To produce TEM-116 extended-spectrum beta-lactamase (ESBL) from recombinant bacteria in a cost-effective way, we purified and renatured the recombinant TEM-116 ESBL from the inclusion bodies by Ni(2+)-NTA affinity and gel filtration chromatography through subcloning the bla(TEM-116) into expression vector pET28a(+), transforming into Escherichia coli BL21(DE3) and inducing with IPTG. We characterized the purified protein that had the molecular weight of 30 kDa and specific activity of 476 IU/mg. The recombinant TEM-116 ESBL showed higher efficiency in eliminating penicillin and cephalosporin in vitro and in vivo. Specifically, the recombinant TEM-116 ESBL could eliminate 7000 mg penicillin G (PG) when used at 10.0 IU in 1 L fermentation medium. When used at 320.0 IU, it could also degrade a mix of PG, ampicillin and cefazolin each at 200 mg in 1 L of urine. In milk, 1.0-2.5 IU of the recombinant enzyme could remove 80 U/L of PG. The recombinant enzyme was fully active at the temperature ranged from 4 degrees C to 37 degrees C. Furthermore, the recombinant enzyme used at 2.0x10(4)-2.3x10(4) IU/(kg bw) (body weight) eliminated 8.0x10(4)-9.1x10(4) microg/(kg bw) PG in mouse models in vivo. The recombinant TEM-116 ESBL has the potential as a tool enzyme in food and environmental protection to eliminate harmful residues of antibiotics.
Animals ; Cephalosporins ; antagonists & inhibitors ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Mice ; Penicillins ; antagonists & inhibitors ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; pharmacology ; beta-Lactamases ; biosynthesis ; genetics ; isolation & purification

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.
Disk Diffusion Antimicrobial Tests ; Escherichia coli/*enzymology/genetics/isolation & purification ; Escherichia coli Proteins/classification/*genetics ; Humans ; Inhibitory Concentration 50 ; Polymerase Chain Reaction ; beta-Lactamases/biosynthesis/genetics/*metabolism

BACKGROUND: Despite increasing importance of Acinetobacter baumannii in nosocomial infections and rapid development of multi-antimicrobial resistance in this strain, the resistance mechanisms of beta-lactam antimicrobials in A. baumannii were not clearly defined. In order to observe the resistance mechanisms against beta-lactams and carbapenem, we characterized the production of beta-lactamases and outermembrane protein (OMP) profiles for the 44 clinical isolates of A. baumannii. METHODS: The MICs of antimicrobials were determined by agar dilution test. The secondary beta-lactamases were characterized by isoelectric focusing, polymerase chain reactions and nucleotide sequencing, and the production of chromosomal beta-lactamases was quantitated by spectrophotometric method. For two strains with an elevated MIC of carbapenem, outermembrane protein (OMP) profile was analyzed by ultracentrifugation of the sonicated bacteral cells and SDS-PAGE. RESULTS AND CONCLUSION: Twenty two or 4 of 44 strains produced TEM-1-like beta-lactamase or PER-1 extended-spectrum beta-lactamase, respectively. However, when we analyzed the MICs of several beta-lactams with the beta-lactamase production, the resistance level of beta-lactam was mainly determined by the production of chromosomal beta-lactamase, not by the secondary beta-lactamases in the clinical isolates of A. baumannii. In two strains with an elevated MIC of imipenem, a decrease or loss of about 35 kDa and 22 kDa proteins in OMP was observed, which suggested that the change of OMP played a role in carbapenem resistance.
Acinetobacter/*drug effects/isolation & purification/metabolism ; Acinetobacter Infections/drug therapy/microbiology ; Antibiotics, Lactam/*pharmacology ; Bacterial Outer Membrane Proteins/biosynthesis ; Carbapenems/pharmacology ; Cross Infection/drug therapy/microbiology ; Drug Resistance, Bacterial ; Human ; beta-Lactamases/biosynthesis

BACKGROUND: Acinetobacter baumannii is an aerobic, gram-negative, glucose-nonfermenting bacterium, which has emerged as a serious opportunistic pathogen. In recent years, the increasing instance of carbapenem-resistant A. baumannii producing metallo-beta-lactamases (MBLs) or OXAtype beta-lactamases is causing a serious clinical problem. In this study, we investigated the prevalence of Ambler class A, B, and D beta-lactamases and their extended-spectrum derivatives in carbapenem-resistant A. baumannii isolates. METHODS: A total of 31 consecutive, non-duplicate, carbapenem-resistant A. baumannii were isolated from three university hospitals in the Chungcheong province of Korea. The modified Hodge and inhibitor-potentiated disk diffusion tests were conducted for the screening of carbapenemase and MBL production, respectively. PCR and DNA sequencing were performed for the detection of beta-lactamase genes. We also employed the enterobacterial repetitive intergenic consensus (ERIC)-PCR method for the epidemiologic study. RESULTS: Twenty-three of 31 isolates harbored bla(OXA-2) (51.6%), bla(OXA-23) (22.6%), bla(IMP-1) (48.4%),and bla(VIM-2) (3.2%). All of the OXA-2-producing strains also evidenced MBLs. The strains that harbored bla(OXA-23) were isolated only in hospital C, and only in a limited fashion. The ERIC-PCR pattern of the five OXA-23 strains indicated that the isolates were closely related in terms of clonality. The six strains producing IMP-1 isolated from hospital A were confirmed to be identical strains. CONCLUSIONS: A. baumannii strains harboring IMP-1 or OXA-type beta-lactamases are currently widely distributed throughout the Chungcheong province of Korea. The most notable finding in this study was that a bla(OXA-2)-producing A. baumannii harboring MBL, which has not been previously reported, can also lead to outbreaks.
Acinetobacter Infections/microbiology ; Acinetobacter baumannii/drug effects/*enzymology/genetics ; Anti-Bacterial Agents/*pharmacology ; Carbapenems/*pharmacology ; Disk Diffusion Antimicrobial Tests ; Drug Resistance, Multiple, Bacterial ; Humans ; Polymerase Chain Reaction ; beta-Lactamases/biosynthesis/genetics/*metabolism

Production of extended-spectrum beta-lactamase (ESBL) is one of the most important resistance mechanisms that hamper the antimicrobial treatment of infections caused by Enterobacteriaceae. ESBLs are classified into several groups according to their amino-acid sequence homology. While TEM and SHV enzymes were the most common ESBLs in the 1990s, CTX-M enzymes have spread rapidly among Enterobacteriaceae in the past decade. In addition, some epidemiological studies showed that organisms producing CTX-M enzymes had become increasingly prevalent in the community setting in certain areas in the world. Several novel enzymes with hydrolyzing activity against oxyimino-cephalosporins, albeit with additional enzymatic characteristics different from those of original TEM and SHV ESBLs (e.g., inhibitor-resistance), have been discovered and pose a problem on the definition of ESBLs. Although several methods to detect the production of ESBL are available in clinical laboratories, existence of other factors contributing resistance against beta-lactams, e.g., inducible production of Amp-C beta-lactamase by some species of Enterobacteriaceae, or inhibitor-resistance in some ESBLs may hinder the detection of ESBLs with these methods. Carbapenems are stable against hydrolyzing activity of ESBLs and are regarded as the drug of choice for the treatment of infections caused by ESBL-producing Enterobacteriaceae. Although several other antimicrobial agents, such as fluoroquinolones and cephamycins, may have some role in the treatment of mild infections due to those organisms, clinical data that warrant the use of antimicrobial agents other than carbapenems in the treatment of serious infections due to those organisms are scarce for now.
Anti-Bacterial Agents/*pharmacology/therapeutic use ; Carbapenems/pharmacology/therapeutic use ; Disk Diffusion Antimicrobial Tests ; Enterobacteriaceae/drug effects/*enzymology/genetics ; Enterobacteriaceae Infections/*drug therapy/microbiology ; Fluoroquinolones/pharmacology/therapeutic use ; Humans ; Microbial Sensitivity Tests/methods ; beta-Lactamases/*biosynthesis/metabolism ; beta-Lactams/*pharmacology/therapeutic use

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.
Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/biosynthesis/*genetics ; DNA, Bacterial/chemistry/genetics ; Drug Resistance, Bacterial/*genetics ; Enterobacteriaceae/enzymology/*genetics/isolation & purification ; Enterobacteriaceae Infections/microbiology ; *Genetic Variation ; Hospitals, University ; Humans ; Microbial Sensitivity Tests ; Plasmids/genetics/*metabolism ; Quinolones/*pharmacology ; 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