Genome-wide Genetic Marker Analysis and Genotyping of Escherichia fergusonii strain OTSVEF–60

Poultry originated Escherichia fergusonii (POEF), an emerging bacterial pathogen, causes a wide range of intestinal and extra-intestinal infections in the poultry industry which incurred significant economic losses worldwide. Chromosomal co-existence of antibiotics and metal resistance genes has recently been the focal point of POEF isolates besides its pathogenic potentials. This study reports the complete genome analysis of POEF strain OTSVEF-60 from the poultry originated samples of Bangladesh. The assembled draft genome of the strain was 4.2 Mbp containing 4,503 coding sequences, 120 RNA (rRNA = 34, tRNA = 79, ncRNA = 7), and three intact phage signature regions. Forty one broad range antibiotic resistance genes (ARGs) including dfrA12, qnrS1, blaTEM-1, aadA2, tet(A) and sul-2 along with multiple efflux pump genes were detected, which translated to phenotypic resistant patterns of the pathogen to trimethoprim, fluoroquinolones, β-lactams, aminoglycoside, tetracycline, and sulfonamides. Moreover, 22 metal resistance genes were found co-existing within the genome of the POEF strain, and numerous virulence genes (VGs) coding for cit (AB), feo (AB), fep (ABCG), csg (ABCDEFG), fliC, ompA gadA, ecpD etc were also identified throughout the genome. In addition, we detected a Class I integron gene cassette harboring dfrA12, ant (3″)-I and qacEΔ-sul2) genes, 42 copies of insertion sequence (IS) elements, and two CRISPR arrays. The genomic functional analysis revealed overexpression of several metabolic pathways related to motility, flagellar assembly, epithelial cell invasion, quorum sensing, biofilm formation, and biosynthesis of vitamin, co-factors, and secondary metabolites. We herein for the first time detected multiple ARGs, VGs, mobile genetic elements, and some metabolic functional genes in the complete genome of POEF strain OTSVEF-60, which might be associated with the pathogenesis, spreading of ARGs and VGs, and subsequent treatment failure against this emerging avian pathogen with currently available antimicrobials.

Machine generated data was transferred to the Ion Torrent server, where data were 1 4 7 processed through signal processing, base calling algorithms, and adapter trimming to produce 1 4 8 mate-pair reads in FASTQ format. The FASTQ reads quality was assessed by the FastQC tool 24 1 4 9 followed by trimming of low quality reads and reads less than 200 bp using the Trimmomatic 1 5 0 tool 24 , while the quality cut off value was Phred-20. Reads were assembled with the SPAdes, 1 5 1 (version 3.5.0) genome assembler 25 . Generated assembled reads were mapped to, and reordered Draft genome of POEF OTSVEF-60 strain was initially annotated using RAST (Rapid and Genomes) Automatic Annotation Server (KAAS) 28 . The RAST server provided data on the showing the distinct differences between multiple genomes were performed by BLAST Ring respectively. Moreover, clusters of regularly interspaced short palindromic repeats (CRISPR)-1 7 6 Cas system of POEF OTSVEF-60 strain was characterized based on annotation by CRISPR one, better visualization, and bootstrap values were reported for each branch. The complete genome sequence, annotated POEF OTSVEF-60 chromosome, and The whole genome sequence (WGS) analysis of the study isolate, OTSVEF-60 using Kmer 1 9 7 Finder 3.1 identified the isolate as E. fergusonii, while the pathogenicity of the isolate was 1 9 8 confirmed (0.936 out of 1.00 (near the pick value indicating higher pathogenicity)) by 1 9 9 PathogenFinder 1.1, as also supported by the biofilm assay (OD=0.431). Besides, the isolate was  plasmids (IncX1 and p0111) in the genome (Table 1). According to RASTtk annotation, only 2 0 7 37% of the genes were located in the generated subsystems, and majority (63%) of the annotated 2 0 8 genes remained out of the subsystem list. The genome was found to bearing 4,503 coding Although, the BLAST and SEED close strain analyses revealed that the genome had serotypes, and clustered separately (Fig. 2). In silico analysis of the genome using housekeeping complete genome assembly, and annotations are summarized in Table 1, and circular graphical 2 2 1 map of the genome is shown in Fig. 3 and Fig. 4. We did not find any similarities to the allelic  OTSVEF-60 according to the MIxS recommendations are presented in Table 2. We identified six prophages in the WGS of the POEF-OTSVEF-60 strain, of which lambda-2 2 9 like prophages were predominantly abundant. The OTSVEF-60 strain carried three intact 2 3 0 prophage sequences (region 1, 2, 5) along with two incomplete (region 4 and 6), and one 2 3 1 questionable prophage (region 3) sequences (Fig. 5). The resemblance of the intact prophage 2 3 2 sequences was found with Salmon_ sal5 (region 5), and Entero _ fiAA91 (region 1), and  sequences of phages best matched either with phage proteins or with hypothetical proteins ( Table   2  3  7 3). The most common subtype of CRISPR-Cas system found in the POEF-OTSVEF-60 was I-A, There was a wide distribution of antibiotic resistance genes (ARGs) including both antibiotic 2 4 7 inactivating enzyme and efflux pump encoding genes in the genome of POEF OTSVEF-60. with the findings of in vitro antibiotic susceptibility tests ( Supplementary Fig. 1, Table 6).
fergusonii ATCC 34569 except tolA, csg (ABCDFG) ( Table 5). We found no plasmid-encoded 2 9 1 VGs in the study genome. Metabolic pathway analysis of POEF-OTSVEF-60 genome revealed a number of 2 9 4 complete and incomplete pathways possibly associated with the AMR, and virulence properties 2 9 5 of the isolate (Fig. 1). The detected functional pathways were distributed under five major KEGG 2 9 6 orthologies (KO) such as cellular process, metabolism, environmental information processing, 2 9 7 genetic information processing, and human diseases (Fig. 1). A deeper look into these KEGG 2 9 8 pathways disclosed that they were associated with the motility, flagellar assembly, quorum degradation (pobA) related to amino benzoate degradation were also identified in this genome.

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Insertion sequence (IS) elements 3 1 3 Forty-two copies of IS elements were identified in the OTSVEF-60 genome 3 1 4 (Supplementary Table 2). The IS Finder software identified 10 families of IS elements, of which 3 1 5 IS3 and Tn3 were most predominant followed by ISKra4, IS200/IS605, IS110, ISAs1, IS5, IS6, 3 1 6 IS1, IS4 family. Moreover, 23 truncated transposases were also found in the OTSVEF-60 3 1 7 genome (Supplementary Table 4). This study to the best of our knowledge, reports the first isolation and characterization  gene sequencing based identification method frequently failed to distinguish it from E. coli 39 .

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The growing concern of this organism as a zoonotic pathogen insisted to analyze the whole 3 2 6 genome of the poultry isolate superbug E. fergusonii OTSVEF-60 for understanding its 3 2 7 pathogenicity and antimicrobial resistance. prophage-like elements, and genomic islands. The VGs also included various curli fimbriae 3 3 1 genes to be associated with virulence, and biofilm formation (BF) 40 . Moreover, the Flagellin, 3 3 2 adhesion, fimbria adhesion, and BF regulatory genes were detected in the genome of POEF- in a distinct category of pathogenic E. fergusonii, which signifies its possible role behind the 3 3 8 outbreak in the poultry farm.

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The results of the in vitro resistance assay corroborated with the genomic resistance ARGs of the study isolate also included efflux pumps mediated resistance genes: resistance of being physically linked to other resistance genes 15 . However, the isolate was susceptible to to 22 antibiotics supporting our current findings 11 . Interestingly, unlike the previous and first 3 5 4 report of the E. fergusonii whole genome, along with antibiotic resistance genes, multiple metal 3 5 5 resistance and tolerance genes conferring resistance to copper, chromium, iron, potassium, 3 5 6 cobalt, nickel, and zinc were found in the OTSVEF-60 strain. Metals have been found to interfere with several bacterial cellular functions such as protein activity, oxidation, nutrient