In vitro selection of Escherichia coli mutants with decreased susceptibility to norfloxacin

In 10 clinical isolates of Escherichia coli the frequency of spontaneous mutation to high levels of resistance to nalidixic acid was greater than 300-fold higher than that to norfloxacin. Norfloxacin resistant mutants could not be detected (mutation frequency: less than 10(-12). However, nalidixic acid resistant mutants of E. coli developed decreased susceptibility to norfloxacin at a rate of approximately 10(-9). Mutants with decreased susceptibility to norfloxacin could also be obtained when the same clinical isolates of E. coli were exposed to norfloxacin in 2 steps. However, mutants with MIC values greater than 8 micrograms/ml for norfloxacin were never obtained even after repeated exposure. Widespread use of cinoxacin or nalidixic acid may select resistant strains and reduce the efficacy of norfloxacin and other 4-quinolones.     

Mechanisms of reduced susceptibility to ciprofloxacin in Escherichia coli isolates from Canadian hospitals

OBJECTIVE:

To determine whether plasmid-mediated quinolone resistance (PMQR) determinants play a role in the increasing resistance to fluoroquinolones among Escherichia coli isolates in Canadian hospitals, and to determine the mechanisms of reduced susceptibility to ciprofloxacin in a recent collection of 190 clinical E coli isolates.

METHODS:

E coli isolates (n=1702) were collected as part of the 2007 Canadian Hospital Ward Antibiotic Resistance Surveillance (CANWARD) study. Antimicrobial susceptibility testing was performed by Clinical and Laboratory Standards Institute (CLSI) broth microdilution. Using a representative subset of isolates (n=190), the mechanisms of reduced susceptibility to ciprofloxacin were detected by polymerase chain reaction and sequencing of the quinolone resistance-determining regions (QRDR) of chromosomal gyrA and parC genes, and by polymerase chain reaction for the PMQR genes: qnr, aac(6′) Ib-cr and qepA.

RESULTS:

2.1% and 1.1% of E coli harboured aac(6′)Ib-cr and qnrB, respectively. Single amino acid substitutions in the QRDR of gyrA were observed among isolates with ciprofloxacin minimum inhibitory concentrations as low as 0.12 μg/mL. As the ciprofloxacin minimum inhibitory concentration increased to 1 μg/mL (which is still considered to be susceptible by the CLSI), the vast majority of isolates demonstrated both gyrA and parC mutations.

CONCLUSION:

PMQR determinants and QRDR mutants among clinical E coli isolates with reduced susceptibility to ciprofloxacin demonstrates the need for increased surveillance and the need to re-evaluate the current CLSI breakpoints to prevent further development of fluoroquinolone resistance.     

Short report: characterization of enteroaggregative Escherichia coli isolates from Iranian children

We have previously shown that enteroaggregative Escherichia coli (EAEC) is an important pathogen among Iranian infants and children. To better understand the characteristics of EAEC in Iran, we analyzed EAEC isolates for the presence of pAA plasmid-borne factors. Ninety-eight E. coli strains that displayed the aggregative adherence (AA) pattern on HeLa cells were hybridized with the CVD432 (AA) probe and with genes encoding enteroaggregative heat-stable enterotoxin-1 and aggregative adherence fimbriae (AAF) I and II. Our data suggest that AAF/II is common in this population and that AAF/I and AAF/II can sometimes be detected in the same E. coli isolate. Surprisingly, we have found that AA probe-negative strains in Iran share virulence factors with AA probe-positive isolates and therefore may be more similar to probe-positive strains than previously believed.     

Phenotypic and genotypic characterization of Rhodococcus equi isolated from sputum

IntroductionRhodococcus equi is an opportunistic pathogen, causing rhodococcosis, a condition that can be confused with tuberculosis. Often, without identifying M. tuberculosis, physicians initiate empiric treatment for tuberculosis. R. equi and M. tuberculosis have different susceptibility to drugs. Identification of R. equi is based on a variety of phenotypic, chromatographic, and genotypic characteristics.ObjectiveThis study aimed to characterize bacterial isolates from sputum samples suggestive of R. equi.MethodsThe phenotypic identification included biochemical assays; thin-layer chromatography (TLC) and polymerase chain reaction (PCR) were used for genotypic identification.ResultsAmong 78 Gram-positive and partially acid-fast bacilli isolated from the sputum of tuberculosis-suspected patients, 51 were phenotypically and genotypically characterized as R. equi based on literature data. Mycolic acid analysis showed that all suspected R. equi had compounds with a retention factor (R_f) between 0.4-0.5. Genotypic characterization indicated the presence of the choE gene 959 bp fragments in 51 isolates CAMP test positive. Twenty-two CAMP test negative isolates were negative for the choE gene. Five isolates presumptively identified as R. equi, CAMP test positive, were choE gene negative, and probably belonged to other bacterial species.ConclusionsThe phenotypic and molecular techniques used constitute a good methodological tool to identify R. equi.     

宋内志贺菌对氟喹诺酮类药物敏感性降低的相关耐药基因研究

目的 研究宋内志贺菌对氟喹诺酮类抗菌药物敏感性降低的相关耐药基因的变化情况.方法 采用琼脂稀释法对131株宋内志贺菌进行药物敏感性检测,采用PCR法检测DNA旋转酶A亚单位(gyrA)、拓扑异构酶ⅣC亚单位(parC)基因的喹诺酮类药物耐药决定区(QRDR),并对PCR结果进行测序分析,同时用PCR法对质粒介导喹诺酮类耐药(PMQR)基因(qnr)和氨基糖苷乙酰转移酶变异基因aac(6’)-Ib-cr]进行筛选.结果 131株宋内志贺菌对萘啶酸的耐药率达100％,而对诺氟沙星、环丙沙星、左氧氟沙星全部敏感,对四环素、复方磺胺甲(噁)唑和氨苄西林的耐药率分别为93.9％、92.8％和93.2％.94％的萘啶酸耐药宋内志贺菌株对氟喹诺酮类药物敏感性出现下降.萘啶酸耐药菌株gyrA均发生单点83位丝氨酸→亮氨酸(Ser→ Leu)突变,但parC未发生突变;未检出qnr和aac(6')-Ib-cr基因.结论 萘啶酸耐药宋内志贺菌对氟喹诺酮类药物敏感性降低,其产生的主要机制为gyrA发生83位Ser→Leu替换.     

Characterization of Staphylococcus aureus isolates with decreased susceptibility to vancomycin and teicoplanin: isolation and purification of a constitutively produced protein associated with decreased susceptibility.

"Derivative isolates" with 4- to 8-fold and 8- to 16-fold increases in MICs of vancomycin and teicoplanin, respectively, were selected from 2 susceptible clinical isolates of Staphylococcus aureus by serial incubation in low-level vancomycin. A protein of approximately 39 kDa was demonstrable in the cytoplasmic fraction and occasionally in the membrane fraction by SDS-PAGE of both derivatives. This protein was purified by DEAE chromatography, preparative SDS-PAGE, and electroelution. Derivative bacteria were larger on transmission electron microscopy, had thicker cell walls, and had changes in colony morphology on solid media. Further evidence for cell wall reorganization included loss of phage and capsular typing, decreased susceptibility to lysostaphin/lysozyme killing, and changes in condition for detection of optimal coagulase activity. The mechanism of decreased susceptibility to glycopeptide antibiotics among S. aureus derivative isolates is uncertain. The production of the approximately 39-kDa cytoplasmic protein and cell wall reorganization may mediate changed affinity of glycopeptide-peptidoglycan binding or impairment of glycopeptide access to its cell wall target.     

Presence of colonization factor antigens on fresh isolates of fecal Escherichia coli: a prospective study

In Dhaka, Bangladesh, fresh isolates of Escherichia coli from 197 patients with diarrhea were investigated for production of enterotoxin and possession of colonization factor antigen (CFA) I or II. Enterotoxigenic E. coli (ETEC) was isolated from 34% of the patients, and of the 67 enterotoxin-positive strains, 75% carried CFAs. Among 68 healthy control persons no strains positive for both enterotoxin and CFA were found. The CFAs in general were restricted to certain serotypes of E. coli. In a subgroup of patients, part of an ongoing surveillance study, mixed infection was seen in 23% of those from whom recognized pathogens were identified. There was a tendency to more severe dehydration when the two virulence factors, enterotoxin and CFA, were simultaneously present.     

Phenotypic and genotypic characteristics of recently adapted isolates of Plasmodium falciparum from Thailand.

The drug sensitivity characteristics and Plasmodium falciparum pfmdr1 status of five isolates of P. falciparum recently isolated from patients presenting for treatment from the Thailand/Myanmar border have been investigated. The aim of the study was to avoid the criticisms of some earlier studies by focusing on newly collected isolates from a specific geographic location. Three of the isolates studied exhibited clear resistance to chloroquine similar to that observed in the K1 Thai standard isolate obtained in the 1970s, and the other two isolates were of intermediate sensitivity to chloroquine with concentrations of drug that inhibit parasite growth by 50% of 50 and 43 nmol. The sensitivity of all isolates was enhanced by verapamil but we found no clear association between chloroquine sensitivity and gene copy number or intra-allelic variation of pfmdr1. In contrast, clear cross-resistance was seen between mefloquine and halofantrine, with the most sensitive isolates carrying the K1 mutation in pfmdr1.     

Phenotypic and genotypic characterization of Helicobacter pylori from patients with and without peptic ulcer disease

BACKGROUND: Helicobacter pylori plays an important role in peptic ulcer disease, although not all H. pylori-infected persons will develop a peptic ulcer. Currently, H. pylori strains cannot be divided into commensals and pathogens. METHODS: Fifty H. pylori strains were cultured from patients divided into five groups on the basis of upper endoscopic findings: gastric ulcer, duodenal ulcer, gastritis, esophagitis, or normal. The ultrastructural adherence pattern in vivo, autoagglutination, hemagglutination, adhesion to human gastric adenocarcinoma (AGS) cells, and the lipopolysaccharide (LPS) profile of H. pylori strains were recorded; randomly amplified polymorphic DNA (RAPD) and urease gene typing were performed and correlated with diagnostic groups. RESULTS: Electron micrographs showed that H. pylori strains from patients with gastric ulcers adhered more frequently through filamentous strands and were less frequently found free in mucus than any other diagnostic group (P < 0.0001). Neither median hemagglutination titer nor median adhesion capacity to a human gastric adenocarcinoma cell line was related to endoscopic findings. Nevertheless, H. pylori strains from patients with gastric ulcers were more prone to autoagglutinate than were strains from the other diagnostic groups (P = 0.03). H. pylori strains from gastric ulcer patients were found to be more homogeneous, as determined by RAPD and urease gene typing, than strains from the other diagnostic groups (P < 0.01). In addition, a positive correlation was found between a patient's age and the adhesion to AGS cells of the patient's H. pylori strain (P = 0.006). CONCLUSION: A combination of an H. pylori autoagglutination test, RAPD, and urease gene typing may be useful in separating gastric ulcer-related strains from duodenal ulcer-related and non-ulcer dyspepsia-related strains.     

Phylogenetic origin and virulence genotype in relation to resistance to fluoroquinolones and/or extended-spectrum cephalosporins and cephamycins among Escherichia coli isolates from animals and humans

In Escherichia coli infection, the implications of fluoroquinolone (FQ) and extended-spectrum cephalosporin plus cephamycin (AmpC) resistance for phylogenetic origin and virulence potential are undefined, as is the influence of ecological context on these associations. Accordingly, 106 E. coli isolates exhibiting FQ and/or AmpC resistance and 98 susceptible isolates were compared with regard to phylogenetic background and virulence profiles, stratified by host group (104 predominantly extraintestinal human isolates and 100 predominantly intestinal cattle and swine isolates). Although resistant isolates exhibited significant shifts in phylogenetic distribution and virulence profiles, human and animal isolates exhibited different phylogenetic shifts, and only among human isolates did resistance predict reduced virulence. Evidence for similar strains being resistant versus susceptible was scant. The O15:K52:H1 clonal group and the closely related "clonal group A" featured prominently among resistant and susceptible human isolates, respectively. Thus, in E. coli, antibiotic resistance predicts phylogenetic background and virulence potential in a complex, context-dependent fashion.     

Safety and effectiveness of switching from infliximab to etanercept in patients with rheumatoid arthritis: results from a large Japanese postmarketing surveillance study

Finding an effective treatment strategy for rheumatoid arthritis (RA) patients who have not benefited from previous tumor necrosis factor–α antagonist treatment is important for minimizing RA disease activity and improving patient outcomes. The aim of this study was to compare the safety and effectiveness of etanercept in patients with and without infliximab (IFX) treatment experience. Patients (n?=?7,099) from a large postmarketing observational study of etanercept use in Japan were divided into 2 cohorts based on previous IFX use (pre-IFX and non-IFX). Baseline characteristics were assessed in each cohort. Adverse events (AEs) and European League Against Rheumatism (EULAR) responses were monitored every 4?weeks for 24?weeks. At baseline, pre-IFX patients were younger and had fewer comorbidities and a shorter RA duration than non-IFX patients. During the study, pre-IFX patients received concomitant methotrexate more often than non-IFX patients. The incidence of AEs and serious AEs were significantly lower in pre-IFX patients, as was the percentage of patients who discontinued treatment. Both cohorts had significant improvement (P?<?0.001) in EULAR responses at the end of the treatment period. This study demonstrated that etanercept was effective and well tolerated in active RA patients with and without prior IFX treatment.     

重庆地区耐多药结核分枝杆菌对氟喹诺酮类药物耐药的相关基因特征分析

目的 分析重庆地区耐多药结核分枝杆菌对氟喹诺酮类(FQs)药物耐药的相关基因特征,以及与结核分枝杆菌基因型的相关性。 方法 收集2015年1月至2017年6月重庆市39个区(县)967例耐多药结核病(MDR-TB)可疑患者的所有耐多药(MDR)结核分枝杆菌临床分离株229株,通过微孔板Alamar blue显色法检测4种FQs药物[氧氟沙星(Ofx)、左氧氟沙星(Lfx)、莫西沙星(Mfx)、加替沙星(Gfx)]的耐药性,用PCR测序方法对FQs药物耐药相关基因gyrA、gyrB进行分析,并采用实时荧光定量熔解曲线方法进行北京基因型鉴定。 结果 在229株MDR菌株中,94株(41.0%,94/229)对任一FQs药物耐药。其中,Ofx耐药率最高(41.0%,94/229);Lfx、Mfx耐药率居中,分别为31.4%(72/229)、30.6%(70/229);Gfx耐药率最低(20.1%,46/229)。94株对FQs耐药菌株中,81株(86.2%,81/94)发生gyrA基因突变,第94位密码子突变最常见(60.6%,57/94);10株(10.6%,10/94)发生gyrB基因突变。7株gyrA基因双位点突变均显示为高水平耐药;9株gyrA与gyrB联合突变中,2株为高水平耐药。重庆地区对FQs耐药菌株中北京基因型80株(85.1%,80/94),其中,现代北京基因型占60.0%(48/80)。 结论 重庆地区MDR结核分枝杆菌 对FQs耐药的菌株以现代北京基因型为主,其耐药相关基因突变主要发生于gyrA基因。     

Evidence for an epidemic trimethoprim-resistance plasmid in fecal isolates of Escherichia coli from citizens of the United States studying in Mexico

A previous study revealed the emergence of high-level resistance to trimethoprim-sulfamethoxazole in strains of Escherichia coli isolated from 95% of students from the United States who were taking either trimethoprim alone or trimethoprim-sulfamethoxazole for prophylaxis of travelers' diarrhea while in Guadalajara, Mexico. Many of these strains were subsequently demonstrated to cotransfer resistance to trimethoprim along with that to streptomycin and ampicillin. The present study demonstrated that at least 12 (60%) of 20 transferable (tra+) trimethoprim-resistance plasmids studied possessed both an identical Hind III restriction pattern and type I dihydrofolate reductase genes. Donor strains were shown to be distinct; this finding suggested that a common tra+ trimethoprim-resistance plasmid was widely disseminated among E. coli strains in Guadalajara. These results may explain in part the surprising degree of resistance encountered in trimethoprim-consuming persons in that region.     

Antimicrobial susceptibility profile of community acquired and nosocomial isolates of Escherichia coli from clinical blood culture specimens at a Nigerian university teaching hospital

ObjectiveTo ascertain the antibiotic susceptibility patterns of Escherichia coli recovered from blood culture specimens in Calabar, Nigeria.MethodsThe study was retrospective in nature and was carried out at University of Calabar Teaching Hospital (UCTH) Calabar. Data generated from blood culture specimens over a five year period (Feb. 2004-Feb. 2009) was compiled, relevant information such as age, sex, organism recovered and antibiotic susceptibility patterns were obtained from patients records. Samples were collected, transported, stored and processed using standard laboratory procedures. Data obtained was analysed using Epi Info 6 statistical software.ResultsEscherichia coli was responsible for 15.3% (31/203) of the blood infections being the third most common microorganism encountered. The community acquired (CA) isolates of the organism were significantly less resistant (P<0.05), compared to the nosocomial (NC) isolates against ampicillin, cloxacillin, amoxicillin, tetracycline, co-trimoxazole, chloramphenicol and erythromycin. The sensitivity of both the NC and CA isolates of Escherichia coli to amikacin, augmentin, ofloxacin, ciprofloxacin, ceftazidime, cefuroxime, ceftriaxone and rifampicin was generally high (80-100%) with no significant difference (P>0.05). Majority (>95.0%) of the NC isolates of Escherichia coli were resistant to six of the antibiotics tested.ConclusionsControl mechanisms for hospital acquired infections should be stepped up so as to limit the spread of the highly resistant bacterial strains. Also the sale and consumption of antibiotics by the public need to be regulated.     

Phenotypic and genotypic analysis of clinical HIV-1 isolates reveals extensive protease inhibitor cross-resistance: a survey of over 6000 samples

OBJECTIVE: To evaluate in HIV-1 the extent of phenotypic and genotypic antiretroviral drug resistance and cross-resistance towards the protease inhibitors (PIs) saquinavir, ritonavir, indinavir and nelfinavir among a set of patient samples originating from European and US routine clinical practice and submitted for phenotypic drug resistance testing and/or genotypic analysis. The mutational pattern(s) underlying both resistance and cross-resistance to PIs was investigated. METHOD: Over 6000 patient isolates with plasma viral load greater than 1000 copies/ml plasma were analysed. Phenotypic resistance was evaluated by a recombinant virus assay. Phenotypic resistance is expressed as the fold-increase of the 50% inhibitory concentration (IC50) value of a compound for a patient-derived recombinant virus isolate compared with that for a wild-type laboratory virus. Genotypic analysis is reported as amino acid changes at positions in the HIV-1 protease compared to a wild-type reference. RESULTS: Phenotypic resistance to any single PI was observed in 17 to 25% of the clinical isolates investigated. Phenotypic cross-resistance among PIs (> 10-fold increase in IC50 value) was detected in 59 to 80% of the samples resistant (> 10-fold increase in IC50 value) to at least one PI. The prevalent mutations in PI-resistant isolates involved substitutions at codons 10, 36, 46, 54, 71, 77, 82 and 90. The most frequent mutational pattern in samples with PI cross-resistance involved combined substitutions at positions 10 and 90, extended with substitutions at positions 54, 71, 77, 82 or 84. CONCLUSIONS: Extensive use of first-generation PIs leads to the emergence of HIV-1 isolates possessing cross-resistance to all members of this class. Identification of particular mutational profiles among these isolates may assist in the design of new generation inhibitors with specific activity against protease-mutant HIV strains.     

Phenotypic and genotypic analysis of mononuclear cells from patients with Felty''s syndrome.

Phenotypic and genotypic characteristics of the peripheral blood mononuclear cells in nine patients with Felty's syndrome have been examined. One patient had an increased number and percentage of peripheral blood mononuclear cells with the phenotype CD3+ Leu-7+ CD16+ and showed a clonal rearrangement of the T cell receptor B chain gene. The remaining eight patients all showed a germline configuration of the T cell receptor B chain gene. In two patients an increased proportion of CD3+ Leu-7+ CD16- peripheral blood mononuclear cells (45 (SD 11)% of peripheral blood mononuclear cells) were found, while the remaining six patients had proportions of CD3+ Leu-7+ cells similar to those of patients with uncomplicated rheumatoid arthritis. These data confirm that patients with Felty's syndrome are heterogeneous, with at least three different peripheral blood mononuclear cell phenotypic subsets. One subset is characterised by a clonal expansion of an unusual lymphocyte subpopulation, another by polyclonal expansion, and the third subset has the same proportions of peripheral blood mononuclear cells as patients with uncomplicated rheumatoid arthritis.