Mastitis significantly threatens the dairy industry, causing substantial revenue losses globally. Other than S. aureus, the non-aureus Staphylococcus and Mammaliicoccus (NASM) are largely responsible for subclinical and clinical mastitis. The disease progression involves a dynamic process shaped by multiple factors, such as the host's genetics, host as well as bacterial resistance mechanisms, host immune response, geographical influences, virulence factors (VFs) and the genetic variability of the bacterium. A comprehensive genome analysis of mastitis-associated NASM strains from diverse geographical regions could enhance our understanding of these factors, aiding in assessing pathogenic potential and infection risk, disease manifestation, and transmission dynamics.
We conducted a comparative genomic study on 22 strains of NASM that caused bovine mastitis in three states of India. We sequenced their whole-genomes and identified STs specific to each species, along with the distribution of AMR genes and virulence factors. In previous studies, an ANI threshold of < 96% was suggested for identifying NASM at the species level (Kim et al., 2014). By comparing the nucleotide sequences of all the 22 strains, we found that members of the same species had ANI values consistently above 96%, indicating significant genomic similarity. On the other hand, members of different species had less than 96% ANI, indicating a clear genomic distinction between different NASM species. Our study supports the 96% ANI threshold value for species differentiation. This threshold helps understand and classify microbial diversity within this group, making it a valuable criterion for delineating species boundaries. The genome-wide SNP-based phylogeny analysis supported the ANI-based analysis, and a similar phylogenetic classification was observed.
The minimum spanning tree (MST) based on SNPs can help us understand the complex relationships among closely related strains. It can reveal patterns of co-evolution, host specialization, and potential transmission pathways. We noticed that strains associated with bovine mastitis clustered together as separate subclades in all the NASM genomes. Previous studies have reported that S. chromogenes ST-1 and ST-6 strains are associated with bovine mastitis (Huebner et al., 2021) (Persson Waller et al., 2023). Our observation also documents that S. chromogenes ST-1 and ST-6 are associated with bovine mastitis. Additionally, we found that S. epidermidis genotypes ST111 and ST59 are associated with bovine mastitis, which is consistent with another earlier report (Frey et al., 2013). Similar sub-clusters unique to particular hosts have been observed in other pathogens (Grillová & Picardeau, 2020). In several European countries, S. aureus CC8 strains have been linked with bovine mastitis. Recently, we reported that S. aureus CC97 strains isolated from India were also associated with mastitis (Sivakumar et al., 2023). Further research is needed to understand the relevance of subclade-specific SNPs and their possible association with bovine mastitis to better understand their pathogenicity.
The increase in drug-resistant strains makes it challenging to treat bovine mastitis with antimicrobial intervention. In our study, we found 32 AMR genes in the 22 NASM genomes. Only one of the 22 strains had mecA and other mec-related genes. Previous studies have documented the presence of mecA-positive S. epidermidis strains in bovine milk samples and the clonal dissemination of multi-drug-resistant S. epidermidis strains carrying mecA within herds (Sawant et al., 2009). The emergence of methicillin-resistant S. epidermidis (MRSE) in cattle highlights the need for increased attention, with some researchers suggesting that animals infected with methicillin-resistant NASM should be culled (Gentilini et al., 2002). The S. epidermidis strain K4.3 investigated in this study exhibited resistance to methicillin, classifying it as MRSE. However, the other 21 NASM strains analyzed were methicillin-sensitive staphylococcus (MSS) and did not contain mecA, mecR1, or mec1 genes in their genomes.
The blaZ gene was discovered in 6 out of 22 NASM strains. This gene is responsible for the production of penicillinase, which is the primary mechanism of penicillin resistance in NASM (Olsen et al., 2006). Multi-drug resistant (MDR) efflux pumps were dispersed throughout the strains and were not associated with specific species or STs. The regulation of MDR efflux pumps is a complex process, requiring multiple regulators to express these elements (Costa et al., 2013; Antiabong et al., 2017). Therefore, the presence of these efflux pumps may not necessarily translate to an AMR phenotype.
We have identified 53 virulence-associated genes among the 22 NASM genomes. Of these, 15 genes are critical for adherence and biofilm development. The ica operon, which produce polysaccharide intercellular adhesins (PIA) and is a commonly found genetic component in biofilm formation (McKenney et al., 1998) stands out as a crucial player. The ica gene was present in S. xylosus and M. sciuri strain K14. The impact of ica genes is particularly noticeable within NASM species linked to the human environment (O’Gara, 2007). The diversity of studies examining different variants of the ica genes poses a challenge in understanding the involvement of this gene in biofilm formation (Tremblay et al., 2013). All of our NASM strains, excluding Mammaliicoccus spp., have genes that code for lipases. Cell-wall-associated proteins and enzymes play a significant role in the pathogenesis of staphylococci and are essential targets for drug development (Hu et al., 2012). Lipases in S. aureus, known as SAL, have been identified in community-associated methicillin-resistant strains (Cadieux et al., 2014; Rosenstein, 2000). .
The spa gene, a major antigen of S. aureus, was exclusively detected in S. pseudintermedius. The sspA gene was found in 12 strains, while the sspB gene was found exclusively in S. epidermidis. In S. epidermidis, the sspA and sspB genes were found to coexist, indicating a potential interplay or cooperative function. The initial player in the staphylococcal proteolytic cascade is aureolysin, a metalloprotease. This enzyme undergoes a rapid process of autocatalytic activation. Subsequently, the activated aureolysin plays a crucial role in activating the sspA serine protease, which in turn, serves as a critical activator for the sspB cysteine protease (Massimi et al., 2002; Nickerson et al., 2008). Such cascades are often pivotal in regulating various cellular processes and contribute to the pathogenicity and adaptability of members of the genus Staphylococcus.
The type VII secretion system (T7SS) has been recently identified and selectively distributed in various pathogens, including Mycobacterium tuberculosis and S. aureus. The T7SS plays an essential role in the virulence of human pathogens. In the case of M. tuberculosis, the T7SS is very important for bacterial access to the host cytosol. In S. aureus, T7SS exports several virulence-associated proteins (Lopez et al., 2017). The presence of T7SS cluster was reported in S. lugudensis, in addition to S. aureus (Lebeurre et al., 2019). Staphylococci possess genes enabling the production of capsular polysaccharides that form a protective shield against phagocytosis by the host's immune cells. This capsule enhances virulence and bacterial persistence, highlighting encapsulation as a crucial mechanism for evading immune detection and contributing to pathogenicity. The capB and capC genes were detected in all NASM species except Mammaliicoccus spp. and S. pseudintermedius.
Prophages are responsible for the horizontal gene transfer, which in turn contributes to virulence (Bae et al., 2006; Smirnova et al., 2017). The prophage Staphylococcus StB20 was found in S. xylosus strain SMG24. This prophage is reported to exhibit specific proteolytic cleavages in the carboxy-terminal degradation of its tail tape measure proteins (TMP) in S. aureus (Tallent et al., 2007). However, the roles of prophages NASM strains are largely unknown.
Genomic islands (GIs) are horizontally transferred regions carrying specific genes that confer certain traits to bacteria. These traits include metabolic processes, pathogenicity, antibiotic resistance, and symbiosis. GIs help bacteria establish mutually beneficial relationships with eukaryotic hosts. They often carry genetic material related to virulence or adaptive traits and are commonly located near tRNA or transposase genes at one end of the island. Antibiotic-resistant genes found in GIs make them carriers for the spread of antibiotic resistance, enhancing bacterial species' survival when exposed to antibiotics (Citti et al., 2020). Coupling virulence or adaptive traits with antibiotic resistance in GIs plays a significant role in shaping bacterial populations' resilience and adaptability to environmental challenges, especially when exposed to antibiotics. In the 22 NASM that we studied, most islands carried the two-component regulatory system, which includes the walRK gene. This gene plays a crucial role in regulating the expression of genes associated with cell wall metabolism, influencing autolysis, biofilm formation, and virulence. WalK is also involved in sensing the D-Ala-D-Ala moiety of Lipid II, serving as a signal for active cell wall synthesis. The genes yycH and yycI are co-transcribed with walRK and modulate its activity. In S. aureus, disrupting yycH and yycI genes downregulated the walRK regulon (Gajdiss et al., 2020). The importance of these regulatory components and their roles in governing cell wall-related processes vary across bacterial species. The fosB, blaZ, and several AMR genes were identified among the GIs, suggesting the possibility of transfer of these traits among the mastitis-associated pathogens.