医源性产ESBLs耐热性大肠菌群及其耐药基因在水环境中的转归规律研究
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摘要
近年来,水环境中耐药菌种类、数量、多重耐药率的不断增加日益受到国内外学者的关注。研究结果显示,耐药菌存在于各种各样的水体中,包括污水、矿泉水、管网水、饮用水、海水。
自然界中各种细菌广泛存在,相互联系;细菌具有多种在种内或种间进行自主转移或诱动转移的遗传因子如质粒、转座子、噬菌体等;质粒、转座子上还有募集和表达外源耐药基因的整合子,通过接合、转化及转导等方式,细菌间相互间交换所携带的一些基因,可使菌群更好地适应环境。
水环境作为耐药基因传播的媒介,其庞大的耐药基因库,为进入该环境中的致病菌及条件致病菌提供获得大量耐药基因的机会,一旦这些致病菌再次入侵人体,引起感染,其治疗将非常困难。
产ESBLs(Extended-Spectrumβ-Lactamases,超广谱β-内酰胺酶)菌在水环境中的出现应引起高度重视。ESBLs主要由肠杆菌科产生,大多为TEM-1、TEM-2、SHV-1的突变酶。其特点是能水解青霉素类、头孢菌素类及单环类抗生素,因其作用底物广泛被称作超广谱β-内酰胺酶。ESBLs主要由质粒介导,目前已成为介导革兰阴性杆菌对新型广谱β-内酰胺类抗生素耐药的主要原因。根据2005年CLSI规定,如果临床出现产ESBLs菌株,则对第三代头孢菌素(头孢噻肟、头孢他啶、头孢哌酮、头孢曲松等)耐药,同时对单环酰胺类抗生素(氨曲南)耐药。Sharma等在印度马德里的水体中检测出产ESBLs小肠结肠炎耶尔森菌; Cernat等也在水生环境中分离岀7株产ESBLs阴沟肠杆菌,且通过RAPD和PFGE将其分为两种亚型。本室刘小云前期分离岀的耐热性大肠菌群中产ESBLs初筛实验阳性率为18.75%,提示在国内水体中也存在产ESBLs的菌株。如果这些水环境中的产ESBLs菌株耐药基因能在致病菌和条件致病菌之间传播,人群一旦通过饮用或开放性伤口被水体中产ESBLs菌株感染,将导致治疗和控制上的极大困难。
目前为止,国内污水环境中的耐药菌生存情况及特点,产ESBLs菌在该环境中的流行情况,分子水平特点,不同水环境的生存规律以及各种因素对其生长的影响等情况还不清楚。
本课题第一部分以重庆市某医院污水污染水环境中的耐热性大肠菌群为研究对象,对污水排放后所造成的水环境中菌群耐药性变化规律进行了研究。第二部分对产ESBLs耐热性大肠菌群进行筛选,对其质粒、基因组、耐药基因及其转移进行研究。第三部分在实验室条件下研究产ESBLs菌在不同水体中的生长规律,对其生存末期质粒、基因组和耐药基因变化情况进行研究,观察在不同作用时间和有效氯含量产ESBLs菌对氯制剂的抵抗力。
实验方法
1.以医院污水排放口为参照,分别采集排放口上游100m、排放口、下游150m、下游300m水样,每个点平行采集水样3份,每份500ml。采用0.45μm孔径的滤膜进行样本的富集,滤膜置于m-FC培养基上37℃培养5h,44.5℃19h,将4个采样点的菌落通过滤膜法在m-FC培养基上进行计数。随机挑选上游100m10株,下游150m 20株,下游300m 20株进行纯化培养。于2005年8-11月期间从该医院患者标本中分离大肠杆菌40株(尿液23株、痰及咽拭子12株、分泌物5株),用APILabPlus微生物分析系统、API20E试剂盒进行菌种鉴定,选用氨苄西林、庆大霉素、丁胺卡那霉素、环丙沙星、氯霉素、头孢噻肟、头孢他啶、氨曲南、亚胺培南、头孢曲松、头孢泊肟等11种常用抗生素进行药敏实验(Kirby-Bauer法)。
2.根据CLSI2005标准筛选产ESBLs菌,采用质粒抽提盒和基因组抽提盒进行质粒和基因组的抽提。设计国内常见3种产ESBLs菌耐药基因引物(TEM、CTX-M、SHV)以11株产ESBLs菌的质粒为模板进行PCR,产物进行凝胶电泳。选用GC含量50-80%的随机引物对基因组进行RAPD(随机引物扩增)分析,筛选适宜的引物,将11株菌进行基因分型。以产ESBLs菌为供体菌(头孢噻肟抗性,m-FC培养基上呈现蓝色),非产ESBLs菌为受体菌(头孢噻肟敏感,m-FC培养基上呈现黄色),LB液为培养介质,含头孢噻肟60μg/ml的m-FC培养基作为鉴别培养基,进行耐药基因的转移实验。
3.选用33号菌作为产ESBLs菌的代表,设计5种水体观察其生存情况,5种水体依次为:灭菌河水、未灭菌河水、灭菌蒸馏水、灭菌自来水、未灭菌自来水。在加入菌后2h,6h,12h,24h,1.5天,2天,2.5天,3天,3.5天,以后每隔24h,一直到47天,滤膜法计数33号菌。至33号菌在各水体的生存末期,提取质粒和基因组,比较质粒条带变化,通过RAPD比较基因组的变化,同时进行33号菌固有的耐药基因PCR。利用正交设计助手Ⅱ3.1V软件进行3因素(温度、pH值、颗粒物)3水平(温度:15℃、25℃、35℃;pH值:6.7、7.2、7.5;颗粒物:1mg/100ml、2 mg/100ml、3 mg/100ml)设计,于1、2、4、6、8、10、15天滤膜法计数9种水样的细菌数。氯消毒法首先用简易定量滴定法和余氯测定计法对优氯净有效氯含量进行测定,按照消毒技术规范2002版规定,采用悬液定量鉴定实验进行杀菌实验。配制有效氯含量依次为0.4、0.8、1.2、2.4、3.2、4mg/L的溶液,加入33号菌液至终浓度为1×104-1×105CFU/ ml,于1、5、10、30、50min分别进行菌落计数(滤膜法)和游离氯含量测定(余氯测定计)。
实验结果
1.医院污水污染水环境中分离得到的50株耐热性大肠菌群中,大肠埃希菌为优势菌,占82%。医院污水排放后造成水环境中耐热性大肠菌群绝对数量由0.72×109/100ml增加至1.71×109/100ml(P<0.05),其百分率由42%增加到66%(P<0.05)。与排放口上游比较,其耐药性升高较为显著的为氨苄西林(P<0.01),环丙沙星、庆大霉素、氯霉素、氨曲南、头孢曲松、头孢噻肟(P<0.05),多重耐药率由0上升到50%。临床分离的大肠埃希菌与该水环境的耐热性大肠菌群对抗生素的耐药率相关性良好(r=0.943, P<0.01),前者耐药率高于后者。
2.该环境分离到的11株产ESBLs菌(22%)与非产ESBLs菌比较,环丙沙星、头孢噻肟、头孢曲松、头孢他啶、头孢泊肟的耐药率上显著升高(P<0.01)。11株产ESBLs菌有10株携带片断大小介于1,375bp到21,226bp之间的质粒,其耐药基因分型以TEM-1型为主(6株),CTX-M(2株)和SHV型(1株)为辅,33、34号菌株同时具有TEM型和CTX-M型,有4株菌属于这3种型以外的型别。GC含量为60%的随机引物能够将11株菌分为10种基因型。TEM耐药基因能够以接合的方式从产ESBLs大肠埃希菌转移到房峰哈夫尼菌,其转移率为8.21×10-4。
3. 33号菌在5中水体中生存时间由长到短依次为:灭菌河水>未灭菌河水>灭菌蒸馏水>灭菌自来水>未灭菌自来水,生存时间最短的为未灭菌自来水(3天),最长的为灭菌河水,在47天菌落数仍然维持在106CFU/ml左右。灭菌河水和未灭菌河水生长曲线包括4个时相点:适应期、对数生长期、平台期、衰亡期;灭菌蒸馏水、灭菌自来水和未灭菌自来水仅包括适应期和衰亡期。33号菌在各水体生存末期质粒、基因组及耐药基因特点为:未灭菌河水和灭菌蒸馏水中的33号菌质粒和基因组图谱与原代菌株一致,但灭菌河水、灭菌自来水和自来水中33号菌质粒图谱在2027bp和3000bp左右明显多出两条条带,在1200bp左右缺少1条条带,基因组图谱在1,000bp和低于500bp处出现1条条带,在600bp处缺少1条条带,33号菌所携带的TEM和CTX-M耐药基因在生存末期仍然稳定检岀饺蛩卣皇笛榻峁撼?1天pH值在三因素中对细菌生长影响最大以外,其余检测时间结果均为温度影响作用最大,颗粒物影响作用最小。游离氯(Y)和有效氯含量(X)的一元线性回归方程为:Y=-0.293+0.714X (R2=0.995,P=0.000<0.01);以杀菌率(Y)为因变量,有效氯含量(X1)和作用时间(X2)为自变量,建立多元线性回归方程Y=0.508+0.097X1+0.013X2 (R2=0.709, P=0.000<0.05)。根据公式推导,当有效氯含量为1.05mg/L,即游离氯量为0.46mg/L时,作用30min,对33号菌能够有效杀灭。按照现有的标准,出厂水所要求的游离氯含量(0.3mg/L)杀灭率为97.9%,对于管网水所出现的二次污染,管网末梢水中游离氯含量0.05mg/L更不足以满足杀菌要求(杀灭率为94.5%)。
结论
1.医院污水排放入水环境后造成耐热性大肠菌群绝对数量和相对数量的升高,其耐药谱具有以β-内酰胺酶抗性为主,多重耐药率高(50%)的特点;医院污水排放可能是造成该水环境中微生物耐药性增加的主要原因。
2.医院排放污水污染水环境中的产ESBLs菌比例较高(22%),具有多重耐药,耐药率及质粒携带率高的特点,耐药基因分型以TEM型为主;RAPD技术能够快速有效地对水环境中产ESBLs菌进行基因分型(11株菌分为10种亚型),并能运用于水体中耐药菌株的同源性分析。产ESBLs菌的耐药基因能够在不同菌之间发生转移。
3. 33号菌通过调节自身质粒和基因组水平的变化,能够在多种水体中生长繁殖,以河水最适,自来水最差。耐药基因在各水体的生长末期均非常稳定。温度对细菌生长影响最大,pH值其次,颗粒物影响最小。若该环境中的产ESBLs菌(104CFU/ml左右)污染管网水系统时,在有效氯含量为1.05mg/L,即游离氯量为0.46mg/L时,作用30min,对33号菌能够有效杀灭。按照现有的标准,出厂水所要求的游离氯含量(0.3mg/L)杀灭率为97.9%,对于管网水所出现的二次污染,游离氯含量0.05mg/L更不足以满足杀菌要求(杀灭率为94.5%),应适当延长作用时间或提高余氯含量。
问题与展望
本实验的研究仅针对一家医院的污水排放环境进行研究,选取的菌株较少(50株),且采样季节为冬天,产ESBLs菌的比例和基因分型结果对于研究总体样本的规律来说具有一定的局限性,扩大采样范围和耐热性大肠菌群的标本量,选择不同的时间点进行检测是下一步实验需要解决的问题。
按《生活饮用水卫生标准》出厂水消毒要求,最低游离氯含量为0.3mg/L时对以33号菌为代表的产ESBLs菌的杀灭率为97.9%,按管网末梢水标准,最低游离氯含量为0.05mg/L时对33号菌的杀灭率仅为94.7%,未达到完全杀灭标准,在作用时间为30min,要达到100%的杀菌效果,有效氯含量应达到1.05mg/L(游离氯含量为0.46mg/L)。结果提示目前的标准可能不适于水环境中的产ESBLs大肠埃希菌,由于实验菌株较少,尚需扩大标本量进行重复实验,对这一推测进行验证。
由于产ESBLs菌的耐药基因多携带于质粒上,运用于水环境中耐药菌质粒消除的有效方法至今未见报道,值得进一步开展实验进行研究。该研究的成功将能有效控制水环境中耐药基因的传播,为水环境中日益严重的耐药情况的控制提供解决途径。
鉴于产ESBLs耐药基因在实验室条件下的可转移性,其在人体的定殖和转移研究将具有非常重要的意义。
There has been growing concern about the highest resistance conditions of bacterium found in aquatic environment and antibiotic resistance strains existed in all manner of water environment such as sewge, mineral water, strainer water, drinking water and salt water.
Bacterium in nature environment had genetic factor such as plasmid, transposon and phage which could transfer between or across species by conjugation, transformation and transduction to adapt the environment. Plasmid and phage harbored integron which could recruit and express the antibiotic resistance gene.
The aquatic environment had become a major reservoir for antibiotic-resistant microbes. With global travel and widespread commerce, antibiotic-resistant microbes could spread to all parts of the world. Water-borne bacterial pathogens could lead to disease outbreaks that may have serious medical and economic implications. This problem was further serious by the increasing incidence of pathogens with antibiotic resistance.
We must pay more attention to the detection of ESBLs-producing strains in aquatic environment. When producing broad-spectrum plasmid-encoded enzymes, organisms became highly effective at inactivating penicillin, most cephalosporins, and aztreonam. Sharma detected ESBLs-producing Yersinia enterocolitica from the river of Indian Madrid. Cernat isolated 7 Aerobacter cloacae which produced extended spectrumβ-lactamases from aquatic environment and separated them into two subtype by RAPD and PFGE. Xiaoyun Liu of our department detected 18.75% ESBLs-producing bacterium in aquatic environment. It suggested that the ESBLs-producing bacterium had already existed in water environment of our country. Since the water environment could provide opportunity to ESBLs-producing bacterium to live and transfer the resistance gene, once human was infected by these resistance pathogenic microbes through drink or open wound, it would be very difficult to treatment and control.
Although previous researches displayed that the highest resistant rates were found in aquatic environment and the aquatic environment had become a major reservoir for antibiotic-resistant microbes, the ecological significance and importance of environmental gene transfer and survival of ESBLs-producing microorganisms have been relatively poorly understood. Knowledge about how antibiotic resistance arises, how resistant strains and resistance genes survive and spreading in water environment and the significance of this for humans and nature is far from complete. There are not enough data available to draw a final conclusion especially with respect to the input of already resistant bacterium into water environment.
Thermotolerant coliform isolated from this water environment contaminated by the hospital sewage was used to investigate antibiotic resistance condition in part 1. ESBLs-producing bacterium was screened and plasmid, genome, antibiotic resistant gene and gene transfer were studied in part 2. Survival and stability of plasmid and resistant gene of ESBLs-producing bacterium in different laboratory water environment, the influence sequence of environmental parameters such as temperature, pH value and particle and the impact of Kirbychlor disinfect method were investigated in part 3.
Methods
1. All water samples were collected from 4 different sites situated upstream 100 meters (site A), hospital effluent export (site B), downstream 150 meters (site C) and 300 meters (site D). Water samples were diluted and then filtered through 0.45 mm filter papers. Membrane filters were sticked to m-FC plates and then incubated for 5 hours at 37℃and 19 hours at 45°C. Isolates were identified with API Lab Plus system. Antibiotic susceptibility tests (AST) were performed with Kirby-Bauer disc diffusion method recommended by CLSI (2005). The antibiotics selected were commonly used in patients such as ampicillin, gentamicin, amikacin, ciprofloxacin, chlormycetin, rifampicin, cefotaxime, ceftazidime, aztreonam, imipenem, ceftriaxone and cefpodoxime. Escherichia coli ATCC25922 used as standard strain.
2. ESBLs-producing isolates were detected by the double disc test performed as a standardized disk diffusion assay. 3 primers were designed to detect the commonly antibiotic resistant gene. The plasmid DNA extraction was performed using E.Z.N.A Plasmid Mini KitⅠ(OMEGA) and products were used as templates for the amplification of various ESBLs genes. The plasmids exacted from the isolates and PCR products were all analyzed by gel electrophoresis. 12-mer random primers (CAA TCG CCG T GC) was used for RAPD. Mating experiments were performed on liquid media (Luria Bertani). The ESBLs-producing bacteria were selected to be donors (18, 33, 34, 37) and non-ESBLs -producing bacteria to be recipients (15, 20, 25, 36, 44). The acquisition of resistance by recipient strains was confirmed by colony color and antibiotic resistant difference between donors and recipients. Plasmid profiles were used to detect relocation of plasmid DNA from donor to recipient strains.
3. NO.33 strain was selected to represent ESBLs-producing bacteria to investigate the ability to survive in different water environment which included autoclaved river water (AR), untreated river water(R), autoclaved tap water (AT), untreated tap water (TW) and autoclaved distilled water (AD). The cefotaxime resistance phenotypes and blue color on m-FC agar characteristic of the strain was used as selective markers for selection of indigenous bacteria. The experiments were performed using low bacterial inoculums (105 CFU/ml) with the scope to reproduce the actual conditions. At the end phase of NO.33 suvived in different water environment, plasmid, genome and antibiotic resistant gene were analyzed. Orthogonal design software was used to detect the impact of 3 factors (temperature, pH value and particles) on bacterium survive in 3 levels. Chlorine sterization was operated according to“sterilize technological specification 2002”. Suspension quantitative germicidal test was applied.
Results
1. Escherichia coli was the prevalent species (82%). The others included Citrobacter freundii, Klebsiella pneumonia, Enterobacter cloacae, Serratia odorifera, Hafinia alvei and Morganella morganii. The quanlity of Thermotolerant coliforms increased from 0.72×109/100ml to 1.71×109/100ml(P<0.05)and percentage increased from 42% to 66%. The rate of multi-resistance was in increasing order for Ampicillin(P<0.01), Ciprofloxacin, Gentamicin, Chlormycetin, Aztreonam, Ceftriaxone, Cefotaxime (P<0.05). The rate of multi-resistant increased from 0 to 50%. When comparing the ARPs of the environmental isolates with local clinical isolates from the patients, the resistance patterns from both isolates were strongly correlated (r = 0.943, P<0.01).
2. Compared with non-ESBLs bacterium, the highest resistance rate in ESBLs-producing group (22%) are found for CIP, CTX, CRO and CAZ. All strains except NO.23 strain comprised 1-5 plasmids with molecular weights ranging from 1,375 to 21,226bp. TEM was the major type ofβ-lactamase among ESBLs-producing E. coli, followed by CTX-M and SHV group. 2 strains produced not only TEM type but also CTX-M type. The distribution of resistance gene type was coincidence with this of local hospital isolates. After primers screening and condition optimization, RAPD could be used to distinguish 11 ESBLs-producing strains into 10 types. In laboratory condition, gene transfer took place between E.coli and hafnia alvei by conjugation. The transfer rate was 8.21×10-4.
3. The survival time of strain in different water environment in decrease order was: AR, R, AD, AT, TW. Bacterium lived in R and AR environment displayed a characteristic four-phase pattern of growth curve while two-phase pattern in AT, T and AD. On the end phase of bacterium surviving, few changes were generated on plasmid and genome, but the resistant gene was still stability. Temperature impacted the growth of bacterium much more than pH value and particles. The multiple linear regression equation was Y=0.508+0.097X1+0.013X2(R2=0.709,P=0.000<0.05) (X1= effective chlorine concentration, X2= reaction time, Y=killing rate).
Conclusion
1. The quantity of Thermotolerant coliform in water environment increased significantly after being contaminated by hospital sewage.β-lactamase and multi-resistance (50%) were likely to be widely available in this area. Hospital sewage effluent might be the major reason causing the increase of antibiotic resistance in water environment.
2. The rate of ESBLs-producing bacterium was higher in this area. TEM type was the main resistant gene type of ESBL-producing bacteria. RAPD method could be employed to genotyping and homology analysis. Antibiotic resisance gene could be transferred between species by conjugation.
3. Strain NO.33 could survive in different water environment through accommodation of plasmid and genome. Even in the end phase of survive, antibiotic resistance gene was detected stable. The multiple linear regression equation was Y=0.508+0.097X1+0.013X2 (R2=0.709, P=0.000<0.05) (X1= effective chlorine concentration, X2= reaction time, Y=killing rate). When chlorine residual concentration was 0.05mg/L or 0.3mg/L and 30min reaction time, the killing rate was only 94.7% and 97.9% which was lower than 100% of“Drinking Water hygienic standard 2002”. If we want to get the 100% killing rate, the effective chlorine concentration must be 1.05mg/L (chlorine residual concentration 0.46mg/L) in tap water and 30min reaction time.
Question and Prospect
1. This research only focused on one water environment contaminated by hospital sewage and the quantity of bacterium was only 50. Otherwise the sampling time was only in winter. Those were limited to study the whole condition of ESBLs ratio and genotyping.
2.“Drinking Water hygienic standard 2002”recommended the basic level of chlorine residual concentration in water factory and tap water was 0.3mg/L and 0.05mg/L in 30min reaction time. Our results showed the killing rate was only 97.9% and 94.7% according to the rule. If we want to get the killing rate of 100%, the chlorine residual concentration must be 1.05mg/L (0.46mg/L). These means that the current standard maybe not suitable for ESBLs-producing bacterium in this area.
3. Results showed that most ESBLs-producing strains habored plasmids and antibiotic resistance gene commonly located on plasmid. We could control the antibiotic resistance gene transfer effectively if methods were found to eliminate plasmid.
自然界中各种细菌广泛存在,相互联系;细菌具有多种在种内或种间进行自主转移或诱动转移的遗传因子如质粒、转座子、噬菌体等;质粒、转座子上还有募集和表达外源耐药基因的整合子,通过接合、转化及转导等方式,细菌间相互间交换所携带的一些基因,可使菌群更好地适应环境。
水环境作为耐药基因传播的媒介,其庞大的耐药基因库,为进入该环境中的致病菌及条件致病菌提供获得大量耐药基因的机会,一旦这些致病菌再次入侵人体,引起感染,其治疗将非常困难。
产ESBLs(Extended-Spectrumβ-Lactamases,超广谱β-内酰胺酶)菌在水环境中的出现应引起高度重视。ESBLs主要由肠杆菌科产生,大多为TEM-1、TEM-2、SHV-1的突变酶。其特点是能水解青霉素类、头孢菌素类及单环类抗生素,因其作用底物广泛被称作超广谱β-内酰胺酶。ESBLs主要由质粒介导,目前已成为介导革兰阴性杆菌对新型广谱β-内酰胺类抗生素耐药的主要原因。根据2005年CLSI规定,如果临床出现产ESBLs菌株,则对第三代头孢菌素(头孢噻肟、头孢他啶、头孢哌酮、头孢曲松等)耐药,同时对单环酰胺类抗生素(氨曲南)耐药。Sharma等在印度马德里的水体中检测出产ESBLs小肠结肠炎耶尔森菌; Cernat等也在水生环境中分离岀7株产ESBLs阴沟肠杆菌,且通过RAPD和PFGE将其分为两种亚型。本室刘小云前期分离岀的耐热性大肠菌群中产ESBLs初筛实验阳性率为18.75%,提示在国内水体中也存在产ESBLs的菌株。如果这些水环境中的产ESBLs菌株耐药基因能在致病菌和条件致病菌之间传播,人群一旦通过饮用或开放性伤口被水体中产ESBLs菌株感染,将导致治疗和控制上的极大困难。
目前为止,国内污水环境中的耐药菌生存情况及特点,产ESBLs菌在该环境中的流行情况,分子水平特点,不同水环境的生存规律以及各种因素对其生长的影响等情况还不清楚。
本课题第一部分以重庆市某医院污水污染水环境中的耐热性大肠菌群为研究对象,对污水排放后所造成的水环境中菌群耐药性变化规律进行了研究。第二部分对产ESBLs耐热性大肠菌群进行筛选,对其质粒、基因组、耐药基因及其转移进行研究。第三部分在实验室条件下研究产ESBLs菌在不同水体中的生长规律,对其生存末期质粒、基因组和耐药基因变化情况进行研究,观察在不同作用时间和有效氯含量产ESBLs菌对氯制剂的抵抗力。
实验方法
1.以医院污水排放口为参照,分别采集排放口上游100m、排放口、下游150m、下游300m水样,每个点平行采集水样3份,每份500ml。采用0.45μm孔径的滤膜进行样本的富集,滤膜置于m-FC培养基上37℃培养5h,44.5℃19h,将4个采样点的菌落通过滤膜法在m-FC培养基上进行计数。随机挑选上游100m10株,下游150m 20株,下游300m 20株进行纯化培养。于2005年8-11月期间从该医院患者标本中分离大肠杆菌40株(尿液23株、痰及咽拭子12株、分泌物5株),用APILabPlus微生物分析系统、API20E试剂盒进行菌种鉴定,选用氨苄西林、庆大霉素、丁胺卡那霉素、环丙沙星、氯霉素、头孢噻肟、头孢他啶、氨曲南、亚胺培南、头孢曲松、头孢泊肟等11种常用抗生素进行药敏实验(Kirby-Bauer法)。
2.根据CLSI2005标准筛选产ESBLs菌,采用质粒抽提盒和基因组抽提盒进行质粒和基因组的抽提。设计国内常见3种产ESBLs菌耐药基因引物(TEM、CTX-M、SHV)以11株产ESBLs菌的质粒为模板进行PCR,产物进行凝胶电泳。选用GC含量50-80%的随机引物对基因组进行RAPD(随机引物扩增)分析,筛选适宜的引物,将11株菌进行基因分型。以产ESBLs菌为供体菌(头孢噻肟抗性,m-FC培养基上呈现蓝色),非产ESBLs菌为受体菌(头孢噻肟敏感,m-FC培养基上呈现黄色),LB液为培养介质,含头孢噻肟60μg/ml的m-FC培养基作为鉴别培养基,进行耐药基因的转移实验。
3.选用33号菌作为产ESBLs菌的代表,设计5种水体观察其生存情况,5种水体依次为:灭菌河水、未灭菌河水、灭菌蒸馏水、灭菌自来水、未灭菌自来水。在加入菌后2h,6h,12h,24h,1.5天,2天,2.5天,3天,3.5天,以后每隔24h,一直到47天,滤膜法计数33号菌。至33号菌在各水体的生存末期,提取质粒和基因组,比较质粒条带变化,通过RAPD比较基因组的变化,同时进行33号菌固有的耐药基因PCR。利用正交设计助手Ⅱ3.1V软件进行3因素(温度、pH值、颗粒物)3水平(温度:15℃、25℃、35℃;pH值:6.7、7.2、7.5;颗粒物:1mg/100ml、2 mg/100ml、3 mg/100ml)设计,于1、2、4、6、8、10、15天滤膜法计数9种水样的细菌数。氯消毒法首先用简易定量滴定法和余氯测定计法对优氯净有效氯含量进行测定,按照消毒技术规范2002版规定,采用悬液定量鉴定实验进行杀菌实验。配制有效氯含量依次为0.4、0.8、1.2、2.4、3.2、4mg/L的溶液,加入33号菌液至终浓度为1×104-1×105CFU/ ml,于1、5、10、30、50min分别进行菌落计数(滤膜法)和游离氯含量测定(余氯测定计)。
实验结果
1.医院污水污染水环境中分离得到的50株耐热性大肠菌群中,大肠埃希菌为优势菌,占82%。医院污水排放后造成水环境中耐热性大肠菌群绝对数量由0.72×109/100ml增加至1.71×109/100ml(P<0.05),其百分率由42%增加到66%(P<0.05)。与排放口上游比较,其耐药性升高较为显著的为氨苄西林(P<0.01),环丙沙星、庆大霉素、氯霉素、氨曲南、头孢曲松、头孢噻肟(P<0.05),多重耐药率由0上升到50%。临床分离的大肠埃希菌与该水环境的耐热性大肠菌群对抗生素的耐药率相关性良好(r=0.943, P<0.01),前者耐药率高于后者。
2.该环境分离到的11株产ESBLs菌(22%)与非产ESBLs菌比较,环丙沙星、头孢噻肟、头孢曲松、头孢他啶、头孢泊肟的耐药率上显著升高(P<0.01)。11株产ESBLs菌有10株携带片断大小介于1,375bp到21,226bp之间的质粒,其耐药基因分型以TEM-1型为主(6株),CTX-M(2株)和SHV型(1株)为辅,33、34号菌株同时具有TEM型和CTX-M型,有4株菌属于这3种型以外的型别。GC含量为60%的随机引物能够将11株菌分为10种基因型。TEM耐药基因能够以接合的方式从产ESBLs大肠埃希菌转移到房峰哈夫尼菌,其转移率为8.21×10-4。
3. 33号菌在5中水体中生存时间由长到短依次为:灭菌河水>未灭菌河水>灭菌蒸馏水>灭菌自来水>未灭菌自来水,生存时间最短的为未灭菌自来水(3天),最长的为灭菌河水,在47天菌落数仍然维持在106CFU/ml左右。灭菌河水和未灭菌河水生长曲线包括4个时相点:适应期、对数生长期、平台期、衰亡期;灭菌蒸馏水、灭菌自来水和未灭菌自来水仅包括适应期和衰亡期。33号菌在各水体生存末期质粒、基因组及耐药基因特点为:未灭菌河水和灭菌蒸馏水中的33号菌质粒和基因组图谱与原代菌株一致,但灭菌河水、灭菌自来水和自来水中33号菌质粒图谱在2027bp和3000bp左右明显多出两条条带,在1200bp左右缺少1条条带,基因组图谱在1,000bp和低于500bp处出现1条条带,在600bp处缺少1条条带,33号菌所携带的TEM和CTX-M耐药基因在生存末期仍然稳定检岀饺蛩卣皇笛榻峁撼?1天pH值在三因素中对细菌生长影响最大以外,其余检测时间结果均为温度影响作用最大,颗粒物影响作用最小。游离氯(Y)和有效氯含量(X)的一元线性回归方程为:Y=-0.293+0.714X (R2=0.995,P=0.000<0.01);以杀菌率(Y)为因变量,有效氯含量(X1)和作用时间(X2)为自变量,建立多元线性回归方程Y=0.508+0.097X1+0.013X2 (R2=0.709, P=0.000<0.05)。根据公式推导,当有效氯含量为1.05mg/L,即游离氯量为0.46mg/L时,作用30min,对33号菌能够有效杀灭。按照现有的标准,出厂水所要求的游离氯含量(0.3mg/L)杀灭率为97.9%,对于管网水所出现的二次污染,管网末梢水中游离氯含量0.05mg/L更不足以满足杀菌要求(杀灭率为94.5%)。
结论
1.医院污水排放入水环境后造成耐热性大肠菌群绝对数量和相对数量的升高,其耐药谱具有以β-内酰胺酶抗性为主,多重耐药率高(50%)的特点;医院污水排放可能是造成该水环境中微生物耐药性增加的主要原因。
2.医院排放污水污染水环境中的产ESBLs菌比例较高(22%),具有多重耐药,耐药率及质粒携带率高的特点,耐药基因分型以TEM型为主;RAPD技术能够快速有效地对水环境中产ESBLs菌进行基因分型(11株菌分为10种亚型),并能运用于水体中耐药菌株的同源性分析。产ESBLs菌的耐药基因能够在不同菌之间发生转移。
3. 33号菌通过调节自身质粒和基因组水平的变化,能够在多种水体中生长繁殖,以河水最适,自来水最差。耐药基因在各水体的生长末期均非常稳定。温度对细菌生长影响最大,pH值其次,颗粒物影响最小。若该环境中的产ESBLs菌(104CFU/ml左右)污染管网水系统时,在有效氯含量为1.05mg/L,即游离氯量为0.46mg/L时,作用30min,对33号菌能够有效杀灭。按照现有的标准,出厂水所要求的游离氯含量(0.3mg/L)杀灭率为97.9%,对于管网水所出现的二次污染,游离氯含量0.05mg/L更不足以满足杀菌要求(杀灭率为94.5%),应适当延长作用时间或提高余氯含量。
问题与展望
本实验的研究仅针对一家医院的污水排放环境进行研究,选取的菌株较少(50株),且采样季节为冬天,产ESBLs菌的比例和基因分型结果对于研究总体样本的规律来说具有一定的局限性,扩大采样范围和耐热性大肠菌群的标本量,选择不同的时间点进行检测是下一步实验需要解决的问题。
按《生活饮用水卫生标准》出厂水消毒要求,最低游离氯含量为0.3mg/L时对以33号菌为代表的产ESBLs菌的杀灭率为97.9%,按管网末梢水标准,最低游离氯含量为0.05mg/L时对33号菌的杀灭率仅为94.7%,未达到完全杀灭标准,在作用时间为30min,要达到100%的杀菌效果,有效氯含量应达到1.05mg/L(游离氯含量为0.46mg/L)。结果提示目前的标准可能不适于水环境中的产ESBLs大肠埃希菌,由于实验菌株较少,尚需扩大标本量进行重复实验,对这一推测进行验证。
由于产ESBLs菌的耐药基因多携带于质粒上,运用于水环境中耐药菌质粒消除的有效方法至今未见报道,值得进一步开展实验进行研究。该研究的成功将能有效控制水环境中耐药基因的传播,为水环境中日益严重的耐药情况的控制提供解决途径。
鉴于产ESBLs耐药基因在实验室条件下的可转移性,其在人体的定殖和转移研究将具有非常重要的意义。
There has been growing concern about the highest resistance conditions of bacterium found in aquatic environment and antibiotic resistance strains existed in all manner of water environment such as sewge, mineral water, strainer water, drinking water and salt water.
Bacterium in nature environment had genetic factor such as plasmid, transposon and phage which could transfer between or across species by conjugation, transformation and transduction to adapt the environment. Plasmid and phage harbored integron which could recruit and express the antibiotic resistance gene.
The aquatic environment had become a major reservoir for antibiotic-resistant microbes. With global travel and widespread commerce, antibiotic-resistant microbes could spread to all parts of the world. Water-borne bacterial pathogens could lead to disease outbreaks that may have serious medical and economic implications. This problem was further serious by the increasing incidence of pathogens with antibiotic resistance.
We must pay more attention to the detection of ESBLs-producing strains in aquatic environment. When producing broad-spectrum plasmid-encoded enzymes, organisms became highly effective at inactivating penicillin, most cephalosporins, and aztreonam. Sharma detected ESBLs-producing Yersinia enterocolitica from the river of Indian Madrid. Cernat isolated 7 Aerobacter cloacae which produced extended spectrumβ-lactamases from aquatic environment and separated them into two subtype by RAPD and PFGE. Xiaoyun Liu of our department detected 18.75% ESBLs-producing bacterium in aquatic environment. It suggested that the ESBLs-producing bacterium had already existed in water environment of our country. Since the water environment could provide opportunity to ESBLs-producing bacterium to live and transfer the resistance gene, once human was infected by these resistance pathogenic microbes through drink or open wound, it would be very difficult to treatment and control.
Although previous researches displayed that the highest resistant rates were found in aquatic environment and the aquatic environment had become a major reservoir for antibiotic-resistant microbes, the ecological significance and importance of environmental gene transfer and survival of ESBLs-producing microorganisms have been relatively poorly understood. Knowledge about how antibiotic resistance arises, how resistant strains and resistance genes survive and spreading in water environment and the significance of this for humans and nature is far from complete. There are not enough data available to draw a final conclusion especially with respect to the input of already resistant bacterium into water environment.
Thermotolerant coliform isolated from this water environment contaminated by the hospital sewage was used to investigate antibiotic resistance condition in part 1. ESBLs-producing bacterium was screened and plasmid, genome, antibiotic resistant gene and gene transfer were studied in part 2. Survival and stability of plasmid and resistant gene of ESBLs-producing bacterium in different laboratory water environment, the influence sequence of environmental parameters such as temperature, pH value and particle and the impact of Kirbychlor disinfect method were investigated in part 3.
Methods
1. All water samples were collected from 4 different sites situated upstream 100 meters (site A), hospital effluent export (site B), downstream 150 meters (site C) and 300 meters (site D). Water samples were diluted and then filtered through 0.45 mm filter papers. Membrane filters were sticked to m-FC plates and then incubated for 5 hours at 37℃and 19 hours at 45°C. Isolates were identified with API Lab Plus system. Antibiotic susceptibility tests (AST) were performed with Kirby-Bauer disc diffusion method recommended by CLSI (2005). The antibiotics selected were commonly used in patients such as ampicillin, gentamicin, amikacin, ciprofloxacin, chlormycetin, rifampicin, cefotaxime, ceftazidime, aztreonam, imipenem, ceftriaxone and cefpodoxime. Escherichia coli ATCC25922 used as standard strain.
2. ESBLs-producing isolates were detected by the double disc test performed as a standardized disk diffusion assay. 3 primers were designed to detect the commonly antibiotic resistant gene. The plasmid DNA extraction was performed using E.Z.N.A Plasmid Mini KitⅠ(OMEGA) and products were used as templates for the amplification of various ESBLs genes. The plasmids exacted from the isolates and PCR products were all analyzed by gel electrophoresis. 12-mer random primers (CAA TCG CCG T GC) was used for RAPD. Mating experiments were performed on liquid media (Luria Bertani). The ESBLs-producing bacteria were selected to be donors (18, 33, 34, 37) and non-ESBLs -producing bacteria to be recipients (15, 20, 25, 36, 44). The acquisition of resistance by recipient strains was confirmed by colony color and antibiotic resistant difference between donors and recipients. Plasmid profiles were used to detect relocation of plasmid DNA from donor to recipient strains.
3. NO.33 strain was selected to represent ESBLs-producing bacteria to investigate the ability to survive in different water environment which included autoclaved river water (AR), untreated river water(R), autoclaved tap water (AT), untreated tap water (TW) and autoclaved distilled water (AD). The cefotaxime resistance phenotypes and blue color on m-FC agar characteristic of the strain was used as selective markers for selection of indigenous bacteria. The experiments were performed using low bacterial inoculums (105 CFU/ml) with the scope to reproduce the actual conditions. At the end phase of NO.33 suvived in different water environment, plasmid, genome and antibiotic resistant gene were analyzed. Orthogonal design software was used to detect the impact of 3 factors (temperature, pH value and particles) on bacterium survive in 3 levels. Chlorine sterization was operated according to“sterilize technological specification 2002”. Suspension quantitative germicidal test was applied.
Results
1. Escherichia coli was the prevalent species (82%). The others included Citrobacter freundii, Klebsiella pneumonia, Enterobacter cloacae, Serratia odorifera, Hafinia alvei and Morganella morganii. The quanlity of Thermotolerant coliforms increased from 0.72×109/100ml to 1.71×109/100ml(P<0.05)and percentage increased from 42% to 66%. The rate of multi-resistance was in increasing order for Ampicillin(P<0.01), Ciprofloxacin, Gentamicin, Chlormycetin, Aztreonam, Ceftriaxone, Cefotaxime (P<0.05). The rate of multi-resistant increased from 0 to 50%. When comparing the ARPs of the environmental isolates with local clinical isolates from the patients, the resistance patterns from both isolates were strongly correlated (r = 0.943, P<0.01).
2. Compared with non-ESBLs bacterium, the highest resistance rate in ESBLs-producing group (22%) are found for CIP, CTX, CRO and CAZ. All strains except NO.23 strain comprised 1-5 plasmids with molecular weights ranging from 1,375 to 21,226bp. TEM was the major type ofβ-lactamase among ESBLs-producing E. coli, followed by CTX-M and SHV group. 2 strains produced not only TEM type but also CTX-M type. The distribution of resistance gene type was coincidence with this of local hospital isolates. After primers screening and condition optimization, RAPD could be used to distinguish 11 ESBLs-producing strains into 10 types. In laboratory condition, gene transfer took place between E.coli and hafnia alvei by conjugation. The transfer rate was 8.21×10-4.
3. The survival time of strain in different water environment in decrease order was: AR, R, AD, AT, TW. Bacterium lived in R and AR environment displayed a characteristic four-phase pattern of growth curve while two-phase pattern in AT, T and AD. On the end phase of bacterium surviving, few changes were generated on plasmid and genome, but the resistant gene was still stability. Temperature impacted the growth of bacterium much more than pH value and particles. The multiple linear regression equation was Y=0.508+0.097X1+0.013X2(R2=0.709,P=0.000<0.05) (X1= effective chlorine concentration, X2= reaction time, Y=killing rate).
Conclusion
1. The quantity of Thermotolerant coliform in water environment increased significantly after being contaminated by hospital sewage.β-lactamase and multi-resistance (50%) were likely to be widely available in this area. Hospital sewage effluent might be the major reason causing the increase of antibiotic resistance in water environment.
2. The rate of ESBLs-producing bacterium was higher in this area. TEM type was the main resistant gene type of ESBL-producing bacteria. RAPD method could be employed to genotyping and homology analysis. Antibiotic resisance gene could be transferred between species by conjugation.
3. Strain NO.33 could survive in different water environment through accommodation of plasmid and genome. Even in the end phase of survive, antibiotic resistance gene was detected stable. The multiple linear regression equation was Y=0.508+0.097X1+0.013X2 (R2=0.709, P=0.000<0.05) (X1= effective chlorine concentration, X2= reaction time, Y=killing rate). When chlorine residual concentration was 0.05mg/L or 0.3mg/L and 30min reaction time, the killing rate was only 94.7% and 97.9% which was lower than 100% of“Drinking Water hygienic standard 2002”. If we want to get the 100% killing rate, the effective chlorine concentration must be 1.05mg/L (chlorine residual concentration 0.46mg/L) in tap water and 30min reaction time.
Question and Prospect
1. This research only focused on one water environment contaminated by hospital sewage and the quantity of bacterium was only 50. Otherwise the sampling time was only in winter. Those were limited to study the whole condition of ESBLs ratio and genotyping.
2.“Drinking Water hygienic standard 2002”recommended the basic level of chlorine residual concentration in water factory and tap water was 0.3mg/L and 0.05mg/L in 30min reaction time. Our results showed the killing rate was only 97.9% and 94.7% according to the rule. If we want to get the killing rate of 100%, the chlorine residual concentration must be 1.05mg/L (0.46mg/L). These means that the current standard maybe not suitable for ESBLs-producing bacterium in this area.
3. Results showed that most ESBLs-producing strains habored plasmids and antibiotic resistance gene commonly located on plasmid. We could control the antibiotic resistance gene transfer effectively if methods were found to eliminate plasmid.
引文
1 Mazel D, Davies J. Antibiotic resistance in microbes. Cell Mol Life Sci, 1999,56 (9-10): 742-754.
2 Mudryk ZJ. Occurrence and distribution antibiotic resistance of heterotrophic bacteria isolated from a marine beach. Mar Pollut Bull, 2005, 50 (1): 80-86.
3 Ash RJ, Mauck B, Morgan M. Antibiotic resistance of gram-negative bacteria in rivers, United States. Emerg Infect Dis, 2002, 8 (7): 713-716.
4 Mulamattathil SG, Esterhuysen HA, Pretorius PJ. Antibiotic-resistant gram-negative bacteria in a virtually closed water reticulation system. J Appl Microbiol, 2000, 88 (6): 930-937.
5 刘小云,舒为群,李阳. 地表水中耐热大肠菌群对 10 种常用抗生素的耐药性研究. 解放军预防医学杂志, 2005,23(3):164-167.
6 Thomas Schwartz, Wolfgang Kohnen, Bernd Jansen. Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms. FEMS Microbiology Ecology, 2003, 43(3): 325-335.
7 Messi P, Guerrieri E, Bondi M. Antibiotic resistance and antibacterial activity in heterotrophic bacteria of mineral water origin. Sci Total Environ, 2005, 346 (1-3): 213-219.
8 Guardabassi L, Dalsgaard A, Olsen JE. Phenotypic characterization and antibiotic resistance of Acinetobacter spp. isolated from aquatic sources. J Appl Microbiol, 1999, 87(5): 659-667.
9 Lin J, Biyela PT and Puckree T. Antibiotic resistance profiles of environmental isolates from Mhlathuze River, KwaZulu-Natal (RSA). Water SA, 2004,30(1):23-28.
10 Davison J. Genetic exchange between bacteria in the environment. Plasmid, 1999, 42(2):73-91.
11 Park JC, Lee JC, Oh JY, et al. Antibiotic selective pressure for the maintenance of antibiotic resistant genes in coliform bacteria isolated from the aquatic environment. Water Sci Technol, 2003, 47(3): 249-253.
12 Lin J, Biyela PT. Convergent acquisition of antibiotic resistance determinants amongst the Enterobacteriaceae isolates of the Mhlathuze River, KwaZulu-Natal (RSA). WaterSA, 2005, 31(2): 257-260.
13 Guardabassi L, Lo Fo Wong DM, Dalsgaard A. The effects of tertiary wastewater treatment on the prevalence of antimicrobial resistant bacteria. Water Res, 2002, 36(8): 1955-64.
14 Biyela PT, Lin J, Bezuidenhout CC. The role of aquatic ecosystems as reservoirs of antibiotic resistant bacteria and antibiotic resistance genes. Water Sci Technol, 2004, 50 (1):45-50.
15 D'Costa VM, McGrann KM, Hughes DW, et al. Sampling the antibiotic resistome. Science, 2006, 311(5759): 374-377.
16 Gootz TD. The forgotten Gram-negative bacilli: what genetic determinants are telling us about the spread of antibiotic resistance. Biochem Pharmacol, 2006, 71(7):1073-84.
17 Standard methods for the examination of water and wastewater 20th ed. Washington D.C, 1999: 9221-9225.
18 15th Information, Supplement. Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Susceptibility Testing, 2005. Document M100-S15.
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2. 刘小云,舒为群,李阳。地表水中耐热大肠菌群对 10 种常用抗生素的耐药性研究。解放军预防医学杂志, 2005, 23(3):164-167。
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4. Ash RJ, Mauck B, Morgan M. Antibiotic resistance of gram-negative bacteria in rivers, United States [J]. Emerging Infect Dis, 2002, 8 (7): 713-716.
5. Thomas Schwartz, Wolfgang Kohnen, Bernd Jansen, et al. Detection of antibiotic -resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms [J]. FEMS Microbiology Ecology, 2003, 43(3): 325-335.
6. Mulamattathil SG., Esterhuysen HA, Pretorius P.J. Antibiotic-resistant Gram-negative bacteria in a virtually closed water reticulation system [J]. Journal of Applied Microbiology, 2000, 88(6): 930-937.
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9. Mudryk Z J. Occurrence and distribution antibiotic resistance of heterotrophic bacteria isolated from a marine beach [J]. Mar Pollut Bull, 2005, 50(1): 80-86.
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11. John Davison1. Genetic Exchange between Bacteria in the Environment [J]. Plasmid, 1999, 42(2); 73-91.
12. Akinbowale OL, Peng H, Barton MD. Antimicrobial resistance in bacteria isolated from aquaculture sources in Australia [J]. J-Appl-Microbiol, 2006, 100(5): 1103-13.
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2 Mudryk ZJ. Occurrence and distribution antibiotic resistance of heterotrophic bacteria isolated from a marine beach. Mar Pollut Bull, 2005, 50 (1): 80-86.
3 Ash RJ, Mauck B, Morgan M. Antibiotic resistance of gram-negative bacteria in rivers, United States. Emerg Infect Dis, 2002, 8 (7): 713-716.
4 Mulamattathil SG, Esterhuysen HA, Pretorius PJ. Antibiotic-resistant gram-negative bacteria in a virtually closed water reticulation system. J Appl Microbiol, 2000, 88 (6): 930-937.
5 刘小云,舒为群,李阳. 地表水中耐热大肠菌群对 10 种常用抗生素的耐药性研究. 解放军预防医学杂志, 2005,23(3):164-167.
6 Thomas Schwartz, Wolfgang Kohnen, Bernd Jansen. Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms. FEMS Microbiology Ecology, 2003, 43(3): 325-335.
7 Messi P, Guerrieri E, Bondi M. Antibiotic resistance and antibacterial activity in heterotrophic bacteria of mineral water origin. Sci Total Environ, 2005, 346 (1-3): 213-219.
8 Guardabassi L, Dalsgaard A, Olsen JE. Phenotypic characterization and antibiotic resistance of Acinetobacter spp. isolated from aquatic sources. J Appl Microbiol, 1999, 87(5): 659-667.
9 Lin J, Biyela PT and Puckree T. Antibiotic resistance profiles of environmental isolates from Mhlathuze River, KwaZulu-Natal (RSA). Water SA, 2004,30(1):23-28.
10 Davison J. Genetic exchange between bacteria in the environment. Plasmid, 1999, 42(2):73-91.
11 Park JC, Lee JC, Oh JY, et al. Antibiotic selective pressure for the maintenance of antibiotic resistant genes in coliform bacteria isolated from the aquatic environment. Water Sci Technol, 2003, 47(3): 249-253.
12 Lin J, Biyela PT. Convergent acquisition of antibiotic resistance determinants amongst the Enterobacteriaceae isolates of the Mhlathuze River, KwaZulu-Natal (RSA). WaterSA, 2005, 31(2): 257-260.
13 Guardabassi L, Lo Fo Wong DM, Dalsgaard A. The effects of tertiary wastewater treatment on the prevalence of antimicrobial resistant bacteria. Water Res, 2002, 36(8): 1955-64.
14 Biyela PT, Lin J, Bezuidenhout CC. The role of aquatic ecosystems as reservoirs of antibiotic resistant bacteria and antibiotic resistance genes. Water Sci Technol, 2004, 50 (1):45-50.
15 D'Costa VM, McGrann KM, Hughes DW, et al. Sampling the antibiotic resistome. Science, 2006, 311(5759): 374-377.
16 Gootz TD. The forgotten Gram-negative bacilli: what genetic determinants are telling us about the spread of antibiotic resistance. Biochem Pharmacol, 2006, 71(7):1073-84.
17 Standard methods for the examination of water and wastewater 20th ed. Washington D.C, 1999: 9221-9225.
18 15th Information, Supplement. Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Susceptibility Testing, 2005. Document M100-S15.
19 Brown KD, Kulis J, Thomson B. Occurrence of antibiotics in hospital, residential, and dairy effluent, municipal wastewater, and the Rio Grande in New Mexico. Science of the Total Environment, 2006, 366 (2-3): 772-783.
20 Kummerer K, Henninger A. Promoting resistance by the emission of antibiotics from hospitals and households into effluent. Clin Microbiol Infect, 2003, 9 (12): 1203-1214.
21 Novais C, Coque TM, Ferreira H, et al. Environmental contamination with vancomycin-resistant enterococci from hospital sewage in Portugal. Appl Environ Microbiol, 2005, 71(6): 3364-3368.
22 孙景勇,倪语星. CTX-M-3 超广谱-和 TEM-1 广谱β内酰胺酶基因同时存在于大肠埃希菌. 中华微生物和免疫学杂志, 2001,12(增刊):75-77.
23 陈民钧,徐英春,吴伟元. 一种新 CTX-M 型超广谱 β-内酰胺酶及其临床意义. 中华检验医学杂志, 2001, 24(4): 207-210.
24 魏泽庆,陈亚岗,俞云松. 多重耐药肺炎克雷伯菌分子流行病学及 β 内酰胺酶基因型研究. 中华检验医学杂志, 2002, 25(6): 329-333.
25 Chanawong A, M'Zali FH, Heritage J, et al. Three cefotaximases, CTX-M-9,CTX-M-13, and CTX-M-14, among Enterobacteriaceae in the People's Republic of China. Antimicrob Agents Chemother, 2002, 46(3): 630-637.
26 Yu WL, Winokur PL, Von Stein DL, et al. First description of Klebsiella pneumoniae harboring CTX-M beta-lactamases (CTX-M-14 and CTX-M-3) in Taiwan. Antimicrob Agents Chemother, 2002, 46(4): 1098-1100.
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