Detection of Staphylococcus Isolates and Their Antimicrobial Resistance Profiles and Virulence Genes from Subclinical Mastitis Cattle Milk Using MALDI-TOF MS, PCR and Sequencing in Free State Province, South Africa

Abstract

Staphylococcus species are amongst the bacteria that cause bovine mastitis worldwide, whereby they produce a wide range of protein toxins, virulence factors, and antimicrobial-resistant properties which are enhancing the pathogenicity of these organisms. This study aimed to detect Staphylococcus spp. from the milk of cattle with subclinical mastitis using MALDI-TOF MS and 16S rRNA PCR as well as screening for antimicrobial resistance (AMR) and virulence genes. Our results uncovered that from 166 sampled cows, only 33.13% had subclinical mastitis after initial screening, while the quarter-level prevalence was 54%. Of the 50 cultured bacterial isolates, MALDI-TOF MS and 16S rRNA PCR assay and sequencing identified S. aureus as the dominant bacteria by 76%. Furthermore, an AMR susceptibility test showed that 86% of the isolates were resistant to penicillin, followed by ciprofloxacin (80%) and cefoxitin (52%). Antimicrobial resistance and virulence genes showed that 16% of the isolates carried the mecA gene, while 52% of the isolates carried the Lg G-binding region gene, followed by coa (42%), spa (40%), hla (38%), and hlb (38%), whereas sea and bap genes were detected in 10% and 2% of the isolates, respectively. The occurrence of virulence factors and antimicrobial resistance profiles highlights the need for appropriate strategies to control the spread of these pathogens.

Comprehensive whole genome analysis of Staphylococcus aureus isolates from dairy cows with subclinical mastitis

“…As reported in our previous study ( Khasapane et al, 2024 ) the isolates showed resistance to penicillin 43/50 (86%), ciprofloxacin 40/50 (80%), vancomycin 39/50 (76%), and cefoxitin 26/50 (52%). Observed resistance against gentamycin, ampicillin, tetracycline, and erythromycin was 18/50 (36%), 14/50 (28%), 9/50 (18%), and 9/50 (18%), respectively.…”

“…Isolates were defined as S. aureus on the basis of being gram-positive cocci and catalase positive. Thereafter, matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) and gene sequencing were employed for further identification of the isolates as reported in our previous study ( Ozbey et al, 2022 ; Khasapane et al, 2024 ). Furthermore, we performed phenotypic and genomic antimicrobial resistance based on disk-diffusion and PCR techniques ( Khasapane et al, 2024 ).…”

“…Thereafter, matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) and gene sequencing were employed for further identification of the isolates as reported in our previous study ( Ozbey et al, 2022 ; Khasapane et al, 2024 ). Furthermore, we performed phenotypic and genomic antimicrobial resistance based on disk-diffusion and PCR techniques ( Khasapane et al, 2024 ). To evaluate the susceptibility of Staphylococcus isolates to widely used antimicrobial drugs, the single disk diffusion technique was utilized.…”

Detection of Salmonella Pathogenicity Islands and Antimicrobial-Resistant Genes in Salmonella enterica Serovars Enteritidis and Typhimurium Isolated from Broiler Chickens

“…A PCR assay was conducted with bacterial universal primers (27F: AGA GTT TGA TCM TGG CTC AG and 1492R: GGT TAC CTT GTT ACG ACT T) targeting the 16S rRNA gene [74,75]. The PCR conditions were as follows: 96 • C for 4 min for initial denaturation step, followed by 30 cycles of denaturation at 94 • C for 30 s, annealing at 57 • C for 30 s and extension at 72 • C for 1 min, and one step of final extension at 72 • C for 10 min.…”