| Campylobacter jejuni is one of the most common zoonotic pathogens associated with human diarrhea in both developing and developed countries. Guillain-Barre syndrome is the most common acute flaccid paralysis due to an autoimmune disorder in nature. A considerable number of GBS patients present with a prior history of Campylobacteriosis, and GBS is considered a sequela of infections caused specifically by Campylobacter jejuni. Althongh the pathogenesis of Campylobacter jejuni is still unclear, it’s survival in the environment and a higher incidence are closely related to biofilm formation which plays a significant role in Campylobacter jejuni transmitted infections. A biofilm is a multicellular layer of bacteria attached to surfaces, biological material, or body cavity and embedded within a matrix of extracellular polymeric substances (EPS). Bacteria with biofilm can increase their resistance to external environment, organism’s immunity, hot and cold, cause persistent infection and repeated infection. Therefore, the basic research for biofilm has a very important practical significance. In this study, we first determined the optimal condtion of Campylobacter jejuni biofilm formation, and developed the detection methods for Campylobacter jejuni biofilm formation. The biofilm-forming ability of179isolates were determined. Meanwhile, the biofilm-related genes were also detected in179isolates. Furthermore, two-dimensional gel electrophoresis was used for analysis proteins of biofilm. RT-PCR was aslo used to analysis expression level of mRNA. All this study will provide some useful information for further research on Campylobacter jejuni biofilm.In this study, firstly the amount ofNCTC11168biofilm formed on polystyrene96well plate surface was determined under different incubation time, different oxygen concentration, different temperature and the different medium conditions. The results showed that when it was cultured at37℃,10%O2conditions, Campylobacter jejuni can form the more mature biofilm in the MH broth for72h. Then we used the tube method, microscopy and scanning electron microscopy to observe Campylobacter jejuni biofilm. The results revealed that Campylobacter jejuni can easily form biofilm in the gas-liquid interface, it was clearly observed the biofilm formation using crystal violet staining. Using Congo red/carbol fuchsin staining, the structure of the biofilm was directly observed by microscopy. Using the scanning electron microscopy, the structure of biofilm was more intuitive, three-dimensional, even subtle structure. Different detection methods can be selected depending on the situation of the actual testing.It was quantitatively determined the bio film-forming ability of179isolates from different sources. According to the OD value of the strains, they were divided into weak biofilm-forming ability, medium biofilm-forming ability and strong bio film-forming ability. The results showed that60isolates belong to weak biofilm-forming ability while69are medium ability and50of strong. On the other hand, the distribution of bio film-forming ability from different source was similar to the overall trend, and also there was no significant difference between difference strains.Two sensitive antibiotics of Campylobacter jejuni:cefotaxime and streptomycin, were used to measure the MBC (minimum bactericidal concentration) in planktonic and biofilm of Campylobacter jejuni. The results showed that the MBC of two antibiotics in the biofilm was higher several times than in planktonic. Thus, the formation of biofilms maybe was an important factor of antimicrobial resistance.In this study, according to the literature we choosed9biofilm formation-related genes:the flaA (flagellin gene),fliA (flagellar biosynthesis factor), Luxs (quorum sensing), rpoN(RNA polymerase factor), cmec (outer membrane channel protein), cadF(adhesion), racR (adhesion colonization-related genes), peblA (adhesion protein), Peb4(cell binding protein). These biofilm-related genes were detected in179isolates. The results showed that all nine selected genes had a higher distribution rate among isolates belonging to different group of biofilm formation ability. There was no direct positive and negative expression relationship between single gene and biofilm forming ability. It also revealed that the formation of biofilms maybe the result of co-expression and regulation of multiple genes in a certain extent.Besides, we cultured Campylobacter jejuni in different states:planktonic and biofilm. And the bacterial proteins were extracted using TCA precipitation method for comparative proteomic analysis. The immobilized pH gradient two-dimensional gel electrophoresis was engaged in and got a set of reproducible2-DE gel maps, and then differentiated proteins were filtered out using PDQuest software, harvested from the2-DE gels for MALDI-TOF/TOF-MS analysis. The biological information of predicted proteins was identified based on peptide mass fingerprinting searched in MASCOT database. The10differentiated proteins were successfully obtained. These proteins involve the functions related to biosynthesis, oxidoreductases and transfer enzymes. RT-PCR was aslo used to analysis expression level of mRNA. Both protein and mRNA expression level of groel were higher in biofilm. It may be closely related with biofilm formation. All these results lay a foundation for further study of Campylobacter jejuni biofilm proteomics. |
