Assessment of Clinical and subclinical mastitis Staphylococcus spp. isolates
Staphylococcus chromogenes (28.7%), S. simulans (20.0%). S. aureus (18.7%) and the S. sciuri (10.0%) were the most prevalent clinical mastitis species deposited in the NCBI GenBank database. The remaining 22.5% of the clinical mastitis strains were S. epidermidis (5.00%), S. haemolyticus (5.00%), S. agnetis (2.50%), S. xylosus (2.50%), S. arlettae (1.25%), S. capitis (1.25%), S. cohnii (1.25%), S. devriesei (1.25%), S. gallinarum (1.25%) and S. hominis (1.25%). The most frequent staphylococcal species associated with subclinical mastitis were S. chromogenes (15.6%), S. simulans (6.8%), S. xylosus (6.5%), S. haemolyticus (6.3%), S. cohnii (5.8%), S. epidermidis (5.5%), S. capitis (5.3%), S. sciuri (5.3%), S. gallinarum (5.0%), S. warneri (4.8%), S. equorum (4.5%), S. saprophyticus (4.0%), S. succinus (3.8%), S. arlettae (3.5%), S. agnetis (3.3%), S. aureus (3.3%) and S. hominis (3.0%). The remaining 7.50% of subclinical isolates included S. devriesei (1.76%), S. pasteuri (1.51%), S. vitulinus (1.51%), S. auricularis (0.50%), S. caprae (0.50%), S. fleurettii (0.50%), S. hyicus (0.50%), S. nepalensis (0.50%) and S. kloosii (0.25%) (Fig. 1).
Distribution of Adhesin, biofilm and regulatory genes across clinical and subclinical mastitis Staphylococcus spp. isolates
In the mastitis related staphylococci genomes analyzed (n = 478) the most prevalent genes associated with adhesion and biofilm formation were: ebpS (71.3%), atl (70.9%), sasF (70.7%), sasH (53.3%), araC (52.1%), tcaR (52.1%), sarA (52.1%), sigB (52.1%) pls (44.6%), sasA (37.2%) and sasC (30.8%) (Fig. 2). The icaC (17.3%), icaR (14.0%), sasD (13.8%), sdrE (13.4%), icaA (11.5%), icaB (11.5%), icaD (11.5%), sdrC (11.0%), clfA (10.6%), fnbA (9.62%), spa (9.21%), vWbp (8.79%), fnbB (6.9%), efb (6.07%), coa (5.86%), eap (5.86%), emp (5.65%), clfB (5.44%), aap (5.23%), cna (5.02%), sasG (3.97%), sasK (3.77%) and sdrD (3.14%) genes were detected less frequently. The sasI gene was absent in all isolates (Fig. 2)
In strains associated with clinical mastitis, the ebpS (83.8%), atl (83.8%), sasF (83.8%), sasH (77.5%), atl (56.3%), rbf (56.3%), tcaR (56.3%), sarA (56.3%), sigB (56.3%), pls (55.0%), sasA (47.5%), pls (37.5%) and sasC (30.0%) genes were most frequently detected while sdrE (22.5%), fnbA (21.2%), spa (20.0%), clfA (22.5%) vWbp (18.7%) icaC (17.5%), sdrC (17.5%), efb (17.5%), coa (17.5%), eap (17.5%), emp (17.5%) icaR (16.2%) icaA (16.2%) icaB (16.2%) icaD (16.2%) clfB (16.2%) sasD (15.0%) fnbB (11.2%) sasG (10.0%) sasK (8.75%) sdrD (7.50%) cna (6.25%) and aap (2.50%) were present less often.
The carriage of adhesin/biofilm related genes in isolates associated with subclinical mastitis was less frequent (e.g., ebpS (68.8%), atl (68.3%), sasF (68.1%), atl (51.3%), rbf (51.3%), tcaR (51.3%), sarA (51.3%), sigB (51.3%) sasH (48.5%) pls (42.5%), sasA (35.2%) and sasC (30.9%). Also, a lower frequency of the following genes was also observed: icaC (17.3%) icaR (13.5%) sasD (13.5%) sdrE (11.5%) icaA (10.5%) icaB (10.5%) icaD (10.5%) sdrC (9.80%) clfA (9.05%) fnbA (7.29%) spa (7.04%) vWbp (6.78%) fnbB (6.03%) aap (5.78%) cna (4.77%) efb (3.77%) coa (3.52%) eap (3.52%) emp (3.27%) clfB (3.27%) sasG (2.76%) sasK (2.76%) and sdrD (2.26%). The sasI gene was absent in all subclinical isolates.
Most of the subclinical isolates of S. aureus, S. capitis, S. chromogenes, S. cohnii, S. epidermidis, S. haemolyticus, S. warneri and S. simulans had the ebpS, atl, pls, sasH, sasC and sasF adhesion-associated genes with the clfB and emp genes found only in S. aureus strains.
The PIA production operon icaADBC and its regulator icaR was only present in S. aureus, S. chromogenes, S. capitis, and S. epidermidis; most S. cohnii and S. saprophyticus carried the icaC gene. In the clinical isolates, the icaADBC operon and icaR gene were present only in S. aureus isolates. Other biofilm regulatory genes i.e., rbf, tcaR, sarA and sigB were found in subclinical isolates of S. aureus, S. chromogenes, S. epidermidis and S. haemolyticus, but not in S. simulans isolates.
Phylogenetic analyses reveal no clear relationship between clinical and subclinical isolates showing an uneven distribution of adhesin, biofilm and regulatory genes
Analysis of the 16S RNA genes from the genome sequences of the Staphylococcus spp. from bovine and buffalo mastitis cases revealed that the clinical and subclinical isolates (n = 478) are present in a wide variety of clades and do not show any clear relationship (Supplementary Fig. 1). The 16S RNA gene phylogeny also indicated that the mastitis related S. aureus, S. epidermidis, and S. capitis have a close phylogenetic relationship. These species also possess many adhesion genes (avg. no. = 26, 11, and 17 respectively), followed by S. chromogenes and S. warneri (avg. no.= 9 and 12, respectively). S. capitis has a close phylogenetic relationship to the species that are mainly associated with clinical mastitis (S. aureus and S. epidermidis). S. chromogenes, which was implicated in cases of clinical (n = 23/80) and subclinical mastitis (n = 61/398) is most closely related to S. agnetis and S. hyicus species that were only associated with subclinical mastitis. “Subclinical species” S. saprophyticus, S. xylosus, S. gallinarum and S. arlettae formed a distinct node with few strains involved in clinical mastitis and with most of these strains not carrying known adhesion/biofilm related genes. The “subclinical species” S. warneri and S. pasteuri were also phylogenetically related and carried biofilm/adhesion associated genes (n = 35; avg. no. of genes = 12 and 10, respectively). No specific pattern was observed between clinical and subclinical strains ebpS, rbf, sarA, sasH, sigB, and tcaR gene phylogeny (Supplementary Figs. 2–7, respectively). Overall, the clinical and subclinical strains of most species were in the same clade.
The co-phylogenetic analysis suggests the occurrence of different events horizontal gene transfer (HGT) between virulence genes. For instance, the ebpS gene between clinical and subclinical isolates of S. simulans, S chromogenes and S. aureus (Supplementary Figure S8); the pls, gene from clinical and subclinical isolates of S. haemolyticus, S. chromogenes and S. simulans (Supplementary Figure S9); the rbp gene among clinical and subclinical isolates of S. chromogenes, S. aureus and S. haemolyticus (Supplementary Figure S10), and the sarA gene, between clinical and subclinical isolates of S. chromogenes and S. aureus, respectively (Supplementary Figure S11). Additional evidence of potential HGT were also observed for the sasH gene between S. aureus, S. simulans and S. chromogenes (Supplementary Figure S12), and the tcaR gene among the S. aureus, and S. chromogenes clinical and subclinical isolates (Supplementary Figure S13)
Data analysis indicates adhesion and biofilm genes exclusively related to clinical isolates
Hierarchical clustering analysis based on the presence/absence of the adhesin, biofilm, and regulatory genes revealed 20 different clusters (Supplementary Table 1). One hundred and twenty-seven (26.5%) strains (13 clinical and 114 sub-clinical) of the 478 genomes evaluated lacked the 35 adhesion and biofilm-associated genes identified by the RAST annotation tool. The staphylococcal species lacking these genes included: S. arlettae, S. equorum S. gallinarum S. sciuri, S. succinus, S. vitulinus and S. xylosus. In species heatmaps (Fig. 3), the pattern of adhesion/biofilm genes in clinical isolates differs from that of sub-clinical isolates. The presence of the clfA, clfB, fnbA, spa, sdrC, coa, eap, emp, vWbP, sasD, icaA, icaB, icaC, icaD and icaR genes is highly correlated in clinical isolates, while in subclinical isolates, no specific gene correlations were observed (Spearman coefficient > 0.8). Based on hierarchical matrix clustering, clusters 9 and 10; 19 and 18 and 4 and 5 (Supplementary Table 1) contained most of the strains that harbored a typical pattern of nine genes (rbf, pls, sasF, sarA, atl, sasH, sigB, tcaR and ebpS) in both clinical and subclinical isolates. This pattern is also demonstrated in the heatmap of the gene frequency (Fig. 4).