Characterization of Staphylococcus aureus isolates from raw milk sources in Victoria, Australia

Abstract

Background: Highly pathogenic strains of Staphylococcus aureus can cause disease in both humans and animals. In animal species, including ruminants, S. aureus may cause severe or sub-clinical mastitis. Dairy animals with mastitis frequently shed S. aureus into the milk supply which can lead to food poisoning in humans. The aim of this study was to use genotypic and immunological methods to characterize S. aureus isolates from milk-related samples collected from 7 dairy farms across Victoria.

Results: A total of 30 S. aureus isolates were collected from milk and milk filter samples from 3 bovine, 3 caprine and 1 ovine dairy farms across Victoria, Australia. Pulsed Field Gel Electrophoresis (PFGE) identified 11 distinct pulsotypes among isolates; all caprine and ovine isolates shared greater than 80 % similarity regardless of source. Conversely, bovine isolates showed higher diversity. Multi-Locus Sequence Typing (MLST) identified 5 different sequence types (STs) among bovine isolates, associated with human or ruminant lineages. All caprine and ovine isolates were ST133, or a single allele variant of ST133. Two new novel STs were identified among isolates in this study (ST3183 and ST3184). With the exception of these 2 new STs, eBURST analysis predicted all other STs to be founding members of their associated clonal complexes (CCs). Analysis of genetic markers revealed a diverse range of classical staphylococcal enterotoxins (SE) among isolates, with 11 different SEs identified among bovine isolates, compared with just 2 among caprine and ovine isolates. None of the isolates contained mecA, or were resistant to oxacillin. The only antibiotic resistance identified was that of a single isolate resistant to penicillin; this isolate also contained the penicillin resistance gene blaZ. Production of SE was observed at 16 °C and/or 37 °C in milk, however no SE production was detected at 12 °C.

Conclusion: Although this study characterized a limited number of isolates, bovine-associated isolates showed higher genetic diversity than their caprine or ovine counterparts. This was also reflected in a more diverse SE repertoire among bovine isolates. Very little antibiotic resistance was identified among isolates in this study. These results suggest maintaining the milk cold chain will minimise any risk from SE production and highlights the need to prevent temperature abuse.

Keywords: Antibiotic sensitivity; Dairy farm; MLST; Milk; PFGE; Staphylococcal enterotoxin; Staphylococcus aureus.

Fig. 1

Dendrogram depicting the PFGE profiles of the 13 isolates collected from the Victorian dairy farms. The corresponding farm, sample type, isolation date, animal class and ST are shown

Fig. 2

Minimum Spanning Tree based on the MLST data for each isolate. Numbers indicate ST of each node. Isolates differing by only a single allele are partitioned (grey shading). Novel STs identified in this study were ST3183 and ST3184

Fig. 3

Population snapshot of all bovine isolates. Pink labels represent isolate STs from this study also in the eBURST database, whereas the green label (ST3183) is unique to this study. The putative founding ST is coloured blue, where this could be predicted

Fig. 4

Population snapshot of all ovine and caprine isolates. Pink labels are isolate STs from this study also in the eBURST database, and the green label isolate ST (ST3184) was identified in this study but not in the eBURST database. The putative founding ST is coloured blue, where this could be predicted

Fig. 5

Level of staphylococcal enterotoxin detected in milk at 12 °C for 72 h, 16 °C for 48 h, or at 37 °C for 18 h. Five isolates, each differing by pulsotype, were screened. Green, SEC; blue, SED