Two novel transferable extended-spectrum β-lactamases from Klebsiella pneumoniae in Tunisia

Two novel beta-lactamases conferring multiresistance to antibiotics including oxyimino beta-lactams have been identified in two nosocomial K. pneumoniae strains isolated in Tunis in 1986 and 1988. Both enzymes were encoded by ca. 150-kilobase plasmids. Donor and transconjugant strains producing these enzymes exhibited highly similar pattern of resistance (CTX phenotype) to beta-lactams including penicillins and oxyimino beta-lactams e.g. cefotaxime, ceftriaxone, ceftazidime, and aztreonam. High and variable synergy (16 to 1066-fold) was obtained when combined to 0.1 microgram/ml of clavulanate (beta-lactamase inhibitor). The isoelectric points of these two enzymes were 5.4 and 6.4. These beta-lactamases differed from TEM types by hydrolysis for cefotaxime or ceftriaxone but were inhibited by clavulanate and cloxacillin. DNA hybridization studies suggested that that the genes of these enzymes may be derived from genes encoding TEM-type enzymes.     

同时产多种基因型ESBLs肺炎克雷伯菌的分析

目的分析汕头大学医学院第一附属医院临床分离的肺炎克雷伯菌同时产多种基因型ESBLs的情况。方法采用PCR扩增对产ESBLs的肺炎克雷伯菌初步分型,然后PCR扩增阳性产物克隆测序分析,脉冲场凝胶电泳（PFGE）分型;用浓度梯度法检测10种抗菌药物对同时产多种基因型ESBLs的肺炎克雷伯菌的最低抑菌浓度（MIC）。结果83株产ESBLs的肺炎克雷伯菌中,发现9株同时产TEM、SHV和CTX-M型,测序结果为CTX-M-3、TEM-1及多种SHV型（SHV12及SHV28）。将这9株细菌作PFGE分析,结果共被分成7个型。药敏结果表明,9株菌除对亚胺培南敏感外,对氨曲南、头孢曲松、头孢噻肟、头孢他啶、丁胺卡那和四环素均高度耐药,对哌拉西林／三唑巴坦、头孢吡肟、环丙沙星极少菌株敏感。结论发现CTX—M-3超广谱-及多种SHV型超广谱-和TEM-1广谱β-内酰胺酶基因同时存在该院临床分离的肺炎克雷伯菌中,耐药十分严重,应引起重视。     

The TEM-3 β-lactamase, which hydrolyzes broad-spectrum cephalosporins, is derived from the TEM-2 penicillinase by two amino acid substitutions

Abstract We have determined the nucleotide sequence of the blaT -3 gene of plasmid pCF04 which confers resistance to penicillins and most cephalosporins by mediating the production of TEM-3 β-lactamase. The deduced amino acid sequence of TEM-3 differed in two positions from that of the TEM-2 penicillinase: Lys (TEM-3) for Glu (TEM-2) at position 102, and Ser (TEM-3) for Gly (TEM-2) at position 236 of the unprocessed protein. Examination of the location of the two modified amino acids of TEM-3 in the tertiary structure of class A β-lactamases suggested that they both are part of the substrate binding site. Analysis, by in vitro recombination, indicated that each mutation contributes to the extented substrate range of the enzyme, compared to that of the TEM-type penicillinase, and that the strength of the promoter of blaT -3 is responsible for high-level resistance towards broad-spectrum cephalosporins of strains producing TEM-3.     

Incidence of Extended-Spectrum β-Lactamases and Characterization of Integrons in Extended-Spectrum β-Lactamase-producing Klebsiella pneumoniae Isolated in Shantou, China

This study is concerned with the level of antibiotic resistance of extended-spectrum β-lactamase （ESBL）-producing Klebsiella pneumoniae, isolated in Shantou, China, and its mechanism. Seventy- four non-repetitive clinical isolates of K. pneumoniae producing ESBLs were isolated over a period of 2 years. Antibiotic susceptibility, carried out by Epsilometer test, showed that most of the isolates were multiresistant. Polymerase chain reaction showed that, among the several types of β-lactamases, SHV was the most prevalent, TEM was the second most prevalent, and CTX-M was the least prevalent. Sixty-nine isolates were positive for integrase gene IntI1, but no IntI2 or IntI3 genes were found. The variable region of class 1 integrons were amplified and further identified by sequencing. Thirteen different gene cassettes and 11 different cassette combinations were detected. Dfr and aadA cassettes were predominant and cassette combinations dfrA12, orfF and aadA2 were most frequently found. No gene cassettes encoding ESBLs were found. Integrons were prevalent and played an important role in multidrug resistance in ESBL-producing K. pneumoniae.     

产ESBLs肺炎克雷伯菌β-内酰胺酶基因型分析

目的 了解深圳市人民医院产超广谱β-内酰胺酶(ESBLs)肺炎克雷伯菌的ESBLs和头孢菌素(AmpC)酶基因型分布特点.方法 收集深圳市人民医院产ESBLs肺炎克雷伯菌临床菌株64株,PCR法分别扩增菌株的TEM、SHV、CTX-M基因,并进行DNA测序分型.同时应用多重PCR对其中的头孢西丁耐药株进行AmpC酶基因扩增,DNA序列确定其基因型.结果 64株产ESBLs肺炎克雷伯菌中,61株(95.3％)检出至少一种ESBLs基因.其中51.6％ (33/64)检出SHV-12基因,46.9％(30/64)检出CTX-M-14基因.11株(17.2％)检出AmpC基因,其中10株为DHA-1型,1株为CYM-2型.19株(29.7％)检出2种以上的ESBLs或ESBLs合并AmpC基因.结论 该院产ESBLs肺炎克雷伯菌中,最常见的ESBLs基因型为SHV-12和CTX-M-14型;AmpC酶的主要基因型为DHA-1,菌株中同时产生多种β-内酰胺酶的较多.     

Substitution of Thr for Ala-237 in TEM-17, TEM-12 and TEM-26: alterations in β-lactam resistance conferred on Escherichia coli

Non-naturally occurring mutants of TEM-17 (E104K), TEM-12 (R164S) and TEM-26 (E104K:R164S) extended-spectrum (ES) beta-lactamases bearing threonine at position 237 were constructed by site-specific mutagenesis and expressed under isogenic conditions in Escherichia coli. Quantification of beta-lactamase activities and immunoblotting indicated that Ala-237-->Thr did not significantly affect expression levels of these ES enzymes. Minimum inhibitory concentrations of beta-lactam antibiotics showed that the presence of threonine at position 237 exerted a dominant effect increasing the enzymes' preference for various early generation cephalosporins over penicillins. Activity against broad-spectrum oxyimino-beta-lactams was also changed. The effect of Ala-237-->Thr on the activity against ceftazidime, aztreonam, cefepime and cefpirome of all three ES TEM enzymes was detrimental. Introduction of Thr-237 improved activity against cefotaxime and ceftriaxone in TEM-12 and TEM-26, but not in TEM-17.     

产ESBLs肺炎克雷伯菌的基因型和耐药性研究

超广谱β-内酰胺酶(extended spectrumβ-lactamase,ESBLs)与细菌耐药性密切相关,为了揭示产ESBLs肺炎克雷伯菌的基因型和耐药性,本研究在2017年1月至2018年9月期间,共检测了466例肺炎克雷伯菌感染的患者标本,并分析了产ESBLs肺炎克雷伯菌的基因型和耐药性。研究结果显示,466例患者中共检出562个肺炎克雷伯菌菌株。其中,呼吸内科的检出数最多(212株,37.72%),其次为重症监护科(106株,18.86%)。对于不同标本来源,痰液中检出菌数最多(331株),占总数的58.90%。562株肺炎克雷伯菌中共检测出237株产ESBLs(42.17%)。产ESBLs肺炎克雷伯菌对阿莫西林(89.03%)、替卡西林(87.34%)、氨苄西林(81.43%)的耐药率最高;而对替加环素(9.28%)、阿米卡星(6.75%)、亚胺培南(1.27%)和美罗培南(0.84%)的耐药率最低。237株产ESBLs的肺炎克雷伯菌菌株中,53.59%扩增出CTX-M型,48.95%扩增出TEM型,40.93%增出SHV型。另外,237株产ESBLs的肺炎克雷伯菌菌株中,产两个及以上的ESBLs的菌株共111株(46.84%)。     

连续12年产超广谱β-内酰胺酶肺炎克雷伯菌感染分布及耐药性分析

目的了解产超广谱β-内酰胺酶（ESBLs）肺炎克雷伯菌（KPN）的临床分布及耐药性,为临床合理选用抗菌药物提供依据。方法用常规方法,结合法国梅里埃公司的自动化细菌鉴定分析仪分离鉴定细菌;用K-B纸片扩散法进行药敏试验;用双纸片协同筛选法和酶抑制剂增强纸片法检测ESBLs。对中国医科大学附属盛京医院1999年1月至2010年12月期间临床分离的产ESBLs和非产ESBLs KPN菌株的临床分布及其耐药趋势进行回顾性分析。结果 KPN的ESBLs检出率分别从1999年的19.82%上升至2010年的49.34%;KPN主要分离于痰占52.4%,其次是血液占17.74%,主要来源于外科病区和新生儿病区;产ESBLs菌和非产ESBLs菌对阿米卡星、左氧氟沙星等非β-内酰胺酶类抗菌药物的耐药率均显著增高,差异具有统计学意义（P〈0.01）,对亚胺培南和美罗培南都保持高度敏感,耐药率均低于2%。结论 12年间ESBLs检出率总体呈上升趋势,产ESBLs肺炎克雷伯菌表达对包括非β-内酰胺类在内的抗菌药物多重耐药,对碳青霉烯类抗菌药物仍保持高度敏感性。     

Molecular characterisation by PCR-restriction fragment length polymorphism of TEM β-lactamases

Abstract To rapidly characterise TEM-derived extended-spectrum β-lactamases a fast and easy method using polymerase chain reaction-restriction fragment length polymorphism was developed. This method was validated with ten reference TEM-type extended-spectrum β-lactamases. The mutations involved in TEM-20 and TEM-21, which were previously reported only with biochemical analysis, were then characterised. TEM-20 differed from TEM-19 by a silent mutation at position 925 (A for G), and TEM-21 differed from TEM-3 and TEM-14 by a single mutation (G for A) in an unreported position 660, involving an amino acid substitution, arginine for histidine, at position 153. Moreover, a new extended-spectrum β-lactamase conferring low resistance to ceftazidime (TEM-29), was described. TEM-29 derived from TEM-1, with an amino acid substitution, his-164. Finally, the combination of polymerase chain reaction-restriction fragment length polymorphism and plasmid analysis allowed us to investigate nosocomial outbreaks due to clinical isolates of multi-resistant Klebsiella pneumoniae in three hospitals.     

Treatment options for extended-spectrum beta-lactamase-producers

A review of antibiotic options for the treatment of infections caused by extended-spectrum beta-lactamase-producing isolates is presented. The use of the third-generation cephalosporin, cefotaxime, for infections caused by isolates producing ceftazidimase-type extended-spectrum beta-lactamases is controversial, despite in vitro susceptibility to the antibiotic in many instances. The fourth-generation cephalosporin, cefipime, although active against most extended-spectrum beta-lactamases, is reported to show a marked inoculum effect. The cephamycins, such as cefoxitin. are generally effective against Enterobacteriaceae producing TEM- and SHV-derived extended-spectrum beta-lactamases, but Klebsella pneumoniae strains are prone to cephamycin resistance as a result of porin loss. The use of beta-lactamase inhibitor combinations is variable. Sulbactam is less effective than clavulanate for the inhibition of SHV-derived extended-spectrum beta-lactamases and a marked inoculum effect has been noted, while the efficacy of tazobactam against SHV-derived extended-spectrum beta-lactamase producers is controversial. Furthermore, extended-spectrum beta-lactamases are often encoded by multi-resistant plasmids carrying genes conferring resistance to aminoglycosides, chloramphenicol, sulfonamides, trimethoprim and other antimicrobials, severely limiting even alternative therapies. Extensive susceptibility testing before the institution of antibiotic therapy is thus vital.     

产超广谱β-内酰胺酶肺炎克雷伯菌血流感染的耐药性、危险因素及临床结局分析

本研究旨在分析107例肺炎克雷伯菌血流感染患者的临床资料,探讨产超广谱β-内酰胺酶(extended-spectrumβ-lactamase,ESBL)肺炎克雷伯菌血流感染的耐药特点、危险因素及临床结局,为防治产ESBL肺炎克雷伯菌血流感染提供临床理论参考。选取2012年1月—2016年6月于深圳市南山区人民医院住院且血培养肺炎克雷伯菌阳性的107例患者,根据药敏结果分成产ESBL血流感染组(20例)和非产ESBL血流感染组(87例)。107例患者血流感染主要继发于肺部感染(38.32%)及泌尿系感染(14.02%),细菌对碳青霉烯类抗生素敏感性好。单因素及logistic回归分析结果显示,医院内感染和入院前有外伤史为产ESBL肺炎克雷伯菌血流感染的危险因素。总体肺炎克雷伯菌血流感染病死率为17.76%,产ESBL组与非产ESBL组之间病死率无显著性差异(25%vs.16.09%)。结果提示,产ESBL不是预测肺炎克雷伯菌血流感染患者死亡的独立危险因素。     

Characterization of a novel SHV β-lactamase variant that resembles the SHV-5 enzyme

Abstract An SHV type β-lactamase frequently found in enterobacteria isolated in Greek hospitals was analyzed. The enzyme (SHV-5a) conferred resistance to ceftazidime and aztreonam. The DNA sequence of the structural gene was determined. The deduced amino acid sequence showed that positions 70–73 were occupied by the active site tetrad Ser-Thr-Phe-Lys. As in SHV-5, Ser-238 and Lys-240 were present. However, one deletion (Gly-54) and three substitutions (Arg-140 for Ala, Asn-192 for Lys and Val-193 for Leu) differentiate SHV-5a β-lactamase from SHV-5. Asn-192 and Val-193 have been reported to date only in the R974 plasmid-mediated SHV-1 β-lactamase. Hydrolysis studies with SHV-5a and SHV-5 showed that the enzymes behaved similarly. Additional evidence that they were functionally indistinguishable was provided by the similar MICs of β-lactams when the enzymes were expressed under isogenic conditions. The sequence differences, however, indicate that they are derived from different ancestors.     

Polypeptides of DNA-dependent RNA polymerase of spinach chloroplasts: characterization by antibody-linked polymerase assay and determination of sites of synthesis

Using solid-phase `Sandwich' immunoassays we studied DNA-dependent RNA polymerase of spinach chloroplasts with regard to (i) polypeptide composition of the multimeric enzyme; (ii) immunological cross-reaction with Escherichia coli RNA polymerase; (iii) sites of synthesis of polymerase polypeptides. Our main results are as follows. (i) All polypeptides of isolated chloroplast RNA polymerase (150, 145, 110, 102, 80, 75 and 38 kd) are labeled by an antibody-linked polymerase assay (ALPA), i.e., they are immunologically related to subunits of the holoenzyme. On the other hand differences in the patterns of `ALPA-reactive' polypeptides of a crude RNA polymerase fraction and of the purified enzyme preparation indicate partial proteolytic degradation of polymerase polypeptides during purification. Thus the 80- and 75-kd polypeptides, which had been previously considered as true RNA polymerase polypeptides, probably result from partial proteolytic degradation. (ii) The 150- and 145-kd polypeptides show immunochemical similarities with the β and/orβ' subunits of E. coli RNA polymerase. (iii) Results from solidphase immunoassay of in vitro translated products of both chloroplast RNA and poly(A)⁺ (nuclear) RNA suggest that all chloroplast RNA polymerase polypeptides are coded for by the nucleus.     

血流感染大肠埃希菌和肺炎克雷伯菌的ESBLs基因分型

目的 了解深圳市人民医院致血流感染大肠埃希菌和肺炎克雷伯菌超广谱β-内酰胺酶(ESBLs)的检出率及基因型特点.方法 收集来自临床血液培养标本中的大肠埃希菌和肺炎克雷伯菌115株,采用ESBLs表型确证试验检测菌株的ESBLs,应用PCR扩增产ESBLs菌株TEM、SHV和CTX-M基因,并对阳性扩增产物进行DNA测序分型.结果 115株菌中共检出ESBLs阳性38株,检出率为33.0％;其中大肠埃希菌阳性25株,肺炎克雷伯菌阳性13株.25株产酶肠埃希菌中18株检出CTX-M-14基因,3株检出CTX-M-9基因.13株产酶肺炎克雷伯菌均检出SHV型基因,其中SHV-12阳性10株,SHV-2阳性2株,SHV-59阳性1株;该13株产酶菌中10株同时被检出含CTX-M-14或CTX-M-13基因.结论 该院致血流感染大肠埃希菌产ES-BLs以CTX-M-14为最主要基因型,肺炎克雷伯产ESBLs最常见为SHV-12和CTX-M-14型.     

摩根摩根菌β-内酰胺酶检测及耐药性分析

目的了解摩根摩根菌临床分离株产超广谱β-内酰胺酶（ESBLs）、头孢菌素酶（AmpC）、金属酶（MBLs）、碳青霉烯酶（KPC）情况,并分析其对17种常见抗菌药物的耐药性。方法 ESBLs和AmpC及MBLs采用三维试验检测,碳青霉烯酶采用改良Hodge试验进行检测,并以K-B法测定17种常见抗菌药物的耐药性。结果 102株摩根摩根菌单产ESBLs 15株,检出率为14.71%;单产AmpC 8株,检出率为7.84%;单产金属β-内酰胺酶（MBLs）3株,检出率为2.94%;所有菌株中未检出碳青霉烯酶（KPC）;同产ESBLs及AmpC 6株,检出率为5.88%;未发现其他双产酶菌株。摩根摩根菌非产酶分离株对17种抗生素的耐药率均低于50.0%;摩根摩根菌产酶分离株对亚胺培南、美罗培南培南的耐药率低于15.0%,与非产酶菌株相比,差异无统计学意义（P〉0.05）;对其余抗生素的耐药率均明显高于非产酶菌株（P〈0.05）。同产ESBLs＋AmpC与耐亚胺培南摩根摩根菌分离株呈多重耐药。结论我院摩根摩根菌分离株产生多种β-内酰胺酶,且对常用抗生素耐药性比较严重,建议临床医师合理使用抗生素,以免耐药菌株的产生。