BACKGROUND/AIMS: We examined the prevalence of extended-spectrum beta-lactamase (ESBL) production and the impact of ESBL on clinical outcomes in cancer patients with Enterobacter spp. bacteremia. METHODS: Using prospective cohort data on Enterobacter bacteremia obtained between January 2005 and November 2008 from a tertiary care center, the prevalence and clinical impact of ESBL production were evaluated. RESULTS: Two-hundred and three episodes of Enterobacter spp. bacteremia were identified. Thirty-one blood isolates (15.3%, 31/203) scored positive by the double-disk synergy test. Among 17 isolates in which ESBL genes were detected by polymerase chain reaction and sequencing, CTX-M (n = 12), SHV-12 (n = 11), and TEM (n = 4) were the most prevalent ESBL types. Prior usage of antimicrobial agents (77.4% vs. 54.0%, p = 0.02) and inappropriate empirical antimicrobial therapy (22.6% vs. 3.0%, p < 0.001) were more commonly encountered in the ESBL-positive group than in the extended-spectrum cephalosporin-susceptible ESBL-negative group, respectively. Clinical outcomes did not differ significantly between the two groups (30-day mortality rate, 19.4% vs. 17.0%, p = 0.76; median length of hospital stay, 24.0 days vs. 30.5 days, p = 0.97). Initial presentation of severe sepsis/septic shock, pneumonia, and intra-abdominal infection were independently associated with 30-day mortality. CONCLUSIONS: The prevalence of ESBL-producing isolates was 15.3% in cancer patients with Enterobacter bacteremia. Although inappropriate empirical therapy was more common in the ESBL-positive group, ESBL production was not associated with poorer outcomes.
Adult ; Aged ; Anti-Bacterial Agents/therapeutic use ; Bacteremia/*complications/drug therapy/microbiology ; Child ; Cohort Studies ; Enterobacter/*enzymology/genetics ; Enterobacteriaceae Infections/*complications/drug therapy/microbiology ; Female ; Humans ; Infant ; Male ; Middle Aged ; Neoplasms/*complications ; Prospective Studies ; Treatment Outcome ; beta-Lactamases/*biosynthesis/genetics

OBJECTIVETo investigate the characteristics of New Delhi metallo-beta-lactamase-1 (NDM-1) gene of Klebsiella pneumoniae (K. pneumoniae) emerging in Hunan, and its relationship to antibiotic resistance.

METHODSThe clinical strain was isolated from a sputum sample of a child with severe pnemonia and toxic myocarditis who was admitted into a general hospital of Hunan Province. VITEK-2 compact instrument was used for bacterial identification and antibiotic susceptibility test. Modified Hodge test was used for the screening of carbapenemase. EDTA-synergy test and combination disk diffusion test were used for detection of metallo-&beta;-lactamase (MBL). PCR was performed for amplification of NDM-1 genes and the positive products were sequenced and analyzed with BLAST. Conjugation was also performed to analyze mechanisms of antibiotic resistance. The results of antibiotic susceptibility tests were compared before and after conjugation.

RESULTSThe isolated strain was identified as K.pneumoniae. Modified Hodge test, EDTA-synergy test and combination disk diffusion test were all positive for the strain. The homology between gene sequence of PCR amplification products and NDM-1 gene FN396876.1 in the GenBank was 100%. Transconjugant DNA was used as template for the amplification of NDM-1 gene. The amplification products were sequenced and found to be the same as the NDM-1 gene amplification product of the donor strain. The MIC of transconjugant E.coli J53 (NDM-1) to all the &beta;-lactams increased significantly compared with the recipient strain E.coli J53. The MIC of ertapenem and imipenem increased by more than 8 times, while the MIC of ceftazidime and ceftriaxone increased by more than 64 times.

CONCLUSIONSThis study first identified a strain of K. pneumoniae carrying NDM-1 in mainland China. NDM-1 gene can be transmitted among different strains and causes extensively drug-resistance to &beta;-lactams.

Base Sequence ; China ; Drug Resistance, Bacterial ; Klebsiella pneumoniae ; drug effects ; enzymology ; genetics ; Molecular Sequence Data ; Polymerase Chain Reaction ; beta-Lactamases ; biosynthesis ; genetics

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

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

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: 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: This study was designed to characterize urinary isolates of Escherichia coli that produce extended-spectrum beta-lactamases (ESBLs) and to determine the prevalence of other antimicrobial resistance genes. METHODS: A total of 264 non-duplicate clinical isolates of E. coli were recovered from urine specimens in a tertiary-care hospital in Busan in 2005. Antimicrobial susceptibility was determined by disk diffusion and agar dilution methods, ESBL production was confirmed using the double-disk synergy (DDS) test, and antimicrobial resistance genes were detected by direct sequencing of PCR amplification products. E. coli isolates were classified into four phylogenetic biotypes according to the presence of chuA, yjaA, and TSPE4. RESULTS: DDS testing detected ESBLs in 27 (10.2%) of the 264 isolates. The most common type of ESBL was CTX-M-15 (N=14), followed by CTX-M-3 (N=8) and CTX-M-14 (N=6). All of the ESBL-producing isolates were resistant to ciprofloxacin. PCR experiments detected genes encoding DHA-1 and CMY-10 AmpC beta-lactamases in one and two isolates, respectively. Also isolated were 5 isolates harboring 16S rRNA methylases, 2 isolates harboring Qnr, and 19 isolates harboring AAC(6')-Ib-cr. Most ESBL-producing isolates clustered within phylogenetic groups B2 (N=14) and D (N=7). CONCLUSION: CTX-M enzymes were the dominant type of ESBLs in urinary isolates of E. coli, and ESBL-producing isolates frequently contained other antimicrobial resistance genes. More than half of the urinary E. coli isolates harboring CTX-M enzymes were within the phylogenetic group B2.
Bacterial Proteins/biosynthesis/*genetics ; Bacteriuria/microbiology ; Ciprofloxacin/pharmacology ; Disk Diffusion Antimicrobial Tests ; Drug Resistance, Multiple, Bacterial/*genetics ; Escherichia coli/*drug effects/enzymology/isolation & purification ; Humans ; Methyltransferases/genetics ; Phylogeny ; beta-Lactamases/biosynthesis/*genetics

OBJECTIVE: To determine the relation between resistance of hypermutable Pseudomonas aeruginosa and beta-lactamases produced. METHODS: The bacteria cultured were identified with API 20NE system. Susceptibilities of the bacteria were detected by disk diffusion method. The hypermutable strains were tested with broth dilution assays. The beta-lactamases produced by these strains were characterized by 3-dimensional test and 2-mercaptopropanoic acid inhibited assays. RESULTS: Altogether 120 strains were analyzed and 45 (37.5%) trains were hypermutable.The resistant rates of hypermutable strains were close to or above 60.0% for imipenem, meropenem, cefoperazone/sulbactam, piperacillin/ tazobactam, ceftazidime, cephfime, aztreonam, amikacin and ciprofloxacin.The 3-dimensional test showed that 18 (40.0%) strains produced extended spectrum beta-lactamases (ESBLs), 25 (55.6%) strains produced AmpC enzymes, and 6 (13.3%) strains produced metallo-beta-lactamases. CONCLUSION The resistant rates of hypermutablce strains of Pseudomonas aeruginosa to routine antibiotics are high, which is one of the most important reasons for multi-drug resistance that the hypermutable strains produced ESBLs, AmpC enzymes, and metallo-beta-lactamases.
Adult ; Aged ; Aged, 80 and over ; Bronchitis, Chronic ; microbiology ; Drug Resistance, Multiple, Bacterial ; genetics ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Pseudomonas aeruginosa ; drug effects ; enzymology ; genetics ; Pulmonary Heart Disease ; microbiology ; beta-Lactamases ; biosynthesis