Identification of Staphylococcal species from clinical isolated from hard to treat cases of bovine mastitis using conventional diagnostic techniques and Matrix Assisted Laser Desorption Ionisation – Time of Flight Mass Spectrometry (MALDI-TOF)

Application This study utilised Matrix Assisted Laser Desorption Ionisation – Time of Flight Mass Spectrometry (MALDI-TOF) to determine the species-level identity of Staphylococcus aureus isolates from hard to treat clinical cases of bovine mastitis in Australia. The potential utility of this technology in mastitis disease outbreaks will be discussed.

Introduction Staphylococcus aureus forms part of the normal flora, that is ubiquitous, of the skin and mucus membranes but can also cause both clinical and subclinical mastitis in dairy cattle. S. aureus is a Gram positive, catalase and coagulase positive, haemolytic bacteria which produces toxic products causing hard to treat infections. As well as S. aureus, a number of other streptococcal species are implicated in bovine mastitis including opportunistic and / or environmental pathogens such as S. agalactiae, S. dysgalactiae and S. uberis. The outcome from the treatment of S. aureus mastitis is variable and the cure rates often low. Studies have shown that rates can vary between 4% and 92% for infections caused by S. aureus specifically (Apparao et al., 2009; Barkema et al., 2006; Baskaran et al., 2009) with significant implications therefore for development of antimicrobial resistance in this species. It is possible that misidentification of staphylococcal strains and therefore incorrect treatment. To address this issue, we compared standard morphological and biochemical identification of 56 isolates from clinical cases of bovine mastitis to compare with genus and strain identification using Matrix Assisted Laser Desorption Ionisation – Time of Flight Mass Spectrometry (MALDI-TOF) (Dubois et al, 2010).

Material and methods Bacteria were isolated from infected milk samples after aseptic collection of samples directly from infected cows. Bacterial isolates were compared using standard microbiological identification techniques and then subjected to Matrix Assisted Laser Desorption Ionisation – Time of Flight Mass Spectrometry using a Bruker Ultraflextreme MALDI-TOF/TOF-MS instrument. Spectra were recorded using linear positive mode at a laser frequency of 1000 Hz and within the mass range 2,000 to 20,000 Da. Data was compared to known pathogen standard library for genus and species identification.

Results MALDI-ToF confirmed the identification of 31 S. aureus isolates as defined by standard morphological and biochemical protocols. Of the further 12 Staphylococcus species that could only be determined to a genus level, MALDIFoF confirmed identification of 2 isolates as S. chromogenes, 2 isolates as S. haemolyticus, 4 isolates as S. vitulinus and 3 as S. warneri. A further two isolates that had been previously identified as S. epidermis were confirmed to be S. pseudintermedius. Two isolates were confirmed as Enterococcus faecium and one that had been previously identified as Staphylococcus sp. but without strain identification was confirmed to be Micrococcus luteus. Only one isolate could not be identified by either method. Antimicrobial testing identified multiple resistant strains including MRSA.

Conclusion Together these results indicate that traditional methods of microbial identification may not be sufficient to determine species identify for hard to type bacteria such as less common Staphylococcus species. Misidentification may be misrepresenting antimicrobial resistance or risking this effect where suboptimal antibiotic regmines might therefore be implemented. Use of MALDI-ToF identification for bovine mastitis pathogens can assist in providing clinicians and researchers with a more accurate picture of the major pathogens involved in subclinical and clinical mastitis and assist in better antimicrobial stewardship in the future.