Submitted:
22 April 2025
Posted:
23 April 2025
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Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) in bovine milk poses a significant zoonotic and antimicrobial resistance threat. This study identifies three MRSA strains from subclinical mastitic (SCM) cow milk, carrying resistance genes to β-lactams, lincosamides, macrolides, streptogramin B, and aminoglycosides, underscoring the urgent need for improved hygiene and surveillance in dairy production.
Keywords:
Staphylococcus aureus
; MRSA
; multidrug resistance
; mastitic cow
; antimicrobial resistance
Methicillin-resistant Staphylococcus aureus (MRSA) in bovine milk poses a significant threat to both animal and public health due to its antimicrobial resistance and zoonotic potential (1). Its presence in raw and pasteurized milk increases the risk of clinical management and human transmission, especially among those in close contact with dairy animals or consuming unpasteurized products (2, 3). These concerns underscore the urgent need for improved hygiene practices and robust surveillance in the dairy industry to mitigate MRSA spread. Three subclinical mastitis (SCM) milk samples were collected from the Cumilla district, Bangladesh, and inoculated on 5% bovine blood agar followed by subculture of characteristic colony on Mannitol Salt Agar with incubation at 37 ℃ for 24 h. Staphylococus aureus was confirmed by subsequent Gram staining, biochemical, and nuc gene specific PCR. Antimicrobial sensitivity profiling using twelve antimicrobials representing different classes including oxacillin and cefoxitin resulted in multidrug resistance. Genomic DNA was extracted from 2 to 3 isolated colony on 5% bovine Blood Agar using an AllPrep Bacterial DNA/RNA/Protein Kit (Qiagen, Hilden, Germany), and purity was assessed with a Nanodrop One (Thermo Fisher Scientific, MA, USA). DNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, CA, USA). Genome sequencing was conducted on an Illumina NextSeq2000 platform generating 2 × 150 bp paired end reads. Raw data quality, including read quality, GC content, and adapter contamination, was assessed using FastQC v0.12.1 (4). Trim Galore v0.6.5dev (5) was employed to trim low-quality bases and adapter sequences, followed by normalization of read coverage with bbnorm v39.08 (6) to reduce redundancy. De novo genome assembly was performed using Unicycler v0.4.8 (7) with subsequent polishing using Pilon v1.24 (8) to enhance accuracy. Genome assembly quality was assessed using QUAST v5.2.0 (9), while Samtools v1.13 (10) was used for alignment file manipulation and indexing. The de novo genome assembly conducted through the BV-BRC (11) comprising the abovementioned tools yielded coverage ranging from 146× to 339×, with raw reads between 3,035,549 and 6,710,480. The assembled genomes were annotated using the National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline (PGAP) v6.9 (12) and PATRIC (13) with the in-built RASTtk pipeline to identify the species, Staphylococcus aureus. Transport proteins were identified via TCDB (14), drug targets using DrugBank v6.0 (15), and additional analyses performed for CRISPR arrays and prophages using CRISPR CasFinder (16), plasmids using PlasmidFinder 2.1 (17) and integrative and conjugative elements using ICEFinder (18). All software parameters were set to default, unless specified otherwise. Detailed genomic characteristics are provided in Table 1.
Antimicrobial resistance genotypes were identified using ResFinder 4.6.0 (19). Acquired and chromosomal resistance genes were identified in all three MRSA strains. Methicilin resistant mecA gene and resistance to β-lactam antibiotics were present in all three strains. Additionally, resistance to lincosamide, macrolide, streptogramin b and aminoglycoside were found in BioB-3 and BioB-4 strains (Table 1). This study highlights the need for effective surveillance and control measures to prevent the spread of MRSA within dairy herds and to humans.
Data Availability
The accession numbers for the genomes of the three Staphylococcus aureus strains are shown in Table 1. All genomes are available under BioProject accession number PRJNA1190104.
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Table 1.
Genomic features of the sequenced three Staphylococcus aureus strains isolated from subclinical mastitic cows in Cumilla, Bangladesh.
Table 1.
Genomic features of the sequenced three Staphylococcus aureus strains isolated from subclinical mastitic cows in Cumilla, Bangladesh.
| Strain name | BioB-2 | BioB-3 | BioB-4 | |
| Information title | ||||
| Species | Staphylococcus aureus | Staphylococcus aureus | Staphylococcus aureus | |
| Genome submission ID | SUB14894378 | SUB14894378 | SUB14894378 | |
| Bioproject ID | PRJNA1190104 | PRJNA1190104 | PRJNA1190104 | |
| BioSample accession number | SAMN45025667 | SAMN45025668 | SAMN45025669 | |
| SRA accession number | SRS23333889 | SRS23333890 | SRS23333891 | |
| Genome assembly accession number | GCA_047604675.1 | GCA_047604515.1 | GCA_047604495.1 | |
| GenBank accession number | JBJLRB000000000 | JBJLRA000000000 | JBJLQZ000000000 | |
| FastQC Phred score | 34 | 34 | 34 | |
| Raw reads | 6,710,480 | 4,498,755 | 3,035,549 | |
| Coverage | 339x | 219x | 146x | |
| Contig count | 31 | 21 | 41 | |
| Coarse consistency (%) | 100 | 100 | 100 | |
| Fine consistency (%) | 99.7 | 99.7 | 99.7 | |
| Completeness (%) | 100 | 100 | 100 | |
| Contamination (%) | 0 | 0 | 0 | |
| Genome size (bp) | 2,761,071 | 2,782,581 | 2,797,289 | |
| Contigs N50 (bp) | 219,219 | 329,157 | 137,483 | |
| Contigs L50 | 5 | 4 | 5 | |
| Largest contig (bp) | 402,935 | 536,524 | 380,235 | |
| Guanine–cytosine content (%) | 32.75696 | 32.72947 | 32.637028 | |
| Genes (Total), PGAP | 2,763 | 2,791 | 2,799 | |
| CDS (PGAP; PATRIC) | 2,641; 2,606 | 2,668; 2,624 | 2,634; 2670 | |
| tRNA | 55 | 55 | 57 | |
| rRNA | 4 | 4 | 4 | |
| CDS ratio | 0.9438366 | 0.9430094 | 0.9544956 | |
| Hypothetical CDS | 510 | 507 | 568 | |
| Hypothetical CDS ratio | 0.2943208 | 0.29115853 | 0.31722847 | |
| PLFAM CDS | 2,540 | 2,557 | 0 | |
| PLFAM CDS ratio | 0.9746738 | 0.97446644 | - | |
| Hypothetical proteins | 513 | 507 | 570 | |
| Proteins with functional assignments |
2,093 | 2,115 | 2,100 | |
| Proteins with EC number assignments | 735 | 737 | 740 | |
| Proteins with GO assignments | 607 | 609 | 613 | |
| Proteins with pathway assignments | 532 | 533 | 536 | |
| Proteins with subsystem assignments | 975 | 973 | 974 | |
| Proteins with PATRIC genus-specific family (PLfam) assignments | 2,540 | 2,557 | 0 | |
| Proteins with PATRIC cross-genus family (PGfam) assignments | 2,573 | 2,593 | 2,629 | |
| Proteins with FIGfam assignments | 0 | 0 | 0 | |
| Transporter (TCDB) | 98 | 99 | 100 | |
| Drug target (DrugBank) | 33 | 36 | 34 | |
| Antibiotic resistance genes (PATRIC) | 39 | 40 | 40 | |
| ResFinder 4.6.0 (acquired; chromosomal) | str, mecA; 23S, dfrB, fusA, grlA, grlB, gyrA, ileS, pbp2, pbp4, rpoB, pbp4-promoter | mecA, blaZ, erm(C); 23S, dfrB, fusA, grlA, grlB, gyrA, ileS, pbp2, pbp4, rpoB, pbp4-promoter | mecA, blaZ; 23S, dfrB, fusA, grlA, grlB, gyrA, ileS, pbp2, pbp4, rpoB, pbp4-promoter | |
| AMR phenotype class | β-lactam | β-lactam, lincosamide, macrolide, streptogramin b | β-lactam, aminoglycoside | |
| Plasmid finder 2.1 | rep7a (contig23: 2056..2895) | rep5a (contig13: 16621..17481), rep16(contig13: 18772..19515), rep10(contig18: 1881..2356) | repUS5(contig21: 33881..35455), rep21(contig21: 4961..5956) | |
| Integrative and conjugative elements (ICEs) | 1(2745519..2754895, 9377 bp) | 1(2111899..2114925, 3027 bp) | 2 (2759741..2794168, 34428bp; 1058307..1163904, 105598 bp) | |
| No. of clustered interspaced short repeats (CRISPR) (number of cas clusters) (name of cas genes) | 4(1) (cas4_Type IA) | 6(1) (cas4_Type IA) | 8(1) (cas3_TypeI) | |
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