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.