BACKGROUND/AIMS: The number of urinary tract infections (UTIs) caused by extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) is increasing. In an outpatient setting, there are limited therapeutic options to treat ESBL-producing pathogens. We evaluated the outcomes of amikacin outpatient parenteral antibiotic therapy (OPAT) for UTIs caused by ESBL-EC in patients not pre-treated with carbapenem. METHODS: We retrospectively evaluated the outcomes of amikacin OPAT for UTIs caused by ESBL-EC. RESULTS: From November 2011 to October 2012, eight females, who could not be hospitalized for carbapenem treatment, were treated with amikacin OPAT for nine episodes of non-bacteremic ESBL-EC UTIs. Seven of the eight patients had one or more comorbidities. Of the nine UTI cases, three had symptomatic lower UTIs and six had non-bacteremic upper UTIs. In all of the cases, symptomatic and laboratory improvements were observed following amikacin OPAT. One patient showed a delayed relapse with bilateral microabscesses 3 weeks after treatment cessation; however, a clinical and microbiological cure was eventually reached. All of the patients were able to tolerate amikacin OPAT without any significant nephrotoxicity or ototoxicity. CONCLUSIONS: Amikacin OPAT represents a feasible therapeutic option for non-bacteremic UTIs caused by ESBL-EC in settings with limited resources.
Adult ; Aged ; Aged, 80 and over ; Ambulatory Care ; Amikacin/administration & dosage/adverse effects/*therapeutic use ; Drug Administration Schedule ; Escherichia coli/*drug effects/enzymology/isolation & purification ; Escherichia coli Infections/diagnosis/*drug therapy/microbiology/urine ; Humans ; Microbial Sensitivity Tests ; Middle Aged ; Recurrence ; Remission Induction ; Retrospective Studies ; Time Factors ; Treatment Outcome ; Urinalysis ; Urinary Tract Infections/diagnosis/*drug therapy/microbiology/urine ; Urine/microbiology ; beta-Lactamase Inhibitors/administration & dosage/adverse effects/*therapeutic use ; beta-Lactamases/*metabolism

The tissue-specific and MeJA-induced transcriptional levels of BcUGT3 and BcUGT6 in Bupleurum chinense were analyzed in the present study. The transcriptional levels of BcUGT3 in root, leaf, flower and fruit were similar and they all were higher than those in stem. The transcriptional level of BcUGT6 was the highest in leaf and the lowest in flower among in all tested tissues. With non-treated adventitious roots as control, BcUGT6's transcriptional levels were elevated to nearly 2 folds for 2 h, 8 h, 24 h, 2 d and 4 d in MeJA-treated adventitious roots of B. chinense. It showed that the transcriptional level of BcUGT6 was slightly affected by MeJA. While, BcUGT3's transcriptional levels were gradually elevated, and till 4 d after MeJA treatment, the expression level was about 7 folds than that of non-treated control. Using pET-28a (+), the expressions of two genes was investigated. Induced by IPTG, the target proteins were expressed in E. coli and then purified. All the results obtained in the present study will be helpful for follow-up bio-function analysis of BcUGT3 and BcUGT6.
Acetates ; pharmacology ; Bupleurum ; cytology ; enzymology ; genetics ; Cell Membrane ; metabolism ; Cyclopentanes ; pharmacology ; Escherichia coli ; genetics ; Gene Expression ; Gene Expression Regulation, Plant ; drug effects ; Hexosyltransferases ; chemistry ; genetics ; isolation & purification ; metabolism ; Intracellular Space ; metabolism ; Oxylipins ; pharmacology ; Protein Sorting Signals ; Protein Structure, Secondary ; Protein Transport ; Sequence Analysis ; Transcription, Genetic ; drug effects

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

We report a case of CTX-M-55-type extended-spectrum beta-lactamase (ESBL)-producing Shigella sonnei infection in a 27-year-old Korean woman who had traveled to China. The patient was admitted to the hospital due to abdominal pain, watery diarrhea, and fever (39.3degrees C). S. sonnei was isolated from her stool specimens, and the pathogen was found to be resistant to cefotaxime due to CTX-M-55-type ESBL. Insertion sequence (IS)Ecp1 was found upstream of the blaCTX-M-55 gene. The blaCTX-M-55 gene was transferred from the S. sonnei isolate to an Escherichia coli J53 recipient by conjugation. Pulsed-field gel electrophoresis and Southern blotting revealed that the blaCTX-M-55 gene was located on a plasmid of approximately 130 kb.
Adult ; Anti-Bacterial Agents/pharmacology ; Asian Continental Ancestry Group ; Cefotaxime/pharmacology ; China ; Drug Resistance, Bacterial/drug effects ; Dysentery, Bacillary/diagnosis/*microbiology ; Electrophoresis, Gel, Pulsed-Field ; Escherichia coli/metabolism ; Feces/microbiology ; Female ; Humans ; Plasmids/chemistry/genetics ; Republic of Korea ; Shigella sonnei/enzymology/*isolation & purification ; Travel ; beta-Lactamases/genetics/*metabolism

OBJECTIVETo evaluate the antimicrobial activity of total alkaloids extracted from Sophorea alopecuroides L. (TASA) against clinical isolated extended-spectrum beta-lactamases (ESBLs) producing Escherichia coli (E. coli) strains.

METHODSThe antibacterial activity of TASA either itself or in combination with cefotaxime (CTX) or ceftazidime (CAZ) was investigated by using the microbroth dilution method and phenotypic confirmatory disk diffusion test against three clinical isolated ESBLs-producing E. coli strains; the interactions of TASA and CTX or CAZ were ascertained by evaluating the fractional inhibitory concentration index (FICI).

RESULTSThe antibacterial activity of either TASA itself or in combination with CTX or CAZ was found. The minimum inhibitory concentration (MICs) of TASA against the ESBLs producing isolates was 12.5 mg/mL. In the combinations with a sub-inhibitory concentration of TASA, a synergistic effect on CTX and CAZ against the ESBLs producing isolates was observed. Similarly, the isolates exposed to lower dose of TASA yielded an increased susceptibility to CTX and CAZ by 8-16 folds determined by microdilution assay. Moreover, enzymatic detection of ESBLs demonstrated that TASA induced reversal resistance to CTX and CAZ partially by a mechanism of inhibition of ESBLs activity in these isolates. Additionally, in the tested isolates following the exposure of TASA, molecular analysis verified the SHV-type beta-lactamase encoding ESBL gene in these isolates, and no mutation was introduced into the ESBL gene.

CONCLUSIONSThese results suggest that TASA could be used as a source of natural compound with pharmacological activity of reversal resistance to antimicrobial agent. These findings also indicated that the application of the TASA in combination with antibiotics might prove useful in the control and treatment of infectious diseases caused by the ESBLs producing enterobacteriaceae.

Alkaloids ; isolation & purification ; pharmacology ; Anti-Bacterial Agents ; pharmacology ; Base Sequence ; Cefotaxime ; pharmacology ; Ceftazidime ; pharmacology ; DNA Primers ; Escherichia coli ; drug effects ; enzymology ; Microbial Sensitivity Tests ; Polymerase Chain Reaction ; Sophora ; chemistry ; 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

As a large family of hydrolases, GTPases are widespread in cells and play the very important biological function of hydrolyzing GTP into GDP and inorganic phosphate through binding with it. GTPases are involved in cell cycle regulation, protein synthesis, and protein transportation. Chaperones can facilitate the folding or refolding of nascent peptides and denatured proteins to their native states. However, chaperones do not occur in the native structures in which they can perform their normal biological functions. In the current study, the chaperone activity of the conserved GTPases of Escherichia coli is tested by the chemical denaturation and chaperone-assisted renaturation of citrate synthase and α-glucosidase. The effects of ribosomes and nucleotides on the chaperone activity are also examined. Our data indicate that these conserved GTPases have chaperone properties, and may be ancestral protein folding factors that have appeared before dedicated chaperones.
Citrate (si)-Synthase ; chemistry ; Cloning, Molecular ; Conserved Sequence ; Escherichia coli ; cytology ; enzymology ; GTP Phosphohydrolases ; chemistry ; genetics ; isolation & purification ; metabolism ; Guanosine Diphosphate ; pharmacology ; Guanosine Triphosphate ; analogs & derivatives ; pharmacology ; Molecular Chaperones ; chemistry ; genetics ; isolation & purification ; metabolism ; Protein Denaturation ; drug effects ; Protein Renaturation ; drug effects ; Ribosomes ; metabolism ; alpha-Glucosidases ; chemistry

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

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

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