Detection of extended spectrum beta-lactamases producing Enterobacteriaceae in feces

A study was made of 366 feces for detection of extended spectrum beta-lactamases producing Enterobacteriaceae from feces. The selective agar was used for modified drigalski agar (Eiken Chemical Co., LTD) with 2 micrograms/ml cefotaxime (ESBL screen agar). 92 strains of Enterobacteriaceae, 41 Escherichia coli, 15 Citrobacter freundii, 13 Enterobacter cloacae, 11 Klebsiella pneumoniae, and other 12, were isolated from ESBL screen agar. And, R-plasmid that were selected by 2 micrograms/ml cefotaxime were transferred by conjugation from two of the 92 strains. These strain were E. coli TH9809927 and Proteus mirabilis TH9808262 that were amplified by "Toho-1 type" primer. The clude enzyme from two strains (donor) and transconjugants were especially hydrolysed cepharoridine and cefotaxime. Accordingly, two strains (0.5%) were detected as ESBL producers. We think that the result of our survey suggests the increase of ESBLs producing bacterial infection in Japan, and believe that there is a trend of infection of its by surveilance of the feces.     

产超广谱β内酰胺酶菌株中质粒头孢菌素酶的研究

目的 调查产超广谱β内酰胺酶(ESBLs)的大肠埃希菌和肺炎克雷伯菌中质粒头孢菌素酶(AmpC酶)的发生率及其基因型。方法 收集北京朝阳医院2001年1～12月头孢西丁耐药的产ESBLs的24株大肠埃希菌、8株肺炎克雷伯菌,采用等电聚焦电泳测定β内酰胺酶的等电点;接合试验证实酶基因有无可转移性;脉冲场凝胶电泳(PFGE)确定耐药株的亲缘关系;对AmpC酶基因进行多重PCR及序列分析确定其基因型。结果 2001年北京朝阳医院ESBLs的发生率,大肠埃希菌为16.8％(49／292),肺炎克雷伯菌为160.5％(35／212);ESBLs株中质粒AmpC酶的发生率,大肠埃希菌为2．0％(1／49),肺炎克雷伯菌为17．1％(6／35)。这7株菌均产生DHA-1型AmpC酶;1株肺炎克雷伯菌可将头孢西丁耐药性传给受菌体。该7株菌都产TEM-1酶、5株产CTX-M-3型ESBL、2株产SHV-12型ESBL;该7株菌携带质粒2～5个,且都有约33～36kb的大质粒。PFGE发现这7株菌来自多个不同的克隆株。结论 北京朝阳医院ESBL阳性的大肠埃希菌和肺炎克雷伯菌中,有7株既产DHA-1型质粒AmpC酶,又产CTX-M-3或SHV-12 ESBL。这7株菌来自多个不同的克隆株。     

A case of sepsis caused by Enterobacter cloacae producing both type SHV-12 and type CTX-M-14 ESBLs in a post bone-marrow transplantation patient

Third-generation cephalosporin-resistant Enterobacter cloacae was isolated from the blood culture of a 31-year-old woman after bone-marrow transplantation. Since this strain was resistant to third-generation cephalosporins, the production of extended-spectrum beta-lactamases (ESBLs) was suspected. PCR and a sequence analysis confirmed two ESBL genes, bla(SHV-12) and bla(CTX-M-14). No bacteria were detected after meropenem was administered, and symptoms abated. This is, to our knowledge, the first report in Japan of E. cloacae clinical isolates simultaneously producing both SHV-12 and CTX-M-14 ESBL. In cases where chromosomal AmpC over-production of E. cloacae concomitantly produces ESBL, caution should be exercised due to the potential development of resistance against extended-spectrum beta-lactam agents.     

Clinical and bacterial analysis of pediatric urinary tract infection

We analyzed the clinical and bacterial backgrounds of 120 patients with pediatric urinary tract infection (UTI). Escherichia coli was the main pathogen recovered from 98 patients (81.7%). All causative agents isolated from 50 uncomplicated UTI cases were E. coli. Of 98 cases of E. coli UTI, 71 were treated with second-generation cephems, whose therapeutic effect was equal to that of third and fourth-generation cephems. MIC50 and MIC90 (microg/mL) for E. coli were as follows: cefazolin :2, 4; cefmetazole: < or = 0.5, 2; and ceftazidime: < or = 0.25, < or = 0.25. Yearly decline in susceptibility was not observed, but MIC elevation for third generation cephems (< or = 2 microg/mL) including ceftazidime was seen in six isolates. Careful monitoring of susceptibility trends is therefore necessary for appropriate antimicrobial therapy.     

产超广谱β-内酰胺酶菌院内感染分析

目的：分析产超广谱β－内酰胺酶（ESBL）菌株引起的院内感染的临床特点。方法：收集北京协和医院1999年1－11月ESBL（＋）大肠杆菌和肺炎克菌白菌引起的院内感染50例,随机选择ESBL（－）病例45例作为对照。采用t检验和x^2检验进行分析。结果：住院时间及三代头孢菌素的使用是ESBL菌株感染的危险因素（P＜0．02）;ESBL（＋）组中腹腔、盆腔感染明显高于ESBL（－）组（P＜0．02）,其他感染在两组中无明显差别（P＞0．05）,致病菌分离后72h内选用敏感抗生素治疗组的预后明显好于72h内未选用敏感抗生素治疗组（P＜0．002）;的ESBL菌对亚胺培南敏感,对头孢美唑、阿米卡星和哌拉西林－他唑巴坦的耐药率低;头孢他啶对ESBL菌体外活性高,但临床疗效尚无定论。结论：ESBL菌院内感染病死率高,诊断明确后及时选用敏感抗生素可明显改善预后。     

Extended broad-spectrum beta-lactamases conferring transferable resistance to newer beta-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns

Before 1985 at the Pitié-Salpêtrière Hospital in Paris (2,400 beds), resistance to cefotaxime in clinical isolates of Enterobacteriaceae involved only species producing inducible class 1 beta-lactamase. Between November 1985 and April 1987, however, 62 isolates (57 of Klebsiella pneumoniae and five of Escherichia coli) showed decreased susceptibility to cefotaxime (mean MIC, 8-16 micrograms/mL). The transferability of cefotaxime resistance in E. coli K12 was demonstrated for 15 of 16 selected isolates. By isoelectric focusing using iodometric detection with 20 mg of ceftriaxone/100 mL and determination of substrate and inhibition profiles, three beta-lactamases mediating cefotaxime resistance were identified as SHV-2 (isoelectric point [pI] 7.6), CTX-1 (pI 6.3), and "SHV-2-type" or SHV-3 (pI 6.98). The three beta-lactamases hydrolyzed penicillins and cephalosporins (including cefotaxime and ceftriaxone) and were therefore designated "extended broad-spectrum beta-lactamases" (EBS-Bla). The enzymes conferred to derivatives a high level of resistance to amoxicillin, ticarcillin, piperacillin, and cephalothin and a decreased degree of susceptibility (i.e., MICs increased by 10- to 800-fold) to cefotaxime, ceftriaxone, ceftazidime, and aztreonam. These beta-lactamases did not affect the activity of cephamycins (cefoxitin, cefotetan, moxalactam) or imipenem. Synergy between clavulanate or sulbactam (2 micrograms/mL) and amoxicillin was greater against derivatives producing EBS-Bla than against those producing TEM-1, TEM-2, or SHV-1; this synergy was greater with clavulanate than with sulbactam against derivatives producing SHV-2 and the SHV-2-type enzyme but was similar with clavulanate and sulbactam against those producing CTX-1. A double-disk synergy test performed with cefotaxime and Augmentin disks (placed 30 mm apart, center to center) seemed a useful and specific test for the detection of strains producing EBS-Bla.     

Extended-spectrum beta-lactamase (ESBL) produced by Escherichia coli and Klebsiella pneumoniae isolated from Teikyo University Hospital--the second report

We studied the high-level resistant to cefotaxime (CTX, MIC > or = 512 micrograms/ml) clinical isolates of Escherichia coli and Klebsiella pneumoniae in Teikyo University Hospital. The CTX-resistance could be transferred to E. coli K-12 chi 1037 or ML4903 strains from 30 of the 33 isolates by conjugation at a frequency of 10(-4). When the hydrolysis rate of benzylpenicillin was 100%, the beta-lactamases which were extracted from the transconjugants hydrolyzed CTX, CAZ and AZT at the rate of 38-95%, 0-8.6% and 0-56%, respectively. These results demonstrate that these enzymes should be categorized into ESBL. The nucleotide sequence of CTX-resistant gene was identified to that of the CTX-M2 gene which was first described in Argentina. It was found to have 99.9% homology to Toho-1 gene in Japan and 99.6% homology to CMY-2 gene. Using a PCR methods for the detection of one of ESBL gene such as CTX-M2, Toho-1 or CMY-2, the DNA was amplified from all strains (11 isolates of E. coli and 21 isolates of K. pneumoniae).     

Bacteremia caused by Escherichia coli producing extended-spectrum beta-lactamase: a case-control study of risk factors and outcomes

A case-control study was conducted in order to identify the risk factors associated with bloodstream infection caused by Escherichia coli producing extended-spectrum beta-lactamase (ESBL) and to determine the outcomes of infected patients. Risk factors associated with ESBL production, according to univariate analysis, included a history of recent hospitalization [odds ratio (OR) 4.3, 95% confidence interval (CI) 2.1-8.9; p < 0.001], severe underlying diseases (OR 15, 95% CI 4.4-51.5; p < 0.001), prior exposure to urinary catheters (OR 8.3, 95% CI 3.2-21.7; p < 0.001) and nosocomial (OR 14.1, 95% CI 6.1-32.8; p < 0.001) or urinary (OR 3.6, 95% CI 1.7-7.4; p < 0.001) origin of the bacteria. Multivariate analysis revealed that severe underlying diseases (OR 31.2, 95% CI 6.7-144; p < 0.001) and nosocomial (OR 16.5, 95% CI 5.6-49; p < 0.001) and urinary origins (OR 7.8, 95% CI 2.6-23.8; p < 0.001) of the bacteria were independently associated with ESBL production in bacteremic E. coli. Crude mortality in case patients was more than twice as high as that in controls (p = 0.04). Production of ESBL increased the risk of inappropriate initial therapy (OR 95.6, 95% CI 27.4-334.2; p < 0.001). Treatment failed in 4/7 case patients treated with ceftazidime to which the isolate was susceptible in vitro. Our findings have implications for the choice of empirical therapy in nosocomial urinary tract infection.     

SHV-type extended-spectrum beta-lactamase (ESBL) are encoded in related plasmids from enterobacteria clinical isolates from Mexico

OBJECTIVE: In this work we report the molecular characterization of beta-lactam antibiotics resistance conferred by genes contained in plasmids from enterobacteria producing extended-spectrum beta-lactamases (ESBL). MATERIAL AND METHODS: Fourteen enterobacterial clinical isolates selected from a group of strains obtained from seven different hospitals in Mexico during 1990-1992 and 1996-1998 were analyzed at the Bacterial Resistance Laboratory (National Institute Public Health, Cuernavaca). Molecular characterization included PFGE, IEF of beta-lactamases, bacterial conjugation, PCR amplification and DNA sequencing, plasmid extraction and restriction. RESULTS: Isolates were genetically unrelated. ESBL identified were SHV-2 (5/14) and SHV-5 (9/14) type. Cephalosporin-resistance was transferable in 9 of 14 (64%) clinical isolates with only one conjugative plasmid, DNA finger printing showed a similar band pattern in plasmids. CONCLUSIONS: The dissemination of cephalosporin resistance was due to related plasmids carrying the ESBL genes.     

In vitro antimicrobial activity of rokitamycin, a macrolide antibacterial agent, against clinically isolated strains of Campylobacter and other enteritis-causing bacteria]

We determined the minimum inhibitory concentrations (MICs) of rokitamycin (TMS-19-Q, RKM), a macrolide antimicrobial agent, against strains of various bacterial species isolated from enteritis patients, and compared them with those of josamycin (JM), erythromycin (EM) and ofloxacin (OFLX). MIC90 of RKM against 147 strains of Campylobacter jejuni, and each 25 strains of Shigella spp., Salmonella spp. and diarrheagenic Escherichia coli were 1.56, 200, 800 and 200 micrograms/ml, respectively. There was only one RKM resistant (MIC greater than 100 micrograms/ml) C. jejuni strain, while most of the strains of the other species were resistant to RKM. MIC values of the other drugs were all similar to those of RKM. MIC90 of OFLX against 147 strains of C. jejuni was 0.78 micrograms/ml, lower than other drugs.     

肝硬化合并自发性细菌性腹膜炎医院感染及社区感染病原学研究

目的 探讨肝硬化合并自发性细菌性腹膜炎(SBP)患者中医院感染与社区感染病原菌分布特点及耐药情况.方法 纳入北京地坛医院2001年1月至2008年12月腹水细菌培养阳性的肝硬化合并SBP患者226例,鉴定细菌并行药物敏感试验,数据行卡方检验和t检验.结果 医院感染的SBP患者共86例,占38.0%;社区感染的140例,占62.0%;Child-Pugh分级C级在医院感染和社区感染中各占97.7%和82.8%(x2=11.489,P=0.001),病死率分别为50.0%和30.0%(x2=9.081,P=0.003).腹水细菌培养出病原菌共232株、28种,其中医院感染SBP及社区感染SBP病原菌均以革兰阴性菌为主,分别占77.5%和76.9%,列前两位的均是大肠埃希菌和肺炎克雷伯菌,革兰阳性菌分别占19.1%和21.7%,真菌占3.4%和1.4%(P＞0.05).医院感染的SBP 32株大肠埃希菌和14株肺炎克雷伯菌中,分别有19株和5株产β-内酰胺酶(ESBL);社区感染的SBP60株大肠埃希菌和32株肺炎克雷伯菌中,只有11株大肠埃希菌产ESBL(P＜0.05).医院感染SBP革兰阴性菌对头孢菌素及喹诺酮耐药率明显高于社区感染SBP(P＜0.05),但均对亚胺培南较敏感(P＞0.05);医院感染SBP及社区感染SBP的革兰阳性菌株中,未发现对万古霉素耐药.结论 Child-Pugh C级肝硬化患者更易发生医院感染的SBP,且预后差;医院感染SBP及社区感染SBP病原菌群分布相似,以大肠埃希菌和肺炎克雷伯菌为主,但产ESBL阳性率明显升高.     

Causative agents of liver abscess in HIV-seropositive patients: a 10-year case series in Thai hospitalized patients

Liver abscess is an important tropical gastrointestinal disorder. HIV seropositive patients show relative immunosuppression and are more susceptible to infection, including liver abscess. This retrospective case review was made on 23 patients who were diagnosed as HIV seropositive with liver abscess in Bangkok, Thailand. We demonstrated the high rate of amoebic liver abscess in our series (17.4%) from fresh smear with five cases of tuberculosis and one case of Nocardosis. The rates of positive bacterial culture were 17.4% from blood and 47.8% from pus. Gram-negative aerobes were the major abscess pathogens in our series. Among Gram-negative aerobes, Klebsiella was the most significant microorganism, followed by Escherichia coli and Pseudomonas aeruginosa.     

随机引物PCR法分型大肠艾希菌及其在判断医院感染中的应用

目的对大肠艾希菌(艾希菌)进行随机引物PCR(AP—PCR)法基因分型并应用于医院感染的判断。方法以优化的随机引物、反应体系和扩增条件对临床分离的161株艾希菌进行AP—PCR法基因分型并按指纹图上DNA条带数及片段大小绘制基因分型图谱，同时对艾希菌的医院内感染进行了观察与分析。结果161株艾希菌共得AP—PCR型120种；艾希菌的医院内感染确实存在。结论AP—PCR法可对艾希菌有效分型并可用于艾希菌医院内感染的判断。     

河南省首次从人体内分离出E.coli O26:H11

目的 研究一株从腹泻病人体内分离出的Ecoli O26：H11。方法 血清凝集采用玻片法，生化鉴定采用VITEK32全自动细菌分析系统GNI 卡进行鉴定，并对该株E．coli O26：H11进行多重PCR鉴定、Vero细胞毒性试验、ESBL试验和21种抗生素的药敏试验，并结合NCCLS标准进行判定。结果 血清凝集反应中O26抗原和H11抗原都呈强凝集，且不自凝；生化反应呈现大肠杆菌的典型反应；rfp O157基因、stx1基因和stx2基因均为阴性，hlyA基因和meA基因为阳性；Vero细胞毒性试验阴性；ESBL，试验阳性；对多种抗生素具有耐药性。结论 该株E．coli O26：H11为一不产类志贺氏菌毒素的菌株，但ESBL阳性，具有多重耐药性。     

Multicenter study of the activity of pefloxacin on bacteria isolated in a hospital milieu

Minimal inhibitory concentrations (MICs) of pefloxacin were evaluated by agar dilution for 3422 bacterial strains isolated in nine hospitals. Enterobacteriaceae proved very sensitive to pefloxacin: 62 p. 100 of 1743 strains tested had MIC less than or equal to 0.12 and 90 p. 100 less than or equal to 1 microgram/ml; but the percent of strains with MIC greater than or equal to 2 varied among the different groups of Enterobacteriaceae: 2.7 p. 100 for E. coli to 39 p. 100 for Serratia. 55 p. 100 for Pseudomonas and 81 p. 100 of Acinetobacter were inhibited by 1 micrograms/ml or less (mode MIC 1 and 0.5 micrograms/ml). Haemophilus sp.: 0.03 and 0.06 micrograms/ml and Gonococci were very sensitive to pefloxacin. The spectrum of pefloxacin extended to Gram positive cocci: MIC of Staphylococci were 0.06 to 8 micrograms/ml (mode MIC: 0.5); Enterococci, other Streptococci and Pneumococci were less sensitive: 2 and 4 micrograms/ml for the majority of strains. Concerning anaerobic bacteria, pefloxacin was more active against Clostridium (0.5 to 1 microgram/ml generally), than against Bacteroides (4 to 16 micrograms/ml).     

A case report of siblings with hemolytic uremic syndrome from whose stool E. coli O157:H7 was isolated]

We experienced siblings of hemolytic uremic syndrome which occurred following diarrhea and bloody stool. They were immediately treated with dipyridamole and aspirin, and recovered from hemolytic uremic syndrome in about two weeks. E. coli O157:H7 (verotoxin producing E. coli) which is recently thought to be related to the pathogenesis of hemolytic uremic syndrome was isolated in their stool cultures on admission. As far as we know, this is the first case in Japan from which stool E. coli O157:H7 was detected. Moreover, we reported clinical effectiveness of genomic investigation utilizing polymerase chain reaction method for verotoxin coding region in a rapid diagnosis of this bacterial infection.     

Prevalence of extended-spectrum beta-lactamases (ESBLs) produced in blood isolates of gram-negative bacteria in a teaching hospital in southern Thailand

One hundred and eighty-three strains of gram-negative bacteria were isolated from 177 bacteremic patients during a 6-month period in 2004 at a teaching hospital in southern Thailand. Extended-spectrum beta-lactamases (ESBL) production was detected in K. pneumoniae, E. coli, and E. cloacae, at rates of 16/36 (44.4%), 3/59 (5.1%), and 2/13 (15.4%), respectively. All but one of the screened positive strains were also positive by both the combination disk method and the E-test. All of the K. pneumoniae strains that were resistant to ceftazidime by disk diffusion were demonstrated to be positive for ESBL production by both the combination disk and E-test methods. Most of the ESBL positive strains had a high MIC (more than 32 microg/ml) to ceftazidime. However, all the ESBL positive strains were sensitive to imipenem.     

Molecular characterization of extended-spectrum beta-lactamases produced by clinical isolates of Enterobacter cloacae from a Teaching Hospital in China

Of 59 clinical isolates of Enterobacter cloacae from a teaching hospital in Sichuan, China, 18 isolates were shown to be resistant to oxyimino cephalosporins and aztreonam. Enterobacterial repetitive consensus PCR revealed that these isolates comprised 7 distinct genotypes. The presence of plasmids in the 18 clinical isolates was revealed by conjugational transfer of plasmids from E. cloacae to Escherichia coli with the further isolation of the plasmids in the transconjugants. Subsequent nucleotide sequencing and beta-lactamase isoelectric focusing indicated that the plasmids encoded blaSHV, blaCTX-M and/or blaTEM genes, including genes for CTX-M-22 (13 strains), TEM-1 (12 strains), TEM-29 (1 strain), TEM-141 (1 strain), TEM-157 (1 strain), SHV-5 (1 strain), SHV-12 (1 strain), and SHV-70 (1 strain). The widespread presence of extended-spectrum beta-lactamases in E. cloacae isolated from the southwest of China was likely due to the dissemination of resistance plasmids with the predominant genotype of blaCTX-M-22.     

源自同一患者的二株血和粪便对碳青霉烯类耐药大肠埃希菌

目的 了解对碳青霉烯类抗菌药物耐药大肠埃希菌的耐药机制及与内源性感染的关系.方法 将临床分离的来源于同一患者血培养和粪便培养的大肠埃希菌2株,采用浓度梯度法(E-test)测定亚胺培南、美罗培南的最低抑菌浓度(MIC)值,纸片扩散法测定其他16种抗菌药物的敏感性,等电聚焦电泳(IEF)检测所产生的β-内酰胺酶,PCR及序列分析确定其基因型,接合试验和Southern杂交进行耐药基因定位,脉冲场凝胶电泳(PFGE)分析2株菌株的同源性.结果 亚胺培南、美罗培南对2株大肠埃希菌的MIC值均≥32 mg/L,均产KPC-2(等电点值为6.7)和SHV-12(等电点值为8.2).blaKPC-2基因位于可转移的54 kb质粒上.PFGE显示2株大肠埃希菌为同一克隆株.结论 2株大肠埃希菌的耐药性及染色体DNA酶切图谱均一致,该患者大肠埃希菌败血症极可能是肠道的大肠埃希菌移位引起的内源性感染.