Bearson Shawn M D, Bearson Bradley L, Rasmussen Mark A
US Department of Agriculture, Agricultural Research Service/NADC, 2300 Dayton Ave., Ames, IA 50014, USA.
Appl Environ Microbiol. 2006 Apr;72(4):2829-36. doi: 10.1128/AEM.72.4.2829-2836.2006.
Since the stomach is a first line of defense for the host against ingested microorganisms, an ex vivo swine stomach contents (SSC) assay was developed to search for genes important for Salmonella enterica serovar Typhimurium survival in the hostile gastric environment. Initial characterization of the SSC assay (pH 3.87) using previously identified, acid-sensitive serovar Typhimurium mutants revealed a 10-fold decrease in survival for a phoP mutant following 20 min of challenge and no survival for mutants of rpoS or fur. To identify additional genes, a signature-tagged mutagenesis bank was constructed and screened in the SSC assay. Nineteen mutants were identified and individually analyzed in the SSC and acid tolerance response assays; 13 mutants exhibited a 10-fold or greater sensitivity in the SSC assay compared to the wild-type strain, but only 3 mutants displayed a 10-fold or greater decrease in survival following pH 3.0 acidic challenge. Further examination determined that the lethal effects of the SSC are pH dependent but that low pH is not the sole killing mechanism(s). Gas chromatography analysis of the SSC revealed lactic acid levels of 126 mM. Upon investigating the effects of lactic acid on serovar Typhimurium survival in a synthetic gastric fluid, not only was a concentration- and time-dependent lethal effect observed, but the phoP, rpoS, fur, and pnp genes were identified as involved in protection against lactic acid exposure. These studies indicate a role in gastric survival for several serovar Typhimurium genes and imply that the stomach environment is defined by more than low pH.
由于胃是宿主抵御摄入微生物的第一道防线，因此开发了一种离体猪胃内容物（SSC）检测方法，以寻找对肠炎沙门氏菌鼠伤寒血清型在恶劣胃环境中生存至关重要的基因。使用先前鉴定的酸敏感鼠伤寒血清型突变体对SSC检测（pH 3.87）进行的初步表征显示，在攻击20分钟后，phoP突变体的存活率下降了10倍，而rpoS或fur突变体则无法存活。为了鉴定其他基因，构建了一个签名标签诱变文库，并在SSC检测中进行筛选。鉴定出19个突变体，并分别在SSC和耐酸反应检测中进行分析；与野生型菌株相比，13个突变体在SSC检测中表现出10倍或更高的敏感性，但只有3个突变体在pH 3.0酸性攻击后存活率下降了10倍或更多。进一步研究确定，SSC的致死作用是pH依赖性的，但低pH不是唯一的杀伤机制。对SSC的气相色谱分析显示乳酸水平为126 mM。在研究乳酸对合成胃液中鼠伤寒血清型存活率的影响时，不仅观察到了浓度和时间依赖性的致死作用，而且还确定phoP、rpoS、fur和pnp基因参与了对乳酸暴露的保护。这些研究表明，几个鼠伤寒血清型基因在胃内存活中发挥作用，并暗示胃环境不仅仅由低pH定义。
