食源性革兰氏阴性肠道病原菌PFGE分型和大肠杆菌耐药性研究
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摘要
大肠杆菌、沙门氏菌和空肠弯曲杆菌是目前世界范围内报到最多、引起食源性疾病暴发事件最多的3种致病性革兰氏阴性肠道菌。对暴发病原菌的快速确定和溯源是防止疫情恶化、减少对人们身体和经济带来更大危害的关键。脉冲场电泳(PFGE)分子分型技术由于其高分辨力、实验室间的高度可重复性和易于分析并通过网络进行对比,被誉为病原菌分型的“黄金方法”。越来越严重的病原菌耐药问题,对临床上相关感染的治疗带来极大挑战,成为目前困扰医学的一大难题。肠道菌群因为密度比较大,更容易进行耐药基因的水平传递,作为粪便污染指示菌的大肠杆菌更是被认为是肠道致病菌的耐药基因库。对其耐药性现状、耐药性产生和发展、耐药性转移和相关影响因素的研究,对各种肠道病原菌的多重耐药性的控制、临床治疗具有重要意义。本研究对陕西西安和杨凌地区的食源性大肠杆菌进行分离、鉴定、毒力因子、整合子和药敏性研究,并分别利用6种限制性内切酶对大肠杆菌O157:H7、空肠弯曲杆菌和四个血清型的沙门氏菌进行了PFGE分子分型研究,所得主要结果如下:
1、陕西西安和杨凌地区各种原料肉中,大肠杆菌污染100%存在,其污染程度跟产品种类无关,跟产品来源有一定相关性。凉拌菜样品大肠杆菌分离率86.7%,调味品可能是抑制大肠杆菌在凉拌菜中繁殖的主要原因。在毒力因子检测中,粘附性毒素主要表达基因fimA基因阳性率为56.1%(273株)。通过对STXI、STXII、ST、LT等毒素表达基因的检测,发现产志贺样毒素大肠杆菌阳性率为0.62%,产肠毒素大肠杆菌阳性率是14.4%。
2、陕西食源性大肠杆菌对四环素的耐药率最高,占受试菌株的99.3%(551);耐受率均超过50%的抗生素依次还有链霉素(65.6%)、阿莫西林(61.3%)、萘啶酮酸(57.7%)、氨苄青霉素(55.1%),其余依次是环丙沙星(38.7%)、氯霉素(38.0%)、卡那霉素(34.6%)、庆大霉素(30.8%)、头孢哌酮(23.4%)、头孢西丁(12.4%);试验菌株对阿米卡星最为敏感,有8.8%耐药菌株产生。
3、575株受试菌中,发现2.1%(12)的菌株对12种抗生素全部敏感,其余563(97.9%)株大肠杆菌至少对一种抗生素有耐药性。多重耐药(≥3)率为62.3%(358),37.0%的株菌对7种以上抗生素耐受。多重耐药菌存在最多的是9重耐药菌株,有58株菌, 6重和10重耐药菌次之,均都有49株,其余依次是8(43)、3(35)、4(34)、7(31)、5(27)、11(25)、12(7)。表明多重耐药现象在陕西食源性大肠杆菌中比较严重。
4、大肠杆菌菌株来源不同则耐药性存在很大差异,鸡肉分离株的耐药性和多重耐药率明显高于其它分离株。304株鸡肉分离株100%的耐药,多重耐药(≥3)率为85.5%(260),以9重耐药菌株最多(56),其次是10(47)、8(40)、7(26)、11(25)、6(21)、4(14)、5(14)、3(11)、12(7)重耐药株。对7种以上抗生素耐受率达66.1%(201)。说明陕西鸡肉分离大肠杆菌的耐药性非常严重。
5、118株肉样品分离菌株中,49.1%的菌株携带有750～2000bp的I类整合子。其中鸡肉分离株的整合子率为55.1%。多重耐药菌株(≥3)中,整合子阳性率为59.1%,其中鸡肉多重耐药菌株的整合子阳性率为59.5%,其它肉多重耐药菌株整合子阳性率为55.6%。非多重耐药菌中,I类整合子检出率仅16.7%。在耐药重数≥7的菌株中,66.7%的菌株I类整合子呈阳性。整合子存在与大肠杆菌耐药性之间有一定的相关性。
6、58株携带整合子的菌株,携带约2.0kbI类整合子的菌最多,有29株,有19株菌在750bp左右有扩增带,在1.5kb左右有扩增带的菌12株,其中1株有750bp和1.5kb两条带,2株在750bp和2.0kb均有扩增。
7、在对大肠杆菌O157:H7单酶PFGE分型中,以SpeI效果最好,将43株试验菌株切割后,产生21～32条有效带,产生基因型22个。BlnI/SpeI/PacI3酶结合和XbaI/BlnI/NheI/SpiI/SpeI/PacI 6种酶结合分析做树状图,所得基因型相、束平均数、束菌株百分比、结与菌株比值和SID指数等5个参数完全相同,分别是26、4.4、52%、0.67和0.902。
8、对4个血清型的沙门氏菌S. Heidelberg、S.Kentucky、S.SaintPaul和S.Hadar进行6种酶PFGE分子分型,分型效果最好的酶各不相同,分别是PacI、SpeI、SpiI、NotI;最佳3酶组合分别依次是:SpeI/PacI/BlnI、SpeI/NotI/SfiI、SpeI/BlnI/SfiI和SpeI/NotI/SfiI。6种酶结合使用,血清型不同则产生的分型能力不同。3酶结合就可以达到很好的分型效果。
9、限制性内切酶BamHI是对空场弯曲杆菌PFGE分子分型效果最好的内切酶。对空肠弯曲杆菌,要获得更好的溯源和分型效果,PFGE应该与其它方法结合使用才能达到预期结果。
10、综合分析PFGE分子分型结果,发现与大肠杆菌和沙门氏菌分型结果相比较,PFGE对空肠弯曲杆菌的分型能力要差一些。
Escherichia coli (E.coli) ,Salmonella and Campylobacter jejuni are three major food-borne pathogens which were most frequently reported to cause disease outbreak worldwide. The identification and tracing the source of the pathogens are the keys for preventing deterioration of the epidemic, and economic loss. Because of its high discrimination ability and repeatability, simplicity of analyzing the result and comparing through internet, Pulsed-field gel electrophoresis (PFGE) was named the“gold method”for subtyping pathogens. Multidrug-resistant pathogens are being isolated at an increasing rate in hospital settings and are having a significant impact on clinical practice and overall treatment costs.
Because of the high concentration, it is easy to acquire the resistance genes by horizontal transfer between Enterobacteriaceae. As the indicator bacteria of dejecta contamination, E.coli is regarded as the reservoir of resistance genes. Study on the antibiotic resistance, the antibiotic resistance emergence, the resistance genes transfers and related influencing factors of E.coli have significant impact on controlling the producing of multidrug-resistance pathogens, and clinical practice. We studied the prevalence of E.coli among meats and Chinese salads, the virulence genes, class I integron and antimicrobial susceptibility in these E.coli, and subtyping 43 E.coli O157:H7, 4 serotypes salmonella(114) and 43 Campylobacter jejuni with 6 enzymes PFGE. The main results as following:
1、575 E.coli were isolated from meats (100%) and Chinese salads (86.7%) sampled from Xi’an and Yangling in Shannxi Province. The detection rate of E.coli was unrelated with the species of meat, but resources. The flavorings maybe the main reason in inhibitting E.coli in Chinese salads. The detection rate of fimA genes encoding adhesion toxin was 56.1%(273). Furthermore, we detected the stxI、stxII、st and lt genes encoding intestinotoxin. The detection rate of STEC was 0.62%, and the detection rate of ETEC was 14.4%. This means that the food safety in Xi’an and Yingling is potentially at risk.
2、575 food-borne E.coli isolates were highly resistant to Tetracycline (99.3%), Streptomycin(65.6%), Amoxicillin(61.3%), Nalidixic acid(57.7%), Ampicilllin(55.1%), whose antibiotic resistance rate were above 50%. The others’ordering were Ciprofloxacin (38.7%), Chloramphenicol(38.0%), Kanamycin(34.6%), Gentamicin(30.8%), Cefoperazone(23.4%), Cefoxitin(12.4%). Only 8.8% isolates were resistance to Amikacin.
3、In 575 food-borne E.coli,2.1%(12)isolates were susceptible to all 12 antibiotics used in the study. 563(97.9%)isolates were resistant to one agent at least. Multidrug resistance (≥3)rate was 62.3%(358),and 37.0% E.coli were resistant to 7 or more agents. In 358 multidrug resistance isolates(≥3), 58 were resistant to 9 agents, isolates resistant to 6,10,8,3,4,7,5,11,7 agents were 49,49,43,35,34,31,27,25,7, respectively. All data indicates that the multidrug resistance food-borne E.coli are prevalent in Shaanxi province。
4、304 isolates from Chicken displayed 100% resistance,85.5%(260)isolates resistant to 3 or more antibiotics. 7 isolates with the highest level of antibiotic resistance were resistant to all 12 agents used in the study. 56 isolates were resistant to 9 agents. Isolates resistant to 10,8,7,11,6,4,5,3 antibiotics were 47,40,26,25,21,14,14,11, respectively. 66.1%(201)isolates from chicken were resistant to 7 or more agents. Isolates from different resources had different antimicrobial susceptibility. The isolates from chicken displayed higher resistance level than isolates from other resources.
5、49.1% isolates were PCR positive for class I integron among 118 meat E.coli isolates. 55.1% chicken isolates were PCR positive for class I integron. 59.1% isolates were PCR positive for class I integron among multidrug resistance(≥3) isolates. Isolates resistant to 1 or 2 agents only displayed 16.7% PCR positive for class I integron. Among the isolates resistant to more than 7 agents, 66.7% isolate were PCR positive for class I integron. It demonstrates that antibiotic resistance is associated with class I integron in E.coli.
6、29 isolates had 2.0kb class I integron among 58 class I integrons PCR positive isolates . 19 isolates were 750bp class I integron positive. 12 isolates were 1.5kb class I integron positive. One isolates displayed PCR positive for both 750bp and 1.5kb class I integrons,and two isolates displayed PCR positive for 750bp and 2.0kb class I integron.
7、Enzyme SpeI was most suitable for subtyping STEC O157:H7. There were 21 to 32 bands appeared in the PFGE gel. 43 O157:H7 were divided into 22 subtypes. The dendrograms that resulted from three enzyme combination BlnI/SpeI/PacI and six enzyme combination XbaI/BlnI/NheI/SpiI/SpeI/PacI showed same discrimination, produced 26 subtypes, yielded an average 4.4 isolates per unresolved cluster, 52% isolates unresolved, a node-to-isolates ratio of 0.67, and a SID of 0.902.
8、For 4 serotypes Salmonella -S. Heidelberg, S.Kentucky, S.SaintPaul and S.Hadar, 6 enzymes subtyping results were different in difference serotypes. The enzyme which was most suitable for PFGE subtyping were PacI, SpeI, SpiI and NotI for S. Heidelberg, S.Kentucky, S.SaintPaul and S.Hadar, respectively. For S. Heidelberg, S.Kentucky, S.SaintPaul and S.Hadar, the most suitable three enzyme combination were SpeI/PacI/BlnI, SpeI/NotI/SfiI, SpeI/BlnI/SfiI和SpeI/NotI/SfiI, respectively. 6 enzyme combination PFGE yielded different discrimination for 4 Salmonella serotypes. The simultaneous analysis of 3 enzyme combination proved to be a beneficial scheme for the PFGE-based differentiation of closely related isolates of the four Salmonella serotypes.
9、BamHI was the most suitable Enzyme for Campylobacter jejuni in PFGE subtyping. In order to get higher discrimination ability for subtyping Campylobacter jejuni , PFGE should be used with other methods together.
10、Analyzing all data of PFGE subtyping, the discrimination ability of PFGE subtyping for STEC O157:H7 and Salmonella was better than Campylobacter jejuni.
1、陕西西安和杨凌地区各种原料肉中,大肠杆菌污染100%存在,其污染程度跟产品种类无关,跟产品来源有一定相关性。凉拌菜样品大肠杆菌分离率86.7%,调味品可能是抑制大肠杆菌在凉拌菜中繁殖的主要原因。在毒力因子检测中,粘附性毒素主要表达基因fimA基因阳性率为56.1%(273株)。通过对STXI、STXII、ST、LT等毒素表达基因的检测,发现产志贺样毒素大肠杆菌阳性率为0.62%,产肠毒素大肠杆菌阳性率是14.4%。
2、陕西食源性大肠杆菌对四环素的耐药率最高,占受试菌株的99.3%(551);耐受率均超过50%的抗生素依次还有链霉素(65.6%)、阿莫西林(61.3%)、萘啶酮酸(57.7%)、氨苄青霉素(55.1%),其余依次是环丙沙星(38.7%)、氯霉素(38.0%)、卡那霉素(34.6%)、庆大霉素(30.8%)、头孢哌酮(23.4%)、头孢西丁(12.4%);试验菌株对阿米卡星最为敏感,有8.8%耐药菌株产生。
3、575株受试菌中,发现2.1%(12)的菌株对12种抗生素全部敏感,其余563(97.9%)株大肠杆菌至少对一种抗生素有耐药性。多重耐药(≥3)率为62.3%(358),37.0%的株菌对7种以上抗生素耐受。多重耐药菌存在最多的是9重耐药菌株,有58株菌, 6重和10重耐药菌次之,均都有49株,其余依次是8(43)、3(35)、4(34)、7(31)、5(27)、11(25)、12(7)。表明多重耐药现象在陕西食源性大肠杆菌中比较严重。
4、大肠杆菌菌株来源不同则耐药性存在很大差异,鸡肉分离株的耐药性和多重耐药率明显高于其它分离株。304株鸡肉分离株100%的耐药,多重耐药(≥3)率为85.5%(260),以9重耐药菌株最多(56),其次是10(47)、8(40)、7(26)、11(25)、6(21)、4(14)、5(14)、3(11)、12(7)重耐药株。对7种以上抗生素耐受率达66.1%(201)。说明陕西鸡肉分离大肠杆菌的耐药性非常严重。
5、118株肉样品分离菌株中,49.1%的菌株携带有750～2000bp的I类整合子。其中鸡肉分离株的整合子率为55.1%。多重耐药菌株(≥3)中,整合子阳性率为59.1%,其中鸡肉多重耐药菌株的整合子阳性率为59.5%,其它肉多重耐药菌株整合子阳性率为55.6%。非多重耐药菌中,I类整合子检出率仅16.7%。在耐药重数≥7的菌株中,66.7%的菌株I类整合子呈阳性。整合子存在与大肠杆菌耐药性之间有一定的相关性。
6、58株携带整合子的菌株,携带约2.0kbI类整合子的菌最多,有29株,有19株菌在750bp左右有扩增带,在1.5kb左右有扩增带的菌12株,其中1株有750bp和1.5kb两条带,2株在750bp和2.0kb均有扩增。
7、在对大肠杆菌O157:H7单酶PFGE分型中,以SpeI效果最好,将43株试验菌株切割后,产生21～32条有效带,产生基因型22个。BlnI/SpeI/PacI3酶结合和XbaI/BlnI/NheI/SpiI/SpeI/PacI 6种酶结合分析做树状图,所得基因型相、束平均数、束菌株百分比、结与菌株比值和SID指数等5个参数完全相同,分别是26、4.4、52%、0.67和0.902。
8、对4个血清型的沙门氏菌S. Heidelberg、S.Kentucky、S.SaintPaul和S.Hadar进行6种酶PFGE分子分型,分型效果最好的酶各不相同,分别是PacI、SpeI、SpiI、NotI;最佳3酶组合分别依次是:SpeI/PacI/BlnI、SpeI/NotI/SfiI、SpeI/BlnI/SfiI和SpeI/NotI/SfiI。6种酶结合使用,血清型不同则产生的分型能力不同。3酶结合就可以达到很好的分型效果。
9、限制性内切酶BamHI是对空场弯曲杆菌PFGE分子分型效果最好的内切酶。对空肠弯曲杆菌,要获得更好的溯源和分型效果,PFGE应该与其它方法结合使用才能达到预期结果。
10、综合分析PFGE分子分型结果,发现与大肠杆菌和沙门氏菌分型结果相比较,PFGE对空肠弯曲杆菌的分型能力要差一些。
Escherichia coli (E.coli) ,Salmonella and Campylobacter jejuni are three major food-borne pathogens which were most frequently reported to cause disease outbreak worldwide. The identification and tracing the source of the pathogens are the keys for preventing deterioration of the epidemic, and economic loss. Because of its high discrimination ability and repeatability, simplicity of analyzing the result and comparing through internet, Pulsed-field gel electrophoresis (PFGE) was named the“gold method”for subtyping pathogens. Multidrug-resistant pathogens are being isolated at an increasing rate in hospital settings and are having a significant impact on clinical practice and overall treatment costs.
Because of the high concentration, it is easy to acquire the resistance genes by horizontal transfer between Enterobacteriaceae. As the indicator bacteria of dejecta contamination, E.coli is regarded as the reservoir of resistance genes. Study on the antibiotic resistance, the antibiotic resistance emergence, the resistance genes transfers and related influencing factors of E.coli have significant impact on controlling the producing of multidrug-resistance pathogens, and clinical practice. We studied the prevalence of E.coli among meats and Chinese salads, the virulence genes, class I integron and antimicrobial susceptibility in these E.coli, and subtyping 43 E.coli O157:H7, 4 serotypes salmonella(114) and 43 Campylobacter jejuni with 6 enzymes PFGE. The main results as following:
1、575 E.coli were isolated from meats (100%) and Chinese salads (86.7%) sampled from Xi’an and Yangling in Shannxi Province. The detection rate of E.coli was unrelated with the species of meat, but resources. The flavorings maybe the main reason in inhibitting E.coli in Chinese salads. The detection rate of fimA genes encoding adhesion toxin was 56.1%(273). Furthermore, we detected the stxI、stxII、st and lt genes encoding intestinotoxin. The detection rate of STEC was 0.62%, and the detection rate of ETEC was 14.4%. This means that the food safety in Xi’an and Yingling is potentially at risk.
2、575 food-borne E.coli isolates were highly resistant to Tetracycline (99.3%), Streptomycin(65.6%), Amoxicillin(61.3%), Nalidixic acid(57.7%), Ampicilllin(55.1%), whose antibiotic resistance rate were above 50%. The others’ordering were Ciprofloxacin (38.7%), Chloramphenicol(38.0%), Kanamycin(34.6%), Gentamicin(30.8%), Cefoperazone(23.4%), Cefoxitin(12.4%). Only 8.8% isolates were resistance to Amikacin.
3、In 575 food-borne E.coli,2.1%(12)isolates were susceptible to all 12 antibiotics used in the study. 563(97.9%)isolates were resistant to one agent at least. Multidrug resistance (≥3)rate was 62.3%(358),and 37.0% E.coli were resistant to 7 or more agents. In 358 multidrug resistance isolates(≥3), 58 were resistant to 9 agents, isolates resistant to 6,10,8,3,4,7,5,11,7 agents were 49,49,43,35,34,31,27,25,7, respectively. All data indicates that the multidrug resistance food-borne E.coli are prevalent in Shaanxi province。
4、304 isolates from Chicken displayed 100% resistance,85.5%(260)isolates resistant to 3 or more antibiotics. 7 isolates with the highest level of antibiotic resistance were resistant to all 12 agents used in the study. 56 isolates were resistant to 9 agents. Isolates resistant to 10,8,7,11,6,4,5,3 antibiotics were 47,40,26,25,21,14,14,11, respectively. 66.1%(201)isolates from chicken were resistant to 7 or more agents. Isolates from different resources had different antimicrobial susceptibility. The isolates from chicken displayed higher resistance level than isolates from other resources.
5、49.1% isolates were PCR positive for class I integron among 118 meat E.coli isolates. 55.1% chicken isolates were PCR positive for class I integron. 59.1% isolates were PCR positive for class I integron among multidrug resistance(≥3) isolates. Isolates resistant to 1 or 2 agents only displayed 16.7% PCR positive for class I integron. Among the isolates resistant to more than 7 agents, 66.7% isolate were PCR positive for class I integron. It demonstrates that antibiotic resistance is associated with class I integron in E.coli.
6、29 isolates had 2.0kb class I integron among 58 class I integrons PCR positive isolates . 19 isolates were 750bp class I integron positive. 12 isolates were 1.5kb class I integron positive. One isolates displayed PCR positive for both 750bp and 1.5kb class I integrons,and two isolates displayed PCR positive for 750bp and 2.0kb class I integron.
7、Enzyme SpeI was most suitable for subtyping STEC O157:H7. There were 21 to 32 bands appeared in the PFGE gel. 43 O157:H7 were divided into 22 subtypes. The dendrograms that resulted from three enzyme combination BlnI/SpeI/PacI and six enzyme combination XbaI/BlnI/NheI/SpiI/SpeI/PacI showed same discrimination, produced 26 subtypes, yielded an average 4.4 isolates per unresolved cluster, 52% isolates unresolved, a node-to-isolates ratio of 0.67, and a SID of 0.902.
8、For 4 serotypes Salmonella -S. Heidelberg, S.Kentucky, S.SaintPaul and S.Hadar, 6 enzymes subtyping results were different in difference serotypes. The enzyme which was most suitable for PFGE subtyping were PacI, SpeI, SpiI and NotI for S. Heidelberg, S.Kentucky, S.SaintPaul and S.Hadar, respectively. For S. Heidelberg, S.Kentucky, S.SaintPaul and S.Hadar, the most suitable three enzyme combination were SpeI/PacI/BlnI, SpeI/NotI/SfiI, SpeI/BlnI/SfiI和SpeI/NotI/SfiI, respectively. 6 enzyme combination PFGE yielded different discrimination for 4 Salmonella serotypes. The simultaneous analysis of 3 enzyme combination proved to be a beneficial scheme for the PFGE-based differentiation of closely related isolates of the four Salmonella serotypes.
9、BamHI was the most suitable Enzyme for Campylobacter jejuni in PFGE subtyping. In order to get higher discrimination ability for subtyping Campylobacter jejuni , PFGE should be used with other methods together.
10、Analyzing all data of PFGE subtyping, the discrimination ability of PFGE subtyping for STEC O157:H7 and Salmonella was better than Campylobacter jejuni.
引文
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