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® ) after bacteriological growth. METHODS AND RESULTS: A total of 182 samples were aseptically collected from 119 cows with high somatic cell counts (>2·105 SCC per ml) on 11 farms in Belgium in 2014. The mutiplex qPCR assay was carried out on 350 µl of milk with the PathoProof® 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.

In vitro and in vivo assessment of phage therapy against Staphylococcus aureus causing bovine mastitis.

OBJECTIVE: The aim of this study was to assess the efficacy of lytic bacteriophages on Staphylococcus aureus causing bovine mastitis, by in vitro and in vivo assays using Galleria mellonella and murine mastitis models. METHODS: Between May and December 2016, ten S. aureus (five methicillin-resistant and five methicillin-sensitive) isolates were isolated from milk samples of cattle with mastitis in Belgium and Norway. The isolates were assessed in vitro for their susceptibility to four lytic bacteriophages (Romulus, Remus, ISP and DSM105264) and subsequently in vivo in G. mellonella larvae and in murine mastitis model. RESULTS: Romulus, Remus and ISP showed a lytic activity against the S. aureus isolates in vitro. A larvae survival rate below 50% was observed at 4 days post-inoculation (DPI) in the groups infected with a methicillin-sensitive S. aureus isolate and treated with these three phages in vivo. An incomplete recovery of the mouse mastitis was observed at 48h post-inoculation (HPI) in the groups infected and treated with the ISP phage in vivo. CONCLUSIONS: The observations are much more pronounced statistically between the infected- phosphate buffered saline (PBS)-treated and infected-phage-treated groups in G. mellonella and the murine mastitis model demonstrating an effect of the phages against S. aureus associated with bovine mastitis.