Whole genome sequencing of antimicrobial resistant strains of selected bacterial species from communally grazed cattle of Mogosane village, South Africa

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The gastrointestinal tract of cattle houses several microorganisms that are excreted through faecal material into the environment. Some strains of these microorganisms are known to be zoonotic and carry determinants of antimicrobial drug resistance (AMR). Antimicrobial-resistant organisms from food producing animals pose a significant public health danger globally (Song et al., 2022). This research aimed at conducting whole genome sequencing (WGS) for both generic Escherichia coli (E. coli) and Enterobacter hormaechei (E. hormaechei) isolates from healthy communally grazed cattle faecal samples, and identifying AMR determinants. This study also assessed the phenotypic resistant profiles of E. coli and E. hormaechei isolates from the same study population. In total, 35 faecal samples were collected and traditional microbiological techniques were used to culture and isolate bacteria from faecal samples. Of the 35 samples only three (n=3; 9%) of the samples yielded E. coli isolates and 12 (34%) were positive for E. hormaechei. Confirmed E. coli and E. hormaechei isolates were tested for susceptibility using the NCCL disc diffusion method towards 10 antimicrobials namely: Gentamycin (CN), Streptomycin (S), Neomycin (N), Ciprofoxacin (CIP), Norfloxacin (NOR), Erythromycin (E), Ampicillin (AMP), Chloramphenicol (C), Nalidixic acid (NA) and Tetracycline (T). A selection of isolates exhibiting resistance to more than two antibiotics were sent to the Agricultural Research Council-Onderstepoort Institute (ARC-OVI) for the purpose of extracting DNA. For whole genomic sequencing, Illumina TruSeq Nano DNA Library Preparation kit was used to construct DNA libraries. These libraries were sequenced on an Illumina Hiseq X instrument to generate 2 x 150bp reads. Whole genome assemblies were performed with Abyss software (v.2.2.4-1) at default parameters. Thereafter, the generated raw reads (fastq files) were assembled on the Inqaba Biotech platform. Reference assembly for WGS was done with De novo assembly. Pathogenwatch (https://pathogenwatch/) was used to get possible species as a positive hit per the assembled Contig fasta file. For annotating and reference assembly, bacterial and viral resource center were used (https://www.bv-btc.ord). Blast-ncbi was used (basic local alignment tool: https://blast.ncbi.nlm.nih.gov/Blast.cgi) for accession ID for reference assembly. All E. coli isolates (100%), exhibited resistance against streptomycin and erythromycin, thus a phenotype of S-E. The aforementioned isolates also showed 100% intermediate resistance against NA and AMP. The two E. coli isolates were evidenced as susceptible to all other antimicrobials. These isolates exhibited resistance to two drugs from two antimicrobial classes, indicating the absence of multi-drug resistance (MDR). With regards to AMR genes, “aphA” together with “rpsL” mutation genes were detected in E. coli isolates that showed phenotypic resistance to aminoglycoside. WGS annotations are accessible under BioProject accession number PRJNA1028407. The experimental sequence reads archives (SRA) are accessed under SRR26374259 and SRR26393805 respectively in the National Center for Biotechnology Information (NCBI) platform. Twelve (n=12; 34%) E. hormaechei were isolated from communally grazed HMVC. Of the 12, seven (n=8; 67%) were resistant to E and AMP, and four (n=4; 33%) to S, E and AMP. The observed phenotypes were E-AMP (67%), S-E-AMP (33%). AMR was observed in 67% of these isolates, while 33% were MDR. AMR was observed in 67% of these isolates. Several AMR and MDR determinants were identified and included: “acrA”, “acrB” and “marA” genes amongst many others. WGS annotations are accessible under BioProject accession number PRJNA991313 and the experimental sequence reads archives (SRA) of the 12 E. hormaechei isolates are accessed in the NCBI platform. This study identified possible phenotypic resistance profile of bacterial from communally grazed cattle. The results reported here confirm the presence of antimicrobial drug resistance determinants among cattle in rural areas of South Africa. The obtained data provides support for future research on E. coli and E. hormaechei isolates from communally grazed HMVC. Thus, to prevent the distribution of AMR organisms or their transferable resistance genes, a sensible use of antimicrobials is still necessary in veterinary medicine, animal husbandry, and human health. Further studies on AMR should be undertaken especially in rural areas, where communal farmers and veterinary officials could be using over-the-counter and prescribed medicines when it is not necessary.

Agriculture and  Animal Health

D. Phil. (Agriculture and  Animal Health)