烧伤病房耐碳青霉烯类铜绿假单胞菌耐药机制及分子流行病学研究
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摘要
铜绿假单胞菌（Pseudomonas aeruginosa, PA）是临床最常见的条件致病菌之一，尤其是医院获得性感染的最常见致病菌之一。碳青霉烯类抗菌药物为目前临床治疗铜绿假单胞菌的常用药物，但随着此类药物的广泛使用，耐碳青霉烯类铜绿假单胞菌菌株逐渐增多，已对临床控制医院获得性感染构成了严重威胁，且该菌的流行呈全球化趋势。铜绿假单胞菌耐药机制主要包括抗生素灭活酶或修饰酶的生成、外膜低渗透性、外膜孔蛋白缺失、生物膜形成和主动外排等。
产金属β-内酰胺酶（metallo-β-lactamases, MBL）的生成是铜绿假单胞菌耐碳青霉烯类抗菌药物最主要的耐药机制之一。MBL属于Ambler分子结构分类中的B类和Bush功能分类中的第3组，是一组活性部位为金属离子、且必须依赖金属离子的存在而发挥催化活性的酶类。MBL能水解包括碳青霉烯类在内的一大类β-内酰胺抗菌药物，其活性可被离子螯合物EDTA、菲咯啉以及巯基化合物所抑制，但不被克拉维酸、舒巴坦等常见的β-内酰胺酶抑制剂抑制。
吉林市某医院烧伤病房在2006年10月～12月期间，住院患者暴发多重耐药性铜绿假单胞菌的流行。为探讨该烧伤病房铜绿假单胞菌多重耐药性产生和流行机制，本研究分别采集烧伤患者烧伤患部标本和烧伤病房环境标本，以常规方法分离出18株铜绿假单胞菌。在测定抗菌药物敏感性基础上，检测细菌细胞壁外膜蛋白（OprD2）表达情况，采用PCR、克隆测序技术，转化和接合传递实验，检测细菌携带MBL耐药基因、整合子类型及整合子定位分析，并以质粒谱、脉冲场凝胶电泳和随机引物多态性分析技术检测铜绿假单胞菌株流行机制。
1.烧伤患者和烧伤病房环境标本的铜绿假单胞菌分离培养及血清学鉴定
吉林市某医院烧伤中心共有30张病床，分隔离病房和普通病房两种。2006年10月10日普通病房收治一名4岁农村儿童，腿部烧伤后在当地医院治疗，患部经治疗没有好转反而感染加重入院。入院时采集患部脓汁标本经常规分离培养后，用Viteck系统进行鉴定，证实是铜绿假单胞菌感染（命名为PA1）。入院1周后，该患者出现肺部感染，并继发败血症，从血液标本中分离出PA1。在10月～12月期间，分别住在3个普通烧伤病房7名患者的患部相继发生感染，采集7名患者脓汁标本，经常规分离培养鉴定为铜绿假单胞菌感染（命名为PA2，3，4，5，6，7，8）。经血清学鉴定，8株铜绿假单胞菌均为同一血清型，即O6型。
为调查烧伤病房铜绿假单胞菌来源，对医院烧伤病房环境进行监测。用无菌棉拭子涂擦方式采集医护人员手指、注射室操作台表面、治疗室表面、手推车表面、洗漱室水龙头表面、病房门把手表面、陪护人员手指。用无菌注射器抽取医疗器械浸泡液以及无菌平板采集病房空气各四份样本，共51份标本。结果从7份手推车表面涂抹样中分离出1株PA(14.3%）；从洗漱室5个水龙头表面涂抹样中分离出3株PA(60.0%）；从5名陪护人员手涂抹样中分离出3株PA(60.0%）；从4个病房门把手表面涂抹样中分离2株PA(50.0%）；从4个病房空气样中分离出1株PA(25.0%），共10株PA(18.6%)，分别命名为PA9～18，血清学鉴定分别属于O6，O7和O11三个血清型。
2.铜绿假单胞菌临床分离株耐药性的研究
2.1耐药性检测
采用WHO推荐的K-B法测定分离菌株对15种抗菌药物敏感性。实验时用标准的参考对照菌株与检测菌株平行测定，根据临床实验室标准化研究所（CLSI，2005）规定标准判读结果。大肠埃希菌ATCC25922、铜绿假单胞菌ATCC27853作为药敏质控菌株。采用微量肉汤稀释法对β-内酰胺抗菌药物进行MIC测定。结果显示8株烧伤患者临床标本分离株对氨苄西林、阿米卡星、庆大霉素、妥布霉素、复方磺胺、四环素、氯霉素7种抗菌药物耐药率均为100%，对环丙沙星耐药率为50%。替卡西林、替卡西林/克拉维酸和头孢唑啉MIC均大于256μg/ml。头孢噻肟和头孢吡肟MIC均大于128μg/ml。亚胺培南和氨曲南MIC有所差异：对亚胺培南表现高度耐药，但是PA1、PA2、PA3、PA4的MIC为64μg/ml，PA5、PA6、PA7、PA8的MIC为32μg/ml。PA1、PA2、PA3、PA4对氨曲南表现高度耐药（MIC为64μg/ml），PA5、PA6、PA7、PA8对氨曲南敏感（MIC为8μg/ml）。10株烧伤病房环境分离株对氨苄西林耐药率为100%，对阿米卡星、庆大霉素、妥布霉素、复方磺胺、四环素、氯霉素6种抗菌药物耐药率均为20%，对环丙沙星敏感。微量肉汤稀释法结果显示：替卡西林、替卡西林/克拉维酸和头孢唑啉的MIC均小于16μg/ml。头孢噻肟和头孢吡肟的MIC均小于12μg/ml。亚胺培南和氨曲南的MIC均小于8μg/ml。
2.2MBLs菌株的确定
采用双纸片增效法。将受试菌均匀涂布于M-H琼脂平板，然后在平板上贴两张亚胺培南(IPM，10μg/片)纸片，其中1张纸片上加入适量EDTA，置35℃孵育16～18h，比较两张纸片的抑菌圈大小。加EDTA的IPM纸片比不加EDTA的IPM纸片抑菌圈明显扩大，超过了5mm判断为产酶株。结果显示烧伤患者临床标本分离株均为产MBLs菌株，而10株烧伤病房环境分离株均为未产MBLs菌株。
3.铜绿假单胞菌临床分离株耐药机制的研究
3.1铜绿假单胞菌临床分离株外膜蛋白检测
铜绿假单胞菌细胞壁两侧有内、外两层膜，其中具有异常低通透性的外膜在抗菌药物耐药产生中发挥着重要作用。外膜蛋白oprD2孔道是小分子的亚胺培南选择性快速进入菌体而达高度抗菌活性的特异性通道；碳青霉烯类抗生素必须首先穿透外膜才能与内膜的青霉素结合蛋白结合，发挥抗菌效应。实验采用超声波破碎铜绿假单胞菌，提取细胞壁蛋白，进行SDS-PAGE和Western blotting，结果显示8株烧伤标本铜绿假单胞菌临床分离株OprD2孔蛋白缺失，与亚胺培南耐药相关；10株烧伤环境标本铜绿假单胞菌分离株均具有OprD2孔蛋白，对亚胺培南敏感。
3.2铜绿假单胞菌临床分离株携带MBL基因和第1类整合子
迄今为止发现细菌的获得性MBL有五种基因类型: IMP、VIM、SPM、GIM和AIM，其中最主要的是IMP型和VIM型，根据氨基酸序列不同分为很多亚型。到目前为止，在铜绿假单胞菌中发现了至少38种MBL，最常见的MBL包括：IMP-1、IMP-7、IMP-9、IMP-10、IMP-11和IMP-13型等；VIM型包括VIM-1、VIM-2、VIM-3、VIM-4和VIM-7型等；SPM、GIM和AIM型只有一种，即SPM-1、GIM-1和AIM-1型。这些基因大多数位于整合子上，并随着碳青霉烯类抗菌药物的应用不断有新类型出现，整合子不仅可通过位点特异性重组在细菌中传播，而且具有高效捕获耐药基因的功能，对细菌之间耐药的水平传播起着重要的作用。
实验设计了IMP、VIM、SPM和GIM基因引物及第1类整合酶引物，采用PCR、克隆测序检测MBLs耐药基因类型及整合子特征。PCR产物克隆测序8株烧伤标本铜绿假单胞菌临床分离株均携带VIM-2基因和第1类整合子，而10株烧伤环境标本铜绿假单胞菌分离株均未携带VIM-2基因和第1类整合子。
为明确细菌耐药性扩散的机制及整合子和MBLs基因定位，通过接合转移实验，把携带整合子阳性的质粒转移到工程菌内，进行质粒和转接合子图谱分析。对接合子耐药表型鉴定，发现烧伤标本铜绿假单胞菌临床分离株对氯霉素、环丙沙星、四环素耐药特性通过质粒转移到受体菌内。而β-内酰胺类、磺胺类及氨基糖苷类耐药特性不能转移。PCR检测铜绿假单胞菌转接合子携带MBLs基因和整合子情况，结果以转接合子为模板没有扩增出MBLs基因和第1类整合子，说明MBLs基因和第1类整合子可能位于染色体上，接合传递试验进一步证实VIM-2基因位于染色体上。
4.铜绿假单胞菌临床分离株在烧伤病房流行机制的研究
4.1铜绿假单胞菌临床分离株质粒指纹图谱分析
不同地区耐药菌株有着不同的质粒谱型。不同菌株之间耐药R质粒存在着一定的相互联系，它们的质粒谱型提示着不同地区、医院和病房耐药质粒的流行变化趋势。实验采用碱裂解法和质粒试剂盒提取方法，对18株铜绿假单胞菌临床分离株进行质粒抽提和质粒图谱分析。研究结果显示:8株烧伤标本铜绿假单胞菌临床分离株质粒图谱复杂，携带7个、大小约为1.0～60kb的天然质粒。而10株烧伤环境标本铜绿假单胞菌分离株质粒图谱单一，携带3～5个、大小约为1.0～60kb的天然质粒。18株细菌具有一个共同大小为60kb的质粒。
4.2铜绿假单胞菌临床分离株脉冲场凝胶电泳分析
脉冲场凝胶电泳分析是目前公认的分子流行病学调查首选的方法。结果显示8株烧伤标本铜绿假单胞菌临床分离株在10～800kb间产生12～18条大小不同的DNA酶切片段区带，分离株显示三种基因型的克隆株，克隆株分别来自三个不同烧伤病房。铜绿假单胞菌烧伤环境标本分离株表现出相同的基因型，均为同一克隆株。证明了吉林地区烧伤病房存在铜绿假单胞菌克隆株播散及流行。
4.3铜绿假单胞菌临床分离株同源性分析
提取18株铜绿假单胞菌分离株完整的基因组DNA，进行随机引物扩增多态性分析。烧伤标本铜绿假单胞菌分离株表现出三种基因型：PA1、PA2和PA3为同一克隆株；PA4、PA5和PA6为同一克隆株；PA7和PA8为同一克隆株。烧伤环境标本铜绿假单胞菌分离株表现出相同的基因型，证明了PA9～PA18来自五个不同部位标本，均为同一克隆株。
研究结果揭示了铜绿假单胞菌临床分离株对碳青霉烯类抗菌药物耐药机制及耐药性传递方面的分子流行病学机制，为进一步更好地控制烧伤病房铜绿假单胞菌的流行提供重要的实验依据。
Pseudomonas aeruginosa (PA) is one of the most common clinical conditioned pathogens,in particular, it plays a very important role in hospital-acquired infections. Carbapenem,anantimicrobial drug, plays a major part in the clinical anti-PA infection treatment. However,with its widespread use, PA strains resistant to i carbapenem are increasing and spreading,which have led to a serious threat to the clinical infection control. Moreover, the prevalence ofthe bacteria has showed a trend to spread throughout the world. Resistance mechanisms of PAinclude the generation of antibiotics inactivated enzymes or modified enzymes, the outermembrane with low permeability, the lack of outer membrane protein, biomembranceformation and active efflux, etc.
The generation of metallo-β-lactamases (MBL), also known as metalloenzyme, is one ofthe most important mechanisms through which PA is resistant to carbapenem antibacterialdrugs. MBL, classified as an enzyme in Class B based on Ambler molecular structureclassification and Group3based on Bush functional classification, is a group of enzymes thattheir active sites are metal ions and they can play a catalytic activity only depending on thepresence of metal ions. MBL can hydrolyze a large group of beta-lactam antimicrobial agentsincluding carbapenems, and its activity can be inhibited by EDTA, an ion chelate,phenanthroline and mercapto compounds, but not by clavulanic acid, sulbactam and othercommon beta-lactamase inhibitor.
There was an outbreak of epidemic PA infection among inpatients in burn wards of ahospital in Jilin City from October to December in2006. In order to explore the cause and themechanism of the outbreak of multi-drug resistance of PA, the samples from the burning partsof the burn patients and the environment of the burn wards were collected. The conventionalmethods were used to separate18strains of PA from the samples. Then, on the basis of testingthe antibiotic susceptibility, the expression of outer membrane protein (OprD2) in bacterial cellwall was measured, the researcher detected the resistance genes of bacteria carrying the MBL,the sub-types and location analysis of integron by means of PCR, cloning and sequencingtechnology, transformation and conjugal transfer experiments. Finally, the researcher analyzedthe epidemic mechanism of PA strains with the plasmid profiles, pulsed-field gelelectrophoresis and randomly primer polymorphic DNA assay.
1. The Isolation Culture and Serological Identification of PA from ClinicalSpecimens of Burned Patients
There are30beds altogether in the burn center of the hospital in Jilin city, which weredivided into isolation ward and general ward. On October10,2006, its general ward admitted afour-year-old rural child who had been treated due to his leg burning at the local hospitalinitially after the burning, but the affected part became infected and did not recover after thetreatment for a certain time. After he was hospitalized, the pus specimens from the affectedpart were collected and cultivated with the conventional separation method, and then,Viteck system was applied to confirm the infection of PA (named PA1). One week later, thepatient had lung infection and secondary sepsis. PA1was isolated from the blood samples.There were7patients who suffered from the infection in the three general wards from Octoberto December. The pus specimens from the7patients were collected, which were isolated andcultivated with the conventional method, and finally were identified PA infection (named PA2,3,4,5,6,7,8). By serological identification,8strains of PA were the same serotype, namely,06type.
In order to survey the source of the PA in the burn wards, the environment of the burnwards was monitored. In a wipe way with a sterile cotton swab, specimens from medical staff’sfingers, surfaces of operation desk in the injection room, the treatment room, the handcart, thefaucet in the wash room, the doorknob of the wards, and assistants’ fingers. A sterile syringewas used to extract the soak solution of the medical equipments and a sterile flat was apllied tocollect different4air specimens in the wards. The total specimens were51. The results showedthat1PA strain was isolated in7specimens from the surface of the handcart (14.3%);3PAstrains in5specimens from the surface of faucet (60.0%);3PA strains in5specimens fromthe assistants’ fingers (60.0%);2PA strains in4specimens from the surface of doorknob(50.0%);1PA strain in4air specimens from the4wards (25.0%). There were10strains ofPA totally, which were named PA9～18respectively and confirmed to belong to07,06and011serotype with serological identification.
2. Investigation of the Resistance in Clinical Isolates of PA
2.1Detection of Resistance
The sensitivity of isolates to15antimicrobial drugs was detected by means of K-Bmethod recommended by WHO. Experiments were done by parallel determination of controlstrains and detection strains, and the results were interpreted based on the standard set by theClinical and Laboratory Standards Institute (CLSI,2005). Escherichia coli ATCC25922andPA ATCC27853were taken as the susceptibility quality control strains. At the same time, theMIC determination of beta-lactam antimicrobial agents were done with broth microdilutionmethod, then the results showed that the average resistance rate of8strains of PA to7 antibiotics was100%, including ampicillin, amikacin, gentamicin, tobramycin,sulfamethoxazole, tetracycline, chloramphenicol; the rate to ciprofloxacin was50%; MICs ofticarcillin, ticarcillin/clavulanic acid, cefazolin, cefotaxime and cefepime were greater than256μg/ml, and MICs of cefotaxime and cefepime greater than128μg/ml. But there were somedifferences in MICs of imipenem and aztreonam: a high degree of resistance to imipenemappeared, but the MIC of PA1, PA2, PA3and PA was64μg/ml, and PA5, PA6, PA7and PA8was32μg/ml; a high degree of resistance to aztreonam also was found, that is, the MIC of PA1,PA2, PA3and PA4was64μg/ml, and PA5, PA6, PA7and PA8was8μg/ml; the resistancerate of10isolates from the environment of the burn wards to ampicillin was100%, the rates tothe6antimicrobial agents, including amikacin, gentamicin, tobramycin, complex sulfonamide,tetracyclin and chloramphenicol, all were20%, but the isolates to ciprofloxacin were sensitive.Results from broth micro-dillution method showed that MICs of ticarcillin,ticarcillin/clavulanic acid and cefazolin were less than12μg/ml, and those of imipenem andaztreonam were less than8μg/ml.
2.2Determination of MBLs Strains
The double-disk synergy method was applied. Tested bacteria were evenly distributed onMH agar plates, and then two pieces of paper with imipenem (IMP,10μg/ml) were posted inthe flat. An appropriate amount of EDTA was added on one piece of paper, and the paper wasincubated at35℃for16~8h for comparing the size of the inhibition zone on the two pieces ofpaper. The results showed that the inhibition zone on IPM paper with EDTA was significantlyexpanded compared with that on the paper without EDTA, and the inhibition zone more than5mm was taken as the enzyme-producing strains. As a result, the strains of PA isolated fromthe burn patients were all MBLs ones, but none of10strains from the environment of the nurnwards was MBLs strains.
3. Investigation of the Resistance Mechanisms of Clinical Isolates of PA
3.1Outer Membrane Protein Detection of the Clinical Isolates of PA
The cell wall of PA has inner and outer layers, and the outer membrane structure hasunusually low permeability, which plays a major role in multidrug resistance to antimicrobialdrugs. The outer membrane protein OprD2pore is a specific channel for imipenem with smallmolecules to enter the bacteria quickly to achieve a high degree of antibacterial activity.Carbapenem can exert its antibacterial effect when it must penetrate the outer layer firstly andthen combine with the penicillin-binding protein in the inner layer.
In this experiment, the PA was broken by ultrasonic wave, the cell wall proteins wereextracted, and then, SDS-PAGE and Western blotting were performed. The experimental results revealed the deficiency of OprD2porin protein in8strains of PA, which was related tothe resistance to imipenem; All10strains of PA in the specimens from the environment of theburn ward had OprD2porin protein and they were sensitive to imipenem.
3.2Clinical Isolates of PA Carrying the Metal Gene and Class1Integron
So far five gene types of metalloenzyme of bacteria have been discovered: IMP, VIM,SPM, GIM, and AIM. IMP and VIM are most important, which can be divided into manysubtypes according to the amino acid sequence. By far, at least38kinds of metallic enzymehave been found in PA. The most popular MBL includes IMP-1,7,9,10,11and13; VIMincludes VIM-1,2,3,4and7; SPM, GIM and AIM only one type, namely, SPM-1, GIM-1andAIM-1. Most of these genes appear on integron and new types of these genes continuouslyappear with the application of carbapenem antimicrobial drugs. Integron can not only spread inbacteria through site-specific recombination but also capture the drug-resistant gene efficiently,so it plays a great part in horizontal transmission of resistance among bacteria.
In this experiment, IMP, VIM, the SPM and GIM gene primers were designed, and PCR,cloning and sequencing technology were applied to detect the gene types of MBLs resistancegenes and the characteristics of integron. The results from PCR product cloning andsequencing showed eight strains carrying VIM-2gene and class1integron.
In order to make clear the diffusion mechanism of bacterial resistance, and the genelocation of integron and MBLs, the analysis on the plasmids and zygote mapping was carriedout through transferring the plasmid with positive integron into the engineered bacteria.Zygote resistance profile identification revealed that the resistance of PA to chloramphenicol,ciprofloxacin, and tetracycline could be transferred to recipient strains through plasmid, whilethe resistance to Beta-lactamase, sulfonamides and aminoglycoside could not be transferred.PCR was applied to detect the transfer of the zygote carring MBLs genes and integron in PA.As a result, the MBLs genes and class1integron were not amplified on the template of zygote,indicating that the MBLs genes and class1integron might be located on the chromosome, Thetransformation and conjugal transfer experiments further confirmed that VIM-2gene was onthe chromosome.
4. Investigation of Epidemic Mechanism of Clinical Isolates of PA in theBurn Wards
4.1The Analysis of Plasmid Fingerprinting Spectrum of Clinical Isolates of PA
There are plasmid profiles for resistant strains in different regions, resistance R plasmidsbetween different strains are mutually related to some extent, and the plasmid profiles mayreveal the popular trends of resistance plasmid in different regions, hospitals and wards. In this experiment, the alkaline lysis method and plasmid extraction kit were used to extract theplasmids of the18clinical isolates and analyze the plasmid profile. The results indicated thatthe plasmid profiles of the8strains of PA in specimens from the burned patientswerecomplicated, and the strains carried7natural plasmids with various numbers and size inabout1.0~60kb, while the plasmid profiles of the10strains of PA in specimens from theenvironment of burn wards were simple and the strains carried3-5natural plasmids with1.0~60kb, and The18bacteria all had the same plasmid with the size in60kb.
4.2The Analysis of Pulsed Field Gel Electrophoresis in Clinical Isolates of PA
The analysis of pulsed field gel electrophoresis is the first choice currently accepted formolecular epidemiological investigations. The results indicated that12-18DNA fragmentszones with different sizes in10~800kb were found in the8isolates genome of PA in specimensfrom the burned patients and the isolates showed clone isolates with three kinds of gene types,and the clone isolates came from three different burn wards respectively, while the same genetype was found in the isolates of PA in specimens from the environment of the burn wards andall of them were identical cloning strains, proving that cloning isolates of PA are spread andprevalent in many burn wards of Jilin region.
4.3Homology analysis of Isolates of PA in Clinic
Complete genomic DNA of18PA isolates was extracted and the random primersamplified polymorphism analysis was completed. Three genotypes were found in PA isolatesin specimens from the burned patients: PA1, PA2and PA3, which were the identical cloningisolate; PA4, PA5and PA6, which were the identical cloning isolate; PA7and PA8, which werethe identical cloning isolate. The same gene type was found in the isolates of PA in specimensfrom the environment of the burn wards, indicating that PA9～PA18from the different placesare the identical cloned strain.
The results reveal the resistance mechanism of clinical PA isolates to carbapenemantimicrobial agents and the molecular epidemiological mechanism in the resistancetransmission, which may provide experimental evidences for the efficent control of theprvalence of PA in burn wards.
产金属β-内酰胺酶（metallo-β-lactamases, MBL）的生成是铜绿假单胞菌耐碳青霉烯类抗菌药物最主要的耐药机制之一。MBL属于Ambler分子结构分类中的B类和Bush功能分类中的第3组，是一组活性部位为金属离子、且必须依赖金属离子的存在而发挥催化活性的酶类。MBL能水解包括碳青霉烯类在内的一大类β-内酰胺抗菌药物，其活性可被离子螯合物EDTA、菲咯啉以及巯基化合物所抑制，但不被克拉维酸、舒巴坦等常见的β-内酰胺酶抑制剂抑制。
吉林市某医院烧伤病房在2006年10月～12月期间，住院患者暴发多重耐药性铜绿假单胞菌的流行。为探讨该烧伤病房铜绿假单胞菌多重耐药性产生和流行机制，本研究分别采集烧伤患者烧伤患部标本和烧伤病房环境标本，以常规方法分离出18株铜绿假单胞菌。在测定抗菌药物敏感性基础上，检测细菌细胞壁外膜蛋白（OprD2）表达情况，采用PCR、克隆测序技术，转化和接合传递实验，检测细菌携带MBL耐药基因、整合子类型及整合子定位分析，并以质粒谱、脉冲场凝胶电泳和随机引物多态性分析技术检测铜绿假单胞菌株流行机制。
1.烧伤患者和烧伤病房环境标本的铜绿假单胞菌分离培养及血清学鉴定
吉林市某医院烧伤中心共有30张病床，分隔离病房和普通病房两种。2006年10月10日普通病房收治一名4岁农村儿童，腿部烧伤后在当地医院治疗，患部经治疗没有好转反而感染加重入院。入院时采集患部脓汁标本经常规分离培养后，用Viteck系统进行鉴定，证实是铜绿假单胞菌感染（命名为PA1）。入院1周后，该患者出现肺部感染，并继发败血症，从血液标本中分离出PA1。在10月～12月期间，分别住在3个普通烧伤病房7名患者的患部相继发生感染，采集7名患者脓汁标本，经常规分离培养鉴定为铜绿假单胞菌感染（命名为PA2，3，4，5，6，7，8）。经血清学鉴定，8株铜绿假单胞菌均为同一血清型，即O6型。
为调查烧伤病房铜绿假单胞菌来源，对医院烧伤病房环境进行监测。用无菌棉拭子涂擦方式采集医护人员手指、注射室操作台表面、治疗室表面、手推车表面、洗漱室水龙头表面、病房门把手表面、陪护人员手指。用无菌注射器抽取医疗器械浸泡液以及无菌平板采集病房空气各四份样本，共51份标本。结果从7份手推车表面涂抹样中分离出1株PA(14.3%）；从洗漱室5个水龙头表面涂抹样中分离出3株PA(60.0%）；从5名陪护人员手涂抹样中分离出3株PA(60.0%）；从4个病房门把手表面涂抹样中分离2株PA(50.0%）；从4个病房空气样中分离出1株PA(25.0%），共10株PA(18.6%)，分别命名为PA9～18，血清学鉴定分别属于O6，O7和O11三个血清型。
2.铜绿假单胞菌临床分离株耐药性的研究
2.1耐药性检测
采用WHO推荐的K-B法测定分离菌株对15种抗菌药物敏感性。实验时用标准的参考对照菌株与检测菌株平行测定，根据临床实验室标准化研究所（CLSI，2005）规定标准判读结果。大肠埃希菌ATCC25922、铜绿假单胞菌ATCC27853作为药敏质控菌株。采用微量肉汤稀释法对β-内酰胺抗菌药物进行MIC测定。结果显示8株烧伤患者临床标本分离株对氨苄西林、阿米卡星、庆大霉素、妥布霉素、复方磺胺、四环素、氯霉素7种抗菌药物耐药率均为100%，对环丙沙星耐药率为50%。替卡西林、替卡西林/克拉维酸和头孢唑啉MIC均大于256μg/ml。头孢噻肟和头孢吡肟MIC均大于128μg/ml。亚胺培南和氨曲南MIC有所差异：对亚胺培南表现高度耐药，但是PA1、PA2、PA3、PA4的MIC为64μg/ml，PA5、PA6、PA7、PA8的MIC为32μg/ml。PA1、PA2、PA3、PA4对氨曲南表现高度耐药（MIC为64μg/ml），PA5、PA6、PA7、PA8对氨曲南敏感（MIC为8μg/ml）。10株烧伤病房环境分离株对氨苄西林耐药率为100%，对阿米卡星、庆大霉素、妥布霉素、复方磺胺、四环素、氯霉素6种抗菌药物耐药率均为20%，对环丙沙星敏感。微量肉汤稀释法结果显示：替卡西林、替卡西林/克拉维酸和头孢唑啉的MIC均小于16μg/ml。头孢噻肟和头孢吡肟的MIC均小于12μg/ml。亚胺培南和氨曲南的MIC均小于8μg/ml。
2.2MBLs菌株的确定
采用双纸片增效法。将受试菌均匀涂布于M-H琼脂平板，然后在平板上贴两张亚胺培南(IPM，10μg/片)纸片，其中1张纸片上加入适量EDTA，置35℃孵育16～18h，比较两张纸片的抑菌圈大小。加EDTA的IPM纸片比不加EDTA的IPM纸片抑菌圈明显扩大，超过了5mm判断为产酶株。结果显示烧伤患者临床标本分离株均为产MBLs菌株，而10株烧伤病房环境分离株均为未产MBLs菌株。
3.铜绿假单胞菌临床分离株耐药机制的研究
3.1铜绿假单胞菌临床分离株外膜蛋白检测
铜绿假单胞菌细胞壁两侧有内、外两层膜，其中具有异常低通透性的外膜在抗菌药物耐药产生中发挥着重要作用。外膜蛋白oprD2孔道是小分子的亚胺培南选择性快速进入菌体而达高度抗菌活性的特异性通道；碳青霉烯类抗生素必须首先穿透外膜才能与内膜的青霉素结合蛋白结合，发挥抗菌效应。实验采用超声波破碎铜绿假单胞菌，提取细胞壁蛋白，进行SDS-PAGE和Western blotting，结果显示8株烧伤标本铜绿假单胞菌临床分离株OprD2孔蛋白缺失，与亚胺培南耐药相关；10株烧伤环境标本铜绿假单胞菌分离株均具有OprD2孔蛋白，对亚胺培南敏感。
3.2铜绿假单胞菌临床分离株携带MBL基因和第1类整合子
迄今为止发现细菌的获得性MBL有五种基因类型: IMP、VIM、SPM、GIM和AIM，其中最主要的是IMP型和VIM型，根据氨基酸序列不同分为很多亚型。到目前为止，在铜绿假单胞菌中发现了至少38种MBL，最常见的MBL包括：IMP-1、IMP-7、IMP-9、IMP-10、IMP-11和IMP-13型等；VIM型包括VIM-1、VIM-2、VIM-3、VIM-4和VIM-7型等；SPM、GIM和AIM型只有一种，即SPM-1、GIM-1和AIM-1型。这些基因大多数位于整合子上，并随着碳青霉烯类抗菌药物的应用不断有新类型出现，整合子不仅可通过位点特异性重组在细菌中传播，而且具有高效捕获耐药基因的功能，对细菌之间耐药的水平传播起着重要的作用。
实验设计了IMP、VIM、SPM和GIM基因引物及第1类整合酶引物，采用PCR、克隆测序检测MBLs耐药基因类型及整合子特征。PCR产物克隆测序8株烧伤标本铜绿假单胞菌临床分离株均携带VIM-2基因和第1类整合子，而10株烧伤环境标本铜绿假单胞菌分离株均未携带VIM-2基因和第1类整合子。
为明确细菌耐药性扩散的机制及整合子和MBLs基因定位，通过接合转移实验，把携带整合子阳性的质粒转移到工程菌内，进行质粒和转接合子图谱分析。对接合子耐药表型鉴定，发现烧伤标本铜绿假单胞菌临床分离株对氯霉素、环丙沙星、四环素耐药特性通过质粒转移到受体菌内。而β-内酰胺类、磺胺类及氨基糖苷类耐药特性不能转移。PCR检测铜绿假单胞菌转接合子携带MBLs基因和整合子情况，结果以转接合子为模板没有扩增出MBLs基因和第1类整合子，说明MBLs基因和第1类整合子可能位于染色体上，接合传递试验进一步证实VIM-2基因位于染色体上。
4.铜绿假单胞菌临床分离株在烧伤病房流行机制的研究
4.1铜绿假单胞菌临床分离株质粒指纹图谱分析
不同地区耐药菌株有着不同的质粒谱型。不同菌株之间耐药R质粒存在着一定的相互联系，它们的质粒谱型提示着不同地区、医院和病房耐药质粒的流行变化趋势。实验采用碱裂解法和质粒试剂盒提取方法，对18株铜绿假单胞菌临床分离株进行质粒抽提和质粒图谱分析。研究结果显示:8株烧伤标本铜绿假单胞菌临床分离株质粒图谱复杂，携带7个、大小约为1.0～60kb的天然质粒。而10株烧伤环境标本铜绿假单胞菌分离株质粒图谱单一，携带3～5个、大小约为1.0～60kb的天然质粒。18株细菌具有一个共同大小为60kb的质粒。
4.2铜绿假单胞菌临床分离株脉冲场凝胶电泳分析
脉冲场凝胶电泳分析是目前公认的分子流行病学调查首选的方法。结果显示8株烧伤标本铜绿假单胞菌临床分离株在10～800kb间产生12～18条大小不同的DNA酶切片段区带，分离株显示三种基因型的克隆株，克隆株分别来自三个不同烧伤病房。铜绿假单胞菌烧伤环境标本分离株表现出相同的基因型，均为同一克隆株。证明了吉林地区烧伤病房存在铜绿假单胞菌克隆株播散及流行。
4.3铜绿假单胞菌临床分离株同源性分析
提取18株铜绿假单胞菌分离株完整的基因组DNA，进行随机引物扩增多态性分析。烧伤标本铜绿假单胞菌分离株表现出三种基因型：PA1、PA2和PA3为同一克隆株；PA4、PA5和PA6为同一克隆株；PA7和PA8为同一克隆株。烧伤环境标本铜绿假单胞菌分离株表现出相同的基因型，证明了PA9～PA18来自五个不同部位标本，均为同一克隆株。
研究结果揭示了铜绿假单胞菌临床分离株对碳青霉烯类抗菌药物耐药机制及耐药性传递方面的分子流行病学机制，为进一步更好地控制烧伤病房铜绿假单胞菌的流行提供重要的实验依据。
Pseudomonas aeruginosa (PA) is one of the most common clinical conditioned pathogens,in particular, it plays a very important role in hospital-acquired infections. Carbapenem,anantimicrobial drug, plays a major part in the clinical anti-PA infection treatment. However,with its widespread use, PA strains resistant to i carbapenem are increasing and spreading,which have led to a serious threat to the clinical infection control. Moreover, the prevalence ofthe bacteria has showed a trend to spread throughout the world. Resistance mechanisms of PAinclude the generation of antibiotics inactivated enzymes or modified enzymes, the outermembrane with low permeability, the lack of outer membrane protein, biomembranceformation and active efflux, etc.
The generation of metallo-β-lactamases (MBL), also known as metalloenzyme, is one ofthe most important mechanisms through which PA is resistant to carbapenem antibacterialdrugs. MBL, classified as an enzyme in Class B based on Ambler molecular structureclassification and Group3based on Bush functional classification, is a group of enzymes thattheir active sites are metal ions and they can play a catalytic activity only depending on thepresence of metal ions. MBL can hydrolyze a large group of beta-lactam antimicrobial agentsincluding carbapenems, and its activity can be inhibited by EDTA, an ion chelate,phenanthroline and mercapto compounds, but not by clavulanic acid, sulbactam and othercommon beta-lactamase inhibitor.
There was an outbreak of epidemic PA infection among inpatients in burn wards of ahospital in Jilin City from October to December in2006. In order to explore the cause and themechanism of the outbreak of multi-drug resistance of PA, the samples from the burning partsof the burn patients and the environment of the burn wards were collected. The conventionalmethods were used to separate18strains of PA from the samples. Then, on the basis of testingthe antibiotic susceptibility, the expression of outer membrane protein (OprD2) in bacterial cellwall was measured, the researcher detected the resistance genes of bacteria carrying the MBL,the sub-types and location analysis of integron by means of PCR, cloning and sequencingtechnology, transformation and conjugal transfer experiments. Finally, the researcher analyzedthe epidemic mechanism of PA strains with the plasmid profiles, pulsed-field gelelectrophoresis and randomly primer polymorphic DNA assay.
1. The Isolation Culture and Serological Identification of PA from ClinicalSpecimens of Burned Patients
There are30beds altogether in the burn center of the hospital in Jilin city, which weredivided into isolation ward and general ward. On October10,2006, its general ward admitted afour-year-old rural child who had been treated due to his leg burning at the local hospitalinitially after the burning, but the affected part became infected and did not recover after thetreatment for a certain time. After he was hospitalized, the pus specimens from the affectedpart were collected and cultivated with the conventional separation method, and then,Viteck system was applied to confirm the infection of PA (named PA1). One week later, thepatient had lung infection and secondary sepsis. PA1was isolated from the blood samples.There were7patients who suffered from the infection in the three general wards from Octoberto December. The pus specimens from the7patients were collected, which were isolated andcultivated with the conventional method, and finally were identified PA infection (named PA2,3,4,5,6,7,8). By serological identification,8strains of PA were the same serotype, namely,06type.
In order to survey the source of the PA in the burn wards, the environment of the burnwards was monitored. In a wipe way with a sterile cotton swab, specimens from medical staff’sfingers, surfaces of operation desk in the injection room, the treatment room, the handcart, thefaucet in the wash room, the doorknob of the wards, and assistants’ fingers. A sterile syringewas used to extract the soak solution of the medical equipments and a sterile flat was apllied tocollect different4air specimens in the wards. The total specimens were51. The results showedthat1PA strain was isolated in7specimens from the surface of the handcart (14.3%);3PAstrains in5specimens from the surface of faucet (60.0%);3PA strains in5specimens fromthe assistants’ fingers (60.0%);2PA strains in4specimens from the surface of doorknob(50.0%);1PA strain in4air specimens from the4wards (25.0%). There were10strains ofPA totally, which were named PA9～18respectively and confirmed to belong to07,06and011serotype with serological identification.
2. Investigation of the Resistance in Clinical Isolates of PA
2.1Detection of Resistance
The sensitivity of isolates to15antimicrobial drugs was detected by means of K-Bmethod recommended by WHO. Experiments were done by parallel determination of controlstrains and detection strains, and the results were interpreted based on the standard set by theClinical and Laboratory Standards Institute (CLSI,2005). Escherichia coli ATCC25922andPA ATCC27853were taken as the susceptibility quality control strains. At the same time, theMIC determination of beta-lactam antimicrobial agents were done with broth microdilutionmethod, then the results showed that the average resistance rate of8strains of PA to7 antibiotics was100%, including ampicillin, amikacin, gentamicin, tobramycin,sulfamethoxazole, tetracycline, chloramphenicol; the rate to ciprofloxacin was50%; MICs ofticarcillin, ticarcillin/clavulanic acid, cefazolin, cefotaxime and cefepime were greater than256μg/ml, and MICs of cefotaxime and cefepime greater than128μg/ml. But there were somedifferences in MICs of imipenem and aztreonam: a high degree of resistance to imipenemappeared, but the MIC of PA1, PA2, PA3and PA was64μg/ml, and PA5, PA6, PA7and PA8was32μg/ml; a high degree of resistance to aztreonam also was found, that is, the MIC of PA1,PA2, PA3and PA4was64μg/ml, and PA5, PA6, PA7and PA8was8μg/ml; the resistancerate of10isolates from the environment of the burn wards to ampicillin was100%, the rates tothe6antimicrobial agents, including amikacin, gentamicin, tobramycin, complex sulfonamide,tetracyclin and chloramphenicol, all were20%, but the isolates to ciprofloxacin were sensitive.Results from broth micro-dillution method showed that MICs of ticarcillin,ticarcillin/clavulanic acid and cefazolin were less than12μg/ml, and those of imipenem andaztreonam were less than8μg/ml.
2.2Determination of MBLs Strains
The double-disk synergy method was applied. Tested bacteria were evenly distributed onMH agar plates, and then two pieces of paper with imipenem (IMP,10μg/ml) were posted inthe flat. An appropriate amount of EDTA was added on one piece of paper, and the paper wasincubated at35℃for16~8h for comparing the size of the inhibition zone on the two pieces ofpaper. The results showed that the inhibition zone on IPM paper with EDTA was significantlyexpanded compared with that on the paper without EDTA, and the inhibition zone more than5mm was taken as the enzyme-producing strains. As a result, the strains of PA isolated fromthe burn patients were all MBLs ones, but none of10strains from the environment of the nurnwards was MBLs strains.
3. Investigation of the Resistance Mechanisms of Clinical Isolates of PA
3.1Outer Membrane Protein Detection of the Clinical Isolates of PA
The cell wall of PA has inner and outer layers, and the outer membrane structure hasunusually low permeability, which plays a major role in multidrug resistance to antimicrobialdrugs. The outer membrane protein OprD2pore is a specific channel for imipenem with smallmolecules to enter the bacteria quickly to achieve a high degree of antibacterial activity.Carbapenem can exert its antibacterial effect when it must penetrate the outer layer firstly andthen combine with the penicillin-binding protein in the inner layer.
In this experiment, the PA was broken by ultrasonic wave, the cell wall proteins wereextracted, and then, SDS-PAGE and Western blotting were performed. The experimental results revealed the deficiency of OprD2porin protein in8strains of PA, which was related tothe resistance to imipenem; All10strains of PA in the specimens from the environment of theburn ward had OprD2porin protein and they were sensitive to imipenem.
3.2Clinical Isolates of PA Carrying the Metal Gene and Class1Integron
So far five gene types of metalloenzyme of bacteria have been discovered: IMP, VIM,SPM, GIM, and AIM. IMP and VIM are most important, which can be divided into manysubtypes according to the amino acid sequence. By far, at least38kinds of metallic enzymehave been found in PA. The most popular MBL includes IMP-1,7,9,10,11and13; VIMincludes VIM-1,2,3,4and7; SPM, GIM and AIM only one type, namely, SPM-1, GIM-1andAIM-1. Most of these genes appear on integron and new types of these genes continuouslyappear with the application of carbapenem antimicrobial drugs. Integron can not only spread inbacteria through site-specific recombination but also capture the drug-resistant gene efficiently,so it plays a great part in horizontal transmission of resistance among bacteria.
In this experiment, IMP, VIM, the SPM and GIM gene primers were designed, and PCR,cloning and sequencing technology were applied to detect the gene types of MBLs resistancegenes and the characteristics of integron. The results from PCR product cloning andsequencing showed eight strains carrying VIM-2gene and class1integron.
In order to make clear the diffusion mechanism of bacterial resistance, and the genelocation of integron and MBLs, the analysis on the plasmids and zygote mapping was carriedout through transferring the plasmid with positive integron into the engineered bacteria.Zygote resistance profile identification revealed that the resistance of PA to chloramphenicol,ciprofloxacin, and tetracycline could be transferred to recipient strains through plasmid, whilethe resistance to Beta-lactamase, sulfonamides and aminoglycoside could not be transferred.PCR was applied to detect the transfer of the zygote carring MBLs genes and integron in PA.As a result, the MBLs genes and class1integron were not amplified on the template of zygote,indicating that the MBLs genes and class1integron might be located on the chromosome, Thetransformation and conjugal transfer experiments further confirmed that VIM-2gene was onthe chromosome.
4. Investigation of Epidemic Mechanism of Clinical Isolates of PA in theBurn Wards
4.1The Analysis of Plasmid Fingerprinting Spectrum of Clinical Isolates of PA
There are plasmid profiles for resistant strains in different regions, resistance R plasmidsbetween different strains are mutually related to some extent, and the plasmid profiles mayreveal the popular trends of resistance plasmid in different regions, hospitals and wards. In this experiment, the alkaline lysis method and plasmid extraction kit were used to extract theplasmids of the18clinical isolates and analyze the plasmid profile. The results indicated thatthe plasmid profiles of the8strains of PA in specimens from the burned patientswerecomplicated, and the strains carried7natural plasmids with various numbers and size inabout1.0~60kb, while the plasmid profiles of the10strains of PA in specimens from theenvironment of burn wards were simple and the strains carried3-5natural plasmids with1.0~60kb, and The18bacteria all had the same plasmid with the size in60kb.
4.2The Analysis of Pulsed Field Gel Electrophoresis in Clinical Isolates of PA
The analysis of pulsed field gel electrophoresis is the first choice currently accepted formolecular epidemiological investigations. The results indicated that12-18DNA fragmentszones with different sizes in10~800kb were found in the8isolates genome of PA in specimensfrom the burned patients and the isolates showed clone isolates with three kinds of gene types,and the clone isolates came from three different burn wards respectively, while the same genetype was found in the isolates of PA in specimens from the environment of the burn wards andall of them were identical cloning strains, proving that cloning isolates of PA are spread andprevalent in many burn wards of Jilin region.
4.3Homology analysis of Isolates of PA in Clinic
Complete genomic DNA of18PA isolates was extracted and the random primersamplified polymorphism analysis was completed. Three genotypes were found in PA isolatesin specimens from the burned patients: PA1, PA2and PA3, which were the identical cloningisolate; PA4, PA5and PA6, which were the identical cloning isolate; PA7and PA8, which werethe identical cloning isolate. The same gene type was found in the isolates of PA in specimensfrom the environment of the burn wards, indicating that PA9～PA18from the different placesare the identical cloned strain.
The results reveal the resistance mechanism of clinical PA isolates to carbapenemantimicrobial agents and the molecular epidemiological mechanism in the resistancetransmission, which may provide experimental evidences for the efficent control of theprvalence of PA in burn wards.
引文
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