Resumo

Identification of coagulase-negative staphylococci (CoNS) by phenotypic methods has been challenging in veterinary microbiology. These tests are time-consuming and their accuracy remains uncertain as some species are genetically and phenotypically similar. Genotypic methods, such as sequencing, are costly for routine laboratory. On the other hand, proteomic methods, such as MALDI-TOF MS, have been used to identify bacterial species from different sources. This technique requires expensive equipment, but it has benefits considering high accuracy and low costs for running, and being easy to operate. Considering the advantages of MALDI-TOF MS to identify bacterial species, and the lack of studies using it to identify CoNS isolated from milk, we evaluated its ability to identify 92 strains isolated from bulk tank milk. sequencing of the tuf gene was used for comparison. Isolates were obtained from 30 bulk tank milk samples, in São Paulo State, Brazil, cultured on Baird-Parker agar. Two to four colonies of CoNS in each sample were characterized by Gram stain, catalase and coagulase tube test, and stored at -80°C. Isolates were re-isolated and DNA extraction was performed. The tuf gene was amplified by PCR and sent for sequencing. MALDI-TOF analysis was performed direct from fresh culture on blood agar, after simple partial extraction with formic acid. We identified 87 CoNS, four M. caseolyticus and one coagulase-negative S. aureus. The predominant species identified by tuf sequencing were S. hyicus (n = 32, 34.8%), and S. chromogenes (n = 21, 22.8%), followed by S. epidermidis (n = 9, 9.8%), S. haemolyticus (n = 7, 7.6%), S. sciuri (n = 5, 5.4%), S. warneri (n = 5, 5.4%), M. caseolyticus (n = 4, 4.3%), S. xylosus (n = 4, 4.3%), S. aureus (n = 1, 1.1%), S. capitis (n = 1, 1.1%), S. hominis (n = 1, 1.1%), S. saprophyticus (n = 1, 1.1%), S. simulans (n = 1, 1.1%). MALDI-TOF was able to correctly identify the majority of isolates (98.9%), except for one isolate of S. epidermidis that presented an unreliable identification (score 1.425), even after two repetitions. tuf gene sequencing showed a query coverage ≥98% and maximum identity ≥98% in the majority of isolates, and the unidentified isolate by MALDI-TOF was identified as S. epidermidis with a query coverage of 100% and maximum identity of 97%. Using MALDI-TOF for identification of CoNS from bulk tank milk demonstrated to be feasible and fast, correctly identifying the majority of isolates. FAPESP Post-Doc fellowship 2010/17222-8 and FAPESP International Internship scholarship 2012/05967-4 FAPESP Grant 2011/09106-0