Microbial diversity of selective and non-selective media for Campylobacter

Although Campylobacter is a foodborne pathogen commonly associated with poultry, it can be difficult to isolate because Campylobacter selective media vary in effectiveness. Therefore, using 16S rRNA gene sequencing, the relative effectiveness of selective media was determined in the current study by delineating the microbial composition of the pooled colonies on selective and non-selective media for Campylobacter isolation from poultry post-chill carcass rinsates. Samples were made by aliquoting 30 mL of post-chill rinsates to individual Whirl-Pak bags and inoculating them with 1.00 to 4.00 CFU per mL of either C. jejuni, C. coli and C. lari. After inoculation, 2x Blood free Bolton Broth were added to all inoculated samples and enriched for 20 h at 42 C under microaerophilic conditions. Following incubation, samples were spread plated on Campy-Cefex, mCCDA, and TSA plates and incubated for 48 h under the corresponding conditions. The V4 region of the 16S rDNA of the subsequent pooled colonies was sequenced using an Illumina Miseq. Microbial diversity and microbiota composition (ANCOM) were analyzed in QIIME 2.2021.11. Alpha diversity revealed main effect differences for both species and media. Under Pielous Evenness and Shannons Entropy, C. jejuni had the greatest evenness and richness, followed by C. coli then C. lari. For media, under Pielous Evenness and Shannons Entropy, TSA had the most evenly distributed taxa and most diverse taxa. There was an interaction of species and media on the beta diversity. Using Bray Curtis and Weighted Unifrac pairwise differences were seen between all species pairs. Media also impacted the beta diversity with the Bray Curtis and Weighted Unifrac of TSA and mCCDA and TSA and Campy-Cefex being different. Additionally, among the three Campylobacter spp., ANCOM revealed Campylobacter, Lachnospiraceae, and Oscillospiraceae were significantly more abundant taxa. The only significant more abundant taxa among the three media at the genus level was Staphylococcus. As a result, microbial diversity, and composition of enriched poultry rinsates appear to be affected by media used for enumeration and type of Campylobacter spp. present.

尽管弯曲杆菌（Campylobacter）是一种常与家禽相关的食源性致病菌，但其分离难度较大，原因在于弯曲杆菌选择性培养基的抑菌效果参差不齐。因此，本研究采用16S rRNA基因测序技术，通过分析家禽冷却后胴体冲洗液中用于弯曲杆菌分离的选择性与非选择性培养基上的混合菌落微生物组成，明确了各类选择性培养基的相对抑菌效果。实验样品制备方式为：取30 mL冷却后胴体冲洗液分装至Whirl-Pak无菌采样袋中，每毫升样品接种1.00至4.00 CFU的空肠弯曲杆菌（C. jejuni）、结肠弯曲杆菌（C. coli）或劳氏弯曲杆菌（C. lari）。接种后，向所有接种样品中加入2×无血博尔顿肉汤（2x Blood free Bolton Broth），于42℃微需氧条件下富集培养20小时。培养结束后，将样品涂布接种于Campy-Cefex培养基、mCCDA培养基与胰蛋白胨大豆琼脂（TSA）平板，于对应条件下培养48小时。随后，采用Illumina Miseq测序平台对后续混合菌落的16S rDNA V4区进行测序。使用QIIME 2.2021.11软件分析微生物多样性与菌群组成，采用ANCOM进行差异检验。α多样性分析结果显示，菌种与培养基均存在主效应差异：在皮卢均匀度指数（Pielous Evenness）与香农熵指数（Shannons Entropy）下，空肠弯曲杆菌的菌群均匀度与丰富度最高，其次为结肠弯曲杆菌，劳氏弯曲杆菌位列第三。就培养基而言，胰蛋白胨大豆琼脂（TSA）的菌群分类单元分布最均匀且多样性最高。菌种与培养基对β多样性存在交互作用：基于布雷-柯蒂斯距离（Bray Curtis）与加权UniFrac距离（Weighted Unifrac）的分析显示，所有菌种对之间均存在显著差异。培养基同样对β多样性存在影响，其中胰蛋白胨大豆琼脂（TSA）与mCCDA培养基、胰蛋白胨大豆琼脂（TSA）与Campy-Cefex培养基的菌群差异显著。此外，在三种弯曲杆菌属菌种中，ANCOM分析显示弯曲杆菌属、毛螺菌科（Lachnospiraceae）与颤螺菌科（Oscillospiraceae）为显著富集的分类单元；在属水平上，三种培养基间仅葡萄球菌属（Staphylococcus）为显著富集的分类单元。综上，富集培养后的家禽胴体冲洗液的微生物多样性与菌群组成，会受到用于菌落计数的培养基类型以及所存在的弯曲杆菌菌种类型的影响。