The function of Autoinducer-2 (AI-2) which acts as the signal molecule of LuxS-mediated quorum sensing, is regulated through the lsr operon (which includes eight genes: lsrK, lsrR, lsrA, lsrC, lsrD, lsrB, lsrF, and lsrG). However, the functions of the lsr operon remain unclear in avian pathogenic Escherichia coli (APEC), which causes severe respiratory and systemic diseases in poultry. In this study, the presence of the lsr operon in 60 APEC clinical strains (serotypes O1, O2, and O78) was investigated and found to be correlated with serotype and has the highest detection rate in O78. The AI-2 binding capacity of recombinant protein LsrB of APEC (APEC-LsrB) was verified and was found to bind to AI-2 in vitro. In addition, the lsr operon was mutated in an APEC strain (APEC94Δlsr(Cm)) and the mutant was found to be defective in motility and AI-2 uptake. Furthermore, deletion of the lsr operon attenuated the virulence of APEC, with the LD₅₀ of APEC94Δlsr(Cm) decreasing 294-fold compared with wild-type strain APEC94. The bacterial load in the blood, liver, spleen, and kidneys of ducks infected with APEC94Δlsr(Cm) decreased significantly (p < 0.0001). The results of transcriptional analysis showed that 62 genes were up-regulated and 415 genes were down-regulated in APEC94Δlsr(Cm) compared with the wild-type strain and some of the down-regulated genes were associated with the virulence of APEC. In conclusion, our study suggests that lsr operon plays a role in the pathogenesis of APEC.

Mastitis caused by Streptococcus uberis (S. uberis) is a common and difficult-to-cure clinical disease in dairy cows. In this study, the role of Toll-like receptors (TLRs) and TLR-mediated signaling pathways in mastitis caused by S. uberis was investigated using mouse models and mammary epithelial cells (MECs). We used S. uberis to infect mammary glands of wild type, TLR2^−/− and TLR4^−/− mice and quantified the adaptor molecules in TLR signaling pathways, proinflammatory cytokines, tissue damage, and bacterial count. When compared with TLR4 deficiency, TLR2 deficiency induced more severe pathological changes through myeloid differentiation primary response 88 (MyD88)-mediated signaling pathways during S. uberis infection. In MECs, TLR2 detected S. uberis infection and induced mitochondrial reactive oxygen species (mROS) to assist host in controlling the secretion of inflammatory factors and the elimination of intracellular S. uberis. Our results demonstrated that TLR2-mediated mROS has a significant effect on S. uberis-induced host defense responses in mammary glands as well as in MECs.

The luxS gene is required for autoinducer-2 (AI-2) synthesis in many bacterial species. AI-2 is taken up by a specific receptor to regulate multiple bacterial activities. However, the lack of methods to identify AI-2 receptors has impeded investigations into the roles of AI-2. Here, a luxS mutant of Escherichia coli strain BL21 (DE3) was constructed (named BL21∆luxS), and the recombinant LsrB protein of Salmonella enterica was expressed in BL21∆luxS and BL21 cells, which were named LsrB (BL21∆luxS) and LsrB (BL21), respectively. The results of the activity of recombinant LsrB binding showed that LsrB (BL21) bound to endogenous AI-2 (produced from BL21 strain), while LsrB (BL21∆luxS) did not (as BL21∆luxS cannot produce AI-2). However, the results of recombinant LsrB binding showed that LsrB (BL21∆luxS) can bind exogenous AI-2, which was released from LsrB (BL21∆luxS) at 55 °C for 10 min, while LsrB (BL21) could not bind exogenous AI-2 (due to binding of endogenous AI-2 before). Furthermore, analysis of the thermal stability of AI-2 showed that that AI-2 activity was relatively high at incubation temperatures below 65 °C. These findings will be beneficial for screening of new AI-2 receptors in different bacterial species.

Escherichia coli (E. coli) has the ability to induce clinical and subclinical mastitis in dairy cows, causing a huge loss for the dairy industry. In this study, 51 subclinical mastitis isolates and 36 clinical mastitis isolates from eight provinces of China between 2019 and 2021 were used to investigate the differences in their biological characteristics. The results showed that B1 (52.9%) and A (39.1%) were the predominant phylogroups; R1 (50.6%) was the predominant lipopolysaccharide (LPS) core type; and 44 STs (ST10 and ST58 were the most sequence-prevalent STs) and 2 new STs (ST14828 and ST14829) were identified; however, no significant difference was observed between the clinical and subclinical group strains. To compare the virulence gene differences between the clinical and subclinical mastitis-related isolates, 18 common virulence genes (including afaE, eaeA, papC, saa, sfa, ompA, aer, irp2, iucD, escV, sepD, east1, estB, stx2e, CNF1, cba, hlyA and traT) were determined using the PCR method. The results showed that the detection rates of traT, irp2 and iucD in clinical mastitis isolates were significantly higher than those in subclinical mastitis isolates (p ˂ 0.05). Meanwhile, subclinical-group E. coli had stronger biofilm formation abilities than the clinical group (p < 0.05) in 78 (89.7%) mastitis-related E. coli that could form biofilms. Furthermore, 87 mastitis-related E. coli showed severe resistance against tetracycline (37.9%), ampicillin (36.8%), streptomycin (34.5%) and cotrimoxazole (28.7%); their most prevalent resistance genes were blaCTX-M (33.3%), tetA (27.6%), sul2 (18.4%) and strB (28.7%). It was noteworthy that the clinical-group strains had a higher resistance against ampicillin and possessed higher amounts of the resistance gene blaCTX-M (p < 0.05) compared to the subclinical group. This study aims to provide references for preventing the E. coli isolates from inducing different types of mastitis.

Mastitis caused by Streptococcus uberis is a hazardous clinical disease in dairy animals. In this study, the role of Toll-like receptors (TLRs) and TLR-mediated signaling pathways in mastitis caused by S. uberis was investigated using mouse models and mammary epithelial cells (MECs). We used S. uberis to infect mammary glands of wild type, TLR2 ^−/− and TLR4 ^−/− mice and quantified the adaptor molecules in TLR signaling pathways, proinflammatory cytokines, tissue damage and bacterial count in mammary glands. When compared with TLR4 deficiency, TLR2 deficiency induced more severe pathological changes through myeloid differentiation primary response 88 (MyD88)-mediated signaling pathways during S. uberis infection. In MECs, TLR2 detected S. uberis infection and induced mitochondrial reactive oxygen species (mROS) to assist host control of secretion of inflammatory factors and elimination of intracellular S. uberis . Our results demonstrate that TLR2-mediated mROS have a significant effect on S. uberis -induced host defense responses in mammary glands as well as MECs. Author summary: S. uberis contributes significantly to global mastitis and remains a major obstacle for inflammation elimination due to its ability to form persistent infection in mammary tissue. The Toll-like receptor (TLR) family plays a significant role in identifying infections of intracellular bacteria and further triggering inflammatory reactions in immune cells. However, the detailed molecular mechanism by which TLR is regulated, and whether MECs, as the main cells in mammary gland, are tightly involved in these processes is poorly understood. Here, we used S. uberis to infect mammary glands of wild type, TLR2 ^−/− , TLR4 ^−/− mice and MECs to assess pathogenesis, proinflammatory cytokines, ROS as well as mROS levels during infection. We found that during S . uberis infection, it is TLR2 deficiency that induced more severe pathological changes through MyD88-mediated signaling pathways. In addition, our work demonstrates that mROS mediated by TLR2 has an important role in host defense response to combat S. uberis infection in mammary glands as well as MECs.