The Roles Of Ail And PBP Encoding Genes In Mediating Cefotaxime-induced Enhancement Of Salmonella Biofilm Formation

Biofilm is a membrane-like structure formed by bacteria gathering on biological or non-biological surfaces,and can enhance the tolerance of bacteria to adverse environments.Salmonella is a common and important food-borne pathogen,and often exists in the form of biofilm in natural environment.Antibiotics often form different concentration gradients in production and application,which may lead to exposure of bacteria to sub-minimal inhibitory concentrations(sub-MICs),resulting in a series of adaptive changes in bacteria.Our previous study showed that 1/8 MIC of cefotaxime induced the enhancement of biofilm formation by Salmonella strain SH16SP46.According to the transcriptome results and the mechanism of cefotaxime action,ail gene and the genes encoding three penicillin-binding proteins(PBP)were selected for study.The gene deletion mutants of the strain SH16SP46 were constructed by Red homologous recombination,and such phenotypes as biofilm formation,bacterial morphology,growth characteristics,and cell membrane permeability were determined.The roles of different genes in the enhancement of Salmonella biofilm formation induced by cefotaxime were analyzed.The main results are as follows:1.The role of ail gene in mediating cefotaxime-induced enhancement of Salmonella biofilm formationThe ail gene deletion mutant(Δail)was constructed using the parental Salmonella strain SH16SP46.There was no significant difference in biofilm formation between the wild-type(WT)strain and Δail strain without the exposure of cefotaxime(P > 0.05).However,at the exposure to 1/8 MIC of cefotaxime,biofilm formation abilities by both strains were significantly enhanced(P < 0.05),and the Δail strain exhibited a significantly lower ability to form biofilm than the WT strain(P < 0.01).Growth kinetics results showed that Δail strain did not affect bacterial growth.The determination of microplate adhesion ability showed that when induced by 1/8 MIC of cefotaxime,the adhesion ability of the Δail strain was significantly lower compared with the WT strain(P< 0.001).However,the deletion of ail gene did not affect bacterial cell membrane permeability and the agglutination between bacteria,as well as bacterial morphologic change determined by Gram staining.Further study showed that the abilities of adhesion and to form biofilms by the CΔail strain were increased to a certain extent compared with the Δail strain.2.The role of PBP-encoding genes in mediating cefotaxime-induced enhancement of Salmonella biofilm formationThree deletion mutants Δmrc A,ΔmrcB,Δfts I,targeted to PBP1 a,PBP1b,PBP3-encoding gene,respectively,were constructed using the parental Salmonella strain SH16SP46.At the exposure to 1/8 MIC of cefotaxime,biofilm formation ability of theΔfts I and Δmrc A strains was significantly enhanced(P < 0.001),while no significant difference was observed for the ΔmrcB strain(P > 0.05).Further studies showed that the deletion of mrcB gene did not affect bacterial growth kinetics.Whether or not induced by cefotaxime,there was no significant difference in cell membrane permeability and auto-aggregation rate between the WT strain and ΔmrcB strain(P > 0.05).Both SEM and Gram staining results showed that 1/8 MIC of cefotaxime led to morphologic change of the WT strain from an uninduced short-rod state to a long filamentous state,while the change was not found in the ΔmrcB strain.Further study showed that the complement of mrcB gene in the ΔmrcB strain recovered the enhanced biofilm formation ability and long filamentous morphological change induced by cefotaxime.3.A preliminary study on the mechanism of induction and enhancement of Salmonella biofilm formation by other cephalosporinsIn order to explore whether other cephalosporins also have the phenomenon of enhanced biofilm induction on Salmonella at sub-inhibitory concentrations and the PBP types involved,other four cephalosporins were selected for study.The results of biofilm formation induced by sub-MICs of different drugs showed that biofilm formation by the WT strain could be enhanced by the inducing of 1/4 MIC of cefquinome and 1/8 MIC of cefepime,while not by ceftiofur and ceftazidime.The bacterial morphology was observed,and the morphology of WT was long filamentous under the action of cefquinome,cefepime and ceftazidime,and short rod-shaped under the action of ceftiofur.Under the action of 1/8 MIC cefepime,the ΔmrcB strain lost the ability to induce biofilm formation and the filamentous morphological changes.When the mrcB gene was complemented,the enhanced biofilm formation ability and filamentous morphological changes were restored.Under the action of 1/4 MIC cefquinome,the biofilm formation ability of Δmrc A strain was not significantly enhanced,and the bacterial morphology was filamentous.In summary,our study showed that the ail gene contributed to enhancement of Salmonella adhesion ability and biofilm formation,and the PBP1 b encoded by mrcB gene is the main target of cefotaxime and cefepime to mediate the enhancement of Salmonella biofilm formation and long filamentous morphologic change induced by sub-MICs of both drugs,compared with cefotaxime,cefquinome has different targets and mechanisms.