Effects Of Candida Tropicalis On Physiological States Such As Clostridium Difficile Growth And Toxinization

Diarrhea is the primary factor affecting calf health,reducing production efficiency,and increasing feeding costs.Preventing and treating calf diarrhea is a key scientific and industrial issue that urgently needs to be addressed in dairy production.Clostridium difficile,as an important zoonotic intestinal pathogen,can cause antibiotic related diarrhea and intestinal inflammatory diseases in humans and various animals.Research has shown that the detection rate of Clostridium difficile in the intestines of diarrhea calves is approximately10-40%,with some reaching 60%.Recent studies have shown that the abundance of fungi in the genera Cladosporium and Aspergillus is significantly enriched in asymptomatic infections with Clostridium difficile,suggesting that these fungi may play a role in preventing,alleviating,or treating Clostridium difficile infection.Therefore,the aim of this study is to screen fungi that can inhibit the growth of Clostridium difficile,reveal their impact on the pathogenicity of Clostridium difficile,provide theoretical basis for preventing and treating diarrhea caused by Clostridium difficile in calves,and have important significance for maintaining the health of dairy animals and improving the production efficiency of the livestock industry.Exp.1 In vitro contact co-culture screening fungi that inhibit the growth of Clostridium difficileThis experiment focuses on 9 fungi（2 strains of Pichia＿fermentans,1 strain of Candida＿dubliniensis,4 strains of Candida＿tropicalis,1 strain of Aspergillus＿niger,and 1strain of Aspergillus＿proliferans）and Clostridium difficile ATCC 43255.By co culturing 9fungi with Clostridium difficile in vitro,the effects of each fungus on the growth and toxin production of Clostridium difficile were studied,and the fungi with the most obvious inhibitory effect on Clostridium difficile were selected as the research object of subsequent experiments,Further explore the impact on the pathogenicity of Clostridium difficile.The experiment was divided into a negative control group（pure BHI liquid culture medium）,a separate cultivation group of Clostridium difficile,and a co cultivation group of Clostridium difficile and fungi.Each group had 15 replicates,and 3 replicates were taken at 0,12,24,36,and 48 hours for preservation for testing.Under the premise of no pollution in the negative control group,the OD₆₀₀value,Clostridium difficile count,and m RNA expression levels of Tcd A,Tcd B,Tcd C,and Tcd R were used as evaluation indicators.The results showed that allOD₆₀₀values were consistent with the variation pattern of the strain growth curve over time.P.fermentans（1）,P.fermentans（2）,A.niger,C.tropicalis（1）,C.tropicalis（2）,C.tropicalis（3）fungi can all inhibit the growth of Clostridium difficile,among which C.tropicalis（3）has the most significant inhibitory effect on the growth of Clostridium difficile at 12 hours（P<0.001）,and significantly promotes the m RNA expression levels of Tcd A（24-48 hours,P<0.001）and Tcd B（12-48 hours,P<0.001）.This experiment shows that in vitro contact co cultivation of Clostridium difficile with fungi can inhibit the growth of Clostridium difficile,but promote the expression of Clostridium difficile toxin genes.Therefore,further exploration can be conducted on the impact of C.tropicalis（3）on the pathogenicity of Clostridium difficile.Exp.2 Effect of non-contact co-culture in vitro on the pathogenicity of Clostridium difficileOn the basis of Experiment 1,this experiment used C.tropicalis（3）and Clostridium difficile as the research objects,and conducted non-contact co culture in vitro using Transwell chamber to explore the effect of C.tropicalis（3）on the growth and pathogenicity of Clostridium difficile.The experiment was divided into a negative control group（pure BHI liquid culture medium）,a separate cultivation group of Clostridium difficile,and a co cultivation group of C.tropicalis（3）and Clostridium difficile.Each group had 15 replicates,and 3 replicates were taken at 0,12,24,36,and 48 hours,respectively,for preservation for testing.Under the premise of no pollution in the negative control group,the OD₆₀₀value,the amount of Clostridium difficile,the m RNA expression levels of Tcd A,Tcd B,Tcd C,and Tcd R,as well as the motility,adhesion,and biofilm formation ability of Clostridium difficile were used as evaluation indicators.The results showed that the OD₆₀₀value followed the variation pattern of the growth curve.C.tropicalis（3）had no significant effect on the growth of Clostridium difficile（P>0.05）,but significantly inhibited the m RNA expression levels of Tcd A（12,36 hours,P<0.001;24,48 hours,P<0.01）and Tcd B（12,48 hours,P<0.01;24hours,P<0.05;36 hours,P<0.001）.The 24-hour non-contact co culture group showed a decrease in the motility of Clostridium difficile,After 24 and 48 hours of cultivation,the ability of Clostridium difficile to form biofilm decreased（P<0.01）.This experiment shows that non contact co cultivation of C.tropicalis（3）with Clostridium difficile does not affect the growth of Clostridium difficile,but reduces its pathogenic ability by reducing its toxin gene expression,motility,and biofilm formation ability.In summary,P.fermentans（1）,P.fermentans（2）,A.niger,C.tropicalis（1）,C.tropicalis（2）,C.tropicalis（3）fungi can inhibit the growth of Clostridium difficile through in vitro contact co cultivation.Among them,C.tropicalis（3）co culture has the most significant inhibitory effect on Clostridium difficile,but it can promote the expression of Clostridium difficile toxin genes.Non contact co culture of C.tropicalis（3）with Clostridium difficile in vitro eliminates the inhibitory effect on the growth of Clostridium difficile,but can reduce the expression of Clostridium difficile toxin genes,slow down the motility of Clostridium difficile,and alleviate its pathogenicity.