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

Staphylococcus species are the primary cause of mastitis in dairy cows across the world. Staphylococcus aureus has recently become a pathogen that is zoonotic and multidrug resistant. This study aimed to sequence whole genomes of 38 S. aureus isolates from 55 subclinical mastitis dairy cows of 7 small-scale farmers in the Free State Province, South Africa and document and their antimicrobial and virulence genes. The 38 isolates were grouped by the in silico multi-locus sequencing types (MLST) into seven sequence types (STs), that is (ST 97, 352, 152, 243) and three new STs (ST8495, ST8500, and ST8501). Thirty-three S. aureus isolates were divided into 7 core single-nucleotide polymorphism (SNP) clusters. Among the 9 distinct spa-types that were detected, Spa-types t2883 accounted for the majority of isolates at 12 (31.57%), followed by t416 with 11 (28.94%) and t2844 with 5 (13.15%). The data also revealed the identification of four (4) plasmids, with Rep_N (rep20) accounting for the majority of isolates with 17 (44.73%), followed by Inc18 (repUS5) with 2 (5.26%). These isolates included 11 distinct antimicrobial resistance genes and 23 genes linked to bacterial virulence. Surprisingly, no methicillin resistance associated genes were detected in these isolates. Genome data of the current study will contribute to understanding epidemiology S. aureus genotypes and ultimately aid in developing treatment and control plans to stop the spread of mastitis in the Free State province and South Africa as a whole.

Antibiotic resistance and virulence profiles of Proteus mirabilis isolated from broiler chickens at abattoir in South Africa.

BACKGROUND: Proteus mirabilis has been identified as an important zoonotic pathogen, causing several illnesses such as diarrhoea, keratitis and urinary tract infections. OBJECTIVE: This study assessed the prevalence of P. mirabilis in broiler chickens, its antibiotic resistance (AR) patterns, ESBL-producing P. mirabilis and the presence of virulence genes. METHODS: A total of 26 isolates were confirmed as P. mirabilis from 480 pooled broiler chicken faecal samples by polymerase chain reaction (PCR). The disk diffusion method was used to evaluate the antibacterial susceptibility test, while nine virulence genes and 26 AR genes were also screened by PCR. RESULTS: All 26 P. mirabilis isolates harboured the ireA (siderophore receptors), ptA, and zapA (proteases), ucaA, pmfA, atfA, and mrpA (fimbriae), hlyA and hpmA (haemolysins) virulence genes. The P. mirabilis isolates were resistant to ciprofloxacin (62%) and levofloxacin (54%), while 8 (30.7%) of the isolates were classified as multidrug resistant (MDR). PCR analysis identified the blaCTX-M gene (62%), blaTEM (58%) and blaCTX-M-2 (38%). Further screening for AMR genes identified mcr-1, cat1, cat2, qnrA, qnrD and mecA, 12%, 19%, 12%, 54%, 27% and 8%, respectively for P. mirabilis isolates. The prevalence of the integron integrase intI1 and intI2 genes was 43% and 4%, respectively. CONCLUSIONS: The rise of ciprofloxacin and levofloxacin resistance, as well as MDR strains, is a public health threat that points to a challenge in the treatment of infections caused by these zoonotic bacteria. Furthermore, because ESBL-producing P. mirabilis has the potential to spread to humans, the presence of blaCTX -M -producing P. mirabilis in broilers should be kept under control. This is the first study undertaken to isolate P. mirabilis from chicken faecal samples and investigate its antibiotic resistance status as well as virulence profiles in South Africa.

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.

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.

Three novel sequencing types from seventeen Staphylococcus aureus genomes isolated from dairy cows milk in the Free State Province of South Africa.

Staphylococcus aureus is one of the major pathogens causing bovine mastitis, which results in huge economic losses in the dairy industry worldwide. Here, we report genome sequences of 17 S. aureus strains, with three novel sequencing types (ST8495, ST8500, and ST8501) isolated from the milk of dairy cows with subclinical mastitis.

Prevalence of subclinical mastitis, its associated bacterial isolates and risk factors among cattle in Africa: a systematic review and meta-analysis.

BACKGROUND: Subclinical mastitis (SCM) is one of the most economically important diseases affecting the dairy industry. The SCM does not cause visible changes in the udder or physical changes of the milk as compared to clinical mastitis, and a clear overview of the prevalence and risk factors in the different regions of Africa is still lacking. The objective of this study was to investigate the prevalence of SCM and assess the associated risk factors and dominant bacterial pathogens among cattle in Africa. MATERIALS AND METHODS: We gathered and systematically reviewed literature concerning SCM, published in English from January 2010 through December 2020 in two databases (PubMed and Web of Science), and meta-analysis was conducted using the 'meta' and 'metafor' packages in the R statistical software. RESULTS: A total of 258 studies were retrieved and at the end of the screening, 82 full-texts were eligible for inclusion in the meta-analysis. The prevalence of SCM was reported in 11 countries in five regions of Africa, and the random-effects model showed that the weighted pooled prevalence estimate (PPE) was 48.2% (95% CI: 43.6-52.8%). Heterogeneity was high and statistically significant as I2 (proportion of observed variation) was 98.1% (95% CI: 98.0-98.3%), τ2 (true between-study variance) was 0.0433 (95% CI: 0.0322-0.0611), and the Cochran Q statistic was 4362.8 (p < 0.0001). Subgroup and meta-regression analyses showed that East Africa had significantly (p = 0.0092) the highest PPE of SCM (67.7%, 95% CI: 55.7-78.7) followed by West Africa (50.5%, 95%CI: 31.4-69.5), and the lowest was in North Africa (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. Staphylococcus species (prevalence 43.7%) were the most predominant pathogens, followed by Streptococcus (18.2%) and Escherichia species (9.5%). CONCLUSION: The present study showed a high variation of SCM prevalence in various parts of Africa, although there is a need for more data in some regions. The reported prevalence is a clear sign of inappropriate management practices among cattle herds and an indicator of the threat that SCM poses to the dairy industry. The information about the predisposing factors may guide effective management and control strategies to reduce transmission of the disease.

Application of culture, PCR, and PacBio sequencing for determination of microbial composition of milk from subclinical mastitis dairy cows of smallholder farms.

Mastitis is a cow disease usually signalized by irritation, swelling, and soreness of the udder. It is characterized by physical, chemical, and biological changes in the udder and milk. The aim of this study was to detect and characterize pathogens causing subclinical mastitis (SCM) from the milk of dairy cows of small-scale farmers through culture and molecular techniques. Milk was collected from 32 cows belonging to 8 small-scale farmers around Harrismith District, South Africa. The results showed that screening of SCM by California mastitis test and somatic cell counts (SCC) was 21.87 and 25%, respectively. Culture methods revealed the presence of Staphylococcus aureus at 93% followed by Streptococci spp. and Escherichia coli at 36.4 and 13.3%, respectively. The PCR could only detect E. coli, while single-molecule real-time sequencing showed a total of 2 phyla, 5 families, 7 genera, and 131 species. Clostridiaceae was the most abundant family, while Romboutsia was the most abundant genus followed by Turicibacter spp. The present study has documented the occurrence of SCM causing pathogens in milk collected from cows of small-scale farmers in Harrismith, indicating that SCM may be present at higher levels than expected.