Swine intestinal microbiota perturbation by antibiotic treatment is linked to a reduced number of necrotic epithelial cells and up-regulation of immune-associated pathways using an in vitro Salmonella Typhimurium challenge model

Salmonella enterica serovar Typhimurium is an animal welfare and public health concern due to its ability to parasite livestock and potentially contaminate meat products. In pigs, it is associated with enterocolitis and diarrhea, whereas some individuals may become life-long carriers. To reduce Salmonella shedding and the risk of pork contamination, antibiotic therapy is used and can contribute to antimicrobial resistance. Here we hypothesized that immune system education by the microbiota can play a role in intestinal resilience to infection. We used amoxicillin (15mg/Kg) to modulate the intestinal microbiome of 10 piglets, paired with same age pigs that received a placebo (n=10) from 0 to 14 days of age. Animals were euthanized when 4-weeks old. Each pig donated colon sections for ex vivo culture (n=20 explants/pig). Explants were inoculated with S. Typhimurium, PBS or LPS. The gut bacteriome was characterized by sequencing of the 16S rRNA at 7, 21 days of age, and upon in vitro culture. In vivo antibiotic treatment led to b-diversity differences between groups at all times (P<0.05), while a-diversity did not differ between amoxicillin and placebo groups on day 21 and at euthanasia (P<0.03 on day 7). Explant microbiomes were not different from in vivo. No significant differences in epithelial cell necrosis scores were observed between amoxicillin and placebo groups after exposure to Salmonella, even though median scores were lower in the amoxicillin group. Both groups did have significantly higher necrosis scores than the PBS group (P<0.05). Activation of immune-related cascades in control explants from the amoxicillin group was observed, whereas Salmonella exposure activated specific cellular repair, wound healing and macrophage pathogen inhibition pathways that were significantly different from placebo pigs. These suggest that immune education by the microbiota may be developed as a tool to mitigate intestinal lesions following pathogen exposure.

肠炎沙门氏菌鼠伤寒血清型（Salmonella enterica serovar Typhimurium）可寄生于家畜并可能污染肉类产品，因此成为动物福利与公共卫生领域的重点关注对象。在猪群中，该菌与小肠结肠炎及腹泻相关，部分个体可成为终身带菌者。为减少沙门氏菌排菌量与猪肉污染风险，临床常采用抗生素治疗，但此举会加剧抗菌药物耐药性的产生。本研究提出假说：微生物群介导的免疫系统教育可在肠道抵御感染的过程中发挥作用。
我们使用阿莫西林（amoxicillin，15mg/Kg）调节10头仔猪的肠道微生物组，并设置同月龄的安慰剂对照组仔猪（n=10），处理周期为仔猪0至14日龄。仔猪在4周龄时实施安乐死，每头仔猪采集结肠组织样本用于体外培养（每头仔猪20个外植体（explant））。外植体分别接种鼠伤寒沙门氏菌、磷酸盐缓冲液（PBS）或脂多糖（LPS）。分别在仔猪7日龄、21日龄以及体外培养后，通过16S rRNA（16S核糖体RNA）测序对肠道细菌组进行表征。
体内抗生素处理在所有时间点均导致两组间的β多样性（beta diversity）存在显著差异（P<0.05）；而α多样性（alpha diversity）仅在7日龄时阿莫西林组与安慰剂组存在统计学差异（P<0.03），在21日龄及安乐死时两组无显著差异。外植体的微生物组与体内样本无显著差异。沙门氏菌暴露后，阿莫西林组与安慰剂组的上皮细胞坏死评分未观察到显著差异，尽管阿莫西林组的中位坏死评分更低。两组的坏死评分均显著高于PBS组（P<0.05）。对阿莫西林组对照组外植体的分析显示，其免疫相关级联反应被激活；而沙门氏菌暴露则激活了特异性细胞修复、伤口愈合及巨噬细胞病原体抑制通路，且该通路特征与安慰剂组仔猪存在显著差异。
上述结果表明，基于微生物群的免疫系统教育有望作为一种干预手段，用于减轻病原体暴露后的肠道损伤。