Despite considerable effort to understand and control bovine mastitis, it remains the most costly and common production disease in dairies worldwide. The role and impact of non-aureus staphylococci (NAS) in udder health are substantial but not well understood. These Gram-positive bacteria, the most frequently isolated group of pathogens in intramammary infections (IMI) of dairy cows, cause both clinical and subclinical mastitis. However, there are differences among NAS species in their distribution in IMI, pathogenicity, virulence, host adaptation, and antimicrobial resistance profile. They have distinct effects on the mammary gland microbiome and some may confer protection against other mastitis pathogens. Some are persistent colonizers of skin, teat canal and mammary gland, whereas others seem to be transient visitors of the udder. Understanding all of these factors should provide insights into whether NAS species are interchangeable or specialized pathogens or perhaps act synergistically when they colonize or infect the mammary gland. Characteristics and genetic differences in individual species may explain success in colonizing the mammary gland. Understanding species-level interactions within the microbiome and its interactions with host genetics will clarify the role of NAS in bovine mastitis and udder health.
Staphylococci can be subdivided into two groups, coagulase-positive and coagulase-negative, based on a phenotypic characteristic. This classification is centred on their ability to clot rabbit plasma, a key diagnostic step in clinical and veterinary microbiology. Staphylococcal coagulase is an extracellular protein encoded by the coa gene. Staphylocoagulase-associated clotting involves formation of a bacterial and host enzyme complex that converts fibrinogen into fibrin, forming a clot. Coagulase secretion is a key virulence strategy in pathogenesis, persistence and protection against host defences of staphylococcal pathogens (1) and has often been used to distinguish S. aureus from the other staphylococci. However, the terms non-aureus staphylococci and coagulase-negative staphylococci cannot be used synonymously, as the former term better describes certain characteristics that the latter cannot. Some S. aureus isolates of bovine origin react negatively in the standard coagulase test and sometimes lack the coa gene (2, 3). Additionally, the von Willebrand factor-binding protein also exhibits coagulating ability, resulting in S. aureus producing two proteins that coagulate plasma (4). The corresponding gene for the von Willebrand factor-binding protein, vWbp can also be detected in S. agnetis, S. hyicus and S. chromogenes (5), suggesting that using the term coagulase-negative staphylococci, and the ability to coagulate as a diagnostic test, may result in ambiguity in the context of mastitis. Thus, non-aureus staphylococci (NAS) would be a more appropriate term to classify pathogens associated with bovine mastitis, as it provides a clear dichotomy between S. aureus and other staphylococci species.
NAS are often considered pathogens of lesser importance in dairy production, especially compared to S. aureus, streptococci and coliforms. However, they are often the most frequently isolated pathogens from udder quarters with subclinical mastitis (6). In that regard, approximately one in five milk samples from Canadian dairy farms were NAS-positive and the prevalence of NAS in quarters with low somatic cell counts (SCC) (< 200,000 cells/mL), was 43% (7, 8). In another Canada-wide clinical mastitis study (9), NAS was isolated in one of 10 culture-positive samples, whereas in a clinical mastitis study from Wisconsin (10), 6.1% of isolates were NAS. In both studies, milk samples were from quarters with visible symptoms of clinical mastitis. Other studies in the US and Belgium also concluded that NAS are the principal cause of IMI on modern dairy farms (11, 12). Prevalence of IMI with NAS is especially high in virgin and first-lactation heifers (12-16). In addition, it has been argued that modern mastitis control schemes, which focus on major udder pathogens (and are apparently less effective against minor pathogens), are responsible for marked increases in prevalence of IMI with NAS. In dairy farms using modern mastitis control practices, there is a decrease in prevalence of IMI with major pathogens. As a result, NAS in IMI have become relatively more important and are considered the leading cause of subclinical mastitis (17). Still, NAS do not seem to be the underlying cause in herds with significant milk quality problems. Elucidating factors that explain the role of NAS in IMI improve prevention and control of subclinical mastitis.
Species distribution and diversity
Staphylococci have been isolated from many animal species and very few NAS species are host specific (18-22). NAS appear to be very prevalent in bovine IMI, especially in dairy heifers (23). There are 53 species of Staphylococcus, 23 of which have been isolated from bovine milk (6). Recently, a new species, S. debuckii, was identified in this collection (24).
To understand the variety of NAS species isolated from milk, evolutionary relationships among these species have been examined. Recently, the genomes of >400 bovine isolates were sequenced and several methods were used to understand the evolution and relationships between species. Five main clades were identified, each with a varying number of species (25). Earlier studies using single gene sequencing revealed contradicting phylogenies. However, by dividing bovine NAS species into 5 distinct clades, shared biological properties among related species, e.g. virulence and host specificity, were better characterized, providing a basis for studies on the role and significance of individual and related NAS species in udder health.
Why are there so many Staphylococcus species in bovine milk samples? It is unclear if all NAS species fill the same niche (totally interchangeable), whether they are all unique in their interactions with the mammary gland, or if there are synergisms between species. Perhaps a bacterial species only evolves to adapt to a certain niche and genetic mutations are maintained only to provide an advantage. If correct, every NAS species, each with ~2.4 MB genomes and many thousand genetic differences, must have a large array of (micro)behaviours and (micro)characteristics. Besides, each species has a large pan genome (all genes in a collection of isolates of a species), suggesting large strain differences within species. In addition to mutations, the ability of NAS species to persist and colonize other niches may be due to acquired genes that confer selective advantages in their respective environment. For example, S. chromogenes and S. epidermidis are most prevalent in mature cows and neonates, respectively (26).
The observed genetic variation implies differences in virulence and host adaptation genes and gene expression among NAS species and strains, and differences in interactions with other microbes. Additional large-scale studies are needed to provide insight into how large strain variations affect the prevalence and distribution of NAS species in IMI, and the resulting impact on udder health. There is also a need for a more efficient method to distinguish species. MALDI-TOF can correctly identify almost all NAS isolates at the species-level (27).
Dominant NAS species
In a Canada-wide study, 50% of NAS isolates were S. chromogenes. This species had the highest prevalence of any IMI bacteria in milk samples of cattle with subclinical mastitis (and low or high SCC) (6, 23). Staphylococcus chromogenes was also the most prevalent species in the US study (28) and Belgium (29). In Canada, S. chromogenes also has the highest (of any NAS) prevalence in clinical mastitis (in high or low SCC quarters). Of all NAS species, S. chromogenes (next to S. epidermidis, and S. simulans)-positive milk samples had the highest SCC (17, 30-32). S. chromogenes IMI is associated with a significantly higher SCC and is considered an important species in quarters with high SCC, persistent cases and clinical mastitis (23, 31, 33, 34). Furthermore, S. chromogenes increased SCC in cows with persistent subclinical mastitis (35), with greater inflammation and more pronounced clinical signs (36).
The high prevalence of S. chromogenes relative to other NAS species is not explained by antimicrobial resistance, as S. chromogenes has relatively low phenotypic and genotypic prevalence of antimicrobial resistance compared to other NAS species (37). Therefore, we inferred that this species would be more susceptible to antibiotics, resulting in lower prevalence. Interestingly, S. chromogenes is most frequently isolated from milk and skin microbiota (38), but not from other environmental sources and therefore is most likely host-adapted (27, 32, 39-41).
Most prevalent species in various geographical regions
Following S. chromogenes, the most frequently identified NAS are S. simulans, S. xylosus, S. haemolyticus, and S. epidermidis, with the other species collectively representing <10% of NAS isolates, consistent with Canadian findings (6, 23, 37, 42). Interestingly, S. chromogenes consistently appeared in the top three most frequently isolated species across the world, aside from one study in Belgium. Therefore, regional and environmental differences affect the prevalence and distribution of individual NAS species, prompting further work. Interestingly, some Staphylococcus species rarely appear in IMI, e.g. S. rostri (43), a common fecal isolate. Non-milk species may give clues regarding genes enabling NAS to infect and colonize udders.
Impact of NAS species on inflammation in IMI
It has been of great interest to determine if all NAS species cause inflammation and increase SCC. There are conflicting data regarding effects of NAS IMI, related to impact on udder health (44), SCC (45), milk yield (47) and whether NAS is causing IMI (38), prompting large-scale studies with gene sequencing (6) and MALDI (46, 47). Interestingly, when comparing the prevalence of individual NAS spp. between milk samples with low SCC (<200,000 cells/mL) or high SCC (>200,000 cells/mL), all species had higher prevalence in the latter, implicating NAS in higher SCC and inflammation (23). However, in a recent longitudinal study, NAS IMI early in lactation resulted in only a small, yet significant increase in SCC (48).
Virulence and host association
The virulence potential of each Staphylococcus species and the profile of all virulence factors were determined by defining a species-specific virulence gene set for each species and comparing among species (5). Virulence genes may explain why some species are more successful at colonizing and surviving within the mammary gland; products of such genes are considered virulence factors (5). The phylogenetic distribution, sharing and evolution of virulence factors may reveal how species evolved (5). Whether individual NAS species became commensals or if they evolved from a common ancestor is unknown. If the latter scenario were true, then they presumably would have become more aggressive in claiming niches by accumulating virulence factors, leading to their evolution into distinct species (5).
The distribution of 191 virulence factors and their possible associations with pathogenesis in 25 NAS species were determined, along with the relationship between virulence factors and udder health (high SCC and clinical signs of mastitis) (5). The overall number of factors was not associated with disease severity. These findings confirmed a parallel study in which the virulence gene profile or accumulation of virulence genes did not predict the type of mastitis (clinical or subclinical) or the severity of inflammation (49). However, increasing numbers of toxin and host immune evasion genes were associated with more severe disease outcomes. Disease development and the interactions of virulence factors with the host are complex and likely determined by the interplay of virulence genes, rather than just their presence. Some NAS strains associated with mastitis had varying proportions of virulence genes; biofilm formation genes were only detected in a small percentage (50). The contribution of virulence genes on disease outcomes or development can also be affected by environmental differences due to geographical location. One study using NAS isolates from a single Chinese herd reported decreased prevalence of exotoxin and biofilm-associated genes compared to previous reports (51). These findings are the impetus for additional studies on the presence or absence of these genes, and further gene expression studies to determine associations with disease severity. Expression studies will elucidate associations between specific NAS species and NAS IMI, as well as genetic elements responsible for differences in prevalence and distribution among NAS species.
Analyses of the distribution of virulence factors of 25 NAS species demonstrated that all species can be defined as separate and homogenous pathogens (5) (Figure 1). The virulence potential was also associated with various phylogenetic clades. We inferred that virulence potential was developed gradually, as NAS evolved into distinct species. This contrasts with the possibility that some species would have acquired several virulence factors relatively suddenly, turning them into more virulent pathogens or more adapted commensals.
Mechanisms enabling S. chromogenes to be the most prevalent organism in bovine mastitis (and its involvement in persistent IMI and subclinical mastitis) are unknown. However, S. chromogenes (~50% of NAS isolates) had similar virulence profiles as closely related NAS isolates from milk (6). A lack of clear differences in virulence gene profile between S. chromogenes and the other Clade B NAS, despite large differences in species distribution, imply an unknown mechanism makes S. chromogenes the most frequently isolated species in NAS IMI.
Interestingly, S. chromogenes is the only species split into two populations with respect to virulence genes (Figure 1), with a minority of the strains clustering with other members of the clade B, whereas most S. chromogenes strains have a distinct profile. An important caveat is that more S. chromogenes isolates were included in this study than other species, but it is tempting to speculate that the larger population of S. chromogenes might represent a strain that has adapted to the mammary gland. If true, this may contribute to S. chromogenes becoming the dominant NAS species isolated from milk and dairy cattle. However, there was no clear difference between the two S. chromogenes populations with respect to severity of mastitis. The subsequent sections will analyze how virulence factors may explain why S. chromogenes is the only species that diverged into two distinct populations. Regardless, other reasons may include differences in antimicrobial resistance profiles, host adaptation, interactions with host genetics and interactions with the microbiome.
It appears that in some S. chromogenes isolates, capsular genes are missing from the larger cluster, which may have contributed to the population split in this species. In S. aureus, expression of these genes results in the formation of a polysaccharide capsule that resists phagocytic cell uptake, promoting evasion of the host immune response (52). However, there is conflicting evidence on the associations among these capsule genes, biofilm formation and overall virulence of Staphylococcus species and their ability to persist in chronic infections. In a Canadian study, it was suggested that biofilms likely increase the ability of NAS to persist in the intramammary environment (42). Additional in vivo testing is needed to better characterize the associations between pathogenicity and biofilm production in S. chromogenes.
Conversely, the absence of these capsular genes can increase both intracellular survival rates as well as invasion rates of S. aureus (53). This was confirmed in a mouse model study where acapsulated mutants persisted for a longer interval and in higher numbers when compared to their capsulated counterparts (54). Therefore, the ability to persist in chronic infections is strongly associated with acapsulated pathogens. With a majority of S. chromogenes isolates lacking capsule genes, it may be of further interest to study the relationship between acapsulation and the persistence of S. chromogenes in IMI.
There is an apparent correlation between the average SCC of milk samples from which specific NAS species were isolated and the number of exoenzyme, host evasion and iron uptake genes these species carried (5, 6). These virulence genes might hold the key to why certain NAS pathogens cause more inflammation than others. Absence of these virulence genes would result in more insidious, perhaps more host-adapted and maybe even more commensal NAS species. This is somewhat illustrated by S. chromogenes, which is considered a host-adapted NAS, and is in the middle in terms of numbers of these exoenzymes, host evasion and iron uptake.
In Canada, more antimicrobial resistance genes—with a strong correlation between genetic and observable resistance— were identified in NAS rather than S. aureus originating from the same dairy herds (7, 55). This corresponds with previous reports that S. aureus isolated from subclinical and clinical mastitis cases are less resistant than NAS against commonly used antimicrobials (47, 56-58). Therefore, NAS could serve as a reservoir for antimicrobial resistance genes for major mastitis pathogens including S. aureus.
The association between antimicrobial resistance and use in NAS was investigated. Such an association was present when antimicrobials were administered systemically, but not when they were administered via intrauterine or intramammary routes (59). Perhaps systemic antimicrobials caused prolonged exposure to sub-therapeutic antimicrobial concentrations in the mammary gland in cattle systemically treated for non-IMI conditions.
Methicillin-resistant NAS were an important reservoir of antimicrobial resistance and virulence genes in a Belgian study. The presence of antimicrobial resistance genes did not correspond to observable resistance in only a few cases, whereas in most cases, there was an association between presence of these genes and observable resistance (60). Furthermore, some isolates did not have any of the investigated antimicrobial resistance genes, yet were resistant to administered antimicrobials (60). In particular, resistance to several antimicrobials such as erythromycin, clindamycin and streptomycin were not explained by the presence of any of the tested genes in another study from Switzerland (61).
Several studies, including a Swedish one (31), revealed that antimicrobial resistance and virulence gene profiles are species-dependent. In this study, the prevalence of β-lactamase varied among CNS species, being more common in subclinical versus clinical cases (31). β-lactamase, the most common resistance mechanism in staphylococci, despite higher detection in S. epidermidis and S. haemolyticus, was rarely detected in S. chromogenes, the most frequently isolated species in NAS IMI, and S. simulans (31). These findings confirmed inter-species variation in antimicrobial resistance profiles, suggesting the need to continue monitoring co-resistance profiles among NAS populations associated with bovine mastitis cases. Coupled with the possible development of new resistance mechanisms not associated with previously characterized virulence genes, additional studies analyzing antimicrobial resistance in NAS are needed as this presents a challenge in treating bovine mastitis cases.
Niche adaptation and host association
NAS prevalence and distribution is apparently impacted by many environmental factors, e.g. geographical region, climate, water source, access to pasture, barn type, bedding and host factors (parity, quarter location, antibiotic use), prompting us to question the natural habitat of various NAS species. To some extent, this defines whether they should be considered as environmental or contagious pathogens. This also relates to their commensal nature and their level of host adaptation to skin, teat canals and mammary gland.
Host adaptation relates to colonization and persistence, as well as degree of induced inflammation. This adaptation can be quite specific, as species and frequency of isolation of NAS differs between teat canal and milk samples (62). In some studies, the most predominant NAS species, S. chromogenes and S. xylosus, were equally ubiquitous in clinical and subclinical mastitis, skin and environment (31, 47). These two species are also more associated with persistent IMI and subclinical mastitis compared to other NAS species (28). Others report differences in distribution and in genotypes among milk, mammary gland and environment (30, 63). In contrast, from molecular epidemiology studies it is relatively clear that, for example, S. haemolyticus, S. fleurettii and S. equorum are predominantly environmental pathogens (28, 63).
Some NAS species are more associated with IMI than with environmental (parlor-associated) niches (63). Interestingly, S. chromogenes is almost uniquely associated with IMI and largely absent from the environment. An unpublished study comparing isolates identified in milk vs body sites, failed to detect S. chromogenes on other body sites of dairy cattle, whereas it was by far the most frequently isolated species from IMI. Therefore, S. chromogenes is highly adapted to the mammary gland, consistent with its involvement in IMI.
To help guide the debate as to whether NAS species are commensals, opportunistic pathogens or obligate pathogens with respect to the mammary gland, a framework was conceptualized to categorize NAS based on discriminating factors (Figure 2). A first factor represents the nature of the interaction the NAS species has with the mammary gland, from a commensal interaction to a pathogenic interaction. A second factor relates to the strength and specialization behind this interaction, from environmental organism to obligate symbiont (this may help define which NAS is of greatest concern to dairy producers). A third factor is the impact of the NAS species on the milk microbiome and on major mastitis pathogens. NAS comprise a significant fraction of the milk microbiome (64) and also seem to contribute to many of the predicted interactions between milk microbiome members. Additional factors could include antimicrobial resistance and compatibility with host immune genetics and response.
Interactions within the microbiome
It appears that the mammary gland microbiome is distributed over the milk, milk ducts, cistern, teat canal, teat apex and teat skin, with Staphylococci having an important role. S. chromogenes negatively influences the udder microbiome, as it has the most negative connections with other members of the milk microbiota. These negative connections presumably reduce diversity and therefore microbiome stability (64). In general, staphylococci are negatively correlated with diversity of the mammary gland microbiota (64); therefore, perhaps NAS are not commensals, but rather disruptors of the normal milk microbiome. Negative interactions might be due to indirect mechanisms that involve the host, e.g. immune responses, or direct mechanisms such as production of antimicrobial factors (e.g. bacteriocins).
NAS produce many bacteriocins, with capacity to inhibit growth of mostly Gram-positive bacteria, but also some potential to inhibit Gram-negatives (65). Interestingly, the bacteriocin produced by an inhibitory S. chromogenes strain used in this study excelled in its antibacterial activity, inhibiting growth of all mastitis-causing pathogens tested (66). This bacteriocin may hold clues to the success of S. chromogenes as an NAS species in IMI and its associations with major mastitis pathogens such as S. aureus, as antibacterial production is often advantageous for strain colonization in a certain niche (66). These findings are mostly based on in vitro studies. It remains unclear if these bacteriocins play an actual role in modulating the microbiota inside mammary gland or on the skin. However, in a mouse model of mastitis, NAS (S. simulans) prevented S. aureus from colonizing mammary glands, but it was not proven that this was due to the action of the bacteriocin genes of this NAS isolate. Other NAS species also inhibit the growth of major mastitis pathogens. In a recent study, cytoplasmic bacteriocins from S. epidermidis selectively inhibited growth of S. aureus and methicillin-resistant S. aureus strains (67), indicating the need for additional in vivo studies to determine how bacteriocins influence NAS species-level interactions in the milk microbiome in the context of bovine mastitis.
There is also conflicting evidence as to whether NAS increase susceptibility to major pathogens such as S. aureus or prevent it from colonizing mammary glands. Because major pathogens are generally considered more virulent and damaging to the udder than minor mastitis pathogens (e.g. NAS), it would be of interest to clarify impacts of NAS on major pathogens. In several studies, NAS colonization protected quarters against IMI by major pathogens (68-70), whereas another reported that the presence of NAS was a risk factor for acquiring S. aureus IMI (71). Interestingly, S. chromogenes inhibited in vitro growth of all S. aureus, S. dysgalactiae and S. uberis strains. The intensity of inhibition varied amongst target species, with only 2 out of 10 S. chromogenes isolates have consistent inhibitory activity (70). Large-scale studies are needed to resolve the existing conflicting evidence and better characterize associations between NAS and major pathogens, in the context of bovine mastitis.
Effect of host genetics on NAS
A host genetic component may determine which NAS species contribute to the milk microbiome. There were genetic variations in a bovine protein that helps present bacterial antigens to immune cells to mount an immune response. Whether a lactating cow harbored either one or the other version of this gene strongly defined which organisms were ‘accepted’ in the milk microbiome of this animal (72). Each genetic variant seems to promote the presence of a specific NAS species: S. equorum, S. gallinarum, S. sciuri and S. haemolyticus were enriched in microbiota of one of the variants, whereas S. chromogenes was enriched within microbiota of the second variant. These findings spark many hypotheses related to the distribution of NAS species, the predominance of S. chromogenes and the apparent dichotomy between ‘environmental’ and ‘host adapted’ NAS.
Despite research on NAS at the species level, many questions remain. The true nature of each NAS species has yet to be identified, either as commensals or pathogens, or as environmental or contagious pathogens. Interactions between NAS and the remainder of the milk microbiome, as well as its associations with host genetics, need to be elucidated. Interactions in the milk microbiome may influence factors such as antimicrobial resistance or virulence in NAS species, resulting in their success as colonizers of the mammary gland. Further investigations on the role of NAS as an antimicrobial resistance reservoir for major and minor pathogens are required to clarify if NAS can truly prevent mastitis-related pathogens from colonizing mammary glands.
Given S. chromogenes’ dominance as NAS and IMI pathogen in general and its negative impacts, it seems that new control strategies to eliminate S. chromogenes from bovine mammary glands would reduce the prevalence and impact of mastitis in dairy herds. It should be determined which other NAS species fall in this same ‘harmful’ category and which species require less or no attention. It is also important to focus on strain differences related to interactions of NAS with the mammary gland, as they may override species-level differences.
Text: Jeroen De Buck, Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary – Illustrations: Jeroen De Buck and M-teamUGent
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