Comparison of PCR-RFLP, API® 20 Strep and MALDI-TOF MS for identification of Streptococcus spp. collected from sheep and goat milk samples

In this study, a bank of 195 Streptococcus isolates collected in Sardinia (Italy) from sheep and goat milk samples was analysed. The purpose of this research was to compare the accuracy of different methods of identifying Streptococcus at the species level: API (R) 20 Strep, PCR-RFLP and MALDI-TOF MS. For PCR-RFLP analysis, the housekeeping gap gene was used as target gene. After alignment of nucleotide sequences of different Streptococcus species, a primer set was designed to amplify a 945-bp DNA fragment. Amplicons of gap gene were submitted to restriction fragment length polymorphism analysis with the enzyme AluI. When PCR-RFLP patterns were different from those of their reference strains, gap gene products were sequenced for definitive identification. Most of the isolates displayed the same PCR-RFLP profile of Streptococcus uberis type strain (124, corresponding to 63.6%), followed by S. dysgalactiae (18, corresponding to 9.2%) and S. suis (14, corresponding to 7.2%). Overall, in comparison with PCR-RFLP and sequence analysis results, 161 over 195 Streptococcus isolates were identified to the same species levels by means API (R) 20 Strep (82.6% agreement, CI95 = 77.2-87.9) versus 166/195 (85.1% agreement, CI95 = 80.1-90.1) using MALDI-TOF. In conclusion, the PCR-RFLP is the most accurate and reproducible method for the identification of streptococci collected from milk samples, although high cost and technical difficulty hinder its usage in routine diagnosis. In our study, the accuracy of MALDI-TOF MS in detecting streptococci at the species level has not been perfect; however we believe that MALDI-TOF has the advantage in terms of its rapidity, reliability and low cost.