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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。
Objective To explore the significance of Pseudomonas putida in gastrointestinal diseases related to Helicobacter pylori infection. Methods A total of 354 gastric mucosa were collected from 14 C-urea breath test-positive patients, and the bacteria were isolated and cultured. Helicobacter pylori was identified by colony morphology, Gram stain, urease test, and PCR of Helicobacter pylori-specific 16S rRNA gene fragments. Meanwhile, gastric mucosal tissue DNA was extracted and rapidly diagnosed by H. pylori-specific PCR. Non-Helicobacter pylori was planted on nutrient agar medium for rapid urease testing. Urease-positive non-Helicobacter pylori chromosomal DNA PCR amplification, sequencing and sequence alignment using bacterial 16SrRNA gene universal primers. Strains that were sequenced to be Pseudomonas putida were identified using a fully automatic bacterial analyzer. K-B disk diffusion method for drug susceptibility testing. Results 10 strains of Pseudomonas putida, which were gram negative and urease, were isolated from 354 samples. The 16S rRNA gene sequencing and sequence alignment showed that the similarity to Pseudomonas putida in GenBank was ≥98%. Of 10 strains of Pseudomonas putida gastric mucosa specimens, 6 specimens of H. pylori-specific PCR-positive, 4 were negative. KB drug susceptibility test of 7 Pseudomonas putida passaged passages showed that all of them were sensitive to levofloxacin, 1 was sensitive to tetracycline, 5 were resistant to amoxicillin, 6 were resistant to clarithromycin and 7 were resistant to clarithromycin Nitidine, ampicillin are resistant. Seven strains of bacteria were identified by automated biochemical analyzer results were Pseudomonas putida. Conclusions The mucosa of gastric mucosa is positive for urease and resistant to various antibiotics for the treatment of Helicobacter pylori infection. It may result in the false positive of 14 C-urea breath test and the subsequent infection affects the progress of gastrointestinal diseases.