梅花鹿源大肠杆菌ESBLs基因型检测与分析

为分析黑龙江省梅花鹿源产超广谱β-内酰胺类酶(ESBLs)大肠杆菌耐药基因的分布情况,为临床抗微生物药的合理使用提出指导意见。从黑龙江省4个养殖场中分离出130株梅花源大肠杆菌,采用K-B纸片法,结果显示对诺氟沙星、环丙沙星、氧氟沙星的耐药率达到10%以上。采用PCR技术对ESBLs基因进行检测。结果显示,耐药基因OXA、TEM的检出率分别为13.85%(18/130)、76.15%(99/130),CTX-M和SHV均未检出。4个养殖场的ESBLs基因以TEM为主。

Occurrence of K1 and K2 serotypes and genotypic characteristics of extended spectrumβ-lactamases-producing Klebsiella pneumoniae isolated from selected hospitals in Malaysia

Objective:To determine the distribution,phenotypic and genetic background of extended spectrumβ-lactamases(ESBL)-producing Klebsiella(K.)pneumoniae clinical isolates associated with K1 and K2 serotypes in two selected hospitals in Malaysia.Methods:A total of 192 K.pneumoniae isolates were collected and subjected to antibiotic susceptibility,hypermucoviscosity test and multiplex PCR to detect the presence of K1-and K2-serotype associated genes.Multilocus sequence typing(MLST)was performed on ESBL-producing K.pneumoniae isolates presented with K1 and K2 serotypes,followed by phylogenetic analysis.Results:A total of 87 out of 192(45.3%)of the K.pneumoniae isolates collected were ESBL producers.However,only 8.3%(16/192)and 10.9%(21/192)of the total isolates were detected to carry K1-and K2-serotype associated genes,respectively.Statistical analysis showed that K1 and K2 capsular serotypes were not significantly associated with ESBL phenotype(P=0.196).However,they were significantly associated with hypervirulent,as demonstrated by the positive string test(P<0.001).MLST analysis revealed that ST23 as the predominant sequence type(ST)in the K1 serotype,while the ST in the K2 serotype is more diverse.Conclusions:Although the occurrence of ESBL-producing isolates among the hypervirulent strains was low,their coexistence warrants the need for continuous surveillance.MLST showed that these isolates were genetically heterogeneous.

羔羊源产ESBLs与非产ESBLs大肠埃希氏菌耐药性差异比较及基因型分析

为了明确宁夏地区羔羊源大肠埃希氏菌产超广谱β-内酰胺酶(ESBLs)与非产ESBLs大肠埃希氏菌的耐药性差异和相关基因携带情况差异,对实验室分离保存的87株羔羊源大肠埃希氏菌,采用ESBLs表型检测方法筛选产ESBLs的菌株和K-B纸片法进行药物敏感性试验,采用PCR法对分离株进行相关基因的检测。结果显示,87株大肠埃希氏菌中5株为产ESBLs菌株,检出率为5.75%。产ESBLs株对多数试验药物呈耐药性;而非产ESBLs菌株对多数试验药物敏感,仅对恩诺沙星、复方新诺明和红霉素耐药率超过50%。20种药物中,产ESBLs株大肠埃希氏菌对18种药物的耐药率极显著高于非产ESBLs菌株(P<0.01)。产ESBLs菌株携带blaCTX的阳性率极显著高于非产ESBLs菌株(P<0.01),产ESBLs菌株中的blaTEM和blaOXA基因的阳性率均高于非产ESBLs菌株,但差异不显著(P>0.05)。结果表明宁夏地区羔羊源大肠埃希氏菌产ESBLs菌株流行率低,产ESBLs菌株耐药性严重,且宁夏地区羔羊源ESBLs菌株优势基因型为blaCTX和blaTEM。

Comparison of extended spectrum β-lactamasesproducing Escherichia coli with non-ESBLsproducing E.coli:drug-resistance and virulence

BACKGROUND:The virulent factors of Escherichia coli(E.coli) play an important role in the process of pathopoiesis.The study aimed to compare drug-resistant genes and virulence genes between extended spectrum β-lactamases(ESBLs)-producing E.coli and non-ESBLs-producing E.coli to provide a reference for physicians in management of hospital infection.METHODS:From October 2010 to August 2011,96 drug-resistant strains of E.coli isolated were collected from the specimens in Qingdao Municipal Hospital,Qingdao,China.These bacteria strains were divided into a ESBLs-producing group and a non-ESBLs-producing group.Drug sensitivity tests were performed using the Kirby-Bauer(K-B) method.Disinfectant gene,qacEA1-sull and 8 virulence genes(CNF2,hlyA,eaeA,VT1,est,bfpA,elt,and CNF1) were tested by polymerase chain reaction(PCR).RESULTS:Among the 96 E.coli isolates,the ESBLs-producing E.coli comprised 46(47.9%)strains and the non-ESBLs-producing E.coli consisted of 50(52.1%) strains.The detection rates of multiple drug-resistant strain,qacEA1-sull,CNF2,hlyA,eaeA,VT1,est,bfpA,elt,and CNF1 in 46ESBLs-producing E.coli isolates were 89.1%,76.1%,6.5%,69.6%,69.6%,89.1%,10.9%,26.1%,8.7%,and 19.6%,respectively.In the non-ESBLs-producing E.coli strains,the positive rates of multiple drug-resistant strain,qacEA1-sull,CNF2,hlyA,eaeA,VT1,est,bfpA,elt,and CNF1 were 62.0%,80.0%,16.0%,28.0%,64.0%,38.0%,6.0%,34.0%,10.0%,and 24.0%,respectively.The difference in the detection rates of multiple drug-resistant strain,hlyA and VT1 between the ESBLs-producing E.coli strains and the non-ESBLs-producing E.coli strains was statistically significant(P<0.05).CONCLUSION:The positive rate of multiple drug-resistant strains is higher in the ESBLsproducing strains than in the non-ESBLs-producing strains.The expression of some virulence genes hlyA and VT1 varies between the ESBLs-producing strains and the non-ESBLs-producing strains.Increased awareness of clinicians and enhanced testing by laboratories are required to reduce treatment failures and prevent the spread of multiple drug-resistant strains.

肠杆菌科细菌耐药基因NDM和ESBLs的检测及白头翁汤增强其敏感性研究

采用美国临床实验室标准化协会(Clinical and Laboratory Standards Institute,CLSI)推荐的方法,检测了临床分离的44株肠杆菌科细菌产超广谱β-内酰胺酶(Extended-Spectrum Beta-Lactamases,ESBLs)的情况.利用PCR对临床分离菌进行新德里金属β-内酰胺酶(New Delhi metallo-β-lactamase,NDM)以及ESBLs耐药基因的检测.用微量肉汤稀释法测定了分离菌对阿莫西林等14种抗菌药的敏感性,并统计其耐药率.结果表明,临床分离的44株肠杆菌科细菌中,有31株产超广谱β-内酰胺酶,检出率为70.5%.耐药基因检测结果显示,耐药基因NDM-1携带率为13.6%.ESBLs耐药基因TEM,SHV,CTX-M,OXA的携带率分别为100%,43.2%,45.5%,6.8%.同时发现,有6株菌株同时检出了含NDM-1和ESBLs的基因,有9株菌株同时检出含有2种ESBLs基因,3株同时检出含有3种ESBLs基因.敏感性测定结果显示,分离菌大多呈现多重耐药,分离菌除对替加环素(18.2%)和阿米卡星(36.4%)的耐药率较低外,对其他药物呈现出较高的耐药率,其中对阿莫西林的耐药率高达97.7%,对头孢曲松、环丙沙星、土霉素、四环素、红霉素的耐药率均在75.0%以上.此外发现,同时携带多个耐药基因的菌株耐药率较高.中药复方白头翁汤对分离菌表现出了一定的抑菌活性,其MIC值为204.8 g/L.14种抗菌药分别与白头翁汤联用后,耐药率均有不同程度下降,其中头孢类药物、氟喹诺酮类药物与白头翁汤联用后,耐药率下降较为明显,如头孢曲松由84.1%下降至56.8%,环丙沙星由75.0%下降至45.5%.综上,NDM和ESBLs耐药基因在肠杆菌科细菌中已有一定的流行,同时携带NDM和ESBLs多个耐药基因的菌株耐药较为严重.白头翁汤与抗菌药联用能在一定程度增强耐药菌株的敏感性,为临床相关感染的防治提供理论依据.

不同家禽产ESBLs沙门菌的流行分布与耐药分析

了解胶东地区不同家禽携带沙门菌的流行分布及其耐药现状,为有效防控家禽沙门菌的流行传播及遏制其耐药提供依据。采用细菌的常规分离培养、质谱法、PCR法和微量肉汤稀释法等,对2021年8—10月在胶东5个地区3种家禽养殖场采集的3150份泄殖腔拭子进行沙门菌分离鉴定、血清型鉴定、药敏试验及β-内酰胺酶主要耐药基因的检测,利用统计学方法进行差异显著性分析。结果发现:(1)共分离获得194株沙门菌(6.16%)。水禽源沙门菌的携带率(14.36%)比肉鸡(5.04%)、蛋鸡(1.15%)高。(2)较多的血清型为S.Enteritidis(38.14%)、S.Kentucky(21.65%)、S.Senftenberg(14.95%)、S.Indiana(12.37%)、S.Typhimurium(8.76%)为主。肉鸡和水禽(均为7种)的沙门菌血清型较蛋鸡(3种)多,但均以S.Enteritidis为主。(3)110株禽源沙门菌对氨苄西林、异磺胺异噁唑、四环素3种药物耐药相对严重(58.18%~77.27%),未检测到美罗培南的耐药菌株。水禽和肉鸡源菌株较蛋鸡耐药严重,尤其对阿莫西林/克拉维酸(P=0.031)和四环素(P=0.007)的耐药差异显著。(4)81株(73.64%)禽源沙门菌为多重耐药,共有43种耐药谱型,肉鸡(25种)和水禽(22种)源菌株的耐药谱型远多于蛋鸡(8种)。(5)110株沙门菌中检测到3种耐药基因:blaCTX-M(70.00%)、blaTEM(51.82%)和blaOXA(26.36%),常见的耐药基因组合类型为blaCTX-M+blaTEM,且耐药基因与耐药表型间存在一定的相关性(P≤0.001)。由此可见,胶东地区的家禽均携带一定比例的沙门菌,但不同品种家禽的流行分布存在差异。不同家禽之间携带血清型的种类也有一定的差异,但携带最多的血清型均为S.Enteritidis。分离菌株耐药情况较为严重,耐药谱复杂多样。禽源沙门菌对β-内酰胺类药物耐药主要是blaCTX-M+blaTEM基因的表达所致。

中药活性成分对产ESBLs大肠埃希菌增敏效应研究

为探究中药活性成分与抗菌药物联合对产ESBLs大肠埃希菌的体外抗菌效果及增敏效应,本研究分别对其最小抑菌浓度(minimum inhibitory concentration,MIC)进行了测定,并在此基础上将倍比稀释的1/2MIC备选中药联合2MIC抗菌药物对产ESBLs大肠埃希菌进行连续传代培养;通过对联合用药及单独抗菌药物MIC进行比较,来判断中西药联合对产ESBLs菌株的增敏作用效果。结果表明:6种中药提取物中,小檗碱的杀菌效果最佳(16μg/mL),丁香酚(64μg/mL)、黄芩苷(256μg/mL)和没食子酸(256μg/mL)其次,原儿茶酸和异甘草酸抑菌效果较差;在联合应用研究中发现,小檗碱与GEN、APR、MMP联用后其最小抑菌浓度分别降低至1.25、0.02、0.02μg/mL,没食子酸与GEN、APR、MMP联用后其最小抑菌浓度分别降低至1.25、2.50、0.01μg/mL。中药活性成分与抗菌药联合应用中,可提高抗菌药对产ESBLs大肠埃希菌的抗菌效果,增敏效应甚至提高200倍以上,且随着传代次数增加,抗菌药物的MIC逐代降低。该研究为临床上大肠杆菌病的中药防治以及开发新兴的中药抑菌剂提供了参考依据。

Prevalence and Resistance Profile of Muenchen Cefotaximase (CTX-M) Group 1 Extended Spectrum Beta-Lactamase (ESBL)-Producing Uropathogenic Escherichia coli Strains in Dakar, Senegal

Background: Extended-spectrum beta-lactamase (ESBL) producing bacteria are a real public health problem, particularly in Africa. Among these ESBLs, there are the Muenchen Cefotaximase (CTX-M) described all over the world of which the most frequent is the CTX-M of group 1 particularly the CTX-M-15 variant. The objective of this study was to determine the prevalence of CTX-M group 1 ESBL-producing Escherichia coli strains and to test their antibiotics susceptibility profile. Methodology: A retrospective cross-sectional descriptive study was conducted to detect ESBL-secreting Escherichia coli strains by the synergy test. Identification of CTX-M type ESBL from group 1 was performed using the NG-Test CTX-M rapid diagnostic test (NG-Biotech®). Antibiotic susceptibility profile was determined using CA-SFM/EUCAST guidelines 2019. Data entry and statistical analysis were performed with Excel version 2010 and SPSS 20.0 respectively. Results: Eighty-two ESBL-producing Escherichia coli strains were tested. A group 1 CTX-M ESBL was detected in 75.6% of the strains (n = 62). These strains were highly resistant to cefotaxim (100%), aztreonam (100%), ceftazidim (85.4%) and cefepim (66.1%). They were also resistant to quinolones, gentamycin and sulfadoxine-trimethoprim combination. However, these strains showed sensitivity to ertapenem (100%), cefoxitin (69.3%), tigecyclin (66%), and amikacin (66.1%). The combination of piperacillin and tazobactam was active on 30.6% of the strains against 6.4% for the combination of amoxicillin and clavulanic acid. Conclusion: The CTX-M type ESBL of group 1 was present in the majority of ESBL-producing Escherichia colis trains. Despite the production of this enzyme conferring resistance to most beta-lactam antibiotics, some antibiotics remain active to treat infections caused by these germs.

Phenotypic Characterization of Extended Spectrum Beta-Lactamase, Class C Cephalosporinase and Carbapenemase-Producing Klebsiella Species Isolated from Patients Consulted at Four Yaounde-Based Hospitals

Background: Klebsiella spp. are bacteria of medical importance for their role in opportunistic infections which are often difficult to treat because of resistance to one or several antimicrobials. The aim of this study was to determine antimicrobial resistance due to Extended Spectrum Beta-lactamase (ESBL), Class C cephalosporinase (AmpC) and carbapenemase enzymes in Klebsiella spp. isolated from patients consulted at four hospitals. Methodology: The study was cross-sectional and descriptive. A total of 4190 non-repetitive patients specimens from 13 types of clinical specimens were analysed from February to November 2020. Two hundred and twenty-five (225) Klebsiella spp isolates were identified using API 20E and antimicrobial susceptibility testing done according to the Kirby Bauer disc diffusion method. ESBL and AmpC phenotypes were determined by the combination disc method and carbapenemases by double disc synergy method, referenced by EUCAST guidelines for the resistance testing. Results: The frequency of the species was Klebsiella pneumoniae (69%, 155/255), K. oxytoca (14%, 31/255), K. ozaenae (12%, 27/225) and K. rhinoscleromatis (5%, 11/225). Isolates were most resistant to sulphomethoxazole trimethoprim (84%, 189/225), cepaholosporins (80%, 180/225), and least resistant to carbapenems (10.7%, 24/225). Two K. oxytoca and one K. pneumoniae were resistant to all antibiotics tested. Klebsiella pneumoniae had the most multidrug resistant isolates (59.4%, 134/225). Most isolates (83.6%, 188/225) expressed at least one enzyme, while 63.6% (143/225) of the isolates expressed at least two enzymes. Some isolates were ESBL (71.6%, 161/225), carbapenemase (10.7%, 24/225) and AmpC (6.6%, 15/225) producers. Three carbapenemases (Klebsiella pneumoniae carbapenemase-KPC, Metallo-Beta Lactamase-MBL and OXA-48) were detected. Conclusion: These results revealed that resistance of Klebsiella spp. to cephalosporins is high and this may be exacerbated by co-expression of AmpC and carbapenemases aggravating associated patient morbidity and mortality. Monitoring of antimicrobial resistance of local strains is necessary for informed decisions on empirical treatment. .

Evaluation of Disk Potentiation Test (DPT) and Double Disk Synergy Test (DDST) for The Detection of Metallo-β-Lactamases (MBLs) in Clinical Isolates of Bangladesh

Objective: Increasing the emergence of Metallo-β-lactamase (MBL) producing gram-negative bacteria and their dexterous horizontal transmission demands rapid and accurate detection. This study was conducted to determine a suitable method to promptly detect MBL-producing gram-negative bacteria. Methods: A total of 103 gram-negative bacteria were identified from various clinical samples at a tertiary care hospital in Dhaka city. MBL producers were detected by two phenotypic methods, the Disk Potentiation Test (DPT) and the Double Disk Synergy Test (DDST) based on β-lactam chelator combinations where EDTA/SMA has been used as an inhibitor and Imipenem, Ceftazidime as substrates. Results: 103 isolates which were identified as Escherichia coli spp, Klebsiella spp, Pseudomonas spp, Acinetobacter spp, Proteus spp, Providencia spp were found to be multidrug-resistant in antibiogram test. Isolates showed complete resistance (100%) to Imipenem, Meropenem, and Amoxiclav. The highest carbapenem-resistant etiological agents were Acinetobacter spp 40 (38.8%) followed by Pseudomonas spp 27 (26.2%), Klebsiella spp 26 (25.2%), Escherichia coli 8 (7.8%), Proteus spp 1 (1%) and Providencia spp 1 (1%). DPT method detected significantly (p = 0.000009) a higher number of MBL-producers (Imipenem with 0.5 M EDTA n = 61, 59.2% & Ceftazidime with 0.5 M EDTA n = 56, 54.4%) compared to the DDST method (Imipenem -0.5 M EDTA n = 43, 41.7%, Imipenem – SMA n = 38, 36.9% & Ceftazidime -0.5 M EDTA n = 15, 14.6%). Conclusion: Pieces of evidence suggest that DPT is a more sensitive method than DDST and could be recommended for identifying MBL-producing bacteria in Bangladeshi hospitals for the proper management of patients, to reduce time constraints and treatment costs.

探讨直接药敏试验在ESBLs细菌血流感染诊断中的应用评估

用于评估直接药敏试验在ESBLs细菌血流感染诊断中的临床应用价值。方法 收集2021年9月-2022年8月重庆市某三甲医院经过镜检为单一革兰阴性菌的阳性血培养标本,使用临床和实验室标准研究所(CLSI)建立的的标准化直接药敏试验方法进行快速药敏试验,选取ESBLs标本结果并与Vitek2 Compact全自动微生物鉴定与药敏分析仪结果进行比较。结果 本研究经常规鉴定为ESBLs菌共45株,包括肺炎克雷伯菌4株、产酸克雷伯菌1株及大肠埃希菌40株。本研究总分类一致率为90.0% (241/265),极重大错误率为0%,重大错误率为3%(8/265),微小错误率为6.1%(16/285)。其中氨苄西林(AMP)为100% (40/40),氨曲南(ATM)为91.2% (41/45),头孢他啶(CAZ)为71.1% (32/45),头孢曲松(CRO)为93.3% (42/45),妥布霉素(TOB)为95.6% (43/45),复方新诺明(SXT)为95.6% (43/45)。氨苄西林、头孢他啶、氨曲南、头孢曲松和复方新诺明的阳性预测值达100%,氨苄西林和妥布霉素的阴性预测值达100%。结论 经过本研究的验证,CLSI法总体分类一致率为90.9% ,VME为0%,ME为3%,mE为6.1%,符合CLSI M52标准,可被被本实验室接受和应用于日常工作。尽管仍有不足和改进之处,但随着评估工作的进一步深入和发展,CLSI在快速药敏纸片扩散试验适用范围、快速结果判断等内容将会不断完善,标准化直接药敏纸片扩散法将会成为临床实验室对肠杆菌目细菌的一种有价值的抗菌管理方法。

Characteristics of β-Lactamase Synthesis in E. coli and K. pneumanie Strains in Nosocomial Infections

Background: Recently micro-organisms that synthesize extended-spectrum β-lactamase (ESBLs) were increased. The peculiarities of ESBL synthesis of Escherichia coli and Klebsiella pneumoniae strains that cause nosocomial urinary tract infections, surgical site infections and pneumonia in surgical clinic were studied. ESBL synthesis were observed 38.9% of E. coli strains obtained from urine, 92.3% of strains obtained from surgical site infections, and 50% of strains obtained from sputum. ESBL synthesis were observed 37.5% of K. pneumoniae strains obtained from urine, 85.7% of strains obtained from surgical site infections, and 60% of strains obtained from sputum. Different levels of ESBL synthesize of E. coli and K. pneumoniae strains isolated from different pattern is discussed. Conclusion. ESBL synthesis is common in E. coli and K. pneumoniae strains, which cause nosocomial infections. The frequency of occurrence of ESBL s synthesis among of these strains depends on clinical forms of nosocomial infections.

超广谱β-内酰胺酶(ESBLs)检测与耐药性菌株分布的关联研究

超广谱β-内酰胺酶(ESBLs)是一类能够水解广谱β-内酰胺类抗生素的酶,其产生与耐药性菌株的分布密切相关。本研究旨在探讨ESBLs检测与耐药性菌株分布的关联,并为临床治疗和抗菌药物的合理使用提供依据。在本研究中,我们收集了157个临床样本,并进行了ESBLs检测和耐药性菌株分布的分析。结果显示,ESBLs阳性菌株的耐药性明显高于ESBLs阴性菌株,表明ESBLs的检测结果与耐药性菌株的分布存在显著的关联性。进一步的分析发现,不同病原菌种类与ESBLs的产生和耐药性之间存在差异。某些病原菌株更容易产生ESBLs,并且这些菌株具有更高的耐药性水平。因此,ESBLs的检测可以作为评估菌株耐药性的重要指标,有助于临床治疗和预防策略的制定。然而,本研究还存在一些限制,包括样本量有限和研究范围局限于特定地区或医疗机构。未来的研究可以扩大样本规模,并在更广泛的区域开展调查,以更全面地了解ESBLs检测与耐药性菌株分布的关联。综上所述,ESBLs检测与耐药性菌株分布之间存在密切的关联,这对于指导临床治疗和抗菌药物的使用具有重要意义。进一步的研究和努力将有助于深入理解耐药性菌株的形成机制,并制定有效的预防和控制策略,以维护公众健康。

青岛地区产ESBLs鸡源大肠杆菌耐药性调查与优势基因型分析

【目的】通过抗菌药物敏感性测定试验以及超广谱β-内酰胺酶(ESBLs)表型和基因型检测试验,了解青岛地区肉鸡养殖场产ESBLs大肠杆菌菌株的分布情况及耐药特征,分析ESBLs流行基因型和基因亚型,为指导临床合理使用抗菌药物、有效控制产ESBLs菌株的感染和流行提供依据。【方法】采用微量肉汤稀释法对249株大肠杆菌菌株进行抗菌药物敏感性测定试验,采用CLSI推荐方法进行ESBLs表型检测和确证实验,采用PCR方法、序列测定以及生物学分析软件进行ESBLs耐药质粒的DNA扩展和基因型分析,利用SPSS19.0软件对产ESBLs菌株与非产ESBLs菌株的耐药情况进行差异显著性分析。【结果】83.13%(207/249)的鸡源大肠杆菌菌株产ESBLs;产ESBLs菌株对7种抗菌药的耐药率高于非产ESBLs菌株,其中对庆大霉素、大观霉素、氨苄西林、阿莫西林/克拉维酸、头孢噻呋等5种药物差异显著(P<0.05),而产ESBLs菌株对四环素和氟苯尼考2种抗菌药的耐药率显著低于非产ESBLs菌株;产ESBLs菌株多重耐药程度显著高于非产ESBLs菌株,其多重耐药率分别为99.03%和92.86%(P=0.035);CTX-M型、TEM型和OXA型ESBLs菌株的检出率分别为100.00%、99.52%和47.83%,未检测出SHV型ESBLs菌株;产酶菌株分属于10个基因亚型,TEM-1型、CTX-M-65型和CTX-M-55型、OXA-1型是优势基因亚型,并首次从健康家禽中检测到基因重组嵌合体CTX-M-123和CTX-M-64。【结论】产ESBLs鸡源大肠杆菌菌株在青岛地区广泛流行和传播;产ESBLs菌株耐药相对非产ESBLs菌株严重;相比国内其它地区,CTX-M型和TEM型同样成为青岛地区产ESBLs鸡源大肠杆菌菌株的流行基因型,但基因亚型存在差异,TEM-1型、CTX-M-65型和CTX-M-55型、OXA-1型分别是各基因型的优势基因亚型。

阴沟肠杆菌去阻遏持续高产AmpC酶和超广谱β-内酰胺酶(ESBLs)的检测

目的：去阻遏持续高产ＡｍｐＣ酶，或产生超广谱β－内酰胺酶（ＥＳＢＬｓ），或同时高表达这两类酶，是阴沟肠杆菌对多种β－内酰胺类抗生素耐药的主要机制。方法：建立了一种改良的酶提取物三维试验法，在常规ＮＣＣＬＳ纸片扩散法基础上接种大肠杆菌ＡＴＣＣ ２５９２２，在头孢西叮药敏纸片周围放射性切出琼脂小槽，待测菌的粗酶提取物在槽内扩散，与头孢西叮纸片扩散相交。结果：由于ＡｍｐＣ酶水解头孢西叮，出现抑菌圈内生长细菌，这一现象能被加入槽内的邻氯西林抑制，而ＥＳＢＬｓ不能水解头孢西叮，无此现象。因邻氯西林不能抑制ＥＳＢＬｓ活性，同样用头孢由松取代头抱西叮纸片，用邻氯西林抑制槽内ＡｍｐＣ酶，可测出 ＥＳＢＬｓ。使用该法检测分别产 ＡｍｐＣ酶、 ＥＳＢＬｓ的标准株和 ２４株临床分离多重耐药的阴沟肠杆菌。结果各种标准株检测无误，２４株阴沟肠杆菌中，１４株去阻遏持续高产ＡｍｐＣ酶试验阳性，４株产ＥＳＢＬｓ试验阳性，５株此两类酶检测均阳性，１株此两类酶检测均阴性，原因待分析。结论：对于阴沟肠杆菌，这种改良的酶提取物三维试验法能较好地鉴别持续高产 ＡｍｐＣ酶和 ＥＳＢＬ的阴沟肠杆菌，并适用于临床常规检验。

鸡源大肠杆菌分离鉴定及ESBLs与AmpC酶基因型检测及耐药性分析

为了解山西省范围内鸡源产超广谱β-内酰胺酶(ESBLs)和头孢菌素酶(AmpC)大肠杆菌分离株的基因型及其耐药现状,本试验从呈现典型鸡大肠杆菌病临床症状的病料中分离、鉴定出68株符合鸡源大肠杆菌生物特性的流行菌株,采用K-B法与双纸片确认法对分离的68株大肠杆菌分别进行药敏试验和ESBLs、AmpC酶耐药表型的筛选;采用PCR方法检测了分离株中质粒介导的ESBLs、AmpC酶的基因型。结果显示,分离到的68株菌对22种抗菌药物均产生不同程度的耐药性,其中耐药谱达7耐以上的有66,占总分离菌株数的97.06%;呈ESBLs、AmpC酶阳性和两者均阳性的菌株数分别为57(83.82%)、19(27.94%)和16(23.53%)株;检测出ESBLs阳性菌株携带的耐药基因型包括bla TEM、bla OXA和bla CTX-M-1/9,AmpC酶阳性菌株耐药基因型为bla FOX型。研究结果表明,分离的68株鸡源大肠杆菌已对绝大多数种类抗菌药物产生耐药性,且产ESBLs和AmpC酶菌株已普遍流行。山西省鸡源大肠杆菌中流行的ESBLs基因型与国内其他地区相关报道大体一致,而检测到的bla FOX型AmpC酶基因在国内报道相对较少。

鸡、猪大肠杆菌ESBLs基因型检测及耐药性分析

为检测不同动物源大肠杆菌中超广谱β-内酰胺酶(ESBLs)的基因型,探究ESBLs对大肠杆菌耐药性的影响,指导兽医临床有效防治大肠杆菌病。采用NCCLS标准方法和双纸片协同法,对102株大肠杆菌菌株进行抗菌药物敏感性测定和和ESBLs检测,设计合成4对引物,用PCR方法进行了ESBLs耐药质粒的DNA扩增和基因型分析。结果表明:以产ESBLs细菌的耐药质粒为模板扩增出了与预期片段大小相符的TEM、CTX-M型ESBLs产物,但均未扩增出SHV、OVA型ESBLs产物。鸡源、猪源产ESBLs菌株检出率分别为81%、6%。CTX-M、TEM型为山东莱西地区动物源产ESBLs大肠杆菌菌株的流行基因型,且鸡源产ESBLs大肠杆菌菌株分布广泛,应加强当地产酶耐药菌的监测和研究,以有效防治此类细菌引发的疾病。

贵州部分地区猪源大肠杆菌耐药性分析及ESBLs基因型检测

为了解贵州地区规模养猪场大肠杆菌菌株耐药情况和ESBLs基因型的流行情况,试验采用CLSI推荐的方法对采自贵州省4个地区规模养猪场的164株大肠杆菌进行药物敏感性试验和产ESBLs菌的检测,并用PCR方法对TEM、SHV、OXA-1和CTX-M-1 4种常见ESBLs基因进行检测。结果显示,164株大肠杆菌对10种常用抗菌药物耐药率分别是头孢噻呋93.29%、氨苄西林87.19%、四环素86.59%、庆大霉素81.10%、链霉素53.66%、多黏菌素51.83%、环丙沙星53.05%、卡那霉素47.56%、金霉素34.76%和氟苯尼考21.95%,且大多为多重耐药,其中检测出ESBLs阳性菌株137株,阳性率为83.54%,各个地区的检出率不同;137株产ESBLs大肠杆菌中,TEM、SHV、OXA-1和CTX-M-1基因的检出率分别为90.51%、70.07%、51.82%和43.07%,且多为复合基因型耐药菌株,各地区的各种基因检出率不同。试验结果表明,贵州部分地区的猪源大肠杆菌耐药现象严重,ESBLs菌株的检出率很高,耐药基因的检出率也极高,且多为复合基因型耐药菌株,应加强当地产酶耐药菌的监测和研究,有效防制此类细菌引发的疾病。

临床产ESBLs细菌的耐药特征和基因分型的研究

目的了解产超广谱β-内酰胺酶(ESBLs)细菌的流行和耐药特点,初步对产ESBLs细菌进行基因分型,确立耐药基因的位点和种类.方法收集分离鉴定菌株,通过K-B法药敏实验初筛以确认产ESBLs菌株,并对ESBLs基因进行初步分型.结果分离到产ESBLs细菌88株,检出率为38.9%,其中肺炎克雷伯菌阳性率为43.1%、大肠埃希菌阳性率为33.3%;产ESBLs细菌对青霉素类、氨曲南及头孢菌素类耐药率约为90%～100%,加用酶抑制剂克拉维酸或他唑巴坦后耐药率降为18.7%～46.8%;PCR初步分型结果表明:大多数产ESBLs细菌携带TEM型和SHV型β-内酰胺酶基因,其中单独携带TEM型基因的占42%,单独携带SHV型基因的占12.5%,携带两种基因的占31.8%.结论产ESBLs细菌具有多重耐药的特点;产ESBLs的大肠埃希菌和肺炎克雷伯菌大都携带TEM和(或)SHV型β-内酰胺酶基因,其中绝大部分产TEM型β-内酰胺酶.

ESBLs耐药菌株分子流行病学调查

目的 :为了研究超广谱 β-内酰胺酶 (extendspectrum β-lactamase ,ESBLs)耐药菌株在儿科病房的分子流行情况 ,及时切断医院感染的传播途径 ,预防医院感染可能引起的暴发流行。方法 :对新生儿科、呼吸科、心脏内科、肾脏科、血液科以及儿童外科、ICU住院患儿进行痰、脐分泌物、脓、咽拭子培养 ,菌体药物敏感实验。分离筛选出ESBLs大肠埃希氏和肺炎克雷伯氏菌54株 ,进行质粒提取琼脂糖电泳分析。结果 :ESBLs在各科所占的百分率分别为46.3 %、12.9 %、5.6 %、12.9 %、5.6 %、11.1 %、5.6 %。在质粒谱的分析中发现有3种质粒谱带 ,新生儿科ESBLs菌所占比例较大 ,均有2株为相同质粒谱。结论