产超广谱β-内酰胺酶大肠埃希菌的耐药机制研究

目的了解贵阳地区医院内检出的大肠埃希菌产超广谱β-内酰胺酶(ESBLs)菌株的耐药性及基因型,为指导临床合理用药提供依据。方法由贵州医科大学附属白云医院ATB系统完成菌株鉴定及药敏试验检测,采用纸片扩散法进行ESBLs表型确证试验,采用碱裂解法提取产ESBLs菌的质粒DNA,采用聚合酶链反应扩增TEM型、SHV型、CTX-M-1组和CTX-M-9组基因。结果产ESBLs大肠埃希菌阳性率为66.7%(279/418);对阿米卡星耐药率为4.3%;对青霉素类,头孢菌素第1、2代药物耐药率均为100.0%;庆大霉素、妥布霉素、喹诺酮类、磺胺类药物耐药率在60.6%~84.9%;对亚胺培南和美罗培南仍保持较高的敏感度。116株产ESBLs大肠埃希菌基因型有113株(97.4%)为CTX-M型,其中CTX-M-9亚型68株(58.6%),CTX-M-1亚型67株(57.8%);TEM型有86株(74.1%);未检出SHV型。结论产ESBLs阳性的大肠埃希菌的耐药率明显高于产ESBLs阴性的大肠埃希菌,且具有多重耐药性。贵州医科大学附属白云医院产ESBLs大肠埃希菌的基因型以CTX-M型为主;碳青霉烯类为最敏感的药物,但本研究中发现亚胺培南耐药菌株,需引起临床重视。     

大肠埃希菌和肺炎克雷伯菌耐药性分析

目的 了解我院2005-2008年产超广谱β内酰胺酶(ESBLs) 大肠埃希菌和肺炎克雷伯菌的检出率及耐药情况.方法 收集我院2005年1月-2008年12月临床分离到的大肠埃希菌和肺炎克雷伯菌分别2 557株和1 883株,采用纸片扩散法(K-B法)对分离株进行抗菌药物敏感试验和ESBLs筛选和确证试验.对其中60株产ESBLs菌用PCR和测序检测该类酶的基因型.结果 4年中大肠埃希菌ESBLs检出率分别为48.0%、63.0%、65.8%和59.8%,肺炎克雷伯菌ESBLs检出率分别为40.9%、53.9%、56.8%和47.3%.产ESBLs大肠埃希菌和肺炎克雷伯菌对氨苄西林、头孢克洛、头孢呋辛、头孢唑林、头孢丙烯和哌拉西林等耐药率超过90%,对美罗培南和亚胺培南的敏感率为97%～100%.29株大肠埃希菌TEM基因检出率为75.8 %(22/29),SHV基因检出率为0,CTX-M基因检出率为86.2%(25/29).31株肺炎克雷伯菌TEM基因检出率为41.9 %(13/31),SHV基因检出率为64.5%(20/31),CTX-M基因检出率为48.4%(15/31).35株(58.3%)菌同时检测到2种以上基因型.序列分析证实TEM 扩增产物均属于TEM-1基因,CTX-M 扩增产物均属于CTX-M-3基因,而SHV 扩增产物则分别属于SHV-1、SHV-5、SHV-11、SHV-12和SHV-28.结论 我院2005-2008年大肠埃希菌和肺炎克雷伯菌ESBLs检出率较高.CTX-M-3、SHV-11和SHV-12是我院产ESBLs 菌株的主要基因型.     

Activation of multiple antibiotic resistance in uropathogenic Escherichia coli strains by aryloxoalcanoic acid compounds

Clofibric and ethacrynic acids are prototypical pharmacological agents administered in the treatment of hypertrigliceridemia and as a diuretic agent, respectively. They share with 2,4-dichlorophenoxyacetic acid (the widely used herbicide known as 2,4-D) a chlorinated phenoxy structural moiety. These aryloxoalcanoic agents (AOAs) are mainly excreted by the renal route as unaltered or conjugated active compounds. The relatedness of these agents at the structural level and their potential effect on therapeutically treated or occupationally exposed individuals who are simultaneously undergoing a bacterial urinary tract infection led us to analyze their action on uropathogenic, clinically isolated Escherichia coli strains. We found that exposure to these compounds increases the bacterial resistance to an ample variety of antibiotics in clinical isolates of both uropathogenic and nonpathogenic E. coli strains. We demonstrate that the AOAs induce an alteration of the bacterial outer membrane permeability properties by the repression of the major porin OmpF in a micF-dependent process. Furthermore, we establish that the antibiotic resistance phenotype is primarily due to the induction of the MarRAB regulatory system by the AOAs, while other regulatory pathways that also converge into micF modulation (OmpR/EnvZ, SoxRS, and Lrp) remained unaltered. The fact that AOAs give rise to uropathogenic strains with a diminished susceptibility to antimicrobials highlights the impact of frequently underestimated or ignored collateral effects of chemical agents.     

产ESBLs大肠埃希菌和肺炎克雷伯菌的耐药性变化

目的分析产ESBLs大肠埃希菌和肺炎克雷伯菌的耐药性变化。方法收集我院2004年1月—2005年12月期间临床分离的大肠埃希菌和肺炎克雷伯菌,对其产ESBLs及耐药情况与2000年1月—2002年10月进行比较研究。结果以头孢噻肟为底物检测ESBLs,2004—2005年产ESBLs大肠埃希菌和肺炎克雷伯菌分别为35.4%和21.1%,与2000—2002年度比较显著增加(P<0.01)。2004—2005年产ESBLs大肠埃希菌和肺炎克雷伯菌占尿标本41.4%(133/321),呼吸道分泌物占29.0%(93/321),血液占5.9%(19/321),伤口分泌物占12.8%(41/321),体液或引流液占8.1%(26/321)。产ESBLs大肠埃希菌对氨曲南敏感性2004—2005年较前有显著下降(P<0.05),对其他抗生素的敏感性差异无统计学意义。产ESBLs肺炎克雷伯菌对抗菌药物的敏感性2004—2005年较2000—2002年除左氧氟沙星外均有下降趋势,并且头孢他啶、头孢哌酮-舒巴坦敏感性显著下降(P<0.05)。结论产ESBLs细菌近2年有增加趋势,并对多种抗生素的敏感性下降。     

Impact of antibiotic resistance and of adequate empirical antibiotic treatment in the prognosis of patients with Escherichia coli bacteraemia

BACKGROUND: Escherichia coli is the most frequent Gram-negative organism causing bacteraemia. There are few data about prognostic factors of bloodstream infections due to E. coli. In particular, the consequences of antibiotic resistance and of adequate empirical antibiotic treatment on outcome remain broadly unknown. METHODS: We conducted a retrospective cohort study of patients with E. coli bacteraemia between January 1997 and June 2005 to identify any association between antibiotic resistance, adequacy of empirical antibiotic therapy and mortality. RESULTS: Of 663 patients with E. coli bacteraemia, 36 (5.4%) died. Patients with multidrug-resistant (MDR) E. coli bacteraemia had a significantly lower frequency of correct empirical antibiotic treatment than patients with non-MDR E. coli bacteraemia [relative risk (RR) 0.53; 95% confidence interval (CI) 0.48-0.67], and also had a significantly higher mortality (RR 3.31; 95% CI 1.72-6.36). An association between the number of antibiotics to which E. coli was resistant with adequacy of empirical antibiotic (P < 0.001) and with mortality (P < 0.001) was detected. After adjustment for other significant risk factors and confounders, the inadequacy of empirical antibiotic treatment was associated with an increased mortality (adjusted OR 2.98; 95% CI 1.25-7.11). When the adequacy of empirical treatment was excluded from the model, the presence of MDR E. coli in blood cultures was also associated with the prognosis (adjusted OR 3.11; 95% CI 1.3-7.44). In multivariate analysis, other variables associated with the outcome were age, the presence of severe sepsis or shock, Charlson index score and a non-urinary origin of the bacteraemia. CONCLUSIONS: Adequacy of empirical antibiotic treatment is an independent risk factor for mortality in patients with E. coli bacteraemia. MDR E. coli bacteraemia had a worse prognosis due, at least in part, to a lower frequency of correct empirical treatment.     

Integron-associated antibiotic resistance and phylogenetic grouping of Escherichia coli isolates from healthy subjects free of recent antibiotic exposure

The study of integrons in 181 Escherichia coli isolates from three groups of healthy subjects who lived in communities and had not taken antibiotics for at least 1 month showed that the presence of integrons was associated with antibiotic resistance and phylogenetic grouping of the bacterial host and dependent on a subject's living environment.     

Association between antibiotic resistance and the expression of Dr adhesin among uropathogenic Escherichia coli

BACKGROUND: Urinary tract infections (UTIs) are a significant health problem and Escherichia coli has been exported to be the primary pathogen in approximately 80% of cases. E. coli express structures called adhesins, fimbriae or pili that help them bind to specific tissue receptors. One such adhesin is Dr, which binds to the complement decay-accelerating factor (CD55) on the host cell. The purpose of the present study was to review the epidemiology and antimicrobial sensitivity spectrum of Dr adhesin-bearing E. coli isolates in women with UTI. METHODS AND RESULTS: A total of 337 uropathogenic E. coli isolates were collected, and mannose-resistant hemagglutination was observed in 43%, while 12.4% expressed Dr adhesin. All 337 uropathogenic E. coli isolates were found to be resistant to penicillin, oxacillin, bactericin and cloxacillin. None of the isolates was resistant to quinolones. The resistance to sulfamethoxazole was most common (90%), followed by nalidixic acid (51%) and ampicillin (41%). Interestingly, uropathogenic E. coli expressing Dr adhesin had a high incidence of ampicillin resistance (83%), and only 17% of the Dr-bearing isolates were found to be resistant to an ampicillin/sulbactum combination. CONCLUSION: These results indicate that UTIs may be successfully treated with ampicillin in combination with a beta-lactamase inhibitor, so that selective colonization by Dr adhesin-bearing uropathogenic E. coli is prevented, which is probably promoted by treating these patients with ampicillin alone.     

Non-target gene mutations in the development of fluoroquinolone resistance in Escherichia coli

Mutations in loci other than genes for the target topoisomerases of fluoroquinolones, gyrA and parC, may play a role in the development of fluoroquinolone resistance in Escherichia coli. A series of mutants with increasing resistance to ofloxacin was obtained from an E. coli K-12 strain and five clinical isolates. First-step mutants acquired a gyrA mutation. Second-step mutants reproducibly acquired a phenotype of multiple antibiotic resistance (Mar) and organic solvent tolerance and showed enhanced fluoroquinolone efflux. None of the second-step mutants showed additional topoisomerase mutations. All second-step mutants showed constitutive expression of marA and/or overexpressed soxS. In some third-step mutants, fluoroquinolone efflux was further enhanced compared to that for second-step mutants, even when the mutant had acquired additional topoisomerase mutations. Attempts to circumvent the second-step Mar mutation by induction of the mar locus with sodium salicylate and thus to select for pure topoisomerase mutants at the second step were not successful. At least in vitro, non-target gene mutations accumulate in second- and third-step mutants upon exposure to a fluoroquinolone and typically include, but do not appear to be limited to, mutations in the mar or sox regulons with consequent increased drug efflux.     

临床大肠埃希菌第Ⅰ类整合子检测及耐药基因盒分析

目的对来自临床腹泻患者大肠埃希菌的第Ⅰ类整合子分布及其耐药基因盒的结构特征进行分析。方法应用聚合酶链反应（PCR）方法检测第Ⅰ类整合酶基因intⅠ阳性菌株，并对其整合的耐药基因进行测序及序列分析。结果在40株大肠埃希菌中有28株（70％）第Ⅰ类整合酶基因阳性。耐药基因盒扩增结果，13株菌得到1664bp的扩增产物，10株得到1586bp的产物，3株得到1009bp的产物，2株得到1009bp和1586bp两种不同产物。序列分析结果表明，1664bp携带aadA5和dfr17，1586bp携带aadA1和dhfrⅠ，均为对氨基糖苷类抗生素壮观霉素、链霉素和磺胺类药物甲氧苄氨嘧啶产生耐药的基因盒；1009bp为aadA23b，为对氨基糖苷类抗生素壮观霉素、链霉素产生耐药的基因盒。结论测出的28株第Ⅰ类整合子阳性菌株携带耐氨基糖苷类抗生素的基因，应从基因水平上对细菌耐药及其传播进行监测。     

Temporal interplay between efflux pumps and target mutations in development of antibiotic resistance in Escherichia coli

The emergence of resistance presents a debilitating change in the management of infectious diseases. Currently, the temporal relationship and interplay between various mechanisms of drug resistance are not well understood. A thorough understanding of the resistance development process is needed to facilitate rational design of countermeasure strategies. Using an in vitro hollow-fiber infection model that simulates human drug treatment, we examined the appearance of efflux pump (acrAB) overexpression and target topoisomerase gene (gyrA and parC) mutations over time in the emergence of quinolone resistance in Escherichia coli. Drug-resistant isolates recovered early (24 h) had 2- to 8-fold elevation in the MIC due to acrAB overexpression, but no point mutations were noted. In contrast, high-level (≥ 64× MIC) resistant isolates with target site mutations (gyrA S83L with or without parC E84K) were selected more readily after 120 h, and regression of acrAB overexpression was observed at 240 h. Using a similar dosing selection pressure, the emergence of levofloxacin resistance was delayed in a strain with acrAB deleted compared to the isogenic parent. The role of efflux pumps in bacterial resistance development may have been underappreciated. Our data revealed the interplay between two mechanisms of quinolone resistance and provided a new mechanistic framework in the development of high-level resistance. Early low-level levofloxacin resistance conferred by acrAB overexpression preceded and facilitated high-level resistance development mediated by target site mutation(s). If this interpretation is correct, then these findings represent a paradigm shift in the way quinolone resistance is thought to develop.     

Genetic relationship between soxRS and mar loci in promoting multiple antibiotic resistance in Escherichia coli.

Multiple antibiotic resistance in Escherichia coli has typically been associated with mutations at the mar locus, located at 34 min on the E. coli chromosome. A new mutant, marC, isolated on the basis of a Mar phenotype but which maps to the soxRS (encoding the regulators of the superoxide stress response) locus located at 92 min, is described here. This mutant shares several features with a known constitutive allele of the soxRS gene, prompting the conclusion that it is a highly active allele of this gene. The marC mutation has thus been given the designation soxR201. This new mutant was used to examine the relationship between the mar and sox loci in promoting antibiotic resistance. The results of these studies indicate that full antibiotic resistance resulting from the soxR201 mutation is partially dependent on an intact mar locus and is associated with an increase in the steady-state level of mar-specific mRNA. In addition, paraquat treatment of wild-type cells is shown to increase the level of antibiotic resistance in a dose-dependent manner that requires an intact soxRS locus. Conversely, overexpression of MarA from a multicopy plasmid results in weak activation of a superoxide stress response target gene. These findings are consistent with a model in which the regulatory factors encoded by the marA and soxS genes control the expression of overlapping sets of target genes, with MarA preferentially acting on targets involved with antibiotic resistance and SoxS directed primarily towards components of the superoxide stress response. Furthermore, compounds frequently used to induce the superoxide stress response, including paraquat, menadione, and phenazine methosulfate, differ with respect to the amount of protection provided against them by the antibiotic resistance response.     

大肠埃希菌对氟喹诺酮类的质粒介导耐药机制研究

目的了解近年来发现的质粒介导耐药机制在临床分离大肠埃希菌中对氟喹诺酮类耐药所起的作用。方法用MIC琼脂稀释法筛选耐左氧氟沙星大肠埃希菌;采用聚合酶链反应对qnrA、qnrS、qnrB、aac(6′)-Ib基因进行扩增,并进行PCR扩增产物直接双向测序。用接合实验了解是否存在水平传播的氟喹诺酮耐药性传播机制。结果 90株耐氟喹诺酮类大肠埃希菌中7株检出aac(6′)-Ib-cr基因,未检出qnrA、qnrS、qnrB基因。78株符合供体菌标准的大肠埃希菌中有2株接合成功,接合率2.6%。结论该地区存在质粒介导的氟喹诺酮类耐药性的水平传播。     

Fleroxacin resistance in Escherichia coli.

Spontaneous fleroxacin-resistant mutants of Escherichia coli K-12 were isolated at a frequency of 10(-10) to 10(-11) mutants per CFU plated. All mutants exhibited quinolone-resistant replicative DNA biosynthesis, and 4 of 11 mutants also had decreased amounts of OmpF or OmpC porin. None of the mutants had changes solely in porin proteins.     

某中医医院大肠埃希菌血流感染临床特征及耐药性分析

摘要 目的 分析某中医医院大肠埃希菌血流感染的易感因素、临床特点及细菌耐药性。方法 回顾性调查该院3年间从血培养中分离出大肠埃希菌的132例患者的临床资料,分析其性别、年龄、科室分布、基础疾病、临床特征、抗生素耐药等情况。结果 大肠埃希菌血流感染以ICU患者发生率最高（48例,36.36%）;最常见首发症状为发热,占91.67%（121例）;93.18%的患者患有各类基础性疾病;产超广谱β内酰胺酶（ESBLs）43株（32.58%）,耐碳氢酶烯类2例（1.52%）,其他抗菌药物耐药率为11.62%～100%。结论 年龄>40岁、有基础性疾病、ICU患者和侵袭性操作是大肠埃希菌血流感染的易患因素,产ESBLs菌株耐药性较高。应做好产ESBLs的检测及常用抗菌药物的药敏试验,指导临床合理用药。     

近6年产超广谱β-内酰胺酶大肠埃希菌耐药性动态观察分析

目的动态观察并分析我院2001年1月至2006年12月6年间产超广谱β-内酰胺酶(ESBLs)大肠埃希菌的检出率及耐药性变化,为临床准确合理地进行治疗提供依据。方法采用美国临床实验室标准化委员会或研究所(NCCLS/CLSI)推荐的纸片扩散确证法检测ESBLs,纸片扩散法做药敏试验,WHONET5.3分析软件对药敏试验进行统计,并对产和非产ESBLs菌株的药敏试验数据进行对比分析。结果1 053株大肠埃希菌中ESBLs阳性共445株,占42.3%,且6年来阳性率逐年显著性增高。体外药敏试验结果显示,亚胺培南、头孢西丁、阿米卡星、哌拉西林-他唑巴坦、头孢哌酮-舒巴坦对产ESBLs大肠埃希菌有较好的抗菌活性。产ESBLs大肠埃希菌的耐药率明显高于非产ESBLs菌株,且具有多重耐药性。结论本院大肠埃希菌产ESBLs率逐年升高,治疗时应考虑首选亚胺培南、头孢西丁、阿米卡星、哌拉西林-他唑巴坦、头孢哌酮-舒巴坦。     

从患者胆汁分离的大肠埃希菌中质粒介导的氟喹诺酮类耐药基因的检测

目的 分析浙江省衢州市开化县第二人民医院消化内科胆汁分离的大肠埃希菌的耐药特点并调查质粒介导的喹诺酮类耐药基因的分布情况和流行特点。方法 收集该院消化内科患者2011年1月至2013年6月分离的大肠埃希菌724株,均为非重复性菌株,采用全自动微生物分析仪器VITEK 2 COMPACT进行细菌鉴定及药物敏感性分析,采用聚合酶链反应 (polymerase chain reaction,PCR)分析质粒介导的氟喹诺酮类耐药基因(plasmid-mediated quinolone resistance genes, PMQR) 如qnrA、qnrB、qnrS、aac(6')-Ib-cr和qepA 基因的流行特点。结果 胆源性大肠埃希菌的构成比达18.65%(135/724),其对环丙沙星和左氧氟沙星的耐药率高达61.5%(83/135)和55.6%(75/135),未检出碳青霉烯类抗生素耐药菌株;PCR检测PMQR基因结果显示:135株胆源性大肠埃希菌中,121株 (89.63%) qnrA1 基因阳性,45株(33.33%) qnrB4 基因阳性,32株 (23.70%) qnrB6 基因阳性,43株(31.85%) qnrS1 基因阳性,34株(25.19%) aac(6')-Ib-cr 基因阳性菌株,未检出qepA基因;其中45株(33.33%)同时携带 qnrA1、qnrB4 基因,32株(23.70%)同时携带 qnrA1、qnrB6, 25株(18.52%)同时携带 qnrA1、qnrB4、qnrS1, 21株(15.56%)同时携带 qnrA1、qnrB4、qnrS1、acc(6')-Ib-cr, 12株(100%)同时携带 qnrA1、qnrB6、qnrS1、acc(6')-Ib-cr, 且环丙沙星和左氧氟沙星的耐药率随着PMQR基因组合种类的增多而增多。结论 消化内科胆汁分离的大肠埃希菌氟喹诺酮类抗生素耐药严重,耐药基因主要以 qnrA1为主,qnrB4和qnrB6 次之,且存在多种PMQR基因组合形式,这些潜在播散的氟喹诺酮耐药基因对于临床胆道感染的治疗有很大的挑战。     

Sulphonamide resistance gene sul3 found in Escherichia coli isolates from human sources

OBJECTIVES: The aim of this study was to investigate the molecular basis of an observed increasing resistance to trimethoprim and sulphonamides despite a simultaneous decline in co-trimoxazole consumption. The distribution of sulphonamide resistance genes sul1, sul2 and the recently discovered sul3 was studied in a collection of clinical isolates of Enterobacteriaceae. METHODS: PCR with primers specific for sul1, sul2 and sul3 was used to detect the three known sulphonamide resistance genes in the isolate collection. Sequence analysis was used for confirmation of results. Restriction endonuclease digestion and conjugational transfer assays were used for plasmid analysis. RESULTS: In 64 sulphonamide-resistant isolates, 39 sul1 genes and 48 sul2 genes were detected. Twenty-five isolates carried both sul1 and sul2 and two were negative for both genes. With PCR and sequence analysis these two were shown to harbour the new sulphonamide resistance gene sul3, which was carried by different plasmids. CONCLUSIONS: Sulphonamide resistance gene sul3, which is widespread among pigs in Switzerland, has now also been identified in two different clinical isolates of Escherichia coli, located in urinary tract infections in patients in Sweden.