Combination Of Pan-Genomic Approaches And Experimental Validation For Characterization Of Biofilm-Associated Outer Membrane Proteins In Aeromonas Hydrophila

Aeromonas hydrophila is an emergent bacterial pathogen.As a well-known pathogen of humans and several aquatic species,this bacterium is found in several different aquatic environments,foods,and food processing and storage systems with diverse strains.A.hydrophila is able to persist and propagate in continuously changing environments.A.hydrophila can form biofilms on suitable surfaces,thereby showing increased coordinated behavior under adverse conditions.The existence of multiple-antibiotic-resistant strains of A.hydrophila with multiple plasmids and mobilomes is also of great concern.It is already known that outer membrane proteins(OMPs)can affect the virulence,antibiotic resistance and biofilm formation ability.However,association of OMPs with virulence and antibiotic resistance in A.hydrophila is little known.1.Comparative genome analysis provides deep insights into A.hydrophila taxonomy and virulence-related factors.A.hydrophila belongs to Aeromonads that have very large number of species.With overlapping characteristics,multiple isolates are often mislabelled and misclassified.Moreover,the potential pathogenic factors among the publicly available genomes in A.hydrophila strains of different origins have not yet been investigated.To identify the valid strains of A.hydrophila and their pathogenic factors,we performed a pan-genomic study.It revealed that there were 13 mislabelled strains and 49 valid strains that were further verified by Average nucleotide identity(ANI),digital DNA-DNA hybridization(dDDH)and in silico multiple locus strain typing(MLST).Multiple numbers of phages were detected among the strains and among them Aeromonas phi 018 was frequently present.The diversity in type Ⅲ secretion system(T3SS)and conservation of type Ⅱ and type Ⅵsecretion systems(T2SS and T6SS,respectively)among all the strains are important to study for designing future strategies.Most strains were found resistant to beta-lactamase antibiotics and polymyxins.The comparative analyses of sequence type(ST)251 and other ST groups revealed that there were higher numbers of virulence factors in ST-251 than in other STs group.Publicly available genomes have 13 mislabelled organisms,and there are only 49 valid A.hydrophila strains.This valid pan-genome identifies multiple prophages that can be further utilized.Different A.hydrophila strains harbour multiple virulence factors and antibiotic resistance genes.Identification of such factors is important for designing future treatment regimes.2.In silico analyses of biofilm related beta barrels and their experimental validation through gene knockout of selected beta barrel proteins.In A.hydrophila,surface exposed antigenic proteins e.g.OMPs have been long studied in order to develop vaccines.Beyond that,there is not much known about frequent OMPs found in A.hydrophila and their role in bacterial characteristics.This study identified biofilm associated OMPs via in silico methods and secondly,used homologous recombination technique to construct ompAⅡ and omp38 gene knockout strains.The construction of complementary strains was also performed on the knockout counterparts.The knockout of OMPs affected the biofilm in opposite ways,for example,the ompAⅡdeletion strain has reduced biofilm production whereas the deletion of omp38 has enhanced the biofilm production.Also,the results demonstrated that maximum biofilm associated optical density was observed at 24h interval.In mRNA based gene expression,omp38 expression was lower in biofilm cells than that in planktonic cells.However,the ompAⅡexpression was relatively higher in biofilm cells as compared with planktonic cells.These results help us in validating the in silico methods and role of omp38 and ompAIl in biofilm production at different time intervals and growth phases.3.Effect of omp38 and ompAⅡ deletion in A.hydrophila NJ-35 on virulence and drug sensitivity against various antibiotics.The main aim of this study was to determine the different characteristics of Omp38 and OmpAⅡ along with minimum inhibitory concentrations(MICs)and minimum biofilm eradication concentrations(MBECs)against common antibiotics being used in the fish industry.Furthermore,the role of sub-MICs was also determined in post biofilm formation at different time intervals.The results showed that omp38 and ompAⅡ also affect the swimming motility and adhesion of the cells.On the other hand,no association of these genes was observed with other characteristics specially virulence.It was also demonstrated that there was diversity of response to antibiotic treatment in A.hydrophila depending on planktonic cells or biofilms.Biofilm-forming A.hydrophila bacteria are able to highly resist the in vitro action of commonly used antibiotics.A.hydrophila were sensitive to Florfenicol even after the formation of biofilm.And A.hydrophila showed much greater resistance to Polymyxin in planktonic cultures.Additionally,antibiotics such as Florfenicol,Enrofloxacin and Oxytetracycline can reduce the biofilm even at their sub-MIC concentration.It is concluded that omp38 and ompAⅡ affect the swimming motility and adhesion but there is no role in virulence and antibiotic resistance.4.Epi-gene:An R-Package to observe the pangenome.The main aim of this study was to develop a package that can statistically analyze the genomic data with less time consumption.Epi-gene is presented as a solution to computational time problems.It uses less time and less programming skills in order to work with large number of genomes.In current study,some functions and opportunities of the Epi-gene R-package are described and illustrated by using public data for the species A.hydrophila.Prodigal gene prediction,joining and relabeling functions were used to handle the protein sequences.To calculate the subsets of core genes,accessory genes and unique genes related functions have been used.Heat maps and phylogenetic genome trees were also constructed.Total of 14 genomes were included in the study.This whole procedure was completed in less than half hour.Other functions and packages were also used that were available in R on the A.hydrophila data to highlight these possibilities from within the R computing environment.