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The role of AJB35136 and fdtA genes in biofilm formation by avian pathogenic Escherichia coli
(2023)
Background Infections caused by avian pathogenic Escherichia coli (APEC) result in significant economic losses in poultry industry. APEC strains are known to form biofilms in various conditions allowing them to thrive even under harsh and nutrient-deficient conditions on different surfaces, and this ability enables them to evade chemical and biological eradication methods. Despite knowing the whole genome sequences of various APEC isolates, little has been reported regarding their biofilm-associated genes. A random transposon mutant library of the wild-type APEC IMT 5155 comprising 1,300 mutants was analyzed for biofilm formation under nutrient deprived conditions using Videoscan technology coupled with fluorescence microscopy. Seven transposon mutants were found to have reproducibly and significantly altered biofilm formation and their mutated genes were identified by arbitrary PCR and DNA sequencing. The intact genes were acquired from the wild-type strain, cloned in pACYC177 plasmid and transformed into the respective altered biofilm forming transposon mutants, and the biofilm formation was checked in comparison to the wild type and mutant strains under the same conditions.
Results In this study, we report seven genes i.e., nhaA, fdeC, yjhB, lysU, ecpR, AJB35136 and fdtA of APEC with significant contribution to biofilm formation. Reintroduction of AJB35136 and fdtA, reversed the altered phenotype proving that a significant role being played by these two O-antigen related genes in APEC biofilm formation. Presence of these seven genes across nonpathogenic E. coli and APEC genomes was also analyzed showing that they are more prevalent in the latter.
To increase our understanding of bacterial intestinal colonization in animal populations lacking anthropogenic influence we devised the first objective and studied the diversity of E. coli in pristine wild animal population. Overall, E. coli in cormorants maintained a high diversity under minimal anthropogenic influences. E. coli were isolated from individual birds of two cormorant colonies located on small islands in lakes at least 100 km away from human settlements. Diversity of the isolates was studied using pulsed field gel electrophoresis (PFGE). 137 isolates of cormorant colony-1 and 75 isolates of cormorant colony-2 resulted in 60 and 33 PFGE types, respectively. Representative strains of each PFGE type were analysed in terms of phylogroups, extraintestinal virulence associated genes (exVAGs), adhesion to the chicken intestinal cell line and antimicrobials. Most isolates belonged to phylogroup B1 (68.3%) followed by B2 and E with B2 harbouring the highest total number of exVAGs per isolate. Surprisingly, a PFGE type with relatively few exVAGs displayed the highest isolation frequency, also showing a high adhesion rate. Comparative analysis of exVAGs to other E. coli populations of wildlife origin revealed that the secreted autotransporter toxin encoding sat gene was only present in cormorants.
The second objective was to characterize and identify factors contributing to the novel adhesion phenotype, known as Clumpy adhesion by the strain 4972 E. coli when incubated on human urinary bladder 5637 cell line. Transcriptome and proteome of adhered bacteria clustered together and diverged from bacteria in the supernatant. The clumpy structure was further explored in terms antibiotic stress where sessile structures could tolerate high antibiotic pressure of ampicillin up to 16 mg/ml and gentamicin up to 2 µg/ml. Data analysis unveiled differential expression of 623 genes between bacteria forming clumps and in supernatant. 148 genes were hypothetical and five hypothetical genes i.e., ffp, espX1, pilV, spnT and yggT along with four known genes i.e., dnaK, spy, flgH and fimH were targeted for deletion. Seven genes deletion mutants, three known (i.e., spy, flgH and fimH) and four hypothetical (ffp, pilV, spnT and yggT) genes were successfully generated and analysed for various parameters. ΔflgH showed loss of clumpy phenotype and adhesion ability up to 80% was regenerated by complementation. When antibiotic stress by ampicillin was applied after clump formation, Δffp depicted no effect of high concentration of ampicillin. In case of ΔpilV, adhesion significantly increased to 151% and upon complementation, adhesion was reduced to 53%. ΔspnT during ampicillin stressed adhesion fared almost 8.5 times better than the wild type strain. ΔyggT exhibited high adhesion of 145.5 % and upon reintroduction, adhesion decreased to 13%. Overall, we were able characterize a new phenotype and determined the role of genes contributing to clumpy adhesion phenotype.
Nanopore sequencing, a third-generation sequencing technique that applies nanometre sized pores to transduce the physical and chemical properties of specific nucleobases into measurable electrical signals, shows attractive advantages over conventional next-generation sequencing techniques. However, primarily due to high sequencing error rates this technique has rarely been used so far in clinical laboratory diagnostics. In this cumulative dissertation Nanopore sequencing was established and validated in clinical diagnostics using the example of the molecular diagnosis of Familial Mediterranean fever (FMF) and SARS coronavirus-2 (SARS-CoV-2) infections. First, a novel data analysis pipeline for accurate single nucleotide polymorphism (SNP) genotyping using Nanopore sequencing data was developed and validated with the corresponding sequencing protocol against conventional Sanger sequencing using 47 samples of patients with clinical suspicion of FMF. This method comparison showed a perfect agreement between both methods rendering current Nanopore sequencing in principle suitable for SNP genotyping in human genetics.
The bioinformatic analysis of sequencing data is one of the most challenging parts in Nanopore sequencing experiments and complicates the application in a clinical diagnostic setting. Therefore, six different bioinformatic tools for sequence alignment were evaluated regarding their applicability to Nanopore sequencing data. This evaluation revealed a good suitability of all except one of these tools although differences in quality and performance exist.
Since Nanopore sequencing showed a robust performance in SNP genotyping, a SARS-CoV-2 whole genome sequencing (WGS) protocol was established to enable onside viral WGS in a clinical laboratory. This was especially important for viral molecular biological surveillance during the pandemic as shown by analysing viral genetic data over the course of one year. Applying this approach in a clinical research project to investigate host-virus interaction by aggregating for the first time viral genetic data, serological data and clinical data, showed diverse humoral immune responses to SARS-CoV-2, that appear to be influenced by age, obesity and disease severity. Further, even small viral genetic changes may influence the clinical presentation of the associated disease COVID-19. Additionally, a novel reverse transcriptase (RT)- loop mediated isothermal amplification (LAMP) assay for the detection of SARS-CoV-2 was developed and validated for diagnostic use by method comparison with conventional RT-polymerase chain reaction (PCR). In summary, by presenting advancements of sequencing and bioinformatic workflows with the focus on an application in clinical diagnostics, the results of this thesis may pave the way for a broader application of Nanopore sequencing in laboratory medicine in the near future.