title: Analysis of bovine intramammary bacteriome, resistome and of the bacterial transmission within dairy herds
Creators: name: Romanò, Alicia
Publisher: name: Universität Bern
Year: 2023
Subjects: subject: 500 Science
subject: 630 Agriculture

description: Mastitis is the most important and costly disease in dairy cows worldwide. Bovine intramammary infection (IMI) caused by pathogenic bacteria is common and well understood but very little is known about the bacteria and their resistome present in the mammary gland (= intramammary resistome, IR) of healthy and untreated cows whose milk is regularly delivered for human consumption. The aim that our research project wanted to: i) describe the bacteriome of healthy cows means all the bacteria that we can isolate from the milk of healthy cow, ii) describe the resistome means the antibiotic resistance correlated with the bacteriome isolated with the use of phenotypic and genomic methods iii) comparison of the bacteriome and resistome with the environmental isolates. 
Healthy, untreated cows of nine dairy herds from the Swiss Canton Tessin were analyzed three times within one year to identify the most abundant species of the intramammary bacteriome of healthy animals. Aseptically collected milk samples were cultured and bacteria identified using MALDI-TOF mass spectrometry. To describe the intramammary resistome, 350 strains of the predominant species were selected and subjected to short-read based whole genome sequencing (WGS) combined with phenotypic analyses and antibiotic resistance gene profiling. Both chromosomes and mobile genetic elements were examined for antibiotic resistance genes (ARGs) using in-house and online software tools. ARGs were then associated with phenotypic antibiotic resistance profiling data from minimum inhibitory concentration assays. Furthermore, the phylogeny of the two main species isolated Staphylococcus xylosus (S. xylosus) and Mammaliicoccus sciuri (M. sciuri) was assessed using 70 housekeeping genes and the maximum likelihood approach (PhyML). Additionally statistical analyses were carried out.
Of 256 cows (1024 single quarters) analyzed, 96% were bacteriologically positive and 80% of the quarters were positive for at least one bacterial species. 84.5% of the quarters were healthy with somatic cell counts (SCC) < 200’000 cells/ml, whereas 15.5% of the quarters showed a subclinical mastitis (SCC ≥ 200’000 cells/ml). The 1288 isolates were assigned to 104 different bacterial species including 24 predominant species. Staphylococcaceae were most prevalent (14 different species; 73.5% bacteria-positive quarters) with S. xylosus and M. sciuri accounting for 41.5% of the strains. Our study demonstrates that Staphylococcaceae could be consider part of the healthy milk bacteriome. Furthermore, the different farm-specific patterns of the bacteriome are associated with the use of different bedding in the herd. The non-aureus staphylococci and mammalicocci (NASM) have been regarded as minor mastitis pathogens being the most abundantly observed bacteria in mastitis milk samples. Based on our study, however, their function needs to be reconsidered. We could assume that different subtypes of NASM may colonize the mammary gland whereas other subtypes may cause minor mastitis.
Phenotypic and genomic antimicrobial resistance (AMR) was bacterium-specific whereby resistance to clindamycin and oxacillin was most frequently observed (40% and 30%). In contrast, AMR to penicillin, although massively used for mastitis treatment during last decades, was rarely observed. The phenotypic findings, not in all the cases, could be linked to chromosomal or plasmid-borne ARGs, demonstrating a lack in understanding the mechanisms that lead to the observed phenotypic AMRs in the isolated bacteria. For some species/AMR, the observed phenotypic AMR could be explained. This is true for M. sciuri and its resistance to clindamycin (salA gene) as well as S. xylosus, M. sciuri and a few other bacteria in the case of tetracycline. For all of them, a small plasmid was found carrying the tetK or another tetracycline ARG. The presence of tetracycline AMR was herd dependent and was observed in various isolates of the same farm indicating a possible horizontal gene transfer among the different bacteria on the same farm, particularly among S. xylosus and M. sciuri.
The phylogenetic studies involved S. xylosus and M. sciuri; these bacteria were the most abundant isolated and were collected from both milk and environmental samples. M. sciuri was found predominantly in environmental samples (particularly straw bedding and teat liners before milking). Differently, the S. xylosus strains were mostly isolated from milk samples. Based on these results we could conclude that S. xylosus is mainly udder adapted whereas the habitat of M. sciuri is more environmentally related. Detailed phylogenetic analyses for M. sciuri revealed two main clades whereby the smaller one included the M. sciuri type strain. Typically, the strains forming this clade were almost exclusively isolated from bedding, milk, and teats, whereas those of the other clade were isolated from milk, teats, and liners, but hardly from bedding. These results suggest that M. sciuri circulates clade dependently more among environment and mammary gland (type strain clade) whereas M. sciuri of the other clade circulates more among mammary gland and milking equipment. These findings are confirmed by genotype analysis, inferring MLST, demonstrating that the distribution of the sequence types (STs) between milk and environment revealed only a few common STs. Interestingly, M. sciuri showed a remarkable variability at the STs level with the consequence that a number of different STs were observed on the same farm. In the case of S. xylosus, three main clades were observed. As in M. sciuri, the phylogenetic distances within each clade were very small showing that the taxa/strains evolved minimally and that they are genetically very similar. For all three clades, the taxa were largely milk associated whereas environmental taxa were hardly observed. These findings indicate that S. xylosus circulates primarily among mammary glands and the taxa found in the environment are more the result of a contamination by milk.
As a conclusion, the research project demonstrates that bacteria, particularly NASM, are very common in the mammary gland of healthy cows, a fact that needs to be considered when interpreting bacteriological results obtained from clinical milk samples. Furthermore, AMR in NASM is uncommon, also against penicillin, although it has been massively used for mastitis treatment during last decades. Unfortunately, AMR in these bacteria remains largely unexplained by genomic analyses. Furthermore, AMR against antibiotics used in human medicine is rare. Finally, phylogenetic studies demonstrate that M. sciuri and S. xylosus formed only a few major clades and within each clade the strains were genetically very similar; for M. sciuri the habitat was clade dependent. 
It is essential to get more knowledge about the bacteriome of the mammary glands of healthy and diseased cows to understand the microbiota, hinder pathogens to gain a foothold and, in the long term, prevent the development and spread of resistances. WGS represents an important tool for detecting ARGs but still needs to be associated with phenotypic analysis and with gene and/or protein expression analyses. Screening for new genes associated with AMR and an increase of the ARG databases will be essential, especially for the One Health concept.