Foremost report of the whole genome of Spirabiliibacterium mucosae from India and comparative genomics of the novel genus Spirabiliibacterium.

Several Pasteurella like organisms isolated from various avian species were recently reclassified into new genus based on whole genome sequence analysis. One such Pasteurella like organism, Bisgaard taxon 14 was classified as Spirabiliibacterium mucosae. In the present study, a Gram-negative organism was isolated from ailing pigeons with respiratory infection from a farm in Tamil Nadu, India and the organism was misidentified as Burkholderia mallei by Vitek 2 compact system based on biochemical characterization. Since, B. mallei is highly pathogenic and zoonotic, to further confirm, 16S rDNA sequencing and analysis was carried out which revealed that the strain belonged to Bisgaard taxon 14 (Spirabiliibacterium mucosae). To further confirm the findings, whole genome sequencing of the isolate was performed. Whole genome phylogeny and average nucleotide identity (ANI) analysis showed that the genome was closely matching with Spirabiliibacterium mucosae type strain 20,609 /3. Hence, the strain from pigeon was named as Spirabiliibacterium mucosae TN_CUL_2021 and the genome was submitted in NCBI SRA database. The genome of S. mucosase TN_CUL_2021 is only the second genome available worldwide in the NCBI database. Comparative genome analysis of 26 Pasteurellaceae family strains revealed 1101 genes specific for Spirabiliibacterium mucosae. Similarly, luxS virulence gene was found only in S. mucosae and Bisgaardia hudsonensis strains. Since there are only 2 genomes available in the NCBI genome database, further studies on isolation of S. mucosae needs to be carried out to identify its epidemiology and pathogenesis so as to develop better diagnostic assays and vaccines.

Comparative phylogenomics of Trueperella pyogenes reveals host-based distinction of strains.

Trueperella pyogenes, an opportunistic pathogen causes various ailments in different animals. Different strains from different animals have distinct characters phenotypically and genotypically. Hence understanding the strains in a particular geographical location helps in framing the preventive measures. Comparative genomics of all the available T. pyogenes genome in the NCBI was conducted to understand the relatedness among strains. Whole genome phylogeny showed host associated clustering of strains recovered from swine lungs. Core genome phylogeny also showed host associated clustering mimicking whole genome phylogeny results. MLST analysis showed that there was higher diversity among cattle strains. Multidimensional scaling revealed five swine clusters, two cattle and buffalo clusters. Pangenome analysis also showed that T. pyogenes had an open genome with 57.09% accessory genome. Host specific genes were identified by pangenome analysis, and (R)-citramalate synthase was specific for swine strains of Asian origin. Host specifc genes identified by pangenome analysis can be exploited for developing a molecular assay to specifically identify the strains. The study shows that MLST having higher discriminatory power can be used as an epidemiological tool for strain discrimination of T. pyogenes.

First report of the whole genome of Moraxella bovoculi genotype 1 from India and comparative genomics of Moraxella bovoculi to identify genotype-specific markers.

Moraxella bovoculi has been isolated frequently from cattle with Infectious bovine keratoconjunctivitis (IBK). Two diverse genotypes of M. bovoculi, 1 and 2 were identified based on whole genome sequence analysis. It is essential to discriminate between the two genotypes to frame prevention and control measures. The whole genome of M. bovoculi TN7 was sequenced and compared to other M. bovoculi strains available in the NCBI database. M. bovoculi TN7 was found to be genotype 1, had an RTX toxin operon and pilA gene that are the known virulence factors in related Moraxella sp., but lacked antimicrobial resistance genes. M. bovoculi was found to have an open pangenome with 4051 (75.31%) accessory genes, and the addition of each new genome adds 18 genes to the pangenome. Comparison of pilin protein amino acid sequences revealed three new sequence types. Furthermore, the presence of linx, nagL, swrC and mdtA genes was found to be genotype 1 specific, whereas hyaD, garR, gbsA, yhdG, gabT, iclR, higB2, hmuU, hmuT and hemS were found only in genotype 2. Polymerase Chain Reaction (PCR) primers were designed and evaluated on strain TN7 plus seven additional strains accessible to us that had not been whole genome sequenced. This initial evaluation of the designed primers for the linX and hyaD genes produced the expected banding patterns on PCR gels for genotypes 1 and 2, respectively, among the 8 strains. The genotype-specific genes identified in this study can be used as markers for accurate diagnosis of genotype 1 isolates and this can aid in the development of autogenous or other molecular vaccines for treatment of infectious bovine keratoconjunctivitis (IBK) in resource-limited research settings.

Comparative genomics of Trueperella pyogenes available in the genome database reveals multidrug resistance genomic islands.

OBJECTIVE: An opportunistic pathogen, Trueperella pyogenes can infect cattle, buffalo, pig, goat, cat, dog, forest musk deer, etc., affecting various organs. The aim of this study was to identify the multidrug resistance genomic islands of T. pyogenes genomes available in NCBI database and also in the recently isolated strain TN_CUL_2020. METHODS: The strain TN_CUL_2020 isolated from swine lung abscess was sequenced by Illumina platform, and all the available T. pyogenes genome in NCBI database was retrieved for the comparative analysis. The ABRicate searches was used to identify antimicrobial resistance genes, and genomic islands (GIs) were predicted using IslandViewer 4. RESULTS: The strains SH01, SH02, and TP1 were predicted with maximum number of drug resistance genes. Genomic islands identified had multidrug resistance genes along with the class I integron and/or IS6100 elements in SH01, SH02, TP1. Composite transposons of IS6100 were noted in T2849, T4479, and TP3 intercalating tet(33) resistance genes. Several strains were predicted with phage elements, type IV secretion system, the toxin-antitoxin system in the GIs. CONCLUSION: Swine strains SH01, SH02 were predicted with multidrug resistance genes along with class I integrons. The presence of class I integrons, insertional elements, type IV secretion system, toxin-antitoxin system, and phage elements may aid in the horizontal transfer of antimicrobial resistance genes.

Virulence genes and enterobacterial repetitive intergenic consensus region (ERIC) profiling reveals highly diverse genetic population among avian strains of Pasteurella multocida.

Pasteurella multocida is a multispecies pathogen with certain host specific capsular types but interspecies transmission cannot be overlooked. Knowing the diversity of P. multocida in a geographical location is essential to formulate a vaccination programme. Diversity among the P. multocida isolates from different avian species recovered in the state of Tamil Nadu, India was studied using enterobacterial repetitive intergenic consensus region (ERIC)-PCR and virulence gene profiling (VP). Capsular typing revealed that 44 (97.78%) strains belonged to capsular type A while only one (2.22%) strain belonged to capsular type B. ERIC-PCR analysis showed eight different clusters and four individual strains. The index of discrimination (D value) was found to be 0.8899. Virulence profiling showed that genes fimA, pfhA, hsf-2 and pmHAS were found in 100% of the strains while ompH, omp87, ompA, plpB, sodA, sodC, ptfA, hsf-1, exbB, fur, hgbA and hgbB were found in ≥90% of the strains. Dermonecrotoxin gene toxA was present only in 4.44% of the strains, while nanH in 68.89% and nanB in 88.89% of the strains. One strain each from turkey and Guinea fowl had toxA gene. Correlation analysis revealed a positive correlation between ptfA and hgbA gene, exbB and fur gene, ptfA and sodC gene, exbB and hsf-1 gene, ompA and ompH gene. Majority of duck strains clustered together both in ERIC and virulence gene profiles. Turkey strains were highly diverse with different VPs and ERIC-PCR patterns.

Genome Sequencing and Comparative Genomics of Indian Isolates of Brucella melitensis.

Brucella melitensis causes small ruminant brucellosis and a zoonotic pathogen prevalent worldwide. Whole genome phylogeny of all available B. melitensis genomes (n = 355) revealed that all Indian isolates (n = 16) clustered in the East Mediterranean lineage except the ADMAS-GI strain. Pangenome analysis indicated the presence of limited accessory genomes with few clades showing specific gene presence/absence pattern. A total of 43 virulence genes were predicted in all the Indian strains of B. melitensis except 2007BM-1 (ricA and wbkA are absent). Multilocus sequence typing (MLST) analysis indicated all except one Indian strain (ADMAS-GI) falling into sequence type (ST 8). In comparison with MLST, core genome phylogeny indicated two major clusters (>70% bootstrap support values) among Indian strains. Clusters with <70% bootstrap support values represent strains with diverse evolutionary origins present among animal and human hosts. Genetic relatedness among animal (sheep and goats) and human strains with 100% bootstrap values shows its zoonotic transfer potentiality. SNP-based analysis indicated similar clustering to that of core genome phylogeny. Among the Indian strains, the highest number of unique SNPs (112 SNPs) were shared by a node that involved three strains from Tamil Nadu. The node SNPs involved several peptidase genes like U32, M16 inactive domain protein, clp protease family protein, and M23 family protein and mostly represented non-synonymous (NS) substitutions. Vaccination has been followed in several parts of the world to prevent small ruminant brucellosis but not in India. Comparison of Indian strains with vaccine strains showed that M5 is genetically closer to most of the Indian strains than Rev.1 strain. The presence of most of the virulence genes among all Indian strains and conserved core genome compositions suggest the use of any circulating strain/genotypes for the development of a vaccine candidate for small ruminant brucellosis in India.