Molecular characterization, antimicrobial resistance and virulence genotyping of Staphylococcus isolates from subclinical mastitis dairy cows in the Thabo Mofutsanyane District Municipality, South Africa

Abstract

Mastitis is one of the economically important diseases in the dairy industry. Staphylococcus species are amongst the bacteria that cause bovine mastitis (BM) worldwide. These bacteria produce and contain a spectrum of extracellular protein toxins, virulence factors, and antimicrobial resistant properties that are thought to contribute to the pathogenicity of the organism. The aims of this study were to: 1. To explore the microbiota of milk from dairy cows with subclinical mastitis (SCM) from small-scale dairy farms in the Thabo Mofutsanyane District Municipality of the Free state Province, South Africa. 2. To determine the virulence and antibiotic resistance profiles as well as genetic characterization of Staphylococcus spp., from milk of SCM dairy cows in the Thabo Mofutsanyane District Municipality of the Free State Province, South Africa. Prior to the experimental analyses, a systematic review and meta-analysis of literature concerning SCM published from January 2010 to December 2020 was conducted with the aim of understanding the prevalence of SCM, its associated risk factors, and major causative agents in Africa. The results showed that the prevalence of SCM was 48.2% (95% CI: 43.6-52.8%) in 11 countries of five African regions. Subgroup and meta-regression analyses further showed that East Africa had a significant (p<0.0092) highest pooled prevalence estimate (PPE) of SCM at 67.7% (95% CI: 55.7-78.7), followed by West Africa at 50.5 (95% CI: 31.4-69.5) and North Africa at 40.3% (95%: 32.2-48.6). Other significant moderators for SCM were age (p<0.0001), breed (p<0.0002), lactation stage (p<0.019), and parity (p<0.0008) of cattle. The Staphylococcus species (43.7%) were the most predominant pathogens detected from SCM cow milk, followed by Streptococcus spp. (18.2%) and Escherichia spp. (9.5%). In the current study, a total of 166 dairy cows were randomly screened for SCM from seven (7) small-scale farms using the California mastitis test and somatic cell count assay. The results showed that 33.13% of all cows had SCM at cow level, while the quarter-level prevalence was 54%. Thereafter, representative samples of 10 each from SCM and non-SCM dairy cow milk were sequenced to determine abundance and compare core microbiota through 16S rRNA metabarconding. Alpha-diversity metrics showed significant differences between the SCM cows and their non-SCM counterparts. The beta-diversity metrics on Principal Coordinate Analysis significantly clustered samples by type (PERMANOVA test, P < 0,05), while Non-Metric Dimensional Scaling did not cluster the samples by type (PERMANOVA test, p>0.07). Four phyla, namely Actinobacteriota, Bacteroidota, Firmicutes, and Proteobacteria collectively accounted for more than 97% of all sequencing reads from both the SCM and non-SCM cow milk samples. The most detected bacterial classes were Actinobacteria, Bacilli, Bacteroidia, Clostridia, and Gammaproteobacteria in healthy cow samples, whilst SCM cow milk samples mainly had Actinobacteria, Alphaproteobacteria, Bacilli, Clostridia, and Gammaproteobacteria. The dominant bacterial species in healthy cow samples were Anthropic spp., Pseudomonas azotoformans, Pseuodomonas fragi, Acinetobacter guillouiae, Enterococcus italicus, and Lactococcus lactis, whilst P. azotoformans, Micrococcus bovis, Pseudomonas fragi, Acinectobacter guillouiae, and Pseudomonas koreensis were dominant in the SCM cow milk samples. In addition to the above-mentioned findings, the study sought to detect Staphylococcus spp. from samples of SCM cow milk using MALDI-TOF MS, PCR assays (nuc, tuf and 16S rRNA), and gene sequencing to further determine their virulence and antimicrobial resistance (AMR) profiles. In 50 of 70 samples bacterial isolates were detected and characterised as Staphylococcus spp., and it was found that S. aureus (76%) was the most prevalent species, followed by S. chromogenes (12%), S. epidermidis (4%), and S. haemolyticus (4%) using MALDI-TOF MS. The 16S rRNA and species-specific gene sequences (nuc and tuf) indicated that S. aureus was the dominant species at 76%, followed by S. chromogenes, S. agnetis, S. argenteus, and S. devriesei at 10%, 4%, 4% and 1% respectively. The AMR test showed that 86% of the isolates were resistant to penicillin, followed by resistence to ciprofloxacin (80%) and cefoxitin (52%). Moreover, the detection of antimicrobial resistant and virulence genes showed that the mecA gene was present in 16% of all the isolates, while 52% of all the isolates carried the LgG gene, followed by the coa and spa (x-region) genes at 42% and 40% respectively. Moreover, the hla and hlb genes were detected in 38% of the isolates, whereas the Sea and bap genes were detected in 10% and 2% of the isolates respectively. In the second phase of the study, whole-genome sequencing technique was conducted to genetically characterize 38 mastitis-associated S. aureus. The results revealed that 38 S. aureus isolates appeared in seven sequence types (STs), namely, ST97, ST352, ST152, ST243, ST8495, ST8500 and ST8501, whereby the latter three STs are novel. The 38 strains were divided into seven (7) primary clusters by core genes. The Spa-type t2883 accounted for most isolates at 12 (31.57%), followed by t416 at 11 (28.94%) and t2844 at 5 (13.15%) among the nine (9) distinct spa-types that were detected. The data also revealed the presence of four (4) plasmid replicONS, with Rep_N (rep20) accounting for the majority (17) of the isolates at 44.73%, followed by 2 Inc18 (repUS5). These isolates included 11 distinct antimicrobial resistance (ARGs) genes and 23 genes that were linked to virulence. All the isolates used in this investigation were methicillin susceptible. In conclusion, it was found that diverse microbial populations were detected in the sampled milk from SCM-infected cows. Furthermore, the study also showed pathogenic potential and antimicrobial resistant status of S. aureus and coagulase negative staphylococci (CNS) through detection of virulence and AMR genes. These S. aureus isolates had varying levels of antibiotic responsiveness with some showing multiple drug resistance traits. The findings of this study will contribute significantly improvement of strategies for control and prevention of Staphylococcus-related mastitis on dairy farms.