Antibiotic resistance, characterization and biological control of bovine mastitis caused by Streptococcus uberis

Bovine mastitis still remains a major problem to the dairy industry world wide although there are mastitis control strategies in place. In the last few decades there has been a change in the predominant pathogens causing mastitis. Streptococcus uberis is now the leading cause of bovine mastitis. Accurate and cost effective methods for identification of S. uberis by diagnostic laboratories would assist farm management to reduce the infection. One common practice to control mastitis is the use of antimicrobial agents. Long term antibiotic use can cause antibiotic resistance and there is concern that resistant bacteria could be transferred to humans. Knowledge of current antibiotic susceptibility test patterns for mastitis pathogens could improve therapy protocols for particular dairy farms. An alternative way to eliminate mastitis pathogens is biological control. Recent evidence has shown that non-pathogenic coagulase-negative staphylococci have the ability to produce antibacterial proteins (bacteriocins) that are active against some mastitis pathogens.
One hundred and fifteen isolates of esculin hydrolyzing streptococci from intramammary infections in dairy cows, which are commonly categorized as S. uberis, were further identified using four additional biochemical tests (hippurate hydrolysis, fermentation of inulin and mannitol and growth in 6.5% NaCl). 16S rRNA sequencing was used as a gold standard for species identification. The additional biochemical tests generated seven different patterns. All isolates belonging to pattern 1 (hippurate hydrolysis positive, inulin and mannitol fermentation positive and negative for growth in 6.5% NaCl) were identified by 16S rRNA sequencing as S. uberis (91%) or S. parauberis (9%). Half of the isolates displaying pattern 2 which differed from pattern 1 only in inulin fermentation (negative) were also identified as S. uberis (33%) or S. parauberis (17%) by 16S rRNA sequencing. Differentiation of S. uberis and S. parauberis required genotypic examination. Use of these additional biochemical tests (hippurate hydrolysis, inulin and mannitol fermentation and growth in 6.5% NaCl) resulted in relatively low numbers of misidentification (8.6%). These tests represent an affordable cost and can therefore be considered an adequate method for veterinary diagnostic laboratories for identifying S. uberis isolated from bovine mastitis.

The susceptibility of S. uberis to nine antimicrobial agents was determined by the broth dilution method. The minimal inhibitory concentrations (MICs) at which 90% of isolates were inhibited (MIC90s) for each of ampicillin, penicillin, oxacillin, erythromycin, tetracycline, novobiocin, cephalothin, vancomycin and gentamicin was 1.0, 0.06, 0.25, 0.5, 0.5, 4.0, 0.25, 0.5 and 8.0 µg/mL respectively. No isolates showed resistance to those antibiotics. However the continuous monitoring of antibiotic resistance is essential, as the emergence of resistance strains has become a concern.

A bacteriocin-producing bacterium, Macrococcus caseolyticus (formerly Staphylococcus caseolyticus) was isolated from a cow’s milk sample in Victoria from a farm with a “uberis” problem; i.e. an unacceptable prevalence of clinical and sibclinical cases of mastitis. The bacteriocin was heat stable and remained active over a wide range of pH, with highly specific activity against the genus Streptococcus . Purification of the bacteriocin, included ammonium sulphate precipitation, gel filtration, cation exchange chromatography, extraction from polyacrylamide gel and reverse phase high performance liquid chromatography. The results showed two components which were independently active and the mass spectrometry showed the mass of both was less than 3000 Da. Scanning and transmission electron microscopy showed damage of target cells. It is suggested that the use of bacteriocins could provide effective control and prevention of bovine mastitis caused by S. uberis.