大肠埃希菌、肺炎克雷伯菌、阴沟肠杆菌超广谱β-内酰胺酶的检测及药敏分析

目的:了解本院大肠埃希菌、肺炎克雷伯菌、阴沟肠杆菌中超广谱β-内酰胺酶(ESBLs)细菌的发生率和药敏情况,为防止这3种细菌产ESBLs菌株的传播和抗生素的合理应用提供依据.方法:细菌鉴定用Vitek-32细菌鉴定仪,药敏试验用K-B法,ESBLs初筛用双纸片法,ESBLs确证用NCCLS1999年推荐的确证方法.结果:产ESBLs耐药菌占全部分离菌的33.2%,其中各细菌ESBLs发生率大肠埃希菌为28.6%,肺炎克雷伯菌为34.1%,阴沟肠杆菌为40.5%.对实验所做抗生素,ESBLs阳性株比阴性株具有更高的耐药率和更严重的交叉耐药现象.结论:本院大肠埃希菌、肺炎克雷伯菌和阴沟肠杆菌ESBLs发生率较高,较往年有上升趋势,ESBLs菌感染的治疗仍以亚胺培南为首选.     

健康老年人肠道中肠杆菌科细菌的分布及其耐药机制研究

目的分析健康老年人肠道菌群,探讨本地区健康老年人肠道中肠杆菌科细菌的分布及其耐药情况。方法取粪便标本分别接种于血平板、SS平板、麦康凯平板进行细菌培养,用全自动微生物鉴定仪和质谱仪鉴定细菌到种,采用K-B纸片扩散法进行药敏试验测定其耐药表型,PCR扩增测序再与GenBank上比对其耐药基因。结果在175例研究对象中,有59. 4%（104/175）的健康老年人肠道中只有一种肠杆菌科细菌;细菌种类以大肠埃希菌为主,占88.0%。大肠埃希菌对IPM、CTX、AMC.F0X和CIP的耐药率分别为0% ,27.9%、4. 5% ,1.3%和37. 0% ;肺炎克雷伯菌对IPM、CTX、AMC、FOX和CIP的耐药率分别为1.9%、5.8%、1.9%、1.9%和3.8%;两者相比较,大肠埃希菌对CTX、CIP的耐药率明显高于肺炎克雷伯菌（P〈0. 05）。ESBLs总携带率为17. 4%,其中大肠埃希菌的携带率（24.0%）明显高于肺炎克雷伯菌（5.8%） （P 〈0.05）;产ESBLs菌株的基因型,均为CTX-M型酶,其中以CTX-M-14最多,占57. 8%。结论本研究获得了老年人肠道肠杆菌科细菌的分布特点及其耐药机制。健康老年人肠道中肠杆菌科细菌部分抗生素的耐药率比浙江省内医院肠杆菌科细菌统计结果低,这主要是和其ESBLs携带率较低相关;但同时也已有泛耐药细菌的出现,应提高警惕。     

亚抑菌浓度头孢他啶对大肠埃希菌生物膜形成的影响与其耐药性相关性研究

目的:探讨亚抑菌浓度头孢他啶对大肠埃希菌生物膜形成的影响与细菌耐药性、超广谱β-内酰胺酶(ESBLs)产生及ESBLs基因分型的相关性,为临床生物膜感染的治疗和抗生素的合理使用提供理论依据。方法:大肠埃希菌最低抑菌浓度(MIC)检测采用琼脂平板倍比稀释法,超广谱β-内酰胺酶(ESBLS)表型确证实验采用双纸片协同法,大肠埃希菌ESBLs基因检测采用PCR扩增,生物膜形成能力检测采用96孔板结晶紫染色法。结果:50株大肠埃希菌临床株对青霉素类、氟喹诺酮类、头孢哌酮及复方新诺明具有较高的耐药性,而对阿米卡星、哌拉西林/他唑巴坦敏感性较高。所有菌株均对碳青霉烯类抗菌药物敏感。31株大肠埃希菌为ESBLs阳性菌株。CTX-M、TEM、OXA、SHV和VEB基因阳性率分别为93.5%、83.9%、19.4%、16.1%和3.2%。亚-MIC头孢他啶对9株(18.0%)大肠埃希菌生物膜形成具有抑制作用。亚-MIC头孢他啶对大肠埃希菌生物膜形成的影响与细菌耐药性和ESBLs均无相关性(P0.05)。结论:亚-MIC头孢他啶对大肠埃希菌生物膜形成的调控作用与细菌耐药性、产ESBLs及ESBLs基因分型均无相关性。     

产超广谱β-内酰胺酶大肠埃希菌的耐药性分析

目的:了解产超广谱β-内酰胺酶大肠埃希菌在泌尿系统感染中的流行情况及对12种常用抗菌药物的耐药性,为临床合理使用抗生素提供必要依据.方法:采用K-B纸片扩散法,测定128株大肠埃希菌对12种抗菌药物的耐药性,同时使用E-test方法筛选超广谱β-内酰胺酶阳性菌株.结果:从128株大肠埃希菌中共检出ESBL 26株,产ESBL细菌阳性率为20.3%.在12种抗菌药物中,亚胺培南的体外抗菌活性最好,敏感率为100%,其次为阿米卡星(90.5%).结论:产ESBL细菌多重耐药现象严重,只有正确使用抗生素才能降低ESBL的发生,而对于产ESBL菌株感染的治疗,亚胺培南应作为首选药物.     

肠杆菌科细菌肺部感染病原菌分布与耐药性监测

目的了解浙江龙游县人民医院肠杆菌科细菌肺部感染病原菌分布及耐药性,为临床合理使用抗菌药物提供依据。方法采用纸片扩散法对病原菌进行药敏试验,并进行ESBLs检测,按CLSI 2012年标准判定药敏结果,用WHONET 5.6软件分析结果。结果痰培养共分离出371株肠杆科细菌,主要为肺炎克雷伯菌、大肠埃希菌和阴沟肠杆菌,产ESBLs菌阳性率高,对碳青霉烯类抗菌药物耐药率低,均在10.00%以下,其他药物均有不同程度耐药,测试抗菌药物的耐药率差异有统计学意义（P〈0.05）。结论临床常见肠杆菌科细菌耐药率高,开展病原菌耐药性监测,对指导临床抗感染治疗合理选择抗菌药物具有重要意义。     

产ESBLs肠杆菌科细菌的检测及耐药性

目的对产超广谱β-内酰胺酶(ESBLs)的病原菌分布及耐药性进行分析,进而为临床合理用药提供依据。方法应用西门子MicroScan autoSCAN4全自动细菌鉴定及药敏分析仪对2014年1月-12月在大连市妇幼保健院住院患者送检的标本进行菌株鉴定及药敏分析,对提示产ESBLs的大肠埃希菌和肺炎克雷伯菌进行纸片扩散法确认,统计分析其分布情况及耐药情况。采用ERIC-PCR法检测同源性。结果检测出125株产ESBLs的肠杆菌,包括大肠埃希菌88株,肺炎克雷伯菌37株。其病区分布情况:肺炎克雷伯菌主要分布在新生儿科,占67.6%,且存在流行感染。大肠埃希菌主要分布在各个产科病房,占48.9%。产ESBLs的肠杆菌对18种抗菌药物的分析表明对β-内酰胺类及第三代头孢菌素类抗生素耐药率达100.0%,对氨基糖苷类、喹诺酮类、四环素类和磺胺类药物出现不同程度的耐药,对碳青霉烯类抗生素敏感率较高。结论临床分离的产ESBLs的肠杆菌主要以大肠埃希菌和肺炎克雷伯菌为主,且为多重耐药,甚至广泛耐药,因此临床治疗上应严格合理选用抗生素。     

合肥地区动物源性大肠埃希菌的血清型分布与耐药性分析

目的了解安徽省合肥地区动物源性大肠埃希菌的血清型分布和耐药状况,以期筛选出菌苗株和指导临床合理用药。方法对46份疑似大肠埃希菌病病料进行细菌分离培养、生化编码鉴定和致病性测定。采用玻片凝集试验对分离到的46株致病性大肠埃希菌进行血清型鉴定。同时分别采用K-B纸片琼脂扩散法和双纸片增效法检测致病性大肠埃希菌的耐药性和ESBLs阳性菌株。结果46株致病性大肠埃希菌中,除7株细菌未能定型外,其余39株细菌分布于10个血清型,O127：K63血清型为优势血清型,占定型菌株的33．33％。46株致病性大肠埃希菌对21种抗菌药物均呈现不同程度的耐药性,15个ESBLs阳性菌株表现为多重耐药,对各种抗菌药物的耐药率均高于ESBLs阴性菌株。结论O127：K63血清型为优势血清型,可作为菌苗株。合肥地区动物源性大肠埃希菌耐药性较为严重,尤其是产ESBLs大肠埃希菌多重耐药更为突出。     

血流感染患者大肠埃希菌产超广谱β-内酰胺酶及耐药性分析

目的分析血流感染患者大肠埃希菌产超广谱β-内酰胺酶（Extended—Spectrum Beta Lactamases,ESBLs）的现状及其耐药特征,为临床合理使用抗菌药物提供依据。方法对浙江省上虞市人民医院2011年1月至2012年12月住院患者血培养分离的96株大肠埃希菌,采用纸片扩散表型确证试验进行ESBLs检测,用K．B法做药敏试验。结果血培养的大肠埃希菌分离率2011年、2012年分别为19．48％、17．47％。大肠埃希菌产ESBLs的检出率2011年、2012年分别为60．00％、60．78％。产ESBLs菌株对多种抗菌药物的耐药率显著高于不产ESBLs菌株。无论大肠埃希菌是否产ESBLs,碳青霉烯类抗生素均具有很高的敏感率。结论血流感染患者分离的大肠埃希菌产ESBLs比率高,产ESBLs菌株对多种抗菌药物耐药性高。可经验性使用碳青霉烯类抗生素治疗大肠埃希菌所致的血流感染。     

大肠埃希菌耐药性及其基因同源性分析

目的 研究临床分离的大肠埃希菌对常用抗生索的耐药性及其基因分型,了解其耐药性趋势与传播流行情况,为临床合理治疗大肠埃希菌引起的感染提供参考依据。方法 采用常规鉴定技术鉴定细菌;采用K—B纸片扩散法测定77株大肠埃希菌对19种药物的耐药性;K—B法鉴定产超广谱β-内酰胺酶（ESBLs）;通过脉冲场凝胶电泳（PFGE）法对其进行基因分型以确定菌株之间的亲缘关系;FINGERPRINT Ⅱ软件进行细菌基因指纹图谱分析。结果 大肠埃希菌对青霉素类、喹诺酮类药物和氨曲南的耐药性明显增高,亚胺培南和美罗培南是大肠埃希菌感染患者的首选药物;经ESBLs确证试验,ESBLs阳性率为28．60％（22／77）;产ESBLs大肠埃希菌经PFGE指纹图谱分析,除第62株和第70株相似性系数为78．27％外,其余相似度均低于70．0％;ESBLs大肠埃希菌阴性株中除少数几对菌株相似性系数较高外,其余呈散在分布,且电泳带存有6条以上的不同条带,为流行病学无关的不同克隆。结论 大肠埃希菌对常用抗生索耐药性明显增高,且呈多重耐药趋势;该研究尚不能证明存在大肠埃希菌爆发性流行感染,提示可能存在院内感染大肠埃希菌的优势克隆;PFGE基因分型方法是耐药性与流行状况分析的有效手段。     

产超广谱β-内酰胺酶大肠埃希菌的耐药性分析

目的了解杭州市第一人民医院产ESBLs大肠埃希菌的发生比例及对临床上常用的24种抗菌药物的耐药率变化.方法收集2003 2004年该院各类临床标本中分离的大肠埃希菌,采用NCCLS推荐的表型确证试验方法检测ESBLs菌株;药敏试验采用纸片扩散法.结果2003年与2004年,产ESBLs的大肠埃希菌分离率分别为46.11%（184/399）、57.44%（386/672）（P=0.0003）;2年来,ESBLs阳性菌对临床常用的24种药物表现出较高的耐药性,耐药率上升非常显著（P=0.0005）;非产ESBLs大肠埃希菌对大多数抗菌药物仍保持较高的敏感率;但2004年ESBLs阴性的大肠埃希菌的耐药性,比2003年显著上升（χ^2=37.785,P=0.0005）.结论尽早开展产ESBLs菌的监测,合理使用抗菌药物,对于有效控制产ESBLs菌的播散与流行是一项重要措施.     

Usefulness of Chromogenic CromoCen® AGN agar medium for the identification of the genus Aeromonas: Assessment of faecal samples

Selective screening media for the detection and identification of Aeromonas strains are needed to guide primary isolation procedures in the clinical laboratory. This study compared the selective CromoCen? AGN chromogenic agar medium for the detection and identification of Aeromonas strains that were isolated from various samples against the conventional selective agar media that are commonly used for the isolation of this organism in food, environmental and clinical samples. The Miles and Misra and ecometric methods were used to evaluate the microbiological performance of CromoCen? AGN chromogenic agar medium, which was shown to be satisfactory. A total of 14 reference Aeromonas strains, 44 wild strains and 106 clinical stool specimens were examined using both non-chromogenic selective agars that are commonly used for Aeromonas isolation and CromoCen? AGN agar. The latter exhibited 94.73% sensitivity and 100% specificity for the various samples. On CromoCen? AGN agar medium, Aeromonas formed colonies with light green, greenish and salmon pigments with or without a surrounding wide transparent zone (halo) of 2-3mm in diameter around the entire border. This medium is recommended for the isolation and potential identification of the Aeromonas genus.     

Evaluation of Chromocult coliform agar for the detection and enumeration of Enterobacteriaceae from faecal samples from healthy subjects

The purpose of this study was to examine the use of Chromocult agar medium for isolation and enumeration of Enterobacteriaceae from human faecal samples, to compare it to MacConkey agar and to evaluate its usefulness as a possible alternative selective medium in human faecal studies. The medium was shown to be effective in identifying Escherichia coli and coliforms in faeces without the need for extensive accompanying biochemical tests for confirmation of identity. A positive correlation (r=0.86) was found between the recovery of Enterobacteriaceae on the two media, and no significant difference (P>0.05) between overall mean bacterial counts for the whole study group or at different intervals of faecal collection were observed. Chromocult agar is an effective replacement for MacConkey agar in human faecal studies and has the advantage of differentiating E. coli from other coliforms.     

The performance of a resazurin chromogenic agar plate with a combined disc method for rapid screening of extended‐spectrum‐β‐lactamases,AmpC β‐lactamases and co‐β‐lactamases in Enterobacteriaceae

A promising means of rapid screening of extended‐spectrum‐β‐lactamase (ESBL), AmpC β‐lactamase, and co‐production of ESBL and AmpC that combines resazurin chromogenic agar (RCA) with a combined disc method is here reported. Cefpodoxime (CPD) discs with and without clavulanic acid (CA), cloxacillin (CX) and CA+CX were evaluated against 86 molecularly confirmed β‐lactamase‐producing Enterobacteriaceae , including 15 ESBLs, 32 AmpCs, nine co‐producers of ESBL and AmpC and 30 carbapenemase producers. The CA and CX synergy test successfully detected all ESBL producers (100% sensitivity and 98.6% specificity) and all AmpC producers (100% sensitivity and 96.36% specificity). This assay also performed well in screening for co‐existence of ESBL and AmpC (88.89% sensitivity and 100% specificity). The RCA assay is simple and inexpensive and provides results within 7 hr. It can be performed in any microbiological laboratory, in particular, in geographic regions in which ESBL, AmpC or co‐β‐lactamase‐producing Enterobacteriaceae are endemic.

     

Comparison of media for enumeration of coliform bacteria and Escherichia coli in non-disinfected water

In this work alternative media for detection and enumeration of E. coli and coliform bacteria were compared to the reference method ISO 9308-1 (LTTC) using non-disinfected water samples with background flora. The alternative media included LES Endo agar medium (LES Endo), Colilert-18 with 51-well Quanti-tray (Colilert), Chromocult Coliform agar (CC), Harlequin E. coli/Coliform medium (HECM) and Chromogenic Escherichia coli/Coliform medium (CECM). A total of 110 samples of groundwater, bathing water and spiked water was used. Our results revealed that confirmation of coliform bacteria counts is necessary, not only on lactose-based LTTC and LES Endo media, but also on the chromogenic agar media tested, due to the growth of oxidase positive colonies. LTTC and CC media also allowed the growth of some morphologically typical coliform colonies containing gram-positive bacteria. The recovery of coliform bacteria was lower on LES Endo than on LTTC. In most cases Colilert, CC, HECM and CECM gave higher coliform counts than LTTC. The use of the LTTC medium led to higher E. coli counts than obtained with any of the alternative mediums. There are three explanations for this: (1) high sensitivity of LTTC, (2) false positives on LTTC or (3) false negatives especially with Colilert, but also with chromogenic agar media. Although LTTC was found to be a very sensitive medium, the high degree of background growth of non-disinfected waters disturbed substantially the use of it. In conclusion, our results suggest that Colilert, CC and CECM are potential alternative media for detection of coliform bacteria and E. coli from non-disinfected water.     

Evaluation of novel chromogenic substrates for the detection of bacterial beta-glucosidase

AIMS: To evaluate three previously unreported substrates for the detection of beta-glucosidase activity in clinically relevant bacteria and to compare their performance with a range of known substrates in an agar medium. METHODS AND RESULTS: The performance of 11 chromogenic beta-glucosidase substrates was compared using 109 Enterobacteriaceae strains, 40 enterococci and 20 strains of Listeria spp. Three previously unreported beta-glucosides were tested including derivatives of alizarin, 3',4'-dihydroxyflavone and 3-hydroxyflavone. These were compared with esculin and beta-glucoside derivatives of 3,4-cyclohexenoesculetin, 8-hydroxyquinoline and five indoxylics. All substrates yielded coloured precipitates upon hydrolysis in agar. Alizarin-beta-D-glucoside was the most sensitive substrate tested and detected beta-glucosidase activity in 72% of Enterobacteriaceae strains and all enterococci and Listeria spp. The two flavone derivatives showed poor sensitivity with Gram-negative bacteria but excellent sensitivity with enterococci and Listeria spp. CONCLUSIONS: Alizarin-beta-d-glucoside is a highly sensitive substrate for detection of bacterial beta-glucosidase and compares favourably with existing substrates. beta-glucosides of 3',4'-dihydroxyflavone and 3-hydroxyflavone are effective substrates for the detection of beta-glucosidase in enterococci and Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented allow for informed decisions to be made regarding the optimal choice of beta-glucosidase substrate for detection of pathogenic and/or indicator bacteria.     

False-positive identification of Escherichia coli in treated municipal wastewater and wastewater-irrigated soils

The increasing use of treated wastewater for irrigation heightens the importance of accurate monitoring of water quality. Chromogenic media, because they are easy to use and provide rapid results, are often used for detection of Escherichia coli in environmental samples, but unique levels of organic and inorganic compounds alter the chemistry of treated wastewater, potentially hindering the accurate performance of chromogenic media. We used MI agar and molecular confirmatory methods to assess false-positive identification of E. coli in treated wastewater samples collected from municipal utilities, an irrigation holding pond, irrigated soils, and in samples collected from storm flows destined for groundwater recharge. False-positive rates in storm flows (4.0%) agreed closely with USEPA technical literature but were higher in samples from the pond, soils, and treatment facilities (33.3%, 38.0%, and 48.8%, respectively). Sequencing of false-positive isolates confirmed that most were, like E. coli, of the family Enterobacteriaceae, and many of the false-positive isolates were reported to produce the β-D-glucuronidase enzyme targeted by MI agar. False-positive identification rates were inversely related to air temperature, suggesting that seasonal variations in water quality influence E. coli identification. Knowledge of factors contributing to failure of chromogenic media will lead to manufacturer enhancements in media quality and performance and will ultimately increase the accuracy of future water quality monitoring programs.     

体液病原菌分布及革兰氏阴性杆菌药敏结果分析

目的：分析2005-2006年我院内无菌体液病原菌流行状况及革兰氏阴性杆菌对常用抗生素的敏感性,以便更好地为临床治疗感染性疾病提供用药参考。方法：所有菌株用美国BD公司BBLCRYSTAL AUTOREADER仪器进行鉴定,药敏用K-B法,培养基和药敏纸片为BD公司的。结果：80株致病菌中,肠杆菌科占65%,非发酵菌占12.5%,革兰氏阳性葡萄球菌占17.5%,酵母样真菌占2.5%,厌氧菌占2.5%。大肠埃希菌感染占首位（50株）其次为铜绿假单胞菌（3株）。嗜麦芽窄食单胞菌（3株）。肺炎克雷伯菌（2株）。大肠埃希菌中ESBL阳性菌18%（9/50）ESBL阳性菌和阴性菌对不同抗菌药物的敏感性有显著差异,前者耐药明显高于后者。结论：临床应根据药敏结果合理使用抗生素,减少院内感染的发生。     

Isolation rate of gram negative microflora and its sensitivity to antibiotics in hemoblastosis patients

A total of 67 patients with blood system diseases and infectious complications were examined. During the period of the examination 139 microorganisms were isolated. Of these gram negative microorganisms constituted 51%, gram positive microorganisms--34.8% and fungal flora--14.2%. Most frequently the following gram negative microorganisms were isolated from the patients: Pseudomonas sp. (including P. aeruginosa), Klebsiella pneumoniae, Escherichia coli, Haemophilus influenzae. All isolated microorganisms retained sensitivity to imipenem, with the exception of individual strains of Pseudomonas sp.; the latter exhibited sensitivity to amicacin and ceftazidim. Cefotaxime was active with respect to 75% of K. pneumoniae strains and all E. coli strains, ciprofloxacin was active with respect to 43% of E. coli strains, 80% of K. pneumoniae strains and 83.4% of Pseudomonas sp. strains, cefepim was active with respect to 85.7% of Pseudomonas sp. strains and all E. coli strains, ceftazidim was active with respect to all Pseudomonas sp. and E. coli strains. 75% of K. pneumoniae strains, 77.8% of Pseudomonas sp. strains and 86% of E. coli strains retained sensitivity to amicacin. 25% of K. pneumoniae strains required testing for ESBL production.     

Evaluation of a chromogenic medium supplemented with glucose for detecting Enterobacter sakazakii

A commercial chromogenic agar medium (DFI) was supplemented with glucose (mDFI) to enhance the specificity of Enterobacter sakazakii (E. sakazakii) detection. Escherichia vulneris (E. vulneris), a putative false-positive strain on the DFI medium, produces alpha-glucosidase. The enzyme alpha- glucosidase hydrolyzes a substrate, 5-bromo-4-chloro-3- indolyl-alpha,D-glucopyranoside (XalphaGlc), producing green colonies. E. sakazakii strains produced green colonies on both DFI and mDFI agar, whereas E. vulneris produced green colonies on DFI agar but small white colonies on mDFI agar. E. sakazakii and E. vulneris were also readily differentiated by colony color when the mixed culture of the two strains was plated on mDFI agar and incubated for 24 h at 37 degrees C. The results indicate that the selectivity of the commercial chromogenic agar medium could be improved by a simple supplementation with glucose.     

Evaluation of a new chromogenic agar medium for isolation and identification of Group B streptococci

AIMS: To evaluate a new chromogenic agar as a screening medium for the isolation of Group B streptococci from high vaginal swabs from pregnant women. METHODS AND RESULTS: The medium was evaluated with 195 high vaginal swabs referred for antenatal screening and compared with blood agar and Granada medium. The new chromogenic medium showed 100% sensitivity for the detection of Group B streptococci, and also showed a positive predictive value of 100%. Granada medium also showed excellent sensitivity and specificity and both media were superior to blood agar. CONCLUSIONS: The new chromogenic medium showed excellent performance for the detection of Group B streptococci from clinical samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first chromogenic medium described for the detection of Group B streptococci. The medium offers an effective and convenient alternative to conventional media, currently used in clinical laboratories.