Amy C. Pickering, Andreas F. Haag, José R. Penades, and J. Ross Fitzgerald The Staphylococcus genus is comprised of Gram‐positive coccus‐shaped bacteria that commonly appear in grape‐like clusters or singly, in pairs, and in short chains. Staphylococci belong to the phylum of Bacillota, the class of Bacilli, the order of Caryophanales, and the family of Staphylococcaceae (Parte et al. 2020; Oren and Garrity 2021). More than 70 species and subspecies make up the genus and it is associated with a wide range of host species. Most staphylococci are classified as commensals, but some cause important opportunistic infections in humans or other animals. In this chapter, we focus particularly on the staphylococcal species that are of most relevance to veterinary diseases. These are Staphylococcus aureus, associated with infections of cattle, poultry, and rabbits, Staphylococcus hyicus, the main causative agent of greasy pig disease, and Staphylococcus pseudintermedius, the main causative agent of canine pyoderma. Staphylococci have complex nutritional requirements and are classified as facultative anaerobes that use aerobic respiration or fermentation to produce lactic acid, except for S. aureus subsp. anaerobius, which is microaerophilic, and Staphylococcus saccharolyticus, which is strictly anaerobic. Staphylococci are non‐motile and do not form spores. The classification of novel species to the Staphylococcus genus requires both biochemical and genetic analysis, with more emphasis now placed on genomic differentiation via whole‐genome sequence analysis (Varadi et al. 2017). However, phenotypic characteristics, such as growth on selective media and the ability to produce catalase and coagulate rabbit plasma, are still used in the diagnosis of staphylococcal infections. An efficient bacterial catalase test was developed in the 1960s reflecting the ability of facultative anaerobes to break down hydrogen peroxide into water and oxygen. All staphylococcal species are capable of producing catalase, except for the microaerophilic and anaerobic S. aureus subsp. anaerobius and S. saccharolyticus. In contrast, the ability to coagulate plasma, used diagnostically since the 1940s, differentiated the genus into coagulase‐positive and coagulase‐negative staphylococci, and was originally used to differentiate pathogenic S. aureus from less clinically relevant species. However, since the 1970s it has been recognized that other staphylococci can also coagulate plasma and that these coagulase‐positive staphylococci are responsible for the majority of staphylococcal‐related human and veterinary infections (Hajek 1976). There are now nine recorded coagulase‐positive species, including S. aureus, Staphylococcus argenteus, Staphylococcus coagulans, Staphylococcus cornubiensis, Staphylococcus delphini, Staphylococcus intermedius, Staphylococcus lutrae, S. pseudintermedius, and Staphylococcus schweitzeri. Staphylococcus agnetis, Staphylococcus chromogenes and S. hyicus are classified as coagulase variable, since they exhibit strain‐dependent coagulase activity (Gonzalez‐Martin et al. 2020). The most common coagulase‐positive species relevant for veterinary medicine are S. aureus, S. hyicus and S. pseudintermedius. There is also increasing appreciation for the role of coagulase‐negative species in both human and veterinary medicine, and their role in bovine mastitis is briefly described in the next section. Staphylococcal species have traditionally been defined based on DNA–DNA reassociation analysis that differentiates species with high biochemical similarities. Whole‐genome sequence analysis is now the gold standard, with novel species described based on their average nucleotide identity and digital DNA–DNA hybridization scores, alongside full‐length 16S ribosomal RNA gene sequences, in comparison with the most closely related staphylococcal species (Chun et al. 2018; Ciufo et al. 2018). A comprehensive phylogenetic analysis of the Staphylococcus genus revealed that the coagulase‐positive species represented in three main clades (the S. aureus complex, the Hyicus group and the Intermedius group) all contain the von Willebrand‐binding protein gene (vwb) with the staphylocoagulase gene (coa) limited to the S. aureus complex. Genetic variation in the vwb gene correlates with unique coagulation phenotypes of coagulase‐positive species associated with different host species (Pickering et al. 2021). In addition to vWbp, coagulase‐positive species share some other factors of relevance to pathogenesis. For example, most staphylococci have the ability to generate sugar and/or protein‐mediated biofilms, which promotes colonization of the host and evasion of the immune system, and antimicrobial activity. Families of cell‐wall‐associated proteins promote colonization and immune evasion through interactions with host extracellular matrix and plasma proteins. The cell envelope also contains wall teichoic acid, involved in nasal colonization with the other cell‐wall components, peptidoglycan, and lipoteichoic acid, capable of inducing shock. Secreted toxins are commonly made by coagulase‐positive species including superantigens, cytolytic toxins such as leukocidins involved in innate immune evasion, exfoliative toxins involved in the pathogenesis of skin infections, and a wide array of immune evasion proteins that prevent the bacterial surface being opsonized and therefore inhibit phagocytosis. Additionally, staphylococci secrete a range of proteases and lipases that impact on immune function, as well as acquiring nutrients for the bacteria. The virulence factors known to be of particular importance during veterinary diseases will be described in more detail in the relevant sections below. Even though coagulase‐positive staphylococci are the primary pathogenic staphylococcal species, coagulase‐negative species are now being recognized as important causes of veterinary diseases. For example, they are increasingly being isolated from cases of subclinical bovine mastitis, with some countries reporting that coagulase‐negative species are now the most prevalent bacteria associated with the disease (De Buck et al. 2021). Often, species‐level identification is not performed, and clinical isolates are grouped as coagulase‐negative species, but the most prevalent coagulase‐negative species associated with mastitis are Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus simulans, and Staphylococcus xylosus, as well as the coagulase‐variable S. chromogenes and S. hyicus (De Buck et al. 2021). Even though coagulase‐positive staphylococci are of increasing concern in the management of bovine mastitis, very little is known regarding their ecology or pathogenesis. Some studies suggested that they might prevent infection by the more pathogenic S. aureus, while others highlight that modified herd management and intervention strategies targeting S. aureus infection and transmission could account for the increasing prevalence of coagulase‐positive species in bovine mastitis isolates. Our current understanding and the gaps in our knowledge on the role of coagulase‐positive staphylococci in bovine udder health has recently been reviewed in detail by De Buck et al. (2021). Important staphylococci in animals and their major colonization sites and disease manifestations are shown in Figure 25.1. S. aureus is the best characterized staphylococcal species due to its importance as an opportunistic pathogen for a wide array of human infections, from mild skin infections to septicemia and toxic shock syndrome. S. aureus is well known for its capacity to acquire resistance to antimicrobials with wide distribution of methicillin‐resistant hospital and community‐associated S. aureus clones and increasing appreciation for livestock‐associated clones adapted to non‐human hosts. Population genomic and phylogenetic analysis have demonstrated that non‐human clones of S. aureus have emerged via host‐switching events from humans followed by adaptation to cattle, birds, and pigs via genetic diversification (Richardson et al. 2018). In veterinary medicine, S. aureus is of particular importance as the causative agent of mastitis in cows and small ruminants, bumblefoot (pododermatitis), osteomyelitis, and septicemia of poultry, and a range of infections in rabbits including mastitis, dermatitis, pododermatitis, and skin abscesses (Mcnamee and Smyth 2000; Corpa et al. 2010). Additionally, S. aureus can cause skin infections of horses and pigs, but these are not as commonly encountered (van Duijkeren et al. 2007; Maddox et al. 2010). The primary causative agent of exudative epidermitis (greasy pig disease) is S. hyicus, which can also cause cystitis and joint infections in pigs, as well as flank biting‐associated and ear‐tip necrosis (Mirt 1999). S. hyicus has also been implicated in less common skin infections of cattle, horses, and poultry. S. pseudintermedius is the primary cause of canine pyoderma, a major veterinary burden in small veterinary clinics (Bannoehr et al. 2007). The other members of the closely related S. intermedius group, can also cause veterinary diseases with S. delphini associated with septicemia in poultry as well as dermatitis in mink and horses. Other species of veterinary importance include S. aureus subsp. anaerobius, the causative agent of Morel’s disease, a lymphadenitis in goats and sheep. Evolutionary genomic analysis has demonstrated that this species has become highly niche restricted through genome remodeling, leading to highly fastidious growth requirements (Yebra et al. 2021). S. agnetis is an emerging veterinary pathogen of broiler chickens, having previously been solely associated with bovine mastitis, but now recognized as an important cause of chondronecrosis with osteomyelitis, endocarditis, and septicemia (Szafraniec et al. 2020). The increasing prevalence of antibiotic resistant strains of S. aureus is now of major concern. This is of public health importance for the highly zoonotic S. aureus clones, such as LA‐MRSA ST398, which have emerged in pig farming but which have human pathogenic potential and may be a reservoir for antibiotic resistance genes of relevance to human health. In staphylococci, methicillin‐resistance is encoded by a mobile genetic element (MGE) named the staphylococcal cassette chromosome mec (SCCmec) containing the mecA gene specific for a penicillin‐binding protein 2a (PBP2a), with reduced affinity for β‐lactam antibiotics. SCCmec variants have been identified in a range of staphylococcal species, including S. pseudintermedius (Smith et al. 2020). SCCmec elements that encode a variant mecC gene have been identified, especially in non‐human associated staphylococcal species (Fisher and Paterson 2020). Resistance to other antibiotics is also observed in staphylococci through the acquisition of plasmids and transposons (e.g. macrolide, aminoglycoside, and tetracycline resistance; Turner et al. 2019). Novel therapeutics and/or vaccine development are therefore required for many veterinary‐related staphylococcal diseases. Figure 25.1 Staphylococcal species and their main animal hosts. Colonization sites are indicated by blue hexagons and disease sites by red circles. S. aureus is one of the major causes of mastitis in dairy cows and incurs a significant economic loss to the dairy industry. Mastitis causes reduced yields, and may require veterinary intervention, including antibiotic treatment, and, if treatment is unsuccessful, culling of infected animals. Healthy cows can carry S. aureus on the teat skin, nasal cavity, and rectum (Haag et al. 2019). While S. aureus bacteria do not persist on healthy teat skin, they readily colonize damaged skin and teat lesions increasing the chance for teat canal colonization and udder infection. Transmission from infected animals occurs primarily from udder to udder during milking via contaminated milking machines or farmer’s hands, and less frequently via contaminated bedding and environmental sources (Klaas and Zadoks 2018). The main clonal complexes (CC) and sequence types associated with dairy cows are CC97, CC151, CC126, CC130, CC133, CC398, CC479 and ST425 (Fitzgerald 2012; Schlotter et al. 2012; Richardson et al. 2018; Hoekstra et al. 2020; Thomas et al. 2021). A recent study showed that S. aureus was likely introduced into cattle following several host jumps from humans as early as 3000 years ago (Richardson et al. 2018). Bovine strains have adapted to their host by acquiring MGEs such as phages and phage‐inducible chromosomal islands (PICIs), which encode factors involved in host adaptation (Richardson et al. 2018). The emergence of new methicillin‐resistant bovine S. aureus strains in recent years as well as their ability to frequently jump between bovine and human hosts highlight the potential public health threat (Richardson et al. 2018). The primary disease caused by S. aureus in dairy cows is mastitis, an inflammation of the mammary gland. Such inflammation is the result of an inability of the innate and adaptive immune response to prevent the invasion and establishment of infection by the bacterium once it enters the mammary gland. Mastitis can manifest either as a subclinical or as clinical disease ranging from mild to moderate or severe. Subclinical mastitis can be preceded by an acute phase with elevated body temperatures, reduction of general activity and social behaviors, as well as reduced feed intake up to several days before clinical diagnosis (Sepulveda‐Varas et al. 2016). Alongside these symptoms, leucocytes enter the mammary gland, which can be followed by the appearance of clots in milk. Clinical signs of acute mastitis also include swelling, firmness, warmth, and tenderness of the udder. Severe and peracute clinical mastitis is characterized by the sudden onset of hyperthermia, reduced feed uptake, rapid heart rate, and depression in the animal. In extremis, these symptoms can include the appearance of patches of blue discoloration caused by ischemic gangrene usually around the teats. Death from toxemia or culling of the animal are the normal outcomes but, if the animal survives, the affected tissue then sloughs from the udder. Most commonly, mastitis in dairy cows is subclinical and results in elevated concentrations of leukocytes in milk resulting in increased somatic cell counts. These subclinical infections are often chronic and can persist through continuing lactation and possibly continue in following lactations accompanied by clinical flare‐up episodes. Of note, S. aureus can be shed from infected glands and the contamination of bulk milk can lead to human food poisoning caused by staphylococcal enterotoxins in fermented raw milk products (Le Loir et al. 2003). S. aureus infections in cattle may also occur on the skin and present as folliculitis or impetigo (Foster 2012) with vesicles (occasionally bullae), papules, pustules, and crusts. Treatment of udder impetigo consists of clipping hair from the affected area and washing the skin thoroughly each day until the condition resolves. S. aureus produces a plethora of virulence factors and our understanding of their role in specific veterinary diseases is very limited. Several studies have identified different virulence gene profiles associated with S. aureus strains from bovine mastitis or bulk milk, but the clinical relevance of these gene correlations is uncertain without functional characterization (Le Marechal et al. 2011; Magro et al. 2017; Fursova et al. 2020; Hoekstra et al. 2020). Among the cell‐wall‐anchored proteins, microbial surface components recognizing adhesive matrix molecules (MSCRAMM; Foster 2019), play key roles in cell surface attachment, colonization, and invasion. MSCRAMM can bind to various host proteins such as collagens (Cna), fibrinogen (FnBPA, FnBPB, Bbp, ClfA, ClfB), fibronectin (FnBPB), bone sialoprotein (Bbp), and laminin (Cna), but their role in the pathogenesis of bovine mastitis is not well elucidated. Fibronectin‐binding proteins are key invasins of bovine mammary epithelial cells in the mammary gland and several members of the MSCRAMM family play important roles in immune evasion. The formation of capsular polysaccharides by S. aureus has been linked to their persistence within the host mammary gland, and encapsulated S. aureus are more resistant to macrophages than non‐encapsulated strains. Capsules can confer resistance to phagocytosis by polymorphonuclear leukocytes (PMNs), which are considered the main line of defense against invading pathogens; they can mask the recognition of antibodies directed against the cell wall and prevent complement activation (Zaatout et al. 2020). While important for immune evasion, capsule expression appears to be detrimental for strain invasiveness as it might mask surface adhesins. Thus, strains not expressing capsular polysaccharides are more invasive and can promote subclinical mastitis (Zaatout et al. 2020). However, capsule expression is highly variable between strains, and many bovine S. aureus strains are capsule‐deficient due to loss of function mutations acquired in capsule biosynthesis genes. Most bovine S. aureus strains express staphylococcal protein A (SpA) and the staphylococcal‐binding immunoglobulin (Sbi) protein (Kim et al. 2012). SpA binds to the Fcγ and F(ab)2 portions of immunoglobulin (Ig) G and IgM via the variable region of the F(ab)2 heavy chain preventing immune recognition by masking surface antigens and inhibiting opsonophagocytic killing of S. aureus by PMNs. In addition, these proteins have been proposed to block the normal function of B cells, either by inducing B cell apoptosis (SpA) or by inhibiting receptor interaction of complement factor C3 (Sbi). SpA also plays a role in biofilm formation (Merino et al. 2009), suggesting that it could be involved in establishing primary microcolonies in the epithelial cells of alveoli and lactiferous ducts in infected mammary glands. Secretion of exotoxins that have cytolytic activity is a key contributor to intramammary infections. While hemolysins target erythrocytes, leukocidins will target white blood cells such as PMNs. Of the four hemolysins (α, β, γ and δ), α‐ and β‐hemolysin have been shown to contribute to the pathogenesis of mastitis. Among the leukocidins, LukED and LukMF’ can cause lysis of bovine neutrophils and high‐level expression of the phage‐encoded LukMF’ correlates with the development of severe clinical mastitis (Vrieling et al. 2016). Staphylococcal superantigens (SAg) represent a diverse family of structurally related bacterial toxins that bind to major histocompatibility complex (MHC) class II and T‐cell receptor to stimulate large numbers of T cells. T cells are stimulated to proliferate in an uncontrolled fashion and release excessive amounts of pro‐inflammatory cytokines, which can lead to rashes, fever, multiorgan damage, coma, and death from severe shock. To date, 23 SAgs have been identified to be produced by S. aureus strains. Most S. aureus strains can produce at least five SAgs and many of these appear to be expressed in the mammary gland and contribute to the development of clinical mastitis (Wilson et al. 2018). Superantigen‐like proteins (SSL) are related to SAgs yet are not mitogenic to T cells and do not bind MHC class II. Instead, their function is targeted toward different key elements of innate immunity. Many staphylococcal strains harbor between 10 and 15 SSL‐encoding genes. Both SAgs and SSLs play crucial roles in subverting the host’s immune response by either interfering with adaptive or innate immunity, respectively. A crucial factor for establishing an intramammary infection is the ability of S. aureus to form biofilms. Biofilm formation is a multi‐factorial process with three main stages (Cheung et al. 2021): adhesion, maturation/proliferation and detachment. Initial attachment is facilitated by surface proteins, many of which belong to the MSCRAMMs (Foster 2019). Maturation of the biofilm involves the production of a biofilm matrix that connects cells and consists of the exopolysaccharide PIA/PNA, extracellular DNA, teichoic acids and proteins such as SasG or the PICI‐encoded Bap (Tormo et al. 2005). In intramammary infections, biofilms are important for the persistence of the pathogen and its recalcitrant ability to resist antimicrobial treatment. Detachment from the biofilm leads to the dispersal of the pathogen through the mammary gland and can result in flareups of acute mastitis. Regulation of virulence in S. aureus is complex and the amalgam of the interaction of a substantial number of distinct regulators. We currently do not fully understand how different combinations of these factors contribute to establishing and maintaining disease and how they subvert and exploit the host’s immune system. Nevertheless, there are certain key regulatory circuits that can control the expression of many virulence determinants. The accessory gene regulator (agr) quorum‐sensing two‐component system coordinates the control of expression of a large set of virulence‐associated genes (Haag and Bagnoli 2017). The staphylococcal accessory regulator SarA can modulate both expression of agr as well as other virulence‐related genes (Bronner et al. 2004). Downregulation or inactivation of the agr system promotes colonization and formation of biofilms, a key factor in establishing intramammary infection. For example, the SrrAB two‐component system has been proposed to downregulate agr expression within the low‐oxygen environment of the mammary gland (Pragman et al. 2004). In fact, many of the 16‐conserved two‐component systems control the expression of virulence factors relevant to bovine mastitis (Haag and Bagnoli 2017; Rapun‐Araiz et al. 2020) but their role in the establishment and development of the disease has not been studied in detail. The alternative sigma factor, SigB, controls the expression of genes required after exposure to a range of different stresses. SigB, is involved in biofilm formation upstream of agr and can affect the expression of sarA (Supa‐Amornkul et al. 2019). Loss of SigB expression also results in reduced persistence and an inability to form small colony variant (SCV)‐like colonies. The ability to form SCVs is also considered important for the persistence of S. aureus within different types of host cells (Kahl et al. 2016) and for establishing recurrent infections in the mammary gland (Zaatout et al. 2020). A comprehensive understanding of the interplay of regulatory networks required for intramammary infection in dairy cows will require more research and is likely to reveal strain‐specific regulation that can affect both disease progression and severity of the infection. Infection of the mammary gland with S. aureus occurs via the teat when the bacteria colonize the tip of the teat and enter the teat canal where they can rapidly replicate (Peton and Le Loir 2014). Entry is facilitated by tissue damage, which can be caused by inappropriately maintained milking equipment. Colonization of ductal and alveolar epithelial cells and their extracellular matrix molecules seems to be crucial for establishing infection likely via MSCRAMMs (Foster 2019). The subsequent establishment of a biofilm is important for protecting the bacteria from the action of antimicrobials and the host immune response (Cheung et al. 2021). Several S. aureus secreted enzymes and toxins facilitate this process by exposing the relevant host molecules (Rainard et al. 2018). Degradation of epithelial cells in the cistern, duct and alveoli causes microlesions in the mammary gland tissue resulting in reduced milk production. Persistence is facilitated by several immune evasion strategies such as the expression of SpA, capsular polysaccharides, and the formation of biofilms. Additionally, S. aureus can invade and survive within both professional and nonprofessional phagocytes, which can be the origin of recurrent infections. SCVs are particularly adept at intracellular survival without triggering a cell‐mediated immune response (Zaatout et al. 2020). Secreted enzymes directly prevent opsonophagocytosis and secreted leukocidins (some of them bovine‐associated) can kill host leukocytes (Vrieling et al. 2016). In addition, SAgs and SSLs target different arms of host immunity and together disrupt the immune response. S. aureus infections of the mammary gland do not result in immunity to subsequent infections; the same quarter of an infected cow can be reinfected by the same or a different strain of S. aureus (Rainard et al. 2018). However, infections raise antibodies against staphylococcal toxins such as hemolysins and leukocidins, which likely contributes to the reduced severity of subsequent infections by toxin‐producing strains. As discussed earlier, the host immune response is subverted by an array of immune evasion factors such as exopolymers, SpA, toxins, SAgs, and SSLs targeting both innate and adaptive arms of immunity (see above). Compared with intramammary infection caused by Escherichia coli, the inflammatory response is lower (see Rainard et al. 2018 for a recent review) and chemoattractants for neutrophils (interleukin 8 and C5a), as well as the proinflammatory cytokine tumor necrosis factor α are present at lower levels in S. aureus compared with E. coli‐induced intramammary infection. Furthermore, global transcriptomic profiling of the bovine udder responses immediately following infection with S. aureus showed a failure in nuclear factor κB signaling and the activation of actin‐cytoskeleton rearrangement through modulating Rho GTPase regulated pathways (Zaatout et al. 2020). Additionally, and even though mammary epithelial cells are highly immunocompetent and express Toll‐like receptors, S. aureus does not activate Toll‐like receptor signaling in these cells. Overall, S. aureus appears to elicit an unbalanced immune suppression rather than inflammation and to promote invasion into the epithelial cells of the host causing persistent infection. Contagious mastitis is primarily controlled through herd hygiene management aimed at reducing transmission opportunities (Rainard et al. 2018). Segregation of infected cows from the herd and separate milking has also been shown to significantly reduce the prevalence of S. aureus mastitis and bulk tank somatic cells (Rainard et al. 2018). Maintenance of milking equipment to avoid injury to the teat and to forestall penetration of S. aureus into the teat canal is also of paramount importance. Antimicrobial therapy of infected animals results in highly variable cure rates (4–92%) and depends on factors such as herd transmission rates, cow, pathogen, and treatment regimen (Rainard et al. 2018). Age of the cow and high levels of somatic cell counts or a high bacterial load before treatment, as well as the duration of infection, are negative predictors of a positive treatment outcome and can be used to decide to treat an animal or not. Several pathogen‐specific factors likely contribute to the failure of antimicrobial treatment including the ability of S. aureus to survive within mammary epithelial cells, the ability to form biofilms masking the pathogen from exposure to antimicrobials, and the formation of small colony variants and L‐forms rendering the bacterium metabolically resistant to antimicrobials. Overall, intramammary rather than systemic delivery of antibiotics is preferable to limit exposure of the digestive flora to antibiotics and to prevent the spread of antimicrobial resistance. If an infection is chronic and has already resisted two antibiotic treatments, culling the infected animal is considered the best solution to prevent further spread throughout the herd (Rainard et al. 2018). Vaccination to prevent intramammary infection has the potential to supplement and replace current management and treatment regimens and has the additional advantage of reducing the use of antibiotics. Recent comprehensive reviews of S. aureus mastitis vaccine development have discussed this matter extensively (Scali et al. 2015; Rainard et al. 2018). Ideally, an S. aureus mastitis vaccine should either prevent infection or facilitate the rapid clearance of bacteria from the mammary gland after an intramammary infection. Such a vaccine would therefore eliminate the possibility of the development of long‐term intramammary infections, which frequently serve as a reservoir for infection within the herd. Despite having been studied for decades, only two commercial vaccines against S. aureus
25
Staphylococcus
Introduction
Characteristics of the Organism
Pathogenic Species
Staphylococcus aureus Infections in Cattle
Source of Infection: Ecology, Evolution and Epidemiology
Types of Disease and Pathologic Changes
Virulence Factors
Regulation of Virulence
Pathogenesis
Immunity
Control
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
