一种简化且经济有效的富集方法，可从零售鸡肉中分离弯曲杆菌属，无需微需氧孵育。
A simplified and cost-effective enrichment protocol for the isolation of Campylobacter spp. from retail broiler meat without microaerobic incubation.
机构信息
Department of Biological Sciences, 1627 Hall Street, Alabama State University, Montgomery, AL, USA.
出版信息
BMC Microbiol. 2011 Aug 3;11:175. doi: 10.1186/1471-2180-11-175.
BACKGROUND
To simplify the methodology for the isolation of Campylobacter spp. from retail broiler meat, we evaluated 108 samples (breasts and thighs) using an unpaired sample design. The enrichment broths were incubated under aerobic conditions (subsamples A) and for comparison under microaerobic conditions (subsamples M) as recommended by current reference protocols. Sensors were used to measure the dissolved oxygen (DO) in the broth and the percentage of oxygen (O2) in the head space of the bags used for enrichment. Campylobacter isolates were identified with multiplex PCR assays and typed using pulsed-field gel electrophoresis (PFGE). Ribosomal intergenic spacer analyses (RISA) and denaturing gradient gel electrophoresis (DGGE) were used to study the bacterial communities of subsamples M and A after 48 h enrichment.
RESULTS
The number of Campylobacter positive subsamples were similar for A and M when all samples were combined (P = 0.81) and when samples were analyzed by product (breast: P = 0.75; thigh: P = 1.00). Oxygen sensors showed that DO values in the broth were around 6 ppm and O2 values in the head space were 14-16% throughout incubation. PFGE demonstrated high genomic similarity of isolates in the majority of the samples in which isolates were obtained from subsamples A and M. RISA and DGGE results showed a large variability in the bacterial populations that could be attributed to sample-to-sample variations and not enrichment conditions (aerobic or microaerobic). These data also suggested that current sampling protocols are not optimized to determine the true number of Campylobacter positive samples in retail boiler meat.
CONCLUSIONS
Decreased DO in enrichment broths is naturally achieved. This simplified, cost-effective enrichment protocol with aerobic incubation could be incorporated into reference methods for the isolation of Campylobacter spp. from retail broiler meat.
背景
为简化从零售鸡肉中分离弯曲杆菌属的方法，我们采用非配对样本设计评估了 108 个样本（鸡胸肉和鸡腿肉）。按照现行参考方案的建议，增菌肉汤在有氧条件下（亚组 A）和比较下在微需氧条件下（亚组 M）孵育。传感器用于测量肉汤中的溶解氧（DO）和增菌袋中顶部空间的氧气（O2）百分比。使用多重 PCR 检测法鉴定弯曲杆菌属分离株，并使用脉冲场凝胶电泳（PFGE）进行分型。核糖体基因间隔区分析（RISA）和变性梯度凝胶电泳（DGGE）用于研究 48 小时增菌后亚组 M 和 A 的细菌群落。
结果
当所有样本合并时，A 和 M 的弯曲杆菌阳性亚组数量相似（P = 0.81），且按产品分析时（鸡胸肉：P = 0.75；鸡腿肉：P = 1.00）也是如此。氧气传感器显示，整个孵育过程中，肉汤中的 DO 值约为 6 ppm，顶部空间中的 O2 值为 14-16%。PFGE 表明，在大多数可从亚组 A 和 M 获得分离株的样本中，分离株的基因组相似度很高。RISA 和 DGGE 结果显示，细菌种群存在很大的可变性，这可能归因于样本间的差异，而不是增菌条件（有氧或微需氧）。这些数据还表明，现行的采样方案并未优化，无法确定零售鸡肉中弯曲杆菌阳性样本的真实数量。
结论
增菌肉汤中的 DO 自然减少。这种简化、具有成本效益的有氧孵育增菌方案可纳入从零售鸡肉中分离弯曲杆菌属的参考方法。
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