Phenotypic and genotypic identification of streptococci and related bacteria isolated from bovine intramammary infections

Abstract

BackgroundStreptococcus spp. and other Gram-positive, catalase-negative cocci (PNC) form a large group of microorganisms which can be found in the milk of cows with intramammary infection. The most frequently observed PNC mastitis pathogens (major pathogens) are Streptococcus uberis, Strep. dysgalactiae, and Strep. agalactiae. The remaining PNC include a few minor pathogens and a large nonpathogenic group. Improved methods are needed for the accurate identification and differentiation of PNC.A total of 151 PNC were collected from cows with intramammary infection and conclusively identified by 16S rRNA sequencing as reference method. Nine phenotypic microbiological tests (alpha-hemolysis, CAMP reaction, esculin hydrolysis, growth on kanamycin esculin azide agar and on sodium chloride agar, inulin fermentation, hippurate hydrolysis, leucine aminopeptidase and pyrrolidonyl peptidase activity), multiplex PCR for the three major pathogens (target genes for Strep. uberis, Strep. dysgalactiae and Strep. agalactiae: pauA, 16S rRNA, and sklA3, respectively), and mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) were evaluated for the diagnosis and discrimination of the three clinically most relevant PNC.ResultsThe probability that a strain of Strep. uberis, Strep. dysgalactiae and Strep. agalactiae was correctly identified by combining the results of the 9 phenotypic tests was 92%, 90%, and 100%, respectively. Applying the multiplex PCR, all strains of the three major pathogens were correctly identified and no false positive results occurred. Correct identification was observed for all strains of Strep. uberis and Strep. agalactiae using MALDI-TOF MS. In the case of Strep. dysgalactiae, some variability was observed at the subspecies level, but all strains were allocated to one single cluster.ConclusionsThe results of the present study show that reliable identification of the clinically most relevant PNC (Strep. uberis, Strep. agalactiae and Strep. dysgalactiae) can be obtained by use of a combination of colony morphology, hemolysis type and catalase reaction, and a multiplex PCR with specific primers restricted to these 3 pathogens. The MALDI-TOF MS is a fast method that shows promising results, although identification of Strep. dysgalactiae at the subspecies level is not yet satisfactory.

Phenotypical and Mass Spectral Assessment Methods for Identification of Some Contagious Mastitis Pathogens

“…agalactiae and 72% of CNS were identified by API system. The results tabulated in the current study, supported by similar and widespread data for clinical human and veterinary microbiology (Ilina et al, 2009;Lartigue et al, 2009; Science Publications Marklein et al, 2009;Nagy et al, 2009;Barreiro et al, 2010;Dubois et al, 2010;Ilina et al, 2010;Seibold et al, 2010;Moon et al, 2013;Raemy et al, 2013), confirmed that MALDI-TOF MS contributes in the fast identification of bacteria (one day versus five to eight days for conventional identification) and consequently the chance of an earlier treatment of subclinical and clinical mastitis with suitable antimicrobial agents will be carried out. In addition, in the dairy industry, MALDITOF MS can provides a faster, cheaper and more reliable identification of microorganisms isolated from milk of animals witth clinical and subclinical mastitis.…”

“…While ~ 95% of 39 isolates of S. aureus were properly detected by MALDI-TOF MS (only two strains produced weak signals in the mass spectrum, which led to failure of identification). According to the conditions of the current study, most spectral peaks noticed were in the mass range of m/z 2000-18000 Da, Similar ranges were noticed by (Barreiro et al, 2010;Raemy et al, 2013) who stated that the most spectral peaks resulted from analysis of protein samples were ranged from 2000 to 20000 Da mass range. The difference in mass range of spectra peaks might be due to the variation in techniques of sample preparation.…”

Technological advances in bovine mastitis diagnosis

“…A 2013 study 61 supports the combination of conventional microbiology as first identification for triage and multiplex PCR use for rapid identification of bacteria associated with IMI. Molecular detection assays are a promising avenue in resolving false-negative issues, and several assays have already been developed that can provide solutions to this problem.…”

“…Mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) can also be performed to determine bacterial species 3,61 and bacterial strains 8 within a few minutes. 53 It is a reliable, easy to use, and cost-effective technique that has the potential to replace and/or complement conventional phenotypic identification, reaching sensitivity and specificity values of 100%.…”

Antimicrobial Activity of 1,3 Dihydroxy-2-Methylimidazolium Bis (Trifluoromethylsulfonyl) Imde and Selected Antibiotics Against <i>Streptococcus</i> Species Isolated from Groundwater in the Northwest Province, South Africa

“…The identities of these isolates were confirmed using Gram staining, oxidase test, catalase test and a serological assay. These same methods have been used for identification of Streptococcus species [33,34,35]. However, these tests are supplemented with molecular assays the isolates are identified at species level [35].…”