Staphylococcus epidermidis and Other Coagulase-Negative Staphylococci

Microbiology and Ecology

The staphylococci are members of the family Micrococcaceae, which also includes Micrococcus, Stomatococcus, and Planococcus. These bacteria are catalase-positive, gram-positive cocci that divide in irregular clusters, producing a “grapelike cluster” appearance when viewed under the microscope. Staphylococcus comprises at least 80 defined species and subspecies ( Table 195.1 ). In the clinical microbiology laboratory, staphylococci are typically categorized as those that have the ability to coagulate rabbit plasma (i.e., coagulase-positive staphylococci or Staphylococcus aureus ) and those that do not (i.e., coagulase-negative staphylococci). The most common coagulase-negative staphylococci associated with human disease include S. epidermidis, Staphylococcus saprophyticus, Staphylococcus lugdunensis, and Staphylococcus haemolyticus. However, numerous other species have less commonly been associated with disease (see Table 195.1 ). An alternative to the coagulase test commonly used in clinical microbiology laboratories includes rapid agglutination kits (containing antibody bound to beads) that target specific S. aureus antigens. The ability to identify coagulase-negative staphylococci to the species level correctly is difficult because of the number of biochemical tests required to yield accurate results. However, most phenotypic systems used in clinical laboratories today can accurately identify those species most commonly isolated from human disease, including S. aureus, S. epidermidis, S. haemolyticus, and S. saprophyticus. A simplified scheme used to identify S. lugdunensis from other coagulase-negative staphylococci includes a positive pyrrolidonyl aminopeptidase (PYR) and ornithine decarboxylase test. Owing to complexities of identifying bacteria through phenotypic means, clinical laboratories are increasingly using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to identify bacterial pathogens, including staphylococci. In addition, US Food and Drug Administration (FDA)–approved polymerase chain reaction (PCR) platforms are commonly used to rapidly detect staphylococci (both S. aureus and coagulase-negative Staphylococcus ) from positive blood culture bottles. Indeed, the use of MALDI and rapid PCR identification testing systems will most certainly replace phenotypic identification of Staphylococcus species in the near future. Lastly, research-based PCR assays that identify coagulase-negative staphylococci to the species level primarily target 16S-23S rRNA regions, hsp60, rpoB, sodA, tuf, and transfer RNA intergenic spacer length polymorphisms.

Coagulase-negative staphylococci are normal commensal skin and mucous membrane microbes and are indigenous to a variety of mammalian hosts. Depending on the anatomic site, healthy human skin or mucous membranes support from 10 ¹ to 10 ⁶ colony-forming units (CFUs) per square centimeter of coagulase-negative staphylococci. We are just now beginning to investigate the fundamental biologic significance of staphylococcal skin colonization. For example, recent data demonstrate that S. epidermidis colonization augments the local T-cell response, providing protection against Leishmania major infection in mice. Furthermore, Nakatsuji and colleagues found evidence of bacterial communities extending beneath the dermal layer to the basement membrane, indicating interactions with various cell types. Indeed, future studies to document the biologic function of coagulase-negative staphylococci and other skin commensals may be of utmost importance in the understanding of normal skin development and immunity. Of interest, the S. epidermidis protease Esp has the ability to inhibit biofilm formation of S. aureus, suggesting that some strains of S. epidermidis may be important in the inhibition of S. aureus colonization in humans. More recent studies have found that S. lugdunensis, Staphylococcus hominis, and S. epidermidis produce antimicrobial peptides (AMPs) that, in some cases, synergize with the human AMP LL-37, to inhibit S. aureus growth on skin. These interactions between coagulase negative staphylococci and S. aureus appear to be vital in the pathogenesis of staphylococcal infections in atopic dermatitis. In addition, Horswill and colleagues found that a strain of Staphylococcus caprae was able to inhibit S. aureus dermonecrosis in a mouse model by inhibition of the Agr quorum sensing system via synthesis of its own autoinducing peptide. Thus, the study of coagulase-negative staphylococci and their interactions with other commensal flora (e.g., Cutibacterium acnes ) and potential pathogens, including S. aureus , is a fertile area of investigation that may lead to novel prevention strategies.

Aptly named, S. epidermidis is one of the most prevalent species found on human skin, with the average person consistently carrying 10 to 24 different strains. Because of varying characteristics of human skin, including varying moisture content, nutrient substances, pH range, and temperature, S. epidermidis must adapt to a variety of environmental conditions. Certain species of coagulase-negative staphylococci are well adapted to exist in specialized niches, such as Staphylococcus capitis (human scalp), Staphylococcus auricularis (human ear canal), and S. saprophyticus (human alimentary and genitourinary tracts); however, the loci that function in staphylococcal skin colonization are not well characterized.

Antibiotic Resistance

Coagulase-negative staphylococci isolated from nosocomial environments are almost always resistant to multiple antimicrobial agents. In two large surveillance studies from the United States and North America, 73% to 88% of isolates were resistant to oxacillin, 55% to 66% were resistant to levofloxacin, 70% to 73% were resistant to erythromycin, 35% to 52% were resistant to clindamycin, and 35% to 48% were resistant to trimethoprim-sulfamethoxazole. Similar results were obtained from the United Kingdom. Although resistance determinants have been defined in some cases, little resistance is observed clinically to agents such as linezolid, tedizolid, daptomycin, quinupristin-dalfopristin, tigecycline, ceftaroline, dalbavancin, telavancin, and oritavancin, or to the investigational agents ceftobiprole and iclaprim. A few coagulase-negative staphylococci with van genes have been isolated from nonclinical sources. In addition, isolates with elevated minimal inhibitory concentrations (MICs) to glycopeptide antibiotics, especially within S. haemolyticus, have been reported. To date, “vancomycin creep” as found in S. aureus has not been observed in coagulase-negative staphylococci. The ability to isolate subpopulations of S. epidermidis clinical isolates with markedly elevated vancomycin MICs (>32 g/mL) has not been associated with a clinical consequence to date.

Phenotypic expression of methicillin (oxacillin) resistance in coagulase-negative staphylococci is much more heterotypic than that observed in S. aureus, meaning that the percentage of the population that expresses high-level oxacillin resistance is smaller. To address this expression difference, the MIC breakpoint to detect oxacillin resistance is lower for coagulase-negative staphylococci (except S. lugdunensis ) than S. aureus (≥0.5 µg/mL vs. >4 µg/mL, respectively). Regardless of the degree of heterotypy observed, all isolates containing mecA (the gene conferring oxacillin resistance) are clinically resistant to all β-lactam antibiotics. Alternative methods to detect oxacillin resistance include a cefoxitin disk test, which is used as a surrogate to detect mecA -mediated oxacillin resistance, PCR assay for mecA detection, and commercial assays to detect PBP2A production (gene product of mecA ). However, in some coagulase-negative staphylococci, PBP2A is detected only after oxacillin induction. Note that mecA, and thus oxacillin resistance, is rapidly detected with PCR-based blood culture identification systems.

A particularly onerous aspect of treatment of most coagulase-negative staphylococcal infections is their ability to form biofilms on biomaterials (e.g., catheters, prostheses; see later). Tolerance to antibiotics and persister cells is a common theme with staphylococci and other bacteria growing within a biofilm; these facts need to be taken into consideration during treatment. Studies testing the effectiveness of antibiotics against staphylococci growing in a biofilm have demonstrated that they are significantly less effective than when treating planktonic cells. However, antibiotic combinations containing daptomycin alone or in combination with rifampin seem promising in treating or at least reducing the bacterial burden of staphylococcal biofilm infections. Interesting to note, studies have shown that staphylococci growing in a biofilm lead to the establishment of an antiinflammatory environment. Redirecting this response with proinflammatory macrophages can lead to partial clearance of S. aureus biofilm, suggesting that the combination of antibiotics plus a cell-based therapy could be a promising approach in the future.

Molecular Epidemiology

Pulsed-field gel electrophoresis (PFGE) is the gold standard method for addressing the short-term molecular epidemiology of S. epidermidis and other coagulase-negative staphylococci. There is extreme diversity in pulsed-field patterns when S. epidermidis epidemiology is studied. Therefore the finding of indistinguishable PFGE patterns within the context of an outbreak assessment is highly relevant. Longer-term, population-based relationships and trends are better addressed with multilocus sequence typing (MLST) analysis, which suggests that the population structure of S. epidermidis is epidemic and that nine clonal lineages are disseminated worldwide. One major clone, CC2, represented 74% of isolates worldwide in one study; similar results were found in other MLST studies.

Interesting to note, population structure and the presence or absence of five genetic markers ( icaA, IS256, sesD, mecA, and the ACME pathogenicity island) have the ability to discriminate between hospital and nonhospital sources of S. epidermidis. However, rapid evolution (and thus PFGE patterns) occurs through frequent transfer of mobile genetic elements and recombination, possibly through insertion sequence elements. Other molecular typing methods, including sequence analysis of repeat regions of sdrG/aap genes and MLVA (multiple-locus variable-number tandem repeat analysis), have been developed and have yielded similar discriminatory power as MLST or PFGE.

Pathogenesis

In contrast to S. aureus, which produces an array of toxins and adherence factors, there are few defined virulence factors in S. epidermidis ( Table 195.2 ) and other coagulase-negative staphylococci. However, significant advances made in the past 20 years have helped define the pathogenesis of infections caused by S. epidermidis. The ability of S. epidermidis to adhere and form biofilm on the surface of biomaterials is thought to be the most significant virulence factor associated with this bacterium. However, other factors, such as the secretion of poly-gamma- dl -glutamic acid (PGA) and phenol-soluble modulins (PSMs), appear to complement and increase virulence.

Virulence Factors

Biofilm

Staphylococcal biofilm formation is thought to occur in multiple stages, including adherence to a surface, multiplication, maturation and tower development, and subsequent dispersal ( Fig. 195.1 ). It is well established that bacteria growing within a biofilm are unique compared with those growing exponentially in the planktonic phase. Microarray studies have demonstrated that both S. epidermidis and S. aureus growing in a biofilm state have unique transcriptional responses compared with cells growing exponentially. For example, these experiments have shown that staphylococci growing in a biofilm shift their physiology toward anaerobic or microaerobic metabolism and downregulate protein, cell wall, and DNA synthesis. Although these experiments have been extremely helpful in defining the “average” transcriptional response of biofilm growth (as all cells growing in a biofilm were examined), it is also well established that cells growing within a biofilm have spatial and temporal responses to their immediate environment (e.g., nutrient and oxygen availability and interactions with metabolic waste). It is hypothesized that these unique physiologic states found within a biofilm allow for tolerance to antibiotics and development of persister and/or dormant cells.

Adherence

Biomaterials placed within a human host are rapidly coated with serum matrix proteins, including fibrinogen and fibronectin. Genome and functional analyses have suggested that S. epidermidis possesses at least the ability to bind fibrinogen, fibronectin, collagen, vitronectin, and elastin. Furthermore, there is evidence that S. epidermidis autolysins have the ability to bind directly to plastic and contain matrix protein binding sites. Lipase (GehD), in addition to its enzymatic function, has been shown to bind collagen. Mutants that do not have the ability to bind fibrinogen or produce autolysin are less virulent in pertinent in vivo models, suggesting that initial adherence to serum matrix proteins is critical. Lastly, the bifunctional protein accumulation-associated protein (Aap) has been shown to bind to catheters in a rat catheter model, but the binding partner within serum or host cells is currently unknown.

Maturation

After adherence to the biomaterial, intercellular adherence of the bacteria is primarily mediated by polymeric molecules. It has been shown that extracellular DNA is a major component of both S. aureus and S. epidermidis biofilms, and mutants defective in DNA release produce deficient biofilms. Some clinical strains of S. epidermidis produce an abundance of polysaccharide intercellular adhesin (PIA), but not all strains produce PIA and it is a minor component of an S. aureus biofilm. PIA (or poly- N -acetylglucosamine [PNAG]) is a β-1,6-linked N -acetylglucosamine synthesized by the ica operon gene products. The ica operon is composed of four genes: icaA, icaD, icaB, and icaC. A divergently transcribed repressor, icaR, is found just upstream of ica. PIA appears to be important in S. epidermidis surface colonization, biofilm formation, and immune system evasion. Regulation of icaADBC transcription has been an intense area of study and involves SarA, SarZ, σB, IcaR, and the TCA cycle, among others. In S. aureus, PIA enhances virulence in murine systemic disease models and is a vaccine candidate for both S. aureus and S. epidermidis. It is important to reiterate that approximately 50% of clinically relevant strains of S. epidermidis do not contain the icaADBC operon, and other alternative, proteinaceous biofilm maturation strategies exist (e.g., AAP, Embp, Bap). PIA-positive strains of S. epidermidis may be selected for in niches of high shear stress such as the catheter lumen; indeed, isolates obtained from high-shear environments are more likely to produce PIA-mediated biofilms than those isolates obtained from other sources, suggesting that an additional function of PIA is related to stability of the biofilm structure under high shear. Furthermore, allelic replacement of icaADBC confers increased fitness during colonization of human skin as compared with the isogenic wild-type isolate. Therefore, although production of PIA is highly advantageous to the organism during the infection process, PIA production may be deleterious to the organism during colonization of human skin. An additional well-studied protein that functions in biofilm maturation and accumulation is Aap. Once Aap binds to its target, the N-terminal portion of the protein is cleaved via an S. epidermidis protease, SepA, and intracellular accumulation occurs via Aap proteins on neighboring cells.

Dispersal

The last stage of biofilm development is dispersal and subsequent spread to other potential sites. The production of PSMs by S. epidermidis has been shown in flow cell biofilm experiments to mediate the detachment of the upper layers of the biofilm, although other mechanisms may exist, including nuclease-mediated dispersal. PSMs, which are regulated by the quorum-sensing global regulator agr, act as surfactants, leading to loss of cellular clusters. In addition, S. epidermidis PSMs are proinflammatory and have been shown to recruit, activate, and lyse human neutrophils during infection with S. aureus.

Other Virulence Factors

Phenol Soluble Modulins

S. epidermidis typically produces five PSMs, a family of amphipathic α-helical peptides that have many functions, including spread on epithelial surfaces. However, some PSMs, including those produced by S. aureus, have cytolytic activities. A study by Otto and coworkers found that PSM-mec, which is encoded within SCC mec, the genomic island conferring methicillin resistance within the staphylococci, contributes significantly in a mouse model of sepsis. These data may be particularly relevant to neonatal sepsis, which is typically mediated by methicillin-resistant S. epidermidis strains encoding psm-mec .

Poly-Gamma-dl-Glutamic Acid

A somewhat surprising finding is that in contrast to S. aureus, multiple species of coagulase-negative staphylococci, including S. epidermidis, produce PGA. PGA is a cell surface–associated, antiphagocytic polymer first described as a virulence factor in Bacillus anthracis. PGA appears to have a bifunctional role in S. epidermidis and functions to inhibit innate host defense and facilitate colonization of human skin.

Antimicrobial Peptide Resistance

Although S. epidermidis does not produce multiple toxins to protect against professional phagocytes, several other mechanisms are used to defend against phagocytosis. First, as previously mentioned, biofilm production is itself antiinflammatory, and production of PIA is antiphagocytic. Second, the Aps system can sense the presence of AMPs and upregulate mechanisms to protect the cell against this injury; this includes increased d -alanylation of teichoic acids and lysylation of phospholipids, thus increasing the overall positive charge of the bacterium.

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