Exploring associations between the teat apex metagenome and Staphylococcus aureus intramammary infection risk in primiparous cows under organic directives

The primary objective of this study was to identify associations between teat apex microbiome and Staphylococcus aureus intramammary infection (IMI) risk in primiparous cows during the first 5 weeks after calving. We performed a case-control study using shotgun metagenomics of the teat apex and culture-based milk data collected longitudinally from 710 primiparous cows on 5 organic dairy farms. We observed a strong association between S. aureus DNA in the metagenomic teat apex data prior to parturition and the odds of S. aureus IMI after parturition (OR = 38.9, 95% CI: 14.84-102.21). Differential abundance analysis confirmed this association, with cases having a 23.8 higher log fold change (LFC) in abundance of S. aureus in their samples compared to controls. Of the most prevalent microorganisms in controls, those associated with a lower risk of post-calving S. aureus IMI included Microbacterium phage Min 1 (OR = 0.37, 95% CI: 0.25-0.53), Corynebacterium efficiens (OR = 0.53, 95% CI: 0.30-0.94), Kocuria polaris (OR = 0.54, 95% CI: 0.35-0.82), Micrococcus terreus (OR = 0.64, 95% CI: 0.44-0.93) and Dietzia alimentaria (OR = 0.45, 95% CI: 0.26-0.75). Microcin B17 was the most prevalent antibacterial peptide on the teat apex of cases and controls (99.7% in both groups). The predicted abundance of Microcin B17 was also higher in cases compared to controls (LFC 0.26). Cow and farm random effects often explained a large proportion of the observed variability in the teat apex microbiome, suggesting that our results need to be interpreted within the context of the random effects. IMPORTANCE Intramammary infections (IMI) caused by Staphylococcus aureus remain an important problem for the organic dairy industry. The microbiome on the external skin of the teat apex may play a role in mitigating S. aureus IMI risk, in particular the production of antimicrobial peptides (AMPs) by commensal microbes. However, current studies of the teat apex microbiome utilize a 16S approach, which precludes detection of genomics features such as AMPs. Therefore, further research using a shotgun metagenomic approach is needed to understand what role pre-partum teat apex microbiome dynamics play in IMI risk.


IMPORTANCE
Intramammary infections (IMI) caused by Staphylococcus aureus remain an important problem 50 for the organic dairy industry. The microbiome on the external skin of the teat apex may play a 51 role in mitigating S. aureus IMI risk, in particular the production of antimicrobial peptides 52 (AMPs) by commensal microbes. However, current studies of the teat apex microbiome utilize a 53 16S approach, which precludes detection of genomics features such as AMPs. Therefore, further 54 research using a shotgun metagenomic approach is needed to understand what role pre-partum 55 teat apex microbiome dynamics play in IMI risk.

72
Bovine mastitis is an inflammatory disease of the udder caused by microorganisms that 73 overcome the physical and immunological barriers of the mammary gland (Sordillo et al., 1997). 74 Despite more than a century's worth of research into the etiology of this disease, mastitis remains 75 an issue for dairy cows and producers (Ruegg, 2017). One major pathogen of concern is 76 Staphylococcus aureus, a contagious gram-positive bacteria that results in persistent infections of 77 the mammary gland, and that is notoriously difficult to treat -even with the use of antibiotics 78 (Rainard et al., 2018). This makes bovine mastitis caused by S. aureus an important animal 79 health and welfare issue for dairy cows, and a major financial burden for dairy farmers; 80 therefore, further research is needed to prevent and treat these infections (Ruegg,   The primary objective of this study was to identify associations between microbial biomarkers 129 on the teat apex and risk of S. aureus IMI in the first 5 weeks after calving in primiparous cows. 130 Secondary objectives were to explore the functional capacity of the teat apex microbiome to 131 produce AMPs, and to identify AMPs that may be associated with S. aureus IMI risk. In this case-control study, we collected skin samples from the distal third of the teat apex and 136 quarter milk samples from 710 organically reared dairy cows from 5 farms in Colorado, 137 Minnesota, New Mexico and Texas. Eligibility for farm and cow enrollment has been described  for matching. If more than one control could be matched to a case animal, then one was selected 197 at random using a random number generator. Cows with a milk sample classified as 198 "contaminated" were not eligible to be considered a case in subsequent weeks. Similarly, cows 199 were not eligible to be matched to cases if a pooled milk sample had been classified as

207
Prior to DNA extraction, batches of 12 whirl-pak bags containing gauze squares were removed 208 from -80 °C storage and thawed at room temperature. During thawing, a biosafety cabinet was 209 cleaned with 70% EtOH and a glass-bead sterilizer preheated to 250 °C. Once gauze squares 210 were thawed, whirl-pak bags were moved inside the biosafety cabinet. Each whirl-pak bag was 211 processed in the following way: the bag was opened and the gauze square cut into thirds using 212 metal scissors sterilized with a glass-bead sterilizer; the dirtiest third of the gauze square was

284
Only primary alignments were considered for downstream analysis. Reads with multiple hits to 285 the reference database (indicated by the XA tag in the SAM file) were retained. Alignments to 286 peptides labelled as "synthetic" in the "protein existence" column of the DRAMP reference 287 database were discarded, as were those with a eukaryotic origin.   10 logarithm or centered-log ratio (Aitchison, 1982) Linear mixed effect models were used to investigate the association between the presence of an  samples sequenced from cases was 5 (min = 3; max = 9) and controls was 5 (min = 3; max = 8).

404
The median number of days after calving for when a case became a case was 6 (min = 0, max =  significantly associated with raw sequencing depth ( Fig. 1 A-E, ANOVA P > 0.05). Sequencing 417 batch was not significantly associated with host sequencing depth ( Fig. 1F; i.e., Bos taurus Days in milk, days to infection and weeks to infection in cases did not differ between cases and 423 controls ( Fig. 1 K-M). Clustering of the teat apex microbiome by sequencing batch was not 424 visually apparent using PCA (Fig. 1N); indeed, the amount of variation in the teat apex  Table S1). Aspergillus fumigatus (n = 13, 1.5%).

466
The most abundant microorganisms were bacteria from the NAS, Corynebacterium, 467 Bifidobacterium, Dietzia and Jeotgalicoccus genera, accounting for much of the metagenomic 468 DNA from the teat apex (Fig. 2). The three most abundant species from the metagenomic data The presence of S. aureus and skin-associated viruses in the metagenomic teat apex data 480 prepartum were significant risk factors for postpartum S. aureus IMI (Fig. 3A) Fig. 4A). Across all farms, the 522 abundance of S. aureus was consistently higher in cases compared to controls (Fig. 4B). There 523 was more variation in S. aureus abundance between cows (ICC: 0.66) than farms (ICC: 0.006).

524
The abundance of sequence reads originating from Psychrobacter immobilis (LogFC = 10.23, 525 BH-adjusted p = 0.002) and Bacteroides pyogenes (LogFC 7.34, BH-adjusted p = 0.04) was 526 significantly higher in samples from controls compared to cases (Fig. 4A). Unlike S. aureus, 527 these two bacteria were primarily found on a single farm (Fig. 4B), and this was reflected in their 528 variance components for the farm specific random effects for P. immobilis (ICC: 0.96) and B. between cases and controls ( Fig. 4A and Fig. 4B). bacterial AMPs that we identified from the teat apex were rare, with 64 of 74 (86.4%) being 539 found in fewer than 10 samples (Fig. 5). The mean number of bacterial AMPs found in cases that IMIs could have been acquired prior to calving (Fig. 3D), as was observed by (Roberson et Table 2). Bacteria belonging to the Microbacterium genus have also 664 been reported as common laboratory contaminants (Salter et al., 2014), but this seems to be an 665 unlikely source in our study as we would expect an equal probability of these bacteriophage to be 666 found in both cases and controls since our samples were processed using the same lot of 667 laboratory reagents (adjusted risk in cases = 63.0% in cases, adjusted risk in controls 82.0%).

669
An unexpected finding of our metagenomic study was the difference in abundance of reads that 670 aligned to the Bos taurus genome between cases and controls ( Fig. 1 G- in our study (Fig. 1 I-J). However, this may only provide a partial explanation, as Bos taurus 684 DNA was consistently higher in samples from cases compared to controls throughout the entire 685 sampling period. It may be tempting to attribute this finding to a technical artifact of our study design, but we found no evidence for this, as the elevated levels of B. taurus DNA in cases 687 versus controls was found consistently across all sequencing batches (Fig. 1G) and there was no 688 difference in raw sequencing depth between samples collected from cases and controls ( Fig. 1 B between Pychrobacter immobilis DNA and S. aureus IMI risk, in which the variation between 724 cows (ICC: 0.04) was less than that between farms (ICC: 0.77). Indeed, the latter microbe was 725 primarily isolated within a single farm (high farm ICC), while the former was isolated 726 consistently across each farm (low farm ICC). This is visually apparent when comparing the 727 abundances of these two specific microorganisms between cases and controls across each farm 728 (Fig. 4B). More broadly, this implies that the teat apex microbiome is not likely to be 729 homogenous in animals between farms, and that future studies of the teat apex microbiome Staphylococcus aureus DNA on the teat apex was the strongest risk factor for S. aureus IMI after 739 parturition and exhibited the largest differential abundance between cases and controls. Further The raw sequence data generated in this study has been submitted to the Sequence Read Archive 752 on NCBI (Accession: PRJNA984925).