ABSTRACT Escherichia coli is a common environmental pathogen associated with clinical mastitis (CM) in dairy cattle. There is an interest in optimizing the udder microbiome to increase the resistance of dairy cattle to E. coli CM; however, the details of which members of the healthy udder microbiome may play a role in antagonizing E. coli are unknown. In this study, we characterized the bacterial community composition in raw milk collected from quarters of lactating Holstein dairy cows that developed E. coli CM during lactation, including milk from both healthy and diseased quarters ( n = 1,172). The milk microbiome from infected quarters was compared before, during, and after CM. A combination of 16S rRNA gene amplicon and metagenomic sequencing was used generate data sets with a high level of both depth and breadth. The microbial diversity present in raw milk significantly decreased in quarters experiencing E. coli CM, indicating that E. coli displaces other members of the microbiome. However, the diversity recovered very rapidly after infection. Two genera, Staphylococcus and Aerococcus , and the family Oscillospiraceae were significantly more abundant in healthy quarters with low inflammation. Species of these genera, Staphylococcus auricularis, Staphylococcus haemolyticus, and Aerocussus urinaeequi , were identified by metagenomics. Thus, these species are of interest for optimizing the microbiome to discourage E. coli colonization without triggering inflammation. IMPORTANCE In this study, we show that E. coli outcompetes and displaces several members of the udder microbiome during CM, but that microbial diversity recovers post-infection. In milk from quarters which remained healthy, the community composition was often highly dominated by S. auricularis, S. haemolyticus, A. urinaeequi, and S. marcescens without increases in somatic cell count (SCC). Community dominance by these organisms, without inflammation, could indicate that these species might have potential as prophylactic probiotics which could contribute to colonization resistance and prevent future instances of E. coli CM.
Additional file 6: Table S5. Bacterial species/strains and their relative abundance (%) found in three milk samples from the shotgun metagenomic sequneces
Escherichia coli is one of the most common etiological agents responsible for clinical bovine mastitis. Here, we report the draft genome sequences and annotations of 113 E. coli strains that were isolated from Holstein cows with intramammary infections in Canada.
Staphylococcus aureus is one of the main pathogens leading to both clinical and subclinical bovine mastitis in dairy cattle. Prediction of disease evolution based on the characteristics of Staph. aureus isolates that cause intramammary infections and understanding the host-pathogen interactions may improve management of mastitis in dairy herds. For this study, several strains were selected from each of the 6 major Canadian spa types associated with mastitis (t267, t359, t529, t605, t2445, and t13401). Adherence to host cells and intracellular persistence of these strains were studied using a bovine mammary gland epithelial cell line (MAC-T). Additionally, relative virulence and host response (cytokines production) were also studied in vivo using a mouse model of mastitis. Whole-genome sequencing was performed on all strains and associations between clonal complex, sequence type, and presence of certain virulence factors were also investigated. Results show that spa type t2445 was correlated with persistence in MAC-T cells. Strains from spa t359 and t529 showed better ability to colonize mouse mammary glands. The exception was strain sa3154 (spa t529), which showed less colonization of glands compared with other t359 and t529 strains but possessed the highest number of superantigen genes including tst. All strains possessed hemolysins, but spa types t529 and t2445 showed the largest diameter of β-hemolysis on blood agar plates. Although several spa types possessed 2 or 3 serine-aspartate rich proteins (Sdr) believed to be involved in many pathogenic processes, most t529 strains expressed only an allelic variant of sdrE. The spa types t605 (positive for the biofilm associated protein gene; bap+) and t13401 (bap-), that produced the largest amounts of biofilm in vitro, were the least virulent in vivo. Finally, strains from spa type t529 (ST151) elicited a cytokine expression profile (TNF-α, IL-1β and IL-12) that suggests a potential for severe inflammation. This study suggests that determination of the spa type may help predict the severity of the disease and the ability of the immune system to eliminate intramammary infections caused by Staph. aureus.
Klebsiella pneumoniae is a common, opportunistic bacterial pathogen that can cause severe clinical mastitis in dairy cattle. Optimizing the bovine udder microbiome to resist mastitis pathogens is a growing area of research; however, previous work has not examined which members of the mammary microbiome may have antagonistic interactions with K. pneumoniae. In this study, we collected quarter-level milk samples from Holstein dairy cows in Québec, Canada every two weeks for 14 months and analyzed differences in the milk microbiome between samples that were collected from healthy quarters, quarters that developed subclinical mastitis, and quarters that experienced K. pneumoniae clinical mastitis (KP-CM) (n = 512 milk samples). The occurrence of subclinical mastitis did not cause significant differences in the alpha-diversity of the milk microbiome, nor did subclinical mastitis alter the interactions between taxa in the microbiome. However, the occurrence of KP-CM caused reductions in Shannon diversity in raw milk relative to healthy milk and altered the interactions between taxa. Specifically, K. pneumoniae showed negative interactions with the genus Aerococcus. The negative interactions between Aerococcus spp. and K. pneumoniae in the context of the bovine milk microbiome should be analyzed further.
Antimicrobial resistance is one of the most serious threats to medical science. Food supply is recognized as a potential source of resistant bacteria, leading to the development of surveillance programs targeting primarily poultry, pork, and beef. These programs are limited in scope, not only in the commodities tested, but also in the organisms targeted (
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