gene Expression ; drug effects ; Isopropyl Thiogalactoside ; pharmacology ; Spinacia oleracea ; enzymology ; genetics

LysinB (LysB) in mycobacteriophage D29 was cloned and expressed and its enzymatic properties were analysed. The lysB gene was amplified by PCR from mycobacteriophage D29 genomic DNA and inserted into pET22b vector. The constructed recombinant plasmid was transformed into Escherichia coli BL21(DE3) to express fusion protein, which was purified by Ni-NTA column and enzymatic activity detected. The results showed that expression plasmid pET22b-lysB was constructed successfully. Highly purified recombination protein (His-LysB) was obtained 33.2 mg from 1 L LB culture medium. A screening for His-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. With p-nitrophenyl butyrate as substrate, the thermal stability of the enzyme was poor when the temperature was above 30 degrees C. The enzyme exhibited higher stability at pH 5.0-9.5. The optimum temperature and pH for the lipolytic activity of His-LysB were 23 degrees C and 7.5 respectively. Under the optimum conditions, the specific activity of His-LysB was 1.3 U/mg. Zn2+, CU2+, Mg2+, Mn2+ and phenylmethane sulfonyl fruoride severely inhibited the lipolytic activity of His-LysB. The result provides a new option for tuberculosis drug research and development.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Mycobacteriophages ; enzymology ; Mycobacterium tuberculosis ; drug effects ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Viral Proteins ; biosynthesis ; genetics

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

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

INTRODUCTIONAntibiotic resistance in gram-negative bacilli is an area of increasing importance. This prospective study was performed to survey antibiotic resistance in Escherichia coli (E. coli), Klebsiella spp., Pseudomonas aeruginosa and Acinetobacter spp. over a 1-year period.

MATERIALS AND METHODSNon-duplicate isolates of E. coli, Klebsiella spp., P. aeruginosa and Acinetobacter spp. were collected from participating Singapore hospitals during defined collection periods in 2006 and 2007. Confirmatory identification and antibiotic susceptibility testing were performed at Changi General Hospital. Minimum inhibitory concentrations (MIC) to a defined panel of antibiotics were determined using microbroth dilution methods. The presence of extended-spectrum beta lactamases and AmpC beta-lactamases in Enterobacteriaceae was determined by phenotypic methods, and susceptibility results were defined using current breakpoints from the Clinical Laboratory Standards Institute (CLSI).

RESULTSSeven hundred and forty-six gram-negative bacilli were received for testing. Resistance to extended-spectrum cephalosporins was present in a third of Enterobacteriaceae isolates, and extended-spectrum beta-lactamases (ESBL) carriage was present in 19.6% and 30.1% of E. coli and Klebsiella pneumoniae, respectively. AmpC enzymes were also detected in 8.5% and 5.6% of E. coli and K. pneumoniae isolates respectively. All Enterobacteriaceae were susceptible to imipenem and meropenem. The most active antibiotics against P. aeruginosa were amikacin, meropenem and piperacillin-tazobactam. A third of P. aeruginosa showed reduced susceptibility to polymyxin B. Carbapenem resistance was significantly higher in Acinetobacter baumannii (70.5%) than in other Acinetobacter species (25.0%). The most active antibiotic against A. baumannii was polymyxin B.

CONCLUSIONAntibiotic resistance is prevalent in gram-negative bacilli isolated from Singapore hospitals. The MIC testing surveillance programme complemented susceptibility data from wider laboratory-based surveillance, and has revealed emerging mechanisms of antibiotic resistance.

Acinetobacter Infections ; drug therapy ; Acinetobacter baumannii ; drug effects ; isolation & purification ; Anti-Bacterial Agents ; pharmacology ; therapeutic use ; Bacterial Proteins ; Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; enzymology ; Hospitals ; Humans ; Klebsiella Infections ; drug therapy ; Klebsiella pneumoniae ; drug effects ; enzymology ; Microbial Sensitivity Tests ; Prospective Studies ; Pseudomonas aeruginosa ; drug effects ; isolation & purification ; Singapore ; beta-Lactamases

The argE gene from Escherichia coli coding for N-acety-L-ornithine deacetylase(NAOase), the key enzyme involved in the L-arginine biosynthesis, had been cloned in pET22b and transformed into BL21 (DE3). With 32.5% expression level in the optimal fermentation medium at 37 degrees C, most NAOase was expressed as inclusion bodies. The soluble and active proportion could be slightly increased when expressed at low temperature. The specific activity of soluble NAOase purified by Ni-NTA resin chromatography was 1193.2u/mg. The species and proportions of whole cell proteins varied with induction conditions. The inclusion bodies expressed at 37 degrees C was more pure than 22 degrees C after gradient wash with urea. Inclusion bodies could be partly refolding and reactivated by dilution and dialysis. Low protein concentration and suitable rate of oxidant/reducing agents were important to renaturation. In the optimal conditions 17.78% of Urea-denatured NAOase could be refolding and reactivated by dilution. The purified fusion protein was obtained after wash, solubilization and Ni-NTA resin affinity chromatography purification of inclusion bodies.
Amidohydrolases ; chemistry ; genetics ; metabolism ; Biocatalysis ; drug effects ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; enzymology ; genetics ; Escherichia coli Proteins ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Gene Expression Regulation, Enzymologic ; Inclusion Bodies ; enzymology ; Protein Folding ; drug effects ; Recombinant Proteins ; chemistry ; isolation & purification ; metabolism ; Urea ; pharmacology