胃黏膜组织中恶臭假单胞菌临床意义的探讨

Investigation of clinical significance of Pseudomonas putida isolated from gastric biopsies

目的探索恶臭假单胞菌在幽门螺杆菌感染相关胃肠疾病中的意义。方法采集14 C尿素呼气试验阳性患者病变胃黏膜354例,进行细菌的分离培养。根据菌落形态、革兰染色、尿素酶试验及幽门螺杆菌特异性16SrRNA基因片段的PCR进行幽门螺杆菌的鉴定,同时提取胃黏膜组织DNA,通过幽门螺杆菌特异性PCR进行快速诊断。将非幽门螺杆菌转种于营养琼脂培养基,进行快速尿素酶试验。尿素酶阳性的非幽门螺杆菌染色体DNA利用细菌16SrRNA基因通用引物进行PCR扩增、测序及序列比对。使用全自动细菌鉴定仪对经测序比对为恶臭假单胞菌的菌株进行鉴定。采用K-B纸片扩散法进行药物敏感性试验。结果从354例样本中分离出革兰阴性、尿素酶阳性的恶臭假单胞菌10株,经16SrRNA基因测序和序列比对,与GenBank中恶臭假单胞菌的相似性≥98%。10株恶臭假单胞菌的胃黏膜标本中,6例标本幽门螺杆菌特异性PCR为阳性,4例为阴性。传代存活的7株恶臭假单胞菌的K-B法药物敏感试验显示对左氧氟沙星均敏感,1株对四环素敏感,5株对阿莫西林耐药,6株对克拉霉素耐药,7株对甲硝唑、氨苄青霉素均耐药。7株菌经全自动细菌生化鉴定仪鉴定结果均为恶臭假单胞菌。结论病变胃黏膜中分离出尿素酶阳性且对治疗幽门螺杆菌感染的多种抗生素耐药;可能造成临床上14 C-尿素呼气试验假阳性,并继发感染影响胃肠疾病的进展。

In order to investigate the role of Pseudomonas putidain H.pylori-associated gastrointestinal diseases,354 H.pylori positive cases determined by 14 C-urea breath test were underwent endoscopy for H.pylori isolation and identification.H.pylori and non-H.pylori were identified by colonial morphology,Grams staining,urease test and H.pylori specific 16 SrRNA gene fragment PCR amplification.NonH.pylori were then inoculated on nutrient agar plate under aerobic conditions at 37℃ for 18-24 hours.Single bacterial colony was used for rapid urease test.Simultaneously,total genomic DNA were extracted from gastric mucosal tissue and H.pylori specific 16 SrDNA was amplified by PCR for rapid diagnosis.Urease positive non-H.pylori was selected for genomic DNA extraction and then bacterial 16 SrDNA was amplified by general primers and then determined by sequencing.Sequence comparison was carried out by the BLASTn program and the GenBank databases（http：//blast.ncbi.nlm.nih.gov/Blast.cgi）.Single colony which was identified as Pseudomonas putida by sequence alignment was then picked for subculturing.Drug susceptibility of 7alive strains were detected by K-B disk diffusion method and also identified by automatic bacteria identification instrument.The results of 16 SrDNA sequencing showed that a total of 10 strains were identified as Pseudomonas putidafrom 354 cases.All of them are Grams negative bacteria with urease activity.In these ten cases,6were H.pylori-positive determined by H.pylori specific PCR amplification,and 4cases were H.pylori-negative.All the seven alive strains of Pseudomonas putida were susceptible to levofloxacin,1was susceptible to tetracycline,5were resistant to amoxicillin,6were resistant to clarithromycin and 7 were resistant to both metronidazole and Ampicillin.In conclusion,Pseudomonas putida which were urease-positive and resistant to antibiotic in H.pylori eradication can be isolated from diseased gastric mucosal tissue bacteria.These strains may cause false positive in clinical 14 C-urese breath test,and influence H.pylori-associated gastrointestinal diseases progression.