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.     

Detection of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in effluents and sludge of a hospital sewage treatment plant.

AIMS: To detect ESBL (extended-spectrum beta-lactamase)-producing Klebsiella pneumoniae present in the effluents and sludge of a hospital sewage treatment plant, evaluating the treatment plant's potential to remove these micro-organisms. METHODS AND RESULTS: Twenty samples (crude sewage, UASB reactor effluent, filtered effluent and sludge) were collected in the period from May to December 2006, in order to analyse antimicrobial susceptibility and to check ESBL production, the disc-diffusion and the combined disc methods were used. Total and faecal coliform concentrations were also determined. ESBL-producing K. pneumoniae were detected in all samples analysed, representing 46 x 5% of the total strains isolated. Among the non-ESBL-producing strains, 26% were multiresistant and one strain resistant to eight of the nine antimicrobials tested was detected in the treated effluent. CONCLUSIONS: The hospital wastewater treatment plant did not show a satisfactory efficacy in removing pathogenic micro-organisms, allowing for the dissemination of multiresistant bacteria into the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The inefficacy of hospital wastewater treatment plants can result in routes of dissemination of multiresistant bacteria and their genes of resistance into the environment, thus contaminating water resources, and having serious negative impact on public health.     

Investigation of extended-spectrum beta-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli in Korea

AIMS: Isolates obtained from various regions in Korea in 2002 were identified and their susceptibility to extended-spectrum cephalosporins, monobactams and/or cephamycins was studied along with any production of extended-spectrum beta-lactamases (ESBLs). METHODS AND RESULTS: Bacteria identified by the conventional techniques and Vitek GNI card were Klebsiella pneumoniae and Escherichia coli. Using disk diffusion and double-disk synergy tests, we found that 39.2% of strains produced ESBLs. About 52% of isolates transferred resistance to ceftazidime by conjugation. Banding patterns of PCR amplification with the designed primers showed that 837- and 259-bp fragments specific to bla(TEM) genes were amplified in 63.3% of strains. 929- and 231-bp fragments (bla(SHV)), 847- and 520-bp fragments (bla(CMY)), 597- and 858-bp fragments (bla(CTX-M)) were amplified in 61.5, 17.3 and 7.7% of strains respectively. About 51.9% of strains contained more than two types of beta-lactamase genes. Especially, one strain contained bla(TEM), bla(CMY) and bla(CTX-M) genes. SIGNIFICANCE: Resistance mechanisms to beta-lactams, comprising mostly ESBL production, lead to the resistance against even recently developed beta-lactams in enterobacteria, which is now a serious threat to antibiotic therapy. The high prevalence of bla(CMY) genes and multidrug-resistant genes may also make therapeutic failure and lack of eradiation of these strains by extended-spectrum cephalosporins or cephamycins.     

A preliminary survey of extended-spectrum beta-lactamases (ESBLs) in clinical isolates of Klebsiella pneumoniae and Escherichia coli in Japan

We conducted a survey of extended-spectrum beta-lactamases (ESBLs) among 16805 Escherichia coli and 9794 Klebsiella pneumoniae clinical isolates recovered from 196 separate medical institutions during the period January 1997 to January 1998. Using the criteria for minimal inhibitory concentrations (MICs) of oxyimino-cephalosporins of >/=8 microg ml(-1) and confirmation by double-disk test, we detected 15 E. coli and 34 K. pneumoniae isolates producing ESBLs. Genotypes of ESBLs determined by PCR with type-specific primers included one TEM-derived and 24 SHV-derived ESBLs, in addition to 24 Toho-1-type ESBLs, one of the major types of ESBLs reported in Japan. Nucleotide sequence analysis of SHV-specific PCR products revealed that SHV-12 was the dominant type of SHV-derived ESBL. In addition, we also identified TEM-26 and SHV-2. This is the first report characterizing TEM- and SHV-derived ESBLs in Japan.     

Detection and source identification of airborne extended-spectrum beta-lactamase-producing Escherichia coli isolates in a chicken house

To identify airborne dissemination of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) in a chicken house, airborne E. coli was collected from indoor air of a chicken house using six-stage Anderson sampler, and E. coli from chicken fecal samples were also isolated simultaneously. ESBL-producing E. coli isolates from indoor air and fecal samples were screened by a phenotypic confirmatory test according to CLSI recommendations. And then, the enterobacterial repetitive intergenic consensus polymerase chain reaction was performed to analyze the source of airborne ESBL-producing E. coli. The results showed that the ESBL-positive rates of E. coli isolates from feces and the indoor air were 56 % (18/32) and 40 % (6/15), respectively. Furthermore, airborne ESBL-producing E. coli isolates in the chicken house had 100 % genetic similarities with the strains from chicken feces, indicating that ESBL-producing E. coli from chicken feces could be aerosolized and spread to the air.     

Detection of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of Iberian lynx

Aims: To characterize the diversity of extended‐spectrum beta‐lactamase (ESBL)‐producing Escherichia coli isolates recovered within the faecal microbiota of Iberian lynx. The identification of other associated resistance genes and the analysis of clonal relationship were also focused in this study. Methods and Results: From 2008 to 2010, 128 faecal samples of Iberian lynx (wild and captive animals) were collected. Eleven tested samples contained cefotaxime‐resistant E. coli isolates (all belonging to captive animals) and 10 ESBL‐producing isolates were showed. CTX‐M‐14 and SHV‐12 ESBL‐types were detected and seven different patterns were identified by pulsed‐field gel electrophoresis analysis. Conclusions: The occurrence of unrelated multiresistant E. coli in faecal flora of captive specimens of Iberian lynx, including the presence of ESBLs, resistant genes in integrons and virulence determinants was showed in this study. Significance and Impact of the Study: The results obtained in this study highlight the environmental problem as future reintroductions of Iberian lynx could lead to a spread of resistant bacteria. Additionally, ESBL‐producing bacteria can represent a health problem for this endangered species.     

Chromosomal sequences from Klebsiella pneumoniae flank the SHV-5 extended-spectrum beta-lactamase gene in pACM1

The nucleotide sequence was determined for Anon 13, a 1250-bp SmaI fragment located approximately 2.8 kb downstream from bla(SHV-5) in pACM1. Anon 13 is 99% identical to a segment of the unpublished sequence of the Klebsiella pneumoniae chromosome. Genes of the K. pneumoniae sequence are undefined, but conceptual amino acid translations of two ORFs in Anon 13 are homologous to L-fuculose-1-phosphate aldolase (FucA) and a conserved hypothetical protein present in the chromosomes of several species of bacteria. In addition, restriction mapping indicates that the region of homology between the K. pneumoniae chromosome and pACM1 is as least 7.9 kb and includes both Anon 13 and bla(SHV). These observations demonstrate the chromosomal origin of the bla(SHV-5) on pACM1.     

Potential pathogenicity and host range of extended-spectrum beta-lactamase-producing Escherichia coli isolates from healthy poultry

Thirty of 33 epidemiologically unrelated extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from healthy poultry lacked the virulence genes commonly associated with human-pathogenic strains. The main zoonotic risk is associated with the broad host range of avian E. coli belonging to sequence type complex 10 and of IncN and IncI1 plasmids carrying bla(CTX-M) or bla(SHV).     

临床分离肺炎克雷伯菌耐药性监测

目的了解临床分离肺炎克雷伯菌的耐药性,为临床合理应用抗菌药物提供实验室依据。方法采用微量稀释法对392例临床分离肺炎克雷伯菌进行药物敏感性测定;超广谱β-内酰胺酶(Extendedspectrumbeta-lactamases,ESBLs)检测用微量稀释法初筛,纸片法做确证试验。结果肺炎克雷伯菌对18种抗菌药物的药敏结果中,耐药率大于30%的抗菌药物多达11种;其中氨苄西林-舒巴坦、氨苄西林、头孢噻吩、哌拉西林、复方新诺明和头孢唑啉的耐药率高达20%以上。耐药率低于10%的抗菌药物仅有4种,分别为头孢曲松(7.7%)、头孢噻肟(7.4%)、氨曲南(6.9%)和亚胺培南(3.1%)。其它抗菌药物的耐药率都高于10%。产ESBLs菌株的发生率为32.9%～45.8%,平均为39.8%;产ESBLs菌株对多种抗菌药物的耐药率显著高于非产ESBLs菌株(P<0.05)。结论临床分离肺炎克雷伯菌对多种抗菌药物的耐药率较高,尤其是产ESBLs菌株的高耐药率及多重耐药性更为明显。临床应加强对肺炎克雷伯菌耐药性的监测并预防耐药菌株的传播流行。     

A simple and rapid serum bactericidal assay and its evaluation in clinical isolates of Klebsiella pneumoniae

A simple and rapid assay for the determination of serum bactericidal activity was developed and evaluated in 125 clinical isolates of Klebsiella pneumoniae. The serum reactivity against these isolates was concomitantly determined by the conventional viable count technique in order to compare the efficacy of the two techniques. The rapid assay could be completed within 5-8 h and the results were recorded in terms of visible change of colour of the culture medium. Of the 125 strains tested, more than 50% were found to be resistant to 20% normal human serum. There was an excellent agreement between the two methods. The degree of discordance observed in the results obtained by the two methods was statistically not significant (P>0.05). The conventional technique is labor intensive, cumbersome and time-consuming. The assay described here, on the other hand, is simple, easy and rapid enough to allow testing of a large number of bacterial isolates or recombinant clones in a single day. Thus, the assay can serve as an excellent alternative to the conventional technique for determining the bacterial serum susceptibility.     

Incidence of two virulence factors (aerobactin and mucoid phenotype) among 190 clinical isolates of Klebsiella pneumoniae producing extended-spectrum β-lactamase

Because outbreaks of multiple-resistant Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamases were recently observed in French hospitals, the presence of virulence factors was examined for (i) phenotype by bioassay for aerobactin production and by culture for the mucoid phenotype, and (ii) genotype using intragenic probes of respectively 2-kb BglII and 235-bp BamHI-BglII fragments and dot-blotting among 190 unreplicated K. pneumoniae clinical isolates issued from 25 French hospitals and producing different types of extended-spectrum beta-lactamases (TEM-related enzymes: TEM-3, TEM-4, CAZ-1, CAZ-2, TEM-8, or SHV-related enzymes: SHV-2, SHV-3, SHV-4). Only 3.7% and 7% of K. pneumoniae isolates produced aerobactin and mucoid phenotypes respectively, unrelated to type of beta-lactamase. Only 2% had both factors. No discordance was reported according to the detection method tested. The low prevalence of such virulence factors seems to indicate they were not involved in dissemination of nosocomial K. pneumoniae isolates producing an extended-spectrum beta-lactamase.     

肺炎克雷伯菌临床分离株的庆大霉素耐药性和细菌铁载体的作用

目的 研究临床分离的肺炎克雷伯菌对氨基糖苷类抗生素庆大霉素的耐药性与其产铁载体的关系。方法 采用K-B纸片法和肉汤稀释法确定70株临床分离的肺炎克雷伯菌对庆大霉素的药物敏感性;CAS琼脂实验检测肺炎克雷伯菌是否产铁载体;紫外可见分光光度法确定细菌产铁载体的量,根据中位数法将70株临床分离菌分为铁载体高产组（35株）和低产组（35株）;应用SPSS统计学软件分析抗生素耐药性与其产铁载体是否相关。结果 药物敏感性试验检测出菌株对庆大霉素的耐药率为50.00%（35/70）;铁载体检测实验确定70株肺炎克雷伯菌均产生铁载体,肺炎克雷伯菌对庆大霉素的耐药性与铁载体产量呈正相关关系（r=0.3154,P<0.05）,对庆大霉素耐药菌株铁载体产量明显高于敏感菌株（t=3.1650,P<0.05）,且铁载体高产组耐药率及lgMIC值明显高于低产组（χ2=9.6570,t=3.1360,P<0.05）。结论 70株临床分离的肺炎克雷伯菌均产生铁载体,铁载体可能参与肺炎克雷伯菌对庆大霉素的耐药,干扰庆大霉素的抑菌或杀菌过程。     

临床分离肺炎克雷伯菌Ⅰ类整合子的结构与功能

为了探索细菌多重耐药性的产生和播散的分子机制, 文章对2002～2007年间179株临床分离的肺炎克雷伯菌进行耐药性、I类整合子可变区基因盒结构以及基因盒携带的耐药性基因进行分段克隆和耐药性功能测定。结果显示：65.9%(118/179)的肺炎克雷伯菌表现出对至少两种以上的抗生素(主要为β-内酰胺类、氨基糖苷类和喹诺酮类抗菌药物)的耐药性; 36.3%(65/179)的菌株检出单条或者双条I类整合子基因盒条带; 对整合子阳性组与阴性组的耐药率进行比较发现, 除氨基糖苷类、喹诺酮类和复方新诺明等药物的耐药性存在显著性差异(P<0.01)外, 其余药物的差异不显著; 共发现15种耐药基因构成形式的整合子基因盒, 其中以dfrA17-aadA5最为多见, 实验证明整合子可由接合转移耐药性质粒携带; 对整合子基因盒(dhfr17-orfF-aadA2)分段克隆的耐药性功能研究发现, 3个克隆重组子(pET28a-dhfr17、pET28a-dhfr17-orfF和pET28a-dhfr17-orfF-aadA2)对复方新诺明的抗性(MIC值)均为256 µg/mL, 重组子pET28a-dhfr17-orfF与重组子pET28a-dhfr17对链霉素的抗性无明显区别, 和受体菌一样MIC值均为8 µg/mL, 而pET28a-dhfr17-orfF-aadA2对链霉素的抗性则明显提高, MIC值为256 µg/mL。结果表明, I类整合子在肺炎克雷伯菌中较常见, 携带氨基糖苷类和甲氧苄啶类的耐药基因盒在数量上占优势, 且整合子携带的耐药基因具有耐药性功能, 位于可水平转移耐药性质粒的耐药性基因相关的整合子对病原菌耐药性播散具有重要意义。 目的基因     

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。     

Two different types of fosfomycin resistance in clinical isolates of Klebsiella pneumoniae

Abstract The fosfomycin susceptibility of 100 clinical isolates of Klebsiella pneumoniae and the resistance mechanisms utilized by resistant strains were examined. Washed cells prepared from the strains demonstrating MICs of more than 8 μg ml⁻¹ of fosfomycin inactivated the drug. A crude extract from strain Tf129B, highly resistant to fosfomycin, was used to study the enzymatic properties of the drug-inactivating enzyme. The optimum pH for inactivation was 7.8 and the optimum temperature of the reaction was 37°C. Glutathione was shown to be effective as a cofactor in the inactivation. It was suggested that the inactivating enzyme of Klebsiella pneumoniae was fosfomycin: glutathione-S-transferase, a constitutive enzyme located in the periplasmic space. A good correlation was found between the specific activities of this enzyme and the MIC levels; however, certain strains showed a low level of fosfomycin: glutathione-S-transferase activity which could not account for the increased MIC. Strains Tf129B and Tf408E, both demonstrating MICs of more than 1024 μg ml⁻¹ of fosfomycin carried a transferable resistance plasmid. In strain Tf129B, the mechanism of fosfomycin resistance was due to a high level of enzymic activity. In strain Tf408E, it was determined to be mainly due to the reduced permeability of the cell membrane.     

Phenotypic and genotypic characterization of Klebsiella pneumoniae isolates recovered from nonhuman primates

Klebsiella pneumoniae is a zoonotic, Gram-negative member of the family Enterobacteriaceae and is the causative agent of nosocomial septicemic, pneumonic, and urinary tract infections. Recently, pathogenic strains of K. pneumoniae sharing a hypermucoviscosity (HMV) phenotype have been attributed to multisystemic abscessation in both human and nonhuman primates. Although K. pneumoniae is a well-recognized zoonotic agent, there is a lack of general information including adequate diagnostic methods or treatments for nonhuman primates. In an effort to increase the body of knowledge of this enigmatic pathogen, K. pneumoniae isolates from African green monkeys (Chlorocebus aethiops sabaeus) on the island of St. Kitts, West Indies were genotypically and phenotypically characterized. Genetic fingerprints generated by PCR-mediated genomic fingerprinting, phenotypic characterization, and antimicrobial susceptibility all identified a high degree of similarity between the HMV and non-HMV K. pneumoniae isolates. The results obtained from this work will help establish a baseline for the development of efficacious diagnostic methods and treatment strategies for both human and nonhuman primates.     

Comparative study of immunobiological properties of Klebsiella planticola and Klebsiella pneumoniae strains

The present investigation was based on information on the frequent isolation of K. planticola, inhabiting soil and water ecosystems, from respiratory tract secretions, urine, wound surface and other clinical material in infectious pathology of humans. The comparative study of the main immunobiological properties of K. planticola strain TCXA 91 and K. pneumoniae strain 204 was carried out. The strains were equally virulent for white mice, the infective dose being about 100x10(6) when introduced intraperitoneally in 0.4% agar. These strains exhibited protective cross activity in the animals challenged with K. pneumoniae virulent strain K16. The survival rates of mice for K. planticola TCXA 91 and K. pneumoniae 204 were, respectively, 60% and 70%, the death rate of the control animals being 100%. For the first time K. pneumoniae 204 was found capable of stimulating the growth of lawn grasses in the presence of sodium chloride. The results were evaluated by the germination of seeds, the length of the plantlet and the root. The data obtained in this study are indicative of the similarity of the immunobiological properties of K. planticola TCXA 91 and K. pneumoniae 204.     

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.