AmpC β-内酰胺酶向持续高产型转变的研究

【作者】 涂婉；

【导师】 赵虎；

【作者基本信息】 复旦大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 第一部分目的：了解ampD基因特异突变对阴沟肠杆菌AmpCβ-内酰胺酶由非持续高产型转变为持续高产型的影响作用。方法：通过细菌鉴定、药敏试验,改良头孢西丁三维试验和质粒ampC基因的扩增,筛选染色质介导的产持续高产型AmpCβ-内酰胺酶的阴沟肠杆菌,扩增其染色质ampD基因、测序并比对,确定其有义突变位点。采用定点突变的方法对野生型阴沟肠杆菌ampD基因进行上述特异位点的突变,形成相应的定点突变菌株；然后再用野生型ampD基因代替临床原始菌株的ampD基因,形成回复突变菌株。最后用改良头孢西丁三维试验和AmpCβ-内酰胺酶活性测定实验测定突变前后菌株和回复突变菌株产酶类型的改变。结果：临床收集的121株阴沟肠杆菌中,15株为染色质介导的产持续高产型AmpC酶的菌株,共筛选出ampD基因的8个有义突变位点,形成7株定点突变菌株。其中Ecl MA(274位插入A)、Ecl MC(327位缺失C)和Ecl MF(27位插入G)由非持续高产型转变为持续高产型,而Ecl MB(371位插入T)、Ecl MD(515位缺失C)、Ecl ME(324位C变为A)、Ecl MG(两点同时的突变238C→A302T→A)的产酶类型未发生变化。从氨基酸水平分析,产酶类型发生改变的菌株的氨基酸改变分别为：93-169位全部改变(170／110位终止)、109-132位全部改变(133位终止)、10-25位全部改变(26位终止)；而产酶类型未发生改变的菌株氨基酸的变化为：124-169位全部改变(170位终止)、172-187位全部改变(187位未终止)、108位变为终止密码、80R→S 101M→K。结论：阴沟肠杆菌ampD基因中的3个特异基因突变导致的氨基酸移码突变可使AmpC酶由非持续高产型转变为持续高产型。ampD基因编码的AmpD酰胺酶可分解包括N-乙酰胞壁酰酐三肽在内的粘肽成分,而这种分解作用很可能依赖AmpD酰胺酶的一个假设的活性区域。从我们的研究结果显示,该活性区域很可能起源于ampD基因的25位或以后(即AmpD蛋白的第9个氨基酸或之后),而终止于ampD基因的321位或以前(即AmpD蛋白的第107个氨基酸或之前)。而在上述活性区域内的单个点突变造成的单个氨基酸的突变有可能并不对整个AmpD酰胺酶活性区域产生影响。当然我们也不排除ampD基因突变对AmpCβ-内酰胺酶存在其他的影响机制。上述假设有待进一步研究。第二部分目的：从体内研究的水平揭示诱导型向持续高产型AmpCβ-内酰胺酶转变的耐药菌的转变率,并对其间患者的基础疾病情况和抗菌药物应用情况加以分析,对产AmpCβ-内酰胺酶菌株的临床控制和治疗起到初步提示的作用。方法：筛选携带产诱导型AmpCβ-内酰胺酶的革兰阴性杆菌的患者作为研究对象,定期收集其感染标本,至其携带菌株的AmpCβ-内酰胺酶转化为持续高产型,并对其菌种、基础疾病和期间抗生素使用情况进行数据收集,使用统计分析软件stata 8.0进行统计分析。结果：两家医院共收集成为研究对象的感染患者559人,收集到实验入主菌株714株,其中铜绿假单胞菌527株,摩根摩根菌26株,鲍曼不动杆菌41株,粘质沙雷氏菌25株,阴沟肠杆菌60株,产气肠杆菌35株。这些菌株中有33株的AmpC酶由诱导型转变为持续高产型,转变率为4.6%,各种细菌的转变率分别为铜绿假单胞菌3.4%、摩根摩根菌7.7%、鲍曼不动杆菌4.9%、粘质沙雷氏菌0%、阴沟肠杆菌13.3%、产气肠杆菌占8.6%。脑血管疾病患者的阳性转变率与非脑血管疾病患者相比,具有统计学差异,优势比OR=4.9。在基础疾病等其他条件相同的情况下,头孢菌素类药物应用天数与转变率呈负相关,而碳青霉烯类药物应用天数与转变率呈正相关,其他抗菌药物应用天数与产酶类型阳性转变率未见明显的相关性,而应用β-内酰胺类抗菌药物还是非β-内酰胺类抗菌药物未对阳性率结果产生明显差异。结论：铜绿假单胞菌(73.8%)是引起院内感染的最常见的产诱导型AmpC酶的细菌；阴沟肠杆菌(13.3%)产酶类型的阳性转变率最高。参与诊治脑梗塞或其后遗症患者的医护人员应更加注意感染菌株持续高产型AmpC酶的发生。在其他条件相同的情况下,需要应用头孢菌素类药物治疗患者的院内感染时,应做到足量、足疗程,降低AmpC酶由诱导型向持续高产型转变的阳性转变率；而需要大剂量、长时间的应用碳青霉烯类药物时,应注意细菌是否转变为产持续高产型AmpC酶的菌株,给予及时控制。更多还原

【Abstract】 Part 1Objectives:To reveal the influence of ampD specific mutations on the transformation of AmpCβ-lactamase from non-constitutive expression to constitutive high expression in Enterobacter cloacae.Methods:The chromosome-mediated constitutive high-productive strains of Enterobacter cloacae were selected with identification, drug sensitivity test, modified Cefoxitin three-dimensional test, and plasmid ampC gene amplification of bacteria. Chromosomal ampD was amplified by PCR and sequenced, and specific mutations were confirmed. Site-directed mutagenesis was used to mutate the wild-type Enterobacter cloacae for the above sites, and the ampD gene of clinical strains were complementated by wild-type ampD gene. The change of AmpCβ-lactamase types was detected by the modified Cefoxitin three-dimensional test and the AmpCβ-lactamase activity test.Results:For 121 clinical strains of Enterobacter cloacae,15 were chromosome-mediated constitutive high expression,8 had significant mutations of ampD and 7 strains by site-directed mutagenesis were established. Ecl MA (274 inserting A), Ecl MC (327 missing C) and Ecl MF (27 inserting G) changed from non-constitutive expression to constitutive high expression; but Ecl MB (371 inserting T), Ecl MD (515 missing C), Ecl ME (324 C→A), Ecl MG (238C→A 302T→A) did not change in AmpCβ-lactamase types. From the amino acid level, the amino acid changes of AmpD for the AmpCβ-lactamase type changes were:93-169 all changed (170/110 termination),109-132 all changed (133 termination),10-25 all changed (26 termination); while the AmpCβ-lactamase type did not change by the following amino acid changes:124-169 all changed (170 termination),172-187 all changed (187 no termination),108→termination,80R→S,101M→K.Conclusions:The 3 significant ampD mutations of Enterobacter cloacae that lead to frame-shift changes of amino acid could converse AmpCβ-lactamase from non-constitutive expression to constitutive high expression. AmpD lactamases encoded by ampD gene decomposes the nien-peptide components including N-acetyl-muramyl tripeptide anhydride, and this decomposition is likely to rely on an hypothetical active region of AmpD lactamase. Our study revealed that the active region of ampD probably originated from 25 or later (the 9th amino acid of AmpD, or after), and terminated on or before 321 (the 107th amino acid of AmpD, or before). In the above active region, a single site mutation for a single amino acid change of AmpD may not impact the AmpCβ-lactamase type of Enterobacter cloacae. Of course, other mechanisms of ampD mutations affecting on the AmpCβ-lactamase were not excluded from the study. The above assumptions should be further studied.Part 2Objectives:To reveal the conversion rate of AmpC P-lactamase from inducible high expression to constitutive high expression in vivo, analyze the underlying diseases and the antimicrobial agents application of the patients, and play an initial role in the clinical control and reatment of AmpCβ-lactamase.Methods:The patients carrying gram-negative bacilli with inducible AmpCβ-lactamase were screened as research objects. The same patients’samples were collected and the strains were cultrued untill they changed to the AmpC P-lactamase of constitutive high expression. The bacterial species, the chronic diseases, the use of antimicrobial agents were recorded. Statistic analysis was performed using a statistical software package (Stata 8.0).Results:559 patients from two hospitals as study objects were enrolled in the study, 714 inducible AmpCβ-lactamase-producing gram-negative bacilli were isolated, including 527 strains of Pseudomonas aeruginosa,26 strains of Morganella Morganii,41 strains of Acinetobacter baumannii,25 strains of Serratia marcescens, 60 strains of Enterobacter cloacae,35 strains of Enterobacter aerogenes.33 strains of the above bacilli changed to constitutive high expression, the overall conversion rate was 4.6%. The conversion rates of bacilli were a variety:Pseudomonas aeruginosa 3.4%, Morganella Morganii 7.7%, Acinetobacter baumannii 4.9%, Serratia marcescens 0%, Enterobacter cloacae 13.3%, Enterobacter aerogenes 8.6%.There is a statistically significant difference of the conversion rate between the patients suffering from cerebral vascular disease and the patients without such disease (OR=4.9). When the underlying diseases and other conditions were equal, the number of days of cephalosporins use was negatively correlated with the conversion rate, while the days of carbapenem use was positively correlated with the conversion rate, but the days of application of other antibiotics had no obvious correlation with the conversion rate, the application ofβ-lactam antibiotics or non-β-lactam antibiotics did not produce significant differences in the results.Conclusions:Pseudomonas aeruginosa (73.8%) were the most common cause of nosocomial infections of inducible AmpC-producing bacteria; the conversion rate of Enterobacter cloacae (13.3%) was the highest. health care workers involved in the diagnosis and treatment of cerebral infarction or its complications with infections should pay more attention to emergence of the constitutive high expressed AmpCβ-lactamase. Under the same conditions, when the cephalosporins was necessary in the treatment of patients with nosocomial infection, the dosage ane the treatment should be enough to reduce the conversion rate; while large doses and a long time application of carbapenem was required, whether the AmpCβ-lactamase of the bacteria changed to the constitutive high expression should be payed attention and controled.更多还原