Study on the Correlation Between Multiple Drug Resistance of Acinetobacter baumannii with Efflux Pump and Biofilm Formation
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摘要:
目的 探讨临床感染性疾病所分离鲍曼不动杆菌（acinetobacter baumarmii，AB）的生物膜基因、外排泵基因及生物膜形成能力与耐药性的相关性。 方法 收集非重复鲍曼不动杆菌共119株（多重耐药鲍曼不动杆菌MDR-AB菌株74株，非多重耐药菌株45株），PCR检测鲍曼不动杆菌外排泵基因（adeB、adeJ、adeG、adeS和adeR）和生物膜相关基因（bap、ompA、csuA、csuB、csuC、csuD、csuE、abaI、bfmR、bfmS），应用24孔细胞培养板体外构建细菌生物膜，通过结晶紫染色法定性观察所有菌株是否形成生物膜。 结果 鲍曼不动杆菌对常用抗菌药物均多数为耐药，对替加环素、多粘菌素B、米诺环素较为敏感；生物膜相关基因的总体检出率约80%左右，多重耐药菌中bap、csuB、csuC、csuD基因检出率显著高于非多重耐药菌（P < 0.05）；主动外排泵基因的检出率不到70%，其中adeB、adeJ、adeS和adeR基因在多重耐药菌中的检出率明显高于非多重耐药菌（P < 0.05）；而adeG携带率相反。有3株多重耐药菌株及5株非多重耐药菌株未携带有上述外排泵相关基因，所有菌株均未检出abeM基因；95.80%菌株可观察到生物膜的形成。 结论 医院内检出的鲍曼不动杆菌多呈现出多重耐药特点，多数具有较强的生物膜形成能力，多重耐药菌生物膜基因和外排泵基因携带率更高，可能参与调控形成其多重耐药性。 Abstract:Objective To investigate the correlation between biofilm gene, efflux pump gene, biofilm forming ability and drug resistance of Acinetobacter baumarmii （AB） isolated from the patients of clinical infectious diseases. Methods A total of 119 non-repeating Acinetobacter baumannii isolates （74 MDR-AB strains and 45 non-multi-drug resistant strains） were collected. The effector pump genes （adeB, adeJ, adeG, adeS and adeR） and biofilm-related genes （bap, ompA, csuA, csuB, csuC, csuD, csuE, abaI, bfmR, bfmS） were detected by PCR. Bacterial biofilm was constructed in vitro with 24-well cell culture plates, and the formation of biofilm was qualitatively observed by crystal violet staining. Results Acinetobacter baumannii was resistant to most of the commonly used antibiotics and sensitive to tigacycline, polymyxin B and minocycline. The overall detection rate of biofilm-related genes was about 80%, and the detection rates of bap, csuB, csuC and csuD genes in multi-drug resistant bacteria were significantly higher than those in non-multi-drug resistant bacteria （P < 0.05）. The detection rate of active effector pump genes was less than 70%, and the detection rate of adeB, adeJ, adeS and adeR genes in multidrug-resistant bacteria was significantly higher than that in non-multidrug-resistant bacteria （P < 0.05）. The adeG carrying rate was opposite. Three multidrug-resistant strains and five non-multidrug-resistant strains did not carry the above efflux pump related genes, and no abeM gene was detected in all strains. The formation of biofilm was observed in 95.80% of strains. Conclusion Acinetobacter baumannii in hospital is mostly multidrug-resistant strains, most of which have strong biofilm forming ability. Multidrug-resistant bacteria have higher biofilm gene and effluent-pump gene carrying rate, which may be involved in the regulation and formation of multidrug resistance. -
Key words:
- Acinetobacter baumannii /
- Multiple drug resistance /
- Efflux pump /
- Biofilm
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图 1 MDR-AB与非MDR-AB对临床常用抗菌药物耐药情况
Figure 1. Resistance of MDR-AB and non-MDR-AB to commonly used antimicrobial drugs in clinical practice
图 2 生物膜相关基因及部分外排泵基因PCR扩增电泳图
Figure 2. PCR amplification electropherogram of biofilm-related genes and some efflux pump genes
图 3 外排泵adeG基因PCR扩增电泳图 和16s rRNA基因PCR扩增电泳图
Figure 3. PCR amplification electropherogram of efflux pump adeG gene and 16s rRNA gene
表 1 鲍曼不动杆菌生物膜相关基因引物列表
Table 1. List of gene primers associated with acinetobacter baumannii biofilm
基因名称 引物 核酸系列（5'→3'） 产物长度（bp） Bap F GAGGGAACTTCTGCAAAACTTTC 108 R CAGACGTATGACTGCATTGGT ompA F GAGTCGTATTGCACTTGCTAC 594 R GCAGGCTTCAAGTGACCACC csuA F GAGGGAAGATGATATTCAATCGTG 517 R CCCTTAGATATACGACTACCATCAT csuB F GCAGCAGATCCTCAGCTCAATTCA 354 R GTTTGTAGGTGTTGTAGCAGGC csuC F ATACGGGTAAGACCGATGC 466 R GTTTTCAGTGCCGAAAGACG csuD F GCTGATTTTATTGTCGGGTGGA 464 R GCCCTATACCAGACTGAGCGAC csuE F ACCAATGCTCAGACCGGAG 751 R CTTGTACCGTGACCGTATCTTG abaI F CCGCTACAGGGTATTTGTTGAA 428 R CACGATGGGCACGAAAACC bfmR F GAAGTTGGTGTAGAAACCGATG 557 R GGATTTTCAGGATCATCGCC bfmS F CATTAGTGAAGGAGTCGCTCG 990 R GGTGTACCCTGCTCTAGTTTT 
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表 2 鲍曼不动杆菌外排泵相关基因引物列表
Table 2. List of gene primers associated with effector pump acinetobacter baumannii
引物名称 引物 序列（5'→3'） 产物长度（bp） adeB F TTAACGATAGCGTTGTAACC 541 R TGAGCAGACAATGGAATAGT adeR F ACTACGATATTGGCGACATT 447 R GCGTCAGATTAAGCAAGATT adeS F TTGGTTAGCCACTGTTATCT 544 R AGTGGACGTTAGGTCAAGTT adeJ F ATTGCACCACCAACCGTAAC 453 R TAGCTGGATCAAGCCAGATA abeM F GTAGGTGTAGGCTTATGGA 303 R GTACCGAAGTGACTGAAAT adeG F TTCATCTAGCCAAGCAGAAG 468 R GTGTAGTGCCACTGGTTACT 16S rRNA F GGAGGAAGGTGGGGATGACG 241 R ATGGTGTGACGGGCGGTGTG
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表 3 119株AB对临床常用抗菌药物的耐药情况
Table 3. Resistance of 119 strains of AB to commonly used antimicrobial drugs in clinical practice
药品名称 耐药菌株数 百分比% 氨苄西林/舒巴坦 76 63.9 米诺环素 9 7.6 阿米卡星 57 47.9 哌拉西林 93 78.2 哌拉西林/他唑巴坦 86 72.3 头孢曲松 77 64.7 头孢吡肟 76 63.9 亚胺培南 75 63.0 庆大霉素 51 42.9 妥布霉素 46 38.7 环丙沙星 78 65.5 左氧氟沙星 72 60.5 四环素 33 27.7 头孢他啶 77 64.7 美洛培南 80 67.2 头孢哌酮/舒巴坦 72 60.5 替加环素 2 1.7 多粘菌素B 1 0.8
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