Novel pyridinium cationic pleuromutilin analogues overcoming bacterial multidrug resistance.

A series of pyridinium cation-substituted pleuromutilin analogues were designed, synthesized and evaluated for their antibacterial activities in vitro and in vivo. Most derivatives showed potent antibacterial activities, especially e4 that displayed the highest antibacterial activity against multi-drug resistant bacteria and was subjected to time-kill kinetics, resistance studies, cytotoxicity and molecular docking assays. Molecular docking results, scanning electron microscopy and o-nitrophenyl-ß-galactopyranoside tests showed that e4 not only inhibited bacterial protein synthesis but also disrupted bacterial cell walls. Compound e4 showed an ED50 of 5.68 mg/kg against multi-drug resistant Staphylococcus aureus in infected mice model. In in vivo and in vitro toxicity tests, e4 showed low toxic effects with an LD50 of 879 mg/kg to mice. These results suggest that compound e4 may be considered as a new therapeutic candidate for bacterial infections.

Molecular Epidemiology and Antimicrobial Resistance of Outbreaks of Klebsiella pneumoniae Clinical Mastitis in Chinese Dairy Farms.

Klebsiella pneumoniae is an opportunistic pathogen that causes serious infections in humans and animals. However, the availability of epidemiological information on clinical mastitis due to K. pneumoniae is limited. To acquire new information regarding K. pneumoniae mastitis, data were mined about K. pneumoniae strains on dairy cattle farms (farms A to H) in 7 Chinese provinces in 2021. Hypermucoviscous strains of K. pneumoniae were obtained by the string test. MICs of antimicrobial agents were determined via the broth microdilution method. Ten antimicrobial resistance genes and virulence genes were identified by PCR. The prevalence of K. pneumoniae was 35.91% (65/181), and 100% of the bacteria were sensitive to enrofloxacin. Nine antimicrobial resistance genes and virulence genes were identified and compared among farms. The hypermucoviscous phenotype was present in 94.44% of isolates from farm B, which may be a function of the rmpA virulence gene. Based on these data, the multidrug-resistant strains SD-14 and HB-21 were chosen and sequenced. Genotypes were assayed for K. pneumoniae isolates from different countries and different hosts using multilocus sequence typing (MLST). Ninety-four sequence types (STs) were found, and 6 STs present a risk for spreading in specific regions. Interestingly, ST43 was observed in bovine isolates for the first time. Our study partially reveals the current distribution characteristics of bovine K. pneumoniae in China and may provide a theoretical basis for the prevention and treatment of bovine K. pneumoniae mastitis. IMPORTANCE K. pneumonia is ubiquitous in nature and infects a wide range of hosts, including animals, and humans. It is one of the leading inducements of clinical mastitis (CM) in dairy cows, a prevalent and costly disease that is predominantly associated with bacterial infection. In general, CM caused by Gram-negative bacteria is more difficult to cure than that associated with Gram-positive pathogens, with an average cost per case of 211.03 U.S. dollars (USD) for Gram-negative bacterial infections compared with 133.73 USD for Gram-positive bacterial CM cases. After Escherichia coli, K. pneumoniae is the second most common Gram-negative cause of bovine CM, but it is the most detrimental in terms of decreased milk yield, discarded milk, treatment costs, death, and culling. In view of the economic implications of K. pneumoniae infection in dairy farming, research into population structure and antibiotic resistance is particularly important.

Identification of novel biofilm genes in avian pathogenic Escherichia coli by Tn5 transposon mutant library.

Avian pathogenic Escherichia coli (APEC) is the main pathogens that inflict the poultry industry. Biofilm as the pathogenic factors of APEC, which can enhance the anti-host immune system of APEC and improve its survival in the environment. In order to screen for new genes related to APEC biofilm. The APEC strain APEC81 was used to construct a mutant library by Tn5 insertion mutagenesis. Moreover the 28 mutant strains with severely weakened biofilm were successfully screened from 1500 mutant strains by crystal violet staining, in which 17 genes were obtained by high-efficiency thermal asymmetric interlaced PCR. The reported genes include 3 flagella genes (fliS, fliD, and fliR), 4 curli fimbriae genes (csgD, csgA, csgF, and csgG) and 3 type 1 fimbriae genes (fimA, fimD, and fimC). The novel genes include 3 coenzyme genes (gltA, bglX, and mltF) and 4 putative protein genes (yehE, 07045, 11735, 11255). To investigate whether these 17 genes co-regulate the biofilm, the 17 identified genes were deleted from APEC strain APEC81. The results showed that except for the 11735 and 11255 genes, the deletion of 15 genes significantly reduced the biofilm formation ability of APEC81 (P < 0.05). The result of rdar (red, dry and rough) colony morphology showed that curli fimbriae genes (csgD, csgA, csgF, and csgG) and other functional genes (fimC, glxK, yehE, 07045, and 11255) affected the colony morphology. In particular, the hypothetical protein YehE had the greatest influence on the biofilm. It was predicted to have the same structure as the type 1 fimbria protein. When yehE was deleted, the fimE transcription was up-regulated, and the fimA and fimB transcription were down-regulated, resulting in a decrease in type 1 fimbriae. Hence, the yehE mutant significantly reduced the biofilm and the adhesion and invasion ability to cells (P < 0.05). This study identified 5 novel genes (gltA, bglX, mltF, yehE, and 07045) related to biofilm formation and confirmed that yehE affects biofilm formation by type 1 fimbriae, which will benefit further study of the mechanism of biofilm regulation in APEC.

Effect of nutritional and environmental conditions on biofilm formation of avian pathogenic Escherichia coli.

AIMS: To study the effects of environmental stress and nutrient conditions on biofilm formation of avian pathogenic Escherichia coli (APEC). METHODS AND RESULTS: The APEC strain DE17 was used to study biofilm formation under various conditions of environmental stress (including different temperatures, pH, metal ions, and antibiotics) and nutrient conditions (Luria-Bertani [LB] and M9 media, with the addition of different carbohydrates, if necessary). The DE17 biofilm formation ability was strongest at 25°C in LB medium. Compared to incubation at 37°C, three biofilm-related genes (csgD, dgcC, and pfs) were significantly upregulated and two genes (flhC and flhD) were downregulated at 25°C, which resulted in decreased motility. However, biofilm formation was strongest in M9 medium supplemented with glucose at 37°C, and the number of live bacteria was the highest as determined by confocal laser scanning microscopy. The bacteria in the biofilm were surrounded by a thick extracellular matrix, and honeycomb-like or rough surfaces were observed by scanning electron microscopy. Moreover, biofilm formation of the DE17 strain was remarkably inhibited under acidic conditions, whereas neutral and alkaline conditions were more suitable for biofilm formation. Biofilm formation was also inhibited at specific concentrations of cations (Na+ , K+ , Ca2+ , and Mg2+ ) and antibiotics (ampicillin, chloramphenicol, kanamycin, and spectinomycin). The real-time quantitative reverse transcription PCR showed that the transcription levels of biofilm-related genes change under different environmental conditions. CONCLUSIONS: Nutritional and environmental factors played an important role in DE17 biofilm development. The transcription levels of biofilm-related genes changed under different environmental and nutrient conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings suggest that nutritional and environmental factors play an important role in APEC biofilm development. Depending on the different conditions involved in this study, it can serve as a guide to treating biofilm-related infections and to eliminating biofilms from the environment.

Effect of Antibiotics on the Colonization of Live Attenuated Salmonella Enteritidis Vaccine in Chickens.

Salmonellosis, caused by Salmonella Enteritidis, is a prevalent zoonosis that has serious consequences for human health and the development of the poultry sector. The Salmonella Enteritis live vaccine (Sm24/Rif12/Ssq strain) is used to prevent Salmonella Enteritidis around the world. However, in some parts of the world, poultry flocks are frequently raised under intensive conditions, with significant amounts of antimicrobials to prevent and treat disease and to promote growth. To investigate whether antibiotic use influences the colonization of orally administered Salmonella live vaccines, 240 1-day-old specific pathogen-free chicks were randomly divided into 24 groups of 10 animals for this study. The different groups were treated with different antibiotics, which included ceftiofur, amoxicillin, enrofloxacin, and lincomycin-spectinomycin. Each group was immunized 2, 3, 4, and 5 days after withdrawal, respectively. At 5 days after immunization, the blood, liver, and ceca with contents were collected for the isolation of the Salmonella live vaccine strain. The result showed that no Salmonella vaccine strain was isolated in the blood and liver of the chicks in those groups. The highest number of Salmonella vaccine strains was isolated in the cecum from chicks vaccinated 2 days after ceftiofur withdrawal, and no Salmonella vaccine strain was isolated from the cecum in chicks immunized 3 days after ceftiofur withdrawal. Among the chickens immunized 4 days after the withdrawal of amoxicillin, enrofloxacin, and lincomycin-spectinomycin, the number of Salmonella vaccine colonization in the cecum was the highest, which was higher than that of the chickens immunized at other withdrawal interval (2, 3, and 5 days) groups and was higher than that of the chickens without treatment (P < 0.05). This study provides a reference for the effective use of the Salmonella Enteritidis live vaccine and key antibiotics commonly utilized in the poultry industry.

Regulation of ydiV-induced biological characteristics permits Escherichia coli evasion of the host STING inflammatory response.

Mammary gland-derived Escherichia coli (E. coli) is an important pathogen causing dairy cow mastitis. YdiV, with EAL-like domains, inhibits flagellum biogenesis and motility and affects c-di-GMP (eubacterial signaling molecule) concentration changes in bacteria. However, the pathophysiological role of ydiV in host-pathogen cross-talk still needs to be elucidated. In this study, firstly constructed the ydiV mutant (NJ17ΔydiV) and ydiV complementary (cNJ17ΔydiV) E. coli strains to infect mouse mammary epithelial cells (EpH4-Ev) and macrophages (RAW264.7), as well as mouse mammary glands, respectively. Then biological characteristics, adaptor molecules in related signaling pathways, proinflammatory cytokines and the extent of host cell damage was evaluated. Compared with E. coli NJ17 infected mice, the bacterial load in the mammary gland of NJ17ΔydiV was significantly lower and the extent of the damage was alleviated. Notably, the deletion of ydiV significantly aggravated cell damage in RAW264.7 cells and compared with the wild-type strain, NJ17ΔydiV significantly activated the STING/TBK1/IRF3 pathway in macrophages. In EpH4-Ev cells, although STING did not sense E. coli NJ17 invasion, IRF3 was activated by the NJ17ΔydiV strain. Taken together, ydiV deletion significantly affects a variety of biological characteristics and induces severe cell damage, while the STING/TBK1/IRF3 pathway actively participated in pathogen elimination in the host. This study highlights a new role for ydiV in E. coli infection and provides a foundation for further studies to better understand host-bacteria interactions and potential prophylactic strategies for infectious diseases.

The Deletion of yeaJ Gene Facilitates Escherichia coli Escape from Immune Recognition.

Mammary gland-derived Escherichia coli is an important pathogen causing dairy cow mastitis. Mammary gland mucosal immunity against infectious E. coli mainly depends on recognition of pathogen-associated molecular patterns by innate receptors. Stimulator of interferon (IFN) gene (STING) has recently been the dominant mediator in reacting to bacterial intrusion and preventing inflammatory disorders. In this study, we first proved that the diguanylate cyclase YeaJ relieves mouse mammary gland pathological damage by changing E. coli phenotypic and host STING-dependent innate immunity responses. YeaJ decreases mammary gland circular vacuoles, bleeding, and degeneration in mice. In addition, YeaJ participates in STING-IRF3 signaling to regulate inflammation in vivo. In vitro, YeaJ decreases damage to macrophages (RAW264.7) but not to mouse mammary epithelial cells (EpH4-Ev). Consistent with the results in mouse mammary glands, YeaJ significantly activates the STING/TBK1/IRF3 pathway in RAW264.7 macrophages as well. In conclusion, the deletion of yeaJ facilitates E. coli NJ17 escape from STING-dependent innate immunity recognition in vitro and in vivo. This study highlights a novel role for YeaJ in E. coli infection, which provides a better understanding of host-bacterium interactions and potential prophylactic strategies for infections. IMPORTANCE E. coli is the etiological agent of environmental mastitis in dairy cows, which causes massive financial losses worldwide. However, the pathophysiological role of YeaJ in the interaction between E. coli and host remains unclear. We found that YeaJ significantly influences various biological characteristics and suppresses severe inflammatory response as well as greater damage. YeaJ alleviates damage to macrophages (RAW264.7) and mouse mammary gland. Moreover, these effects of YeaJ are achieved at least partial by mediating the STING-IRF3 signaling pathway. In conclusion, the deletion of yeaJ facilitates E. coli NJ17 escape from STING-dependent innate immunity recognition in vitro and in vivo. This study is the basis for further research to better understand host-bacterium interactions and provides potential prophylactic strategies for infections.

Taurine Alleviates Streptococcus uberis-Induced Inflammation by Activating Autophagy in Mammary Epithelial Cells.

Streptococcus uberis infection can cause serious inflammation and damage to mammary epithelial cells and tissues that can be significantly alleviated by taurine. Autophagy plays an important role in regulating immunity and clearing invasive pathogens and may be regulated by taurine. However, the relationships between taurine, autophagy, and S. uberis infection remain unclear. Herein, we demonstrate that taurine augments PTEN activity and inhibits Akt/mTOR signaling, which decreases phosphorylation of ULK1 and ATG13 by mTOR and activates autophagy. Activating autophagy accelerates the degradation of intracellular S. uberis, reduces intracellular bacterial load, inhibits over-activation of the NF-κB pathway, and alleviates the inflammation and damage caused by S. uberis infection. This study increases our understanding of the mechanism through which taurine regulates autophagy and is the first to demonstrate the role of autophagy in S. uberis infected MAC-T cells. Our study also provides a theoretical basis for employing nutritional elements (taurine) to regulate innate immunity and control S. uberis infection. It also provides theoretical support for the development of prophylactic strategies for this important pathogen.

TLR2 Signaling Pathway Combats Streptococcus uberis Infection by Inducing Mitochondrial Reactive Oxygen Species Production.

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.

Role of Toll-like receptor 2 against Streptococcus uberis infection in primary mouse mammary epithelial cells.

Mammary epithelial cells (MECs) play an important role against Streptococcus uberis infection which is one of the main causes of bovine mastitis and a potential threat to human health. Toll-like receptors (TLRs) and their mediated signaling pathways are critical in both innate and infection responses, yet their roles in anti-S. uberis infection in MECs remains poorly defined. In this work we investigated the regulatory mechanisms of TLR2 in inflammatory responses, where WT and TLR2-/- mice were euthanized at 15-18 days gestation, and mammary gland tissues were collected aseptically. The mouse MECs (MMECs) were isolated by combined digestion with type I collagenase, hyaluronidase and trypsin. We challenged MMECs with S. uberis and quantified antioxidant capacity as well as reactive oxygen species (ROS), proinflammatory cytokines and cell damage at different times. The loss of TLR2 function in MMECs results in more serious cell damage, increased cell adhesion, and significantly decreased ROS and mitochondrial ROS (mROS) with bactericidal function in response to S. uberis infection. Moreover, it was observed that the antioxidant capacity declined, and the production of TLR2-mediated cytokines (except CXC ligand 15) also were reduced. We demonstrated that TLR2 can mediate cellular anti-infective processes in MMECs by regulating the production of ROS and mROS and the secretion of cytokines. The results suggest an unpredicted role of TLR2 in MMECs in response to S. uberis infection.

Lsr operon is associated with AI-2 transfer and pathogenicity in avian pathogenic Escherichia coli.

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.

Development of a multiplex PCR assay for the simultaneous and rapid detection of six pathogenic bacteria in poultry.

Escherichia coli, Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella spp. and Staphylococcus aureus are six bacterial pathogens of avian. However, these pathogens may cause many similar pathological changes, resulting in clinical isolates that are difficult to quickly and simultaneously detect and identify. Here, a multiplex polymerase chain reaction (m-PCR) assay is reported to rapidly identify targets genes (phoA, KMT1, ureR, toxA, invA, and nuc) of these six pathogens in clinical samples. Six pairs of specific primers were designed. The optimal reaction conditions, specificity, and sensitivity of the m-PCR assay were investigated. The results showed that betaine remarkably improved amplification of the target genes. Specific test results showed that all six pathogens were detected by the proposed m-PCR protocol without cross-amplification with viruses or parasites. Sensitivity test results showed that the m-PCR system could amplify the six target genes from bacterial genomes or cultures with template amounts of 500 pg or 2.8-8.6 × 103 colony forming units, respectively. Furthermore, the six bacterial pathogens isolated from the infected tissue samples were successfully identified. The proposed m-PCR assay is a useful tool to monitor and diagnose bacterial infection in birds with high specificity, sensitivity and throughput.

Use of a modified bacterial ghost lysis system for the construction of an inactivated avian pathogenic Escherichia coli vaccine candidate.

Vaccination is an effective strategy to prevent avian colibacillosis. Bacterial ghosts (BGs) are prepared by the controlled expression of the phiX174 gene E, which mediates the lysis of Gram-negative bacteria. Staphylococcal nuclease A may be used to produce BGs for further inactivation of host bacteria and elimination of residual genetic material. In this study, the double promoter lysis plasmid (pUC19-ΔcI857-E-rrnB-pL-SN) was successfully constructed and BGs were prepared at 37 °C. The cleavage efficiency of Escherichia coli BGs was 99.9%. Furthermore, to evaluate the immunological effects of the BG vaccines in chickens, a BG vaccine was prepared using the serotype O2 avian pathogenic Escherichia coli deletion strain (DE17ΔluxSΔaroA). The results showed that the BG vaccine was able to achieve over 90% immune protection against virulent challenge using the same serotype O2 strain (DE17 or CE35), while it showed poor cross-protection against serotypes O1 and O78 (data not shown). The enzyme-linked immunosorbent assay results showed that the antibody levels in the immunized groups were higher than in the control group (p < 0.05), with the BG group being the highest. The cytokine tests showed that the levels of interferon-γ in the BG immune group were higher than in the phosphate-buffered saline (PBS) control group (non-immune) (p < 0.01) and the formalin-inactivated vaccine immune group (p < 0.05), and the levels of tumor necrosis factor-α in the BG group were higher than in the formalin-inactivated vaccine (p > 0.05) and the PBS control groups (p < 0.05). In addition, pathological analysis revealed that the PBS control group showed typical fibrinous pericarditis and perihepatitis, whereas the immune group showed no obvious pathological changes. In summary, our findings provide a new strategy for the prevention and control of avian colibacillosis.

The role of the wzy gene in lipopolysaccharide biosynthesis and pathogenesis of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes severe respiratory and systemic diseases in poultry. The wzy gene encodes the O-antigen polymerase (Wzy), which plays an important role in the synthesis of the lipopolysaccharide (LPS) of bacteria. However, the function of the wzy gene in APEC remains unclear. Hence, in this study, a strain harboring a wzy gene mutant (DE17Δwzy) was constructed and the characteristics of this strain were analyzed. The results showed that mutant of wzy changed the phenotype of the LPS and affected serum agglutination of the O-antigen. Decreased motility and biofilm formation was also observed, but the endotoxin titer of the LPS in APEC was not affected. In addition, the wzy mutation significantly decreased the adherence and invasion to DF-1 cells, especially the survival abilities in duck serum and complement. Furthermore, an LD50 assay revealed that the virulence of mutant strain DE17Δwzy was attenuated 132-fold compared with wild-type strain DE17. Moreover, the bacterial load in the blood, liver, spleen, and kidneys of ducks infected with DE17Δwzy was decreased significantly compared with wild-type strain DE17 (p < 0.0001). These results confirmed that the wzy gene is associated with LPS biosynthesis and bacterial pathogenicity in APEC.

[Comparison of three methods for preparation of bacterial ghosts from avian pathogenic Escherichia coli].

Bacterial ghosts are bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology, which can be used as a new vaccine and delivery vector. In this study, a clinical isolate of avian pathogenic Escherichia coli (APEC) strain DE17 was used to prepare bacterial ghost through three different ways. The results showed that the cleavage efficiency of DE17 bacterial ghost was 99.9% with the lysis plasmid containing the PhiX174 lysis gene E. Scanning electron microscopy showed that transmembrane tunnels were formed in the middle or both ends of the cell envelope of DE17. Furthermore, the DE17 bacterial ghost was prepared with one of cell penetrating peptides (CPPs) named MAP (KLALKLALKALKAALKLA), which will completely inactivate DE17 (OD600=0.1) by 10 µmol/L MAP. The cell envelope showed a gully-like structure and obvious transmembrane tunnels were not found through the SEM. However, the DE17 could not be lysed by importing the lysis plasmid (pBV220-MAP), which was used to express MAP. The present study will benefit for research on bacterial ghost preparation methods and provide a reference for biosafety of bacterial ghost vaccines.

Different activated methyl cycle pathways affect the pathogenicity of avian pathogenic Escherichia coli.

The activated methyl cycle (AMC) regulates the cellular levels of S-adenosyl-l-homocysteine (SAH) in bacteria, which plays a crucial role in bacterial pathogenicity. There are two AMC pathways in bacteria: one is a two-step reaction pathway (named the LuxS/Pfs pathway) in which LuxS and Pfs catalyze the conversion of SAH to l-homocysteine and autoinducer-2 (AI-2), and the other is a one-step reaction (named the SahH pathway) mediated by S-adenosyl-l-homocysteine hydrolase (SahH), which completes this cycle without producing AI-2. In this study, we evaluated the effects of different AMC pathways on the pathogenicity of avian pathogenic Escherichia coli (APEC). The plasmid pSTV-sahH (containing the sahH gene of Pseudomonas aeruginosa) was transformed into the wild-type APEC strain DE17 (containing the LuxS/Pfs pathway) and the pfs mutant strain DE17Δpfs, which lacks the LuxS/Pfs pathway, to create the strains SahH-DE17Δpfs (containing the SahH pathway) and SahH-DE17 (containing the LuxS/Pfs and SahH pathways). The results showed that the different AMC pathways had different effects on the growth rate, AI-2 activity, and motility in APEC. Furthermore, we showed that the 50% lethal doses of the DE17Δpfs and SahH-DE17Δpfs strains were reduced by 650-fold and 52-fold, respectively, in ducklings, compared with that of the DE17 strain. The DE17Δpfs strain exhibited significantly reduced adherence and invasion (p<0.01). In addition, the DE17Δpfs and SahH-DE17Δpfs strains also showed reduced survival in vivo, as evidenced by significant (p<0.01) reductions in their bacterial loads in infected liver, spleen, kidney, and blood. This study suggests that different AMC pathways affect the pathogenesis of APEC.

The role of the ptsI gene on AI-2 internalization and pathogenesis of avian pathogenic Escherichia coli.

The LuxS/AI-2 quorum sensing mechanism can regulate the physiological functions of avian pathogenic Escherichia coli (APEC) through internalization of the small molecule autoinducer-2 (AI-2). The ptsI gene encodes enzyme I, which participates in the phosphotransferase system (PTS) that regulates the virulence and AI-2 internalization of bacteria. The aim of the present study was to determine the effect of ptsI on AI-2 internalization and other pathogenesis process in APEC using a ptsI mutant of the APEC strain DE17 (serotype O2), namely DE17ΔptsI. The results showed that deletion of the ptsI gene changed the rdar (red dry and rough) morphotype and decreased motility and biofilm formation in APEC (p < 0.05). Furthermore, scanning electron microscopy showed that the biofilm structure of DE17ΔptsI became sparse and more extracellular, as compared with the wild-type strain DE17. Moreover, AI-2 assay showed that AI-2 was internalized by DE17ΔptsI, while the recombinant PtsI protein had no AI-2 binding activity. Furthermore, deletion of the ptsI gene in APEC significantly increased adherence to DF-1 cells (p < 0.05). The 50% lethal dose of DE17ΔptsI was decreased by 17.8-fold and the bacterial loads of DE17ΔptsI were decreased by 13600-, 68.5-, 131-, and 3600-fold in the blood, liver, spleen, and kidney, respectively, as compared to the DE17. Moreover, histopathological analysis showed that the mutant DE17ΔptsI was associated with reduced pathological changes in the heart, liver, spleen, and kidney of ducklings, respectively, as compared to the wild-type strain DE17. The results of this study will benefit further studies on the functions of the ptsI in APEC.

LsrB-based and temperature-dependent identification of bacterial AI-2 receptor.

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.

[wzzE does not affect lipopolysaccharide phenotype of avian pathogenic Escherichia coli from clinical ducking].

Objective: wzz is involved in the synthesis of O antigen and plays a role in virulence in many gram-negative bacteria. However, the function of wzzE in avian pathogenic Escherichia coli (APEC) is unclear. The aim of this study is to elucidate the role of wzzE in the synthesis of LPS and virulence. Methods: Mutant strain with wzzE deletation was constructed. Biological characteristics of the wild and mutant strains, such as LD50, adherence and invasion to DF-1 cells, phenotype of LPS and endotoxin titer, were detected. Results: There was no significant difference in growth, adherence and invasion to DF-1 cells, serotype and the endotoxin titer between the wide strain and the mutant. However, compared with the wild-type strain, the virulence of DE17ΔwzzE had dropped by 10 times. Conclusion: wzzE is not involved in the synthesis of LPS in DE17, but participates in the pathogenic process of APEC. However, the mechanism of wzzE in APEC virulence needs to be studied in the future.

Deletion of luxS further attenuates the virulence of the avian pathogenic Escherichia coli aroA mutant.

In this study, an aroA-deletion avian pathogenic Escherichia coli (APEC) mutant (strain DE17ΔaroA) and aroA and luxS double deletion APEC mutant (strain DE17ΔluxSΔaroA) were constructed from the APEC DE17 strain. The results showed that as compared to DE17ΔaroA, the virulence of DE17ΔluxSΔaroA was further attenuated by 200- and 31.7-fold, respectively, in ducklings based on the 50% lethal dose. The adherence and invasion abilities of DE17ΔluxSΔaroA and DE17ΔaroA were reduced by 36.5%/42.5% and 25.8%/29.3%, respectively, as compared to the wild-type strain DE17 (p < 0.05 and 0.01, respectively). Furthermore, in vivo studies showed that the bacterial loads of DE17ΔluxSΔaroA were reduced by 8400- and 11,333-fold in the spleen and blood of infected birds, respectively, while those of DE17ΔaroA were reduced by 743- and 1000-fold, respectively, as compared to the wild-type strain DE17. Histopathological analysis showed both that the mutants were associated with reduced pathological changes in the liver, spleen, and kidney of ducklings, and changes in DE17ΔluxSΔaroA-infected ducklings were reduced to a greater degree than those infected with DE17ΔaroA. Real-time polymerase chain reaction analysis further demonstrated that the mRNA levels of virulence-related genes (i.e., tsh, ompA, vat, iucD, pfs, fyuA, and fimC) were significantly decreased in DE17ΔaroA, especially in DE17ΔluxSΔaroA, as compared to DE17 (p < 0.05). In addition, the deletion of aroA or the double deletion of aroA and luxS reduced bacterial motility. To evaluate the potential use of DE17ΔluxSΔaroA as a vaccine candidate, 50 7-day-old ducklings were divided randomly into five groups of ten each for the experiment. The results showed that the ducklings immunized with inactivated DE17, DE17ΔluxS, DE17ΔaroA, and DE17ΔluxSΔaroA were 70.0%, 70.0%, 70.0, and 80.0% protected, respectively, after challenge with strain APEC DE17. The results of this study suggest that the double deletion of luxS and aroA attenuated APEC pathogenicity and DE17ΔluxSΔaroA was more appropriate for development of a future vaccine against avian colibacillosis than DE17ΔaroA.