奇异变形杆菌可移动耐药基因岛研究
摘要
随着奇异变形杆菌(Proteus mirabilis)耐药菌株不断增加,其耐药机制成为研究人员关注的热点。沙门氏基因组岛(Salmonella Genomic Island 1, SGI1)作为细菌编码抗性的可移动元件,于2007年在P. mirabilis菌株中发现,它在细菌多重耐药基因传播和转移过程中发挥重要的作用。本论文主要研究P. mirabilis中SGI1介导耐药基因的水平传播。通过对广州市天河区食品和临床P. mirabilis菌株的分离,研究这些菌株对17种常用抗生素的耐药性以及Ⅰ类、Ⅱ类和Ⅲ类整合子,耐药基因盒的携带情况。采用PFGE方法分析Ⅰ类整合子阳性菌株、Ⅱ类整合子阳性菌株的同源性以及携带相同耐药基因盒菌株间的亲缘关系。结合耐药基因盒信息,对P. mirabilis分离株中SGI1的携带情况、基因岛的结构与序列进行解析,结果如下:
使用传统分离培养法和建立的靶基因为tuf基因的变形杆菌属PCR方法对肉类产品中分离的可疑菌株和P. mirabilis临床分离株进行鉴定,PCR方法和生化实验结果完全一致,从食品中分离到14株P. mirabilis菌株并验证了46株P. mirabilis临床分离株的准确性。
46株P. mirabilis临床分离株普遍具有多重耐药性,其中耐10种以上抗生素高达69.6%。14株P. mirabilis食品分离株均耐5种以下抗生素,28.6%耐3-5种抗生素。氯霉素、四环素、红霉素、复方新诺明抗菌药物不适合治疗本地区P. mirabilis引起的感染。头孢曲松、头孢西丁的敏感率达到88.3%～91.7 %,可作为首选药物。
Ⅰ类整合子阳性菌株34株(56.7%),其中,4株携带blaP1和aadA2基因盒,对β-内酰胺类和氨基糖苷类药物耐药;19株携带dfrA17和aadA5基因盒,对磺胺类和氨基糖苷类药物耐药;3株分别携带blaP1、dfrA1和aadB基因盒;8株携带dfrA5基因盒。17株(28.3%)菌株携带Ⅱ类整合子和dfrA1、sat2、aadA1基因盒,其中16株(94.1%)携带Ⅰ类整合子且均携带dfrA17和aadA5基因盒,Ⅰ、Ⅱ类整合子阳性率为26.7%。没有检测到Ⅲ类整合子。
56株P. mirabilis成功分型,相似值为0.40～1.00。12组,42株P. mirabilis的脉冲场凝胶电泳(Pulsed-field gel electrophoresis, PFGE)图谱分别相同,表明在病原学上具有同源性,为克隆相关菌株。同时携带Ⅰ类和Ⅱ类整合子的菌株同源性很高,相似值约0.72～1.00。
2株P. mirabilis临床分离株分别携带SGI1-B和SGI1-O。4株P. mirabilis临床分离株携带SGI1,且这4株为克隆相关菌株。5株P. mirabilis临床分离株和3株P. mirabilis食品分离株携带一种新型基因岛变异体SGI1-U,且为相同克隆菌株,提示了该岛从食源到人类水平传播的可能性。前三种基因岛均位于染色体thdF和hipB基因间,而SGI1-U染色体缺失了hipB和hipA基因,插入在thdF和PMI3124基因之间。
综上所述,P. mirabilis菌株携带多种SGI1且携带率很高,研究结果为流行病学研究提供了依据,为了解细菌多重耐药性获得和传播机制奠定了基础。
With Proteus mirabilis resistant isolates increased, the mechanism of antibiotic resistance become focus by researchers in recent years. As a bacterial mobile element encoding resistance, Salmonella genomic island 1, found in the P. mirabilis strains in 2007, which played an important role in transferring resistance genes. The purpose of this study was to determine the key mechanisms in horizontal transfer of antibiotic resistance genes in SGI1. P. mirabilis food and clinical isolates were examined for their susceptibility to 17 antibiotics and the presence of antibiotic resistance integronsⅠ,Ⅱ,Ⅲand gene cassette. After all the P. mirabilis isolates were genotyped by PFGE,the results were analyzed according to integronsⅠ,Ⅱand gene cassettes. The SGI1s were detected in P. mirabilis isolates and their structures were analyzed. The results were as follows:
P. mirabilis food and clinical isolates were detected by plate-culture and the tuf gene PCR methods, and the results of the both methods were in agreement. 14 P. mirabilis strains were isolated from food. The clinical isolates results of PCR and biochemical test were both consistent with the report from the samples donor the first Affiliated Hospital of Jinan University, indicating that the authenticity of P. mirabilis clinical isolates were proved. Forty-six P. mirabilis clinical isolates with multiple drug resistance in general, of which more than 10 kinds of antibiotic-resistant up to 69.6%. 14 P. mirabilis isolates from food were resistant to antibiotics, 28.6% resistant to 3~5 antibiotics. Chloramphenicol, tetracycline, erythromycin and cotrimoxazole were not suitable for infections caused by P. mirabilis in this region. The sensitive rate of ceftriaxone and cefoxitin were 88.3%~91.7%, indicating that the two antibiotics can be used for patients.
IntegronⅠwas found in 34 (56.7%) P. mirabilis isolates, of which 4 isolates carring aadA2 and blaP1 gene cassettes, resistance toβ-lactams and aminoglycosides; 19 isolates carrying dfrA17 and aadA5 gene cassettes, resistance to sulfonamides and aminoglycosides; 3 isolates carring blaP1, dfrA1 and aadB gene cassettes, respectively; 8 isolates carrying dfrA5 gene cassette. IntegronⅡand dfrA1, sat2 and aadA1 gene cassettes were found in 17 (28.3%) P. mirabilis isolates, in which 16 (94.1%) isolates carring integronⅠand dfrA17 and aadA5 gene cassettes. IntegronⅠandⅡwere both found in 26.7% P. mirabilis isolates. IntegronⅢwas not detected in all isolates.
The similarity value of 0.40 to 1.00 in 56 P. mirabilis isolates was shown by PFGE analysis. 12 groups, 42 P. mirabilis isolates had the same PFGE patterns, respectively, indicating that they may be the same clone strains. The value of Phylogenetic relationships of the isolates carring integronⅠandⅡwas up to 0.72 to 1.00.
Two P. mirabilis clinical isolates were detected to carry SGI1-B and SGI1-O, respectively. 4 P. mirabilis clinical isolates which might be clone to each other were detected to carry SGI1. 5 clinical isolates and 3 food isolates were found to carry a new SGI1 variant SGI1-U, and cloning for the same strain, suggesting that the possibility of SGI1-U horizontal transmission from food to human beings. SGI1, SGI1-B and SGI1-O were located between thdF and hipB genes, and SGI1-U was inserted between thdF and PMI3124 genes and the 8 isolates carring SGI1-U were detected missing hipA and hipB genes.
In summary, many P. mirabilis isolates carried a variety of SGI1s and the results were useful to the study on epidemiology, bacterial multi-drug resistance mechanisms and dissemination.
使用传统分离培养法和建立的靶基因为tuf基因的变形杆菌属PCR方法对肉类产品中分离的可疑菌株和P. mirabilis临床分离株进行鉴定,PCR方法和生化实验结果完全一致,从食品中分离到14株P. mirabilis菌株并验证了46株P. mirabilis临床分离株的准确性。
46株P. mirabilis临床分离株普遍具有多重耐药性,其中耐10种以上抗生素高达69.6%。14株P. mirabilis食品分离株均耐5种以下抗生素,28.6%耐3-5种抗生素。氯霉素、四环素、红霉素、复方新诺明抗菌药物不适合治疗本地区P. mirabilis引起的感染。头孢曲松、头孢西丁的敏感率达到88.3%～91.7 %,可作为首选药物。
Ⅰ类整合子阳性菌株34株(56.7%),其中,4株携带blaP1和aadA2基因盒,对β-内酰胺类和氨基糖苷类药物耐药;19株携带dfrA17和aadA5基因盒,对磺胺类和氨基糖苷类药物耐药;3株分别携带blaP1、dfrA1和aadB基因盒;8株携带dfrA5基因盒。17株(28.3%)菌株携带Ⅱ类整合子和dfrA1、sat2、aadA1基因盒,其中16株(94.1%)携带Ⅰ类整合子且均携带dfrA17和aadA5基因盒,Ⅰ、Ⅱ类整合子阳性率为26.7%。没有检测到Ⅲ类整合子。
56株P. mirabilis成功分型,相似值为0.40～1.00。12组,42株P. mirabilis的脉冲场凝胶电泳(Pulsed-field gel electrophoresis, PFGE)图谱分别相同,表明在病原学上具有同源性,为克隆相关菌株。同时携带Ⅰ类和Ⅱ类整合子的菌株同源性很高,相似值约0.72～1.00。
2株P. mirabilis临床分离株分别携带SGI1-B和SGI1-O。4株P. mirabilis临床分离株携带SGI1,且这4株为克隆相关菌株。5株P. mirabilis临床分离株和3株P. mirabilis食品分离株携带一种新型基因岛变异体SGI1-U,且为相同克隆菌株,提示了该岛从食源到人类水平传播的可能性。前三种基因岛均位于染色体thdF和hipB基因间,而SGI1-U染色体缺失了hipB和hipA基因,插入在thdF和PMI3124基因之间。
综上所述,P. mirabilis菌株携带多种SGI1且携带率很高,研究结果为流行病学研究提供了依据,为了解细菌多重耐药性获得和传播机制奠定了基础。
With Proteus mirabilis resistant isolates increased, the mechanism of antibiotic resistance become focus by researchers in recent years. As a bacterial mobile element encoding resistance, Salmonella genomic island 1, found in the P. mirabilis strains in 2007, which played an important role in transferring resistance genes. The purpose of this study was to determine the key mechanisms in horizontal transfer of antibiotic resistance genes in SGI1. P. mirabilis food and clinical isolates were examined for their susceptibility to 17 antibiotics and the presence of antibiotic resistance integronsⅠ,Ⅱ,Ⅲand gene cassette. After all the P. mirabilis isolates were genotyped by PFGE,the results were analyzed according to integronsⅠ,Ⅱand gene cassettes. The SGI1s were detected in P. mirabilis isolates and their structures were analyzed. The results were as follows:
P. mirabilis food and clinical isolates were detected by plate-culture and the tuf gene PCR methods, and the results of the both methods were in agreement. 14 P. mirabilis strains were isolated from food. The clinical isolates results of PCR and biochemical test were both consistent with the report from the samples donor the first Affiliated Hospital of Jinan University, indicating that the authenticity of P. mirabilis clinical isolates were proved. Forty-six P. mirabilis clinical isolates with multiple drug resistance in general, of which more than 10 kinds of antibiotic-resistant up to 69.6%. 14 P. mirabilis isolates from food were resistant to antibiotics, 28.6% resistant to 3~5 antibiotics. Chloramphenicol, tetracycline, erythromycin and cotrimoxazole were not suitable for infections caused by P. mirabilis in this region. The sensitive rate of ceftriaxone and cefoxitin were 88.3%~91.7%, indicating that the two antibiotics can be used for patients.
IntegronⅠwas found in 34 (56.7%) P. mirabilis isolates, of which 4 isolates carring aadA2 and blaP1 gene cassettes, resistance toβ-lactams and aminoglycosides; 19 isolates carrying dfrA17 and aadA5 gene cassettes, resistance to sulfonamides and aminoglycosides; 3 isolates carring blaP1, dfrA1 and aadB gene cassettes, respectively; 8 isolates carrying dfrA5 gene cassette. IntegronⅡand dfrA1, sat2 and aadA1 gene cassettes were found in 17 (28.3%) P. mirabilis isolates, in which 16 (94.1%) isolates carring integronⅠand dfrA17 and aadA5 gene cassettes. IntegronⅠandⅡwere both found in 26.7% P. mirabilis isolates. IntegronⅢwas not detected in all isolates.
The similarity value of 0.40 to 1.00 in 56 P. mirabilis isolates was shown by PFGE analysis. 12 groups, 42 P. mirabilis isolates had the same PFGE patterns, respectively, indicating that they may be the same clone strains. The value of Phylogenetic relationships of the isolates carring integronⅠandⅡwas up to 0.72 to 1.00.
Two P. mirabilis clinical isolates were detected to carry SGI1-B and SGI1-O, respectively. 4 P. mirabilis clinical isolates which might be clone to each other were detected to carry SGI1. 5 clinical isolates and 3 food isolates were found to carry a new SGI1 variant SGI1-U, and cloning for the same strain, suggesting that the possibility of SGI1-U horizontal transmission from food to human beings. SGI1, SGI1-B and SGI1-O were located between thdF and hipB genes, and SGI1-U was inserted between thdF and PMI3124 genes and the 8 isolates carring SGI1-U were detected missing hipA and hipB genes.
In summary, many P. mirabilis isolates carried a variety of SGI1s and the results were useful to the study on epidemiology, bacterial multi-drug resistance mechanisms and dissemination.
引文
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