Antibiotic resistance of coagulase positive staphylococci isolated from milk of South African dairy herds

Abstract

The discovery and the subsequent global use of antibiotics has led to the survival of resistant microorganisms and suppression of susceptible species. This has caused a worldwide interest in antibiotic resistance and its threat to human and animal health. World-wide and particularly in South Africa there is a lack of antibiotic resistance surveillance data specifically for dairy cattle. The routine sampling of the Milk Laboratory Faculty of Veterinary Science at the University of Pretoria, as part of the pro-active udder health programme, has generated such data which needed to be analysed, interpreted and applied in practice. Staphylococcus aureus (S. aureus), one of the biggest problems in the dairy industry, was chosen as the organism to be used as the starting point for this ongoing project. The retrospective antibiotic resistance data (Kirby Bauer) were analysed for S. aureus (n= 2532) to eight commonly used antibiotics available as intramammary remedies for specific mastitis treatments in Southern Africa from 2000 to 2010. While overall antibiotic resistance was generally increasing over time as shown worldwide, antibiotic resistance was in fact decreasing over time for twenty well-managed herds (nineteen in South Africa and one in Zambia). This was attributed mostly to the effects of good management in the herds that were regularly tested as part of the pro-active udder health programme. There were also significant effects of seasons and regions on antibiotic resistance in tested isolates. All of the antibiotics tested, barring cephalosporins, showed a predicted prevalence of resistance of above 50% in most provinces. This is a concern. The lowest prevalence of resistance to the majority of the categories of antibiotics tested was in KwaZulu-Natal Province during spring. The reasons for the differences in antimicrobial rsistance between seasons and provinces are obscure. These differences may be a secondary effect related to the amount of antibiotic usage. The cephalosporins had the lowest levels of prevalence of bacterial resistance in Gauteng Province during winter. Although, mostly unexplained, such effects on antibiotic resistance could possibly be attributed to the different weather conditions in different regions of the country during different seasons. The conventional procedures for the identification of S. aureus led to the identification of coagulase positive and maltose negative staphylococci with doubtful identification of species. This research aimed at confirming the identification of this organism (conventional microbiology), which seemed to be an emerging pathogen, using molecular methods (MALDI-TOF MS, and 16s rRNA sequencing). The isolates of the maltose negative Staphylococcus sp. tested, were confirmed as being S. aureus by both molecular methods (100% correlation). However, it is also important to differentiate between maltose negative and maltose positive S. aureus isolates during routine diagnostics because these organisms react differently and thus need to be treated differently in practice. Also, maltose negative S. aureus tested positive for both malA and malR genes. A stop codon was discovered at position 844 of the malA gene caused by a cytosine to thymine transition which resulted in early termination of the α-glucosidase protein which would most likely be inactivated. This truncated protein may be the cause of the maltose negative phenotype. The discovery of this stop codon proves that maltose negative S. aureus ST 2992 is indeed different to conventionally identified maltose positive S. aureus. Antibiotic resistance of maltose negative S. aureus was analysed using retrospective data of this pathogen (n = 271), from milk samples of 117 farms between 2010 and 2017 (Kirby Bauer). The analysis was done using both the previous system (intermediate grouped with resistant) and more recent system (intermediate grouped with susceptible) CLSI breakpoints. The results between the previous and more recent analysis differed for tylosin, cefalonium, oxy-tetracycline and cloxacillin. Neither the previous system nor more recent system of analysis showed any difference between provinces for the maltose negative S. aureus. Strains of S. aureus which differed on phenotypic identification with the maltose test, also differed in antibiotic resistance patterns over time, per province, per season and SCC category. Further antibiotic susceptibility testing (MIC) was carried out, using the automated broth microdilution method for both maltose positive (n= 57) and maltose negative (n = 57) S. aureus from 34 farms. The MIC results for maltose negative S. aureus confirmed the results of the Kirby Bauer for the products tested. MIC 50 and MIC 90 were susceptible for both maltose negative and maltose positive S. aureus, except for MIC 90 of maltose negative S. aureus. This MIC analysis indicated more resistance in general seen in the maltose negative S. aureus, than in the maltose positive strains. Uncommon resistance patterns such as the resistance to vancomycin, oxacillin and carpapenems were shown for maltose negative S. aureus isolates, implying a possible anthroponosis (previously known as reverse zoonosis or zooanthroponosis). It is still unclear why this is found and how this might be linked to the difference in the phenotypic identification of this organism. The surveillance and monitoring of antibiotic resistance is important in order to assist decision makers, influence legislation, control antibiotic resistance, preserve human and animal health and to promote food security.