Construction And Influence Factors Analysis Of Biofilm In Clinical Isolates Of Streptococcus Suis In Vitro

In this study, the biofilms of Streptococcus suis isolated from clinical were constructed and identified by crystal violet staining, silver staining, scanning electron microscopy, laser scanning confocal microscope in vitro, as well as the impact factors of Streptococcus suis biofilm formation were analyzed. Also the commonly used antimicrobial agents against biofilm formation were studied. All the results can provide a reference for clinical veterinary antimicrobial agents utilizing to be against the biofilm formation and provide some methods for further studying the mechanism of Streptococcus suis drug resistance caused by biofilm. Specific aspects are involved in this study as follows.The first part involved biofilm construction in Streptococcus suis isolates and identification by crystal violet staining, silver staining, scanning electron microscope and laser scanning confocal microscope.The biofilm formation of two Streptococcus suis isolates was detected on the polystyrene surface with microliter plate-crystal violet staining. The results showed that OD550value of Streptococcus suis type2YY060816strain and type9NJ-3strain cultured for1day were significantly different (P<0.01) compared with that of planktonic. The results indicated that both Streptococcus suis strainsYY060816and NJ-3can form biofilms at the bottom of polystyrene ELISA plates.The detected results by silver staining method showed that the blank glass cover slides and planktonic NJ-3glass slides stained by silver were uniforml and no particles and dense black-dyed woven material present. However, the glass cover slips, on which Streptococcus suis NJ-3and YY060816strains were cultured for2days, displayed the distribution of some kind of dense black-dyed woven materials, among which there were some sporadic thallus. The results further proved that the biofilms in the two Streptococcus suis strains have been successfully constructed in vitro.The results of scanning electron microscopy showed that organic membrane was rough on the surface and had obvious hole which was apt to biofilm formation. Planktonic Streptococcus suis YY060816strain was displayed spherical, among which no secretion appears. Streptococcus suis YY060816biofilm cultured for1day showed that the organic membrane surface with multi-layer bacteria were covered with clear, sticky-like substance. SEM image (×6000-fold) of Streptococcus suis NJ-3biofilm have thicker sticky-like substance, under which there are multi-layer coating thallus. Compared with NJ-3strains, the viscous-like substance were not so many as that of YY060816strain.CLSM detection with FITC-ConA and PI staining showed that the sterile coverslips were not dyed either green or red fluorescence, indicating the coverslip had not been contaminated by bacteria and polysaccharides and can be served as an ideal carrier. Planktonic Streptococcus suis NJ-3cultured after12h stained with FITC-ConA were found no green fluorescent material, indicating the planktonic NJ-3did not secrete any polysaccharides among itself. Biofilm caused by Streptococcus suis NJ-3strains were observed green fluorescent material wraped among thallus after stained with FITC-ConA. Also green fluorescent materials of three-days biofilm were denser than that of one-day biofilm, indicating the biofilm formation can be increased gradually with the culture time extended.The second part of the study was about the analysis of influential factors on Streptococcus suis biofilms formation in vitro. The crystal violet staining was used to study the influence of glucose and sodium chloride concentration in medium on the biofilm formation of Streptococcus suis and analyze biofilm-forming ability of46Streptococcus suis clinical isolates. The results showed that the adhesive ability of Streptococcus suis NJ-3and98012on the microliter plate were enhanced with the increase of glucose concentration in medium. When NaCl concentration in medium was at0.5%, the adhesive abilities of two strains were the strongest. However, the adhesion was diminished with the NaCl concentration further increased in the culture. Different Streptococcus suis clinical isolates posses different biofilm-forming ability. There were3strains (10.7%) with OD550exceeding0.1in28type2Streptococcus suis strains, and25strains (89.3%) with OD550less than0.1. In16type9of Streptococcus suis strains,13(81.3%) were with OD550value greater than0.1, and the OD550of three strains (18.7%) were less than0.1. OD550value of one strain of type1and type7strains were less than0.1.The results indicated that glucose and sodium chloride concentration in medium have effect on the formation of biofilm in Streptococcus suis. On the other hand, the serotype of Streptococcus suis was also involved in the biofilm formation. The third part was regarding the inhibition of6antimicrobial agents on biofilm formation in Streptococcus suis strains. Crystal violet staining was used to study6antimicrobial agents against Streptococcus suis biofilm formation in vitro. The results showed that penicillin, erythromycin, azithromycin, ciprofloxacin, ofloxacin, allicin can inhibit biofilm formation of Streptococcus suis at sub-MIC concentration and with increasing antimicrobial agents concentration, the inhibition effect can be gradually improved. Among six antimicrobial agents, the capabilities of ciprofloxacin and ofloxacin inhibited biofilm formation of Streptococcus suis were more stronger than other four antimicrobial agents.