海口市5类食品中蜡样芽胞杆菌药敏和毒力基因检测及分子分型研究

目的 了解海口市5类食品中蜡样芽胞杆菌污染状况、毒力基因携带类型、药物敏感特点和分子分型特征。方法 参照GB 4789.14—2014《食品安全国家标准 食品微生物学检验 蜡样芽胞杆菌检验》对海南粉、海南粉配料、奶粉类、快餐类和糕点类5类食品进行分离鉴定蜡样芽胞杆菌,并应用普通聚合酶链式反应(PCR)方法进行菌群特异性基因groEL和10种毒力基因检测；采用微量肉汤稀释(MIC)法检测菌株对抗生素的敏感性；应用脉冲场凝胶电泳(PFGE)技术对菌株进行分子分型。结果 626份不同食品样品中有197份检出蜡样芽胞杆菌,检出率为31.5%,其中海南粉检出率最高,为63.1%(140/222)。所有菌株均至少携带1种毒力基因,entFM基因携带率最高,为99.0%(195/197),ces基因携带率最低,为2.5%(5/197)；同时携带nheA、nheB和nheC基因的菌株占88.8%(175/197),同时携带hblA、hblC和hblD基因的菌株占13.7%(27/197)。所有菌株对庆大霉素和氯霉素的敏感率均为100.0%(197/197),对万古霉素和环丙沙星敏感率分别为99.5%(196/197)和92.9%(183/197),对青霉素和复方新诺明的耐药率分别为100.0%(197/197)和90.9%(179/197)。PFGE分型结果显示,所有菌株可分为30个簇,117种带型。结论 海口市5类食品均存在蜡样芽胞杆菌污染,其中海南粉污染较重,对食品安全构成潜在的威胁；可依据菌株的毒力基因、分子分型和抗生素敏感性特点进行针对性防控和重点监管。

Study of the drug sensitivity, virulence genes and molecular typing of Bacillus cereus in five foods in Haikou

Objective To study the distribution, virulence genes, drug sensitivity and molecular characteristic of Bacillus cereus in five foods in Haikou area. Methods The Bacillus cereus strains were isolated from different foods including pastries, milk powder, fast food, Hainan rice noodles and its ingredients, collected from Haikou area, according to the standard GB 4789.14-2014. The specific gene groEL and 10 virulence genes were detected by general polymerase chain reaction (PCR) method. The sensitivity to antibiotics of isolates was performed with minimum inhibitory concentration (MIC). The pulsed field gel electrophoresis (PFGE) patterns of strains were analyzed. Results 197 strains of B.cereus were isolated from 626 food samples and the positive rate of B.cereus was 31.5%. In particular, most of B.cereus strains were isolated from Hainan rice noodles, accounting for 63.1%(140/222). All isolates carried at least one virulence gene, the rate of entFM gene was the highest, up to 99.0%(195/197). On the contrary, ces gene was the lowest at 2.5%(5/197) only. There were 175 (88.8%) strains that carried genes of nheA,nheB and nheC simultaneously. There were 27 (13.7%) strains that carried genes of hblA,hblC and hblD simultaneously. The sensitive rate of strains towards gentamicin, chloramphenicol, vancomycin and ciprofloxacin were 100.0%(197/197), 100.0%(197/197), 99.5%(196/197) and 92.9%(183/197), respectively. And the resistance rate of strains towards penicillin and trimethoprim-sulfamethoxazole were 100.0%(197/197) and 90.9%(179/197), respectively. The PFGE result showed 30 clusters and 117 genotypes. Conclusion The positive rate of Bacillus cereus in five foods in Haikou area was relatively high, especially in Hainan rice noodles, which should arouse attention and close observation. Targeted prevention and control measures can be carried out according to the characteristics of virulence genes, molecular typing and antibiotic sensitivity of the strains.