Comparison of quantitative PCR and MALDI-TOF mass spectrometry assays for identification of bacteria in milk samples from cows with subclinical mastitis

Aims: The objective of this study was to compare qualitatively and quantitatively the results of identification of the bacteria present in milk samples from cows with subclinical mastitis using multiplex qPCR assay and matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS (R)) after bacteriological growth. Methods and Results: A total of 182 samples were aseptically collected from 119 cows with high somatic cell counts (>2.10(5) SCC per ml) on 11 farms in Belgium in 2014. The mutiplex qPCR assay was carried out on 350 mu l of milk with the PathoProof (R) Complete-16kit. Ten microlitre of milk was streaked on Columbia blood agar and three selective agar plates. Growing colonies were identified by MALDI-TOF MS. Of the 182 samples, 90 gave positive results with either or both tests for one or two bacterial species/genera. Total qualitative agreement of the bacteria identified was observed in 41 mono- or bi-bacterial samples (46%) and partial agreement in 19 bi-bacterial samples at both or either tests (21%). The results of both tests on those mono- and bi-bacterial samples were not significantly different (McNemar test; P = 0.395) with a fair agreement (Cohen's kappa test; k = 0.375; P = 0.055). Moreover, quantitative correlation between the qPCR intensity and the numbers of growing colonies was observed in half of the 60 samples with qualitative matching results. Conclusions: Both methods give identical qualitative and quantitative results with approximately a half and a quarter of the mono- and bi-bacterial samples respectively. Several reasons can explain the differences. The multiplex qPCR assay only targets the most important mammary gland pathogens and can detect DNA of bacteria both alive and dead. Conversely, bacteria only grow when alive and the MALDI-TOF MS databases do not include all bovine milk-associated bacterial species yet. Significance and Impact of the Study This study further highlights the limitations and complementarity of the genetic and phenotypic tests for the identification of bacteria present in milk samples