Preliminary Study On The Role Of Type ? Secretion System In Intraspecific Competition Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus is a common foodborne pathogenic bacteria in aquatic products,mainly distributed in sea and estuary sediments.It can adhere to the body surface of fish,shrimp,shellfish and other aquatic products,leading to Vibrio disease of aquatic products and harming the aquaculture industry.Food poisoning can also be caused by human ingestion of aquatic products contaminated with Vibrio parahaemolyticus.Food poisoning caused by this bacterium occurs frequently all over the world.It has been considered as one of the main pathogens of microbial food poisoning.Vibrio parahaemolyticus has many pathogenic factors and the secretion system,hemolysin,urease,adhesion factors play different roles in its pathogenicity.At present,the study on the secretory system of Vibrio parahaemolyticus is not mature.Vibrio parahaemolyticus has T3SS1,T3SS2,T6SS1 and T6SS2.However,its distribution in clinical and environmental isolates is mostly limited to structural genes,and the distribution of effector proteins is poorly understood.T6SS is believed to be a factor mediating intraspecific bacterial competition.Whether intraspecific competition is common in Vibrio parahaemolyticus and the role of T6SS in intra-species competition in Vibrio parahaemolyticus are still unclear.1.The distribution of T3SS and T6SS related genes in Vibrio parahaemolyticusT3SS structural protein and effector protein genes of 85 Vibrio parahaemolyticus?44clinical isolates and 41 environmental isolates?from different sources were detected by PCR.The T6SS genes were also detected.The results showed that all strains carried T3SS1 and T6SS2 structural genes.Among the 85 strains tested,34 strains contained all the T6SS genes tested,among which 32 strains were tdh⁺strain and 2 strain was tdh^-strain.The T6SS1-related genes tested showed different degrees of deletion in the T6SS1positive strain.80%of the tdh⁺strains carried T3SS2 genes,while tdh^-strains did not contain T3SS2 genes.Most clinical isolates are strains that carry both T3SS2 and T6SS1structural genes,while most environmental strains do not carry both genes.tdh⁺and tdh^-strain T3SS2 and T6SS1 genes were significantly different,showing a certain correlation.The vop Q and vop A/P genes showed different degrees of deletion in T3SS1 positive and T3SS2 positive strains,respectively.2.Quantity change of Vibrio parahaemolyticus under mixed cultureUsing Real-time PCR technology,the standard curve was established with tdh and trh as target genes.And the relationship between colony number and C_T value was determined.The quantity of Vibrio parahaemolyticus with different virulence genes was determined after mixed culture.The results showed that the strains with more virulence genes were not necessarily the dominant strains after mixed culture.Two survival modes were found in Vibrio parahaemolyticus:?1?One strain inhibits the growth of another;Among them,the number of dominant strains ranged from 9.31 to 9.58 log10 CFU/mL,while the inferior strains were only 3.27 to 3.98 log₁₀ CFU/mL,and the dominant strains were all T6SS1⁺genotype strains.?2?The two strains are stable coexistence.The number of the two strains ranged from 8.12 to 9.14 log₁₀ CFU/mL.Under mixed culture conditions,some strains of Vibrio parahaemolyticus?T6SS1⁺?had significant intraspectional competitive advantages and could inhibit the growth of other strains.3.To explore the effect of temperature,culture time and inoculation quantity on the survival mode.The influence of temperature on this survival mode was explored through co-culture at 30?and 37?respectively.The results showed that it was more advantageous for one strain to inhibit another strain at 30?.After mixed culture for 4h,8h,12h and 24h,Vibrio parahaemolyticus was quantitatively detected,and it was found that the inhibition of Vibrio parahaemolyticus was more obvious after culture for 8h.The results showed that under the condition of increasing the proportion of inferior bacteria,the difference of the number of strains after mixed culture was more obvious.4.Expression of T6SS related genes before and after mixed cultureStudies have shown that in many gram-negative bacteria,T6SS is the main cause of intraspecific competition.It has also been confirmed that intraspecific competition exists in Vibrio parahaemolyticus.The reasons for this phenomenon were preliminarily explored through the expression of T6SS related genes before and after mixed culture.It was found that the expression of T6SS related genes was up-regulated after mixed culture.This provides a clue for the study of the role of T6SS in Vibrio parahaemolyticus mediated intraspecific competition.