Data_Sheet_1_Role of LptD in Resistance to Glutaraldehyde and Pathogenicity in Riemerella anatipestifer.xlsx

Riemerella anatipestifer is a gram-negative bacterium that causes disease in ducks and other birds. Despite being an important pathogen in poultry, the pathogenesis and drug resistance mechanisms of this bacterium are poorly understood. An analysis of our unpublished RNA-Seq data showed that lptD, a gene encoding one of the lipopolysaccharide transport components, is transcribed at higher levels in strain CH-1 than in strain ATCC11845. In addition, strain CH-1 has been shown to display broader drug resistance than strain ATCC11845. Since LptD is involved in LPS biogenesis and drug resistance, we wondered if lptD is associated with increased R. anatipestifer resistance to glutaraldehyde, a disinfectant used in the production industry. In this study, the minimal inhibitory concentration (MIC) of glutaraldehyde for strain CH-1 was determined to be 0.125% (vol/vol), whereas an MIC of 0.05% (vol/vol) was observed for strain ATCC11845. Furthermore, the level of lptD transcription in strain CH-1 was consistently 2-fold higher than that observed in strain ATCC11845. Moreover, lptD transcription was upregulated in both strains at a subinhibitory concentration of glutaraldehyde. The role of lptD in R. anatipestifer was further assessed by constructing an ATCC11845 mutant strain with low lptD expression, R. anatipestifer ATCC11845 lptD−. The growth of R. anatipestifer ATCC11845 lptD− was severely impaired, and this strain was more susceptible than the wild-type strain to glutaraldehyde. Moreover, compared to the wild-type strain, R. anatipestifer ATCC11845 lptD− exhibited decreased biofilm formation and was more sensitive to duck serum. Finally, low lptD expression led to decreased colonization in ducklings. These results suggest that LptD is involved in R. anatipestifer glutaraldehyde resistance and pathogenicity.

鸭疫里默氏杆菌（Riemerella anatipestifer）是一种可引发鸭类及其他禽类感染的革兰氏阴性菌。尽管该菌是家禽养殖业的重要致病菌，但其致病机制与耐药机制仍未被充分阐明。对本课题组未发表的RNA测序（RNA-Seq）数据进行分析后发现，编码脂多糖转运复合体组分的基因lptD在菌株CH-1中的转录水平显著高于菌株ATCC11845。此外，已有研究证实菌株CH-1的耐药谱较ATCC11845更为广泛。鉴于LptD参与脂多糖生物合成与耐药调控过程，本研究旨在探究lptD是否与鸭疫里默氏杆菌对工业生产常用消毒剂戊二醛的抗性增强相关。本研究测定了戊二醛对菌株CH-1的最低抑菌浓度（MIC）为0.125%（体积/体积），而菌株ATCC11845的最低抑菌浓度仅为0.05%（体积/体积）。进一步检测发现，菌株CH-1中lptD的转录水平始终较ATCC11845高出2倍。同时，在亚抑菌浓度戊二醛的处理下，两株菌的lptD转录水平均出现上调。为进一步验证lptD在鸭疫里默氏杆菌中的功能，本研究构建了lptD低表达的ATCC11845突变株（R. anatipestifer ATCC11845 lptD⁻）。实验结果显示，该突变株的生长能力严重受损，且相较于野生型菌株，其对戊二醛的敏感性显著升高。此外，与野生型菌株相比，鸭疫里默氏杆菌ATCC11845 lptD⁻的生物被膜形成能力下降，同时对鸭血清的敏感性更高。最终实验证实，低水平的lptD表达会导致该菌在鸭体内的定植能力显著降低。上述结果表明，LptD参与了鸭疫里默氏杆菌对戊二醛的抗性及致病过程。