Coagulase-Negative Staphylococci and Their Role in Infection

Chapter 43

Coagulase-Negative Staphylococci and Their Role in Infection Curtis G. Gemmell1,2 and Sue Lang3 1

University of Glasgow and Strathclyde, Glasgow, UK, 2University of Strathclyde, Glasgow, UK, 3Glasgow Caledonian University, Glasgow, UK

The differentiation between virulent staphylococci and those of the normal commensal flora has long depended on the production of coagulase and this has for many years formed part of the diagnostic armoury of microbiologists. It was not until the mid-1960s that coagulase-negative, mannitol-negative and DNase-positive staphylococci were isolated from clinical material [1,2]. Such strains were recognized as the aetiological agents of subacute bacterial endocarditis [3]. Since the introduction of the biochemical taxonomy scheme of Kloos and Schleifer [4] for coagulase-negative staphylococci (CNS), rapid progress has also been made in the understanding of infections caused by these organisms. The number of species that can be distinguished now exceeds 20. Some of the new species, such as Staphylococcus xylosus and S. capitis are only occasionally isolated from normal human and animal skin and mucosal surfaces and rarely or never cause infections. Others, such as S. lugdunensis, are increasingly being associated with clinical infection [5]. It is, however, difficult to define the borderline between pathogenic and non-pathogenic microorganisms in the context of modern medicine and surgery, when the immune systems of patients are often suppressed. Following the full sequencing [6,7] of the genomes of two strains of S. epidermidis (ATCC 12228 and ATCC 35984) it has been recognized that the core genomes of them and S. aureus are very similar, although as predicted the S. epidermidis genome encodes fewer virulence factors. This lack of virulence genes corresponds to our inability to detect any of the classical S. aureus toxins in culture supernates of S. epidermidis. However the genome of S. epidermidis does carry a gene capable of producing capsular material similar to that of B. anthracis (polygamma-DL-glutamic acid). The capBCAD operon responsible for the expression of PGA [8] is conserved in several Molecular Medical Microbiology. DOI: http://dx.doi.org/10.1016/B978-0-12-397169-2.00043-3 © 2015 Elsevier Ltd. All rights reserved.

coagulase-negative staphylococcal species [9] other than S. epidermidis (S. capitis, S. caprae, S. haemolyticus, S. warneri, S. saccharolyticus, S. hominis and S. lugdunensis). PGA is not synthesized by S. aureus. It has been suggested that expression of this gene in vivo may offer advantages to these species in terms of their ability to survive and colonize the higher salt concentrations seen on human skin. There would appear to be nine clonal lineages of S. epidermidis worldwide [10] of which one, CC2, comprises almost 75% of isolates. Several recent surveys indicate that S. haemolyticus and S. epidermidis are commonly isolated from a great variety of human tissue infections, while a third species, S. saprophyticus, shows a tropism for the human urinary tract. Other species such as S. simulans and S. hominis have, however, also regularly been isolated from infections. Few attempts have been made to define the virulence potential of such strains, which belong to the skin flora and commonly contaminate clinical specimens, including abscess material and blood cultures.

POSSIBLE SOLUBLE VIRULENCE FACTORS OF COAGULASE-NEGATIVE STAPHYLOCOCCI In one study, 350 clinical isolates of staphylococci were screened for coagulase, deoxyribonuclease (DNase) and several other biochemical characteristics [11]. Of these, 241 were designated as S. aureus and the remainder were identified as S. epidermidis. The biochemical activity patterns of the latter revealed 17 different profiles. Analysis of an additional 500 isolates according to their clinical source and association with disease, showed that 16.8% were coagulase- and mannitol-negative, but DNasepositive. Others have shown that coagulase-negative staphylococci can produce a number of toxins and 793

794 PART | 6 Disseminating Bacterial Infections

aggressins [12
16]. It is noteworthy that coagulasenegative staphylococci can produce some of the toxins and enzymes characteristically associated with S. aureus [17,18]. One reason for the wide variation in toxin/enzyme incidence observed in various studies of CNS may be related to their clinical source. The literature is replete with information about the toxinogenicity of clinical isolates of S. aureus [19], but rather sparse with respect to coagulase-negative staphylococci. Only when a true association between the isolation of such a staphylococcal strain and a clinical situation is revealed, does any insight into their mechanism(s) of pathogenicity emerge. Elek and Levy [20] failed to detect α-, β- or δ-haemolysin in a collection of 77 skin-derived CNS strains, but they reported the presence, in most of these strains, of a hitherto uncharacterized haemolysin, which they designated Σ-toxin. Heczko et al. [21] studied 423 strains of S. epidermidis isolated from the skin, nose and urine of healthy human volunteers, and also 55 CNS isolated from various body fluids of children with septicaemia, purulent skin lesions, urinary tract infections or a ventriculo-atrial shunt for the relief of hydrocephalus. The proportion of strains producing α-haemolysin, DNase and staphylokinase (fibrinolysin) was larger in those isolated from infected patients than in those from healthy individuals. Indeed, more than 80% of the clinically significant strains produced α-haemolysin. An attempt was made by Grigorova and Bailjosov [15] to distinguish between different biotypes of coagulase-negative staphylococci on the basis of the expression of various combinations of toxins and enzymes. Of the strains tested, 145 belonged to biotype 1 as designated by Baird-Parker [22]; over 50% produced DNAase, several proteases, as well as urease and lysozyme. In addition, almost 65% hydrolysed various synthetic lipids (Tween compounds). A critical analysis of the toxinogenicity of 180 CNS strains isolated from proven clinical cases, showed that many produced up to six distinct toxins and aggressins [16]. These included both α- and β-haemolysins, which were clearly distinguishable with rabbit antibody to neutralize haemolysis of rabbit and human erythrocytes, respectively (Table 43.1).

HAEMOLYSIN PRODUCTION In general, human strains of S. aureus produce α- and δ-haemolysins, whereas animal strains produce mainly β-haemolysin [20], but CNS can also produce α- and δ-haemolysins [17,23] and a number of studies have sought to investigate the nature of the latter haemolysin [12,24]. Many strains of CNS produce a haemolysin that resembles the δ-haemolysin of S. aureus, and Marks and Vaughan [25] equated it with the earlier reported Ω-haemolysin [26]. Kleck and Donahue [23] found that this haemolysin was produced by most strains isolated

TABLE 43.1 Toxins and Enzymes Produced by 118 Isolates of Coagulase-Negative Staphylococci Property Measured

Strains with Indicated Activity (n 5 118)

α-haemolysin

69 (58)

δ-haemolysin

70 (59)

DNase

70 (59)

Gelatinase

50 (42)

Egg yolk factor

28 (24)

Succinic oxidase factor

39 (58)

Lipase/esterase

57 (80)

Phosphatase

32 (53)

Values are numbers with percentages in parentheses. Adapted from [16].

either from the nose of healthy carriers or from the blood of patients with endocarditis. By means of a cellophane overlay technique, they showed that all of the former were capable of producing haemolysin, while only 83% of the clinically significant blood culture isolates produced haemolysin, but they were the most prolific producers. Brun et al. [27] found that 20.3% of 354 clinical isolates of CNS were haemolytic for human erythrocytes and, therefore, possible δ-haemolysin producers. When CNS are grown in aerated brain heart infusion broth appreciable amounts of the haemolysin, to which erythrocytes from guinea pigs and human beings were most sensitive, is produced within 5 hours [28]. Partial purification by isoelectric focusing over the pH range 3.5
10.0 revealed two components, one with a pI of 5.5 and the other of 10.0. The latter was the more active and abundant. The haemolysin was not, however, inactivated by heating at 60 C or 100 C, or by exposure to egg yolk lecithin, suggesting that the toxin was not similar to either the α- or δ-haemolysin of S. aureus. Gradient polyacrylamide electrophoresis (PAGE) detected four to six protein bands with molecular weights ranging from 23 000 to .460 000. It was not, however, possible to determine which band(s) corresponded to the haemolysin(s), because of the relatively low biological activity of the preparations. By means of Sephadex gel chromatography followed by isoelectric focusing, it has been possible to isolate the haemolysin as a protein of molecular weight $100 000 and a pI of 4.25 [16]. The haemolysin was neutralized by egg yolk lecithin and by specific antibody. The same antibody also neutralized the δ-haemolysin of S. aureus and formed a precipitin line in agarose gels [29].

Chapter | 43 Coagulase-Negative Staphylococci and Their Role in Infection

The haemolysin was stable at 60 C, making it almost the same as the δ-haemolysin of S. aureus. The relative incidence of this haemolysin in a collection of CNS revealed its presence amongst strains of S. epidermidis, S. saprophyticus and S. haemolyticus [24], with titres ranging from 4 to 512 haemolytic units (HU)/ml (Table 43.2). Haemolysin-positive culture supernates from several strains were subjected to isoelectric focusing in polyacrylamide gels over a pH ranging from 3.5 to 10.0 and the gels were overlaid with a 2.0% suspension of human erythrocytes suspended in agarose. Several bands of haemolytic activity were detected, and, in terms of pI, the major band of haemolytic activity corresponded to the haemolytic product obtained earlier by isoelectric focusing in sucrose gradients. In addition to its haemolytic action, the biological activity of this exoprotein against human embryo lung fibroblasts has been examined (Table 43.3). These cells had previously been used to show biological activity of various other bacterial haemolysins, on the basis of the leakage of intracellular radioactive markers of varying size [30]. Most haemolytic strains of S. epidermidis, S. saprophyticus and S. haemolyticus caused the release of [3H]uridine from human embryonic lung fibroblasts [24,31]. These strains were also active in the colony overlay test (COT) (see later). The haemolysin has not been identified as one of the main staphylococcal toxins (alpha, beta or gamma). Instead it resembles the δ-haemolysin. In the case of S. lugdunensis a DNA probe for the four haemolysins did not hybridize with its extracellular haemolysin using Southern blotting techniques. Most strains produced a heat-stable delta-like haemolysin that shares phenotypic

795

properties with S. aureus δ-haemolysin which is encoded by the hld gene [32]. hld is part of the agr locus in S. aureus; a similar sequence is found in S. lugdunensis. The delta toxin-like haemolysin of S. lugdunensis is mediated by three small peptides produced upon expression of the slush locus which acts synergistically with the β-haemolysin of S. aureus to produce a zone of complete haemolysis on sheep red blood cells [33]. The δ-haemolysin gene (hld) encodes a short and heatlabile delta toxin which is secreted by S. epidermidis (ATCC 12228) and is active against several types of membrane [34]; it is located near the 50 end of RNAIII in the agr locus [35] and RNAIII from S. epidermidis can regulate agr-dependent virulence genes in S. aureus [36]. S. epidermidis can also produce auto-inducing peptides (pheromones) which can inhibit agr responses in other staphylococcal species [37]. The haemolysin is probably also responsible for the cytopathogenic effect seen when various strains of CNS are grown on semi-solid agar over a monolayer of murine skin fibroblasts. Certain strains of coagulase-negative staphylococci produce a cytopathogenic effect similar to that reported with toxinogenic strains of Corynebacterium diphtheriae [38]. All of the isolates for which the culture supernates caused uridine leakage from human embryo lung fibroblasts also showed activity in the colony overlay test (COT). Strains of staphylococci positive in this test caused swelling of the cells and membranous bleb formation, leading to cell lysis within 4
6 hours. A good correlation was obtained between a positive COT assay, leakage of [3H]uridine from lung fibroblasts and haemolytic activity for human erythrocytes. Whether such biological activity in vitro can be reproduced in vivo in clinical infections caused by coagulase-negative staphylococci remains to be determined.

TABLE 43.2 Haemolytic Activity of 50 Clinical Isolates of Coagulase-Negative Staphylococci Species

No. Tested

No. Producing $ 16 HU/ml

TABLE 43.3 Cytolytic Activity of 50 Isolates of Coagulase-Negative Staphylococci

S. epidermidis

21

13

S. saprophyticus

14

9

S. haemolyticus

5

4

Clinical Source of Isolate

No. of Strains Tested

Percent Positive in Cell Leakage Test

Percent Positive in COTa

S. simulans

1

1

50

60

1

1

Blood/ endocarditis

10

S. hominis S. capitis

3

0

21

62

67

S. xylosus

3

0

Urinary tract infections

S. cohnii

1

0

Miscellaneous skin infections

19

37

42

S. warneri

1

0

a

Colony overlay test.

796 PART | 6 Disseminating Bacterial Infections

There is also evidence that the β-haemolysins of S. aureus and S. intermedius are similar but not identical. There is 94% similarity in amino acid sequences of S. epidermidis β-haemolysin and that of S. aureus using BLAST analysis [7].

DEOXYRIBONUCLEASE The close correlation between the ability of strains of S. aureus to produce both coagulase and deoxyribonuclease (DNase) and their pathogenicity for humans and animals has encouraged research into the possibility that coagulase-negative staphylococci may also produce DNase. Many non-pathogenic strains of staphylococci produce a DNase that differs from that produced by S. aureus. A large number of strains of S. aureus were examined by Menzies [39], who found that all produced heat-stable DNase, while only one strain of 307 CNS did so, but 18.2% had some DNase activity. The incidence of the enzyme amongst CNS strains ranges from 8 to 63%, a distribution dependent on the source of the strains examined. In most instances the biological activity remained stable after heat treatment.

PROTEASES Most strains of S. aureus are actively proteolytic. This property is usually demonstrated with a gelatin or casein substrate. This proteolytic activity was long regarded as a homogeneous entity, but this concept needs re-evaluation in the light of the identification of at least three different protease types, designated I, II and III [40]. Based on their ability to hydrolyse casein in the presence or absence of ethylenediaminotetraacetic acid (EDTA) or cysteine and Ca21, these proteases can be classified as serine proteases (I), thiol proteases (II) or metallo-proteases (III). Staphylococcal serine protease cleaves peptide bonds at the C-terminal side of glutamic acid and aspartic acid, while the thiol protease shows no preference for particular peptide bonds, and the metallo-protease cleaves peptide bonds at the N-terminal side of hydrophobic residues of casein. CNS isolates from cases of human infection produce mainly thiol protease (type II) and metalloprotease (type III). The production of the thiol protease by so many strains is unusual, since this enzyme is produced by a minority of S. aureus strains. Like humans, cows are susceptible to infection with CNS. Several studies have shown they are most frequently recovered from cases of bovine mastitis, especially in first lactation and in inbred heifers. The somatic cell counts of CNS-infected cows are generally two- to three-fold higher than those of uninfected cows. CNS strains isolated from mastitis samples have higher

protease, DNase and lecithinase activity than CNS from normal cows. One species in particular, S. chromogenes is as pro-inflammatory as S. aureus, and one study has shown that this species produces a cytotoxin active against several cell lines [41]. A protein of 34
36 kDa was associated with the cytotoxicity and also had metalloprotease activity. By mass spectrometry this metalloprotease was deemed to be different from other staphylococcal proteases. It has been proposed that this cytotoxin causes damage to mammary epithelial and vascular epithelial cells, leading to intravascular fluid leakage and the migration of neutrophils.

STAPHYLOKINASE (FIBRINOLYSIN) Staphylokinase converts plasminogen to plasmin and is indirectly responsible for the fibrinolytic activity of S. aureus. Staphylokinase is found in three forms (pI 5.6, 6.1 and 6.7). Direct enzymatic activity has not been demonstrated with the exoproduct. Staphylokinase is produced by 90% of S. aureus strains, but by only 10
25% of coagulase-negative staphylococci [17,42]. The relatively low incidence of this enzyme among CNS associated with clinical infections makes its contribution to the pathogenicity of coagulase-negative staphylococci of little significance.

LIPASE/ESTERASE Marked differences exist in the ability of various coagulase-negative staphylococci to hydrolyse various lipids and esters (Table 43.4). As with haemolysin production, lipase/esterase activity is more often found among isolates from blood, abscesses and skin lesions than in those from urine. Two distinct molecular forms of the enzyme (pI 6.2 and 5.1) are distributed among the CNS

TABLE 43.4 Lipase Activity of Some Clinical Isolates of Coagulase-Negative Staphylococci Tween Tested (Carbon Chain Length)

% Strains Showing Lipase Activity Isolated From Blood, Abscesses and Wounds (n 5 42)

Urinary Tract Infections (n 5 76)

20 (12C)

57

29

40 (16C)

45

22

60 (18C)

36

16

80 (18C)

12

5

Chapter | 43 Coagulase-Negative Staphylococci and Their Role in Infection

strains, but no strain possessed both. The pI 6.2 enzyme, a protein of molecular weight 44 000 displays biological activity against a wide range of lipid substrates, and corresponds to a similar enzyme produced by S. aureus [43]. It is now recognized that CNS such as S. lugdunensis produce a number of soluble virulence factors including the accessory gene regulator system (agr) and RNAIII coded by agr gene and responsible for the quorum sensing system that acts as a global regulator of other virulence factors [44], SLUSH-A, SLUSH-B and SLUSH-C haemolytic peptides (delta toxin-like) coded by the gene slush and acting as haemolytic peptides [33], and a peptidoglycan O acetyl transferase coded by oatA and responsible for a membrane-bound enzyme conferring resistance to lysozyme [45].

POSSIBLE CELL SURFACE VIRULENCE DETERMINANTS OF COAGULASENEGATIVE STAPHYLOCOCCI Slime production in CNS infections has been intensively studied as a possible virulence factor. The slime produced by these organisms is thought to play a key role in colonization of tissue grafts, catheters and other biomaterials [46,47]. Baddour et al. [47] suggested that capsule production by S. epidermidis, but not by S. hominis, may determine the ability to colonize traumatized vascular walls and endocardium. Numerous studies by scanning and transmission electron microscopy have revealed extensive amorphous accretions surrounding intravascular catheters and other types of plastic devices recovered from patients or experimental animals and colonized by CNS [48]. These accretions are variously called exopolysaccharide, glycocalyx and slime layer [49,50]. The presence of amorphous materials in vivo, in which CNS are embedded, poses the question whether these materials are produced by the cocci themselves during colonization or whether fibrin and other substances from the host blood and other secretions become incorporated into the developing biofilm. When the bacteria attach to host proteins, they may mask cell surface antigens

with host components, such as fibronectin-, fibrinogen- and collagen-binding proteins. Encapsulated CNS are resistant to phagocytic ingestion in non-immune serum and to phagocytic killing. They can persist in the host for days both extra- and intracellularly, indicating an important bacterial defence mechanism [51].

SLIME-ASSOCIATED ANTIGEN The isolation and characterization of extracellular materials produced by S. epidermidis, which may represent the adhesive component of slime, have been reported. Peters and co-workers [52] isolated and partially purified a mannose-rich material from S. epidermidis KH11, and named it extracellular slime substance (ESS) on the basis of its binding of concanavalin A. The expression of ESS is, however, not equivalent to slime production, because most concanavalin A-reactive strains did not produce slime, and most strains that produced slime did not react with concanavalin A. A galactose-rich capsular polysaccharide/adhesin (CPA) has been isolated from S. epidermidis RP62A [53]. Pre-coating a Silastic catheter with CPA inhibited the attachment of RP62A to the catheter surface. While it appears that CPA plays a role in the adherence of S. epidermidis to smooth surfaces, the expression of CPA, like the expression of ESS, is not equivalent to slime production. Several polysaccharide components have been described as chemical markers of slime produced by S. epidermidis [53,54]. In particular, Mack et al. [55] identified hexosamine as the main component of an antigen which may be active in colonization by S. epidermidis. Earlier, Christensen et al. [56] described a slime-associated antigen (SAA) with an analogous function. In their analysis of partially purified SAA, they found the presence of a high content of reducing sugars (64%) plus small amounts of ketoses, uronic acid, N-acetylglucosamine, and glucuronic and galacturonic acid. Later studies by the same group [57] revealed a specific role for SAA in the later stages of biofilm formation (Table 43.5).

TABLE 43.5 Characteristics of S. epidermidis ATCC 35984 and Its Mutant in Terms of SAA and Slime Production Strain

Slime Production (OD570)a

SAA Expression

Associated with Early Adhesion

Associated with Late Accumulation

ATCC 35984

2.89 6 0.02

1

1

1

HAM 892

0.08 6 0.01




1




a

As measured using colorimetry.

797

798 PART | 6 Disseminating Bacterial Infections

Analysis by the phenol sulphuric acid assay indicated that the total sugars amounted to only 5% of the dry weight of purified SAA, whereas with the ferric orcinol assay, which includes a stronger hydrolysis step, the sugar content was 70%. Gas chromatography confirmed the latter result, indicating N-acetylglucosamine as the only detectable component in the purified SAA [57]. Proteins were present only in trace amounts (,0.5%), as was total phosphorus (,0.03 μmol/mg). The hypothesis that at least two adhesive extracellular materials mediate the colonization of inanimate surfaces by CNS is consistent with other microbial adherence systems, which require several different adhesins. It has been proposed that the colonization of dental enamel by S. mutans begins with an initial adherence phase and proceeds through an accumulation phase [58]. Electron microscopy suggest that a similar process applies to S. epidermidis [59].

CAPSULAR POLYSACCHARIDE/ADHESIN Slime is produced in vitro by only 30
60% of clinical CNS isolates [60,61], and slime production does not appear to correlate with the ability of S. epidermidis to cause experimental endocarditis [47]. This may be due to the large contribution of host-derived components to biofilms that are deposited on biomaterials in vivo [62]. Adherence to catheters in vitro correlates with the production of a capsular polysaccharide/adhesin (PS/A), which is produced by most clinical isolates [53]. In rabbits, antibodies to PS/A prevent bacteraemia and endocarditis that results from infected catheters [63]. However, the interrelationship between PS/A and slime and their role in pathogenesis have not been firmly established, because of a lack of isogenic mutants that differ in the production of these factors. To overcome this problem, protoplast fusion was investigated as a means of introducing a temperaturesensitive plasmid, pLTV1, into highly adherent strains of S. epidermidis. pLTV1 is a high-copy-number, 20.6-kb plasmid that contains a temperature-sensitive origin of replication for staphylococci (pE194ts), linked to tetracycline (Tc) resistance. It is maintained in a stable state at 30 C. The actual transposable unit, Tn917-LTV1, is 13.7 kb and encodes erythromycin (Em) and chloramphenicol (Cm) resistance. Tn917-LTV1 has several other important properties: it produces stable Tn insertions after growth at 42 C, it allows rapid cloning of flanking DNA into Escherichia coli by using the ColE1 replicon within the Tn, and analysis of promoter functions of interrupted genes by using the promoterless lacZ gene contained within Tn. PLTV1 has been used successfully to create Tn mutants and to clone the chromosomal DNA that

flanks the Tn insertion from a highly adherent, PS/A- and slime-producing strain of S. epidermidis. Eight slime-negative (sn) Tn mutants of M187 were produced, and in each a single copy of Tn917-LTV1 was transposed into a unique site in the chromosome of Ml87. The sn mutants were characterized as PS/A deficient and poorly adherent to silastic catheters. Similar deficiencies occurred in the other six slime-deficient Tn mutants. Fifteen slime-positive (sp) isolates that contained single Tn inserts were isolated from the biofilms produced in the culture vessels during the enrichments for the sn mutants. Insertion of Tn917-LTV1 into the M187 chromosome at sites differing from those of the sn mutants did not affect PS/A or slime production or initial catheter adherence [64]. All the sn mutants of Ml87 had reduced levels of PS/A production and accumulation into biofilms (slime production), and of the initial phase of adherence to catheter materials, which suggests a close relationship between these three phenotypic characteristics. This supports the observation that PS/A-producing strains of S. epidermidis generally adhere better to plastic biomaterials than PS/A-deficient strains. Mutants deficient in slime production, but not in PS/A production or vice versa, have not been obtained. Biofilm-negative mutants of S. epidermidis obtained by transposon mutagenesis have been studied on the assumption that coagulase-negative staphylococci form biofilms on prosthetic devices in two steps. First, attachment of the bacterial cells to the polymer surface, which occurs in a few minutes and, second, accumulation in multilayered cell clusters in a growth-dependent process. Two classes of mutants were found. Class A mutants were defective in attachment to a polystyrene surface and class B mutants were able to bind to the plastic surface, but unable to accumulate in multilayered cell clusters. It was concluded that the genes for primary attachment and intercellular adhesion are unlinked and represent genetically distinct chromosomal loci. The number of class A mutants that adhered to a polystyrene surface was 50-fold less than those of the wild-type and class B mutants. This phenotype occurred concomitantly with the lack of four surface proteins. Only the 60-kDa protein was still expressed, indicating that this protein is sufficient to mediate attachment to polystyrene. With icaA DNA probes from S. aureus and S. epidermidis and Southern blotting, hybridization was seen with the phylogenetically most closely related Staphylococcus species [65]. The so-called epidermidis phylogenetic group, based on DNA comparisons and some biochemical properties [66], includes S. auricularis and S. capitis, both of which appear to carry a copy of the icaA gene. Other, more distantly related members of this group, S. haemolyticus, S. hominis and S. warneri, failed to cross-hybridize under the conditions used. The coagulase-negative

Chapter | 43 Coagulase-Negative Staphylococci and Their Role in Infection

S. intermedins was so named to reflect a sequence composition that places it phylogenetically between S. aureus and S. epidermidis and accordingly, this species was also able to cross-hybridize with icaA probes. The remaining three icaA-positive species, S. lugdunensis, S. pasteuri and S. piscifermentans have not been classified into any of the larger phylogenetic groupings based on sequence comparisons [67]. Tests on the 22 different Staphylococcus spp. to detect biofilm formation and polysaccharide intercellular adhesin (PIA) production were inconclusive. Strain representatives within the same species behave very differently and a single tested strain from each is unlikely to be representative of the species as a whole. Species that appear not to carry an icaA-like gene, yet are able to form a biofilm in vitro or produce a product that reacts with the anti-S. epidermidis PIA antibody, may well have an entirely different and as yet unidentified mechanism that mediates biofilm formation. Studies that include a larger number of representatives for each species are clearly required in order to show that functional intercellular adhesion, and not only the presence of the ica (intercellular adhesin) locus, occurs in Staphylococcus species other than S. epidermidis and S. aureus. The class A mutant could be complemented by transformation with a 16.4-kb wild-type DNA fragment. The complemented mutant attached to a polystyrene surface, to form a biofilm, and produced all of the missing surface proteins. Subcloning experiments revealed that the 60-kDa protein is sufficient for initial attachment. Immunofluorescence microscopy with an antiserum against the 60-kDa protein showed that this protein is located at the cell surface. The 120-kDa protein missing from the mutant presumably represents the unprocessed amidase and glucosaminidase domain of an autolysin AtlE after proteolytic cleavage of the signal peptide and pro-peptide [68]. The 45- and 38-kDa proteins are probably the degradation products of the 60- and 52-kDa proteins, respectively. AtlE showed vitronectinbinding activity, indicating that AtlE was involved in binding to a naked polystyrene surface as well as to plasma protein-coated polymer surfaces. Staphylococcal adherence to biomaterial is not, however, simple and other antigens are probably involved. McKenny et al. [69] have discovered that the biofilm comprises PS/A, which mediates cell adherence to biomaterials, and another antigen, PIA, which is thought to mediate bacterial accumulation into cellular aggregates. The latter is probably equivalent to the SAA described earlier. PIA is a polymer of β-1,6-linked N-acetylglucosamine residues with a molecular mass of ,30 000 kDa. Recombinant S. carnosus and S. aureus carrying a plasmid with genes of the ica locus, and reported to encode the biosynthetic proteins for production of PIA, were also able to synthesize PS/A. PS/A and a chemically and

799

immunologically identical polysaccharide isolated from S. carnosus carrying the ica genes on plasmid pCN27 were high-molecular-mass (.250 000 kDa), acid-stable polymers of β-1,6-linked glucosamine substituted primarily with succinate on the amino group, although some preparations also contained acetate. Moreover, all recombinant staphylococcal strains with the ica genes had the biological properties previously attributed to PS/A. ica-positive strains readily formed a biofilm in vitro on plastics, adhered three- to ten-fold more to catheters in a 30-minute assay as compared with control strains carrying only the cloning vector, absorbed out antibodies to PS/A from immune serum, and elaborated a capsule observable by immunoelectron microscopy with antisera to PS/A. These properties were also seen with PS/A-producing strains of S. epidermidis but not with transposon mutants that lack PS/A. An antiserum raised against PIA contained high antibody titre to PS/A that was readily absorbed by PS/A-positive strains of S. epidermidis and recombinant strains of staphylococci carrying the ica genes. The ica locus encodes production of PS/A, which triggers bacterial adherence, biofilm formation, and intercellular adhesion in slime-positive strains of S. epidermidis. Two structurally related homoglycans, composed of about 130 residues of β-1,6-linked glucosamine that are mostly ( . 80%) N-acetylated, have been chemically characterized by Mack et al. [55,70]. The major basis for differentiating PIA from PS/A comes from a study by Heilmann et al. [71], who cloned a fragment of DNA from S. epidermidis RP62A into plasmid pCA44. The result was plasmid pCN27, which has been reported to contain four open reading frames comprising the ica locus [72]. After transformation of S. carnosus TM300 with pCN27, the recombinant strain elaborated a material with the reported properties of PIA. It mediated intercellular clumping of bacteria but not the initial adherence of bacteria to polystyrene, as determined in a simple colorimetric assay for biofilm production [71]. The recombinant S. carnosits (pCN27) strain adhered well to glass, which is also a property of PS/A [4,46]. Thus, the reported properties of S. carnosus (pCN27) conform to the model proposed by Mack et al. [55,73], in which adherence of S. epidermidis to biomaterials is a two-step phenomenon involving initial adherence mediated by PS/A and/or one of several proteins [74,75] and then accumulation of cells into the biofilm by elaboration of PIA. Moreover, it is likely that the studies of PIA production among clinical isolates of CNS by Mack et al. [70] and Ziebuhr et al. [76] also measured PS/A production. Mack and colleagues used an antiserum raised to S. epidermidis 1457 to produce antibodies, and absorbed this antiserum with non-isogenic, non-adherent S. epidermidis strains to produce a reagent used to detect antigen elaboration among clinical isolates.

800 PART | 6 Disseminating Bacterial Infections

The enzymatic functions of the ica gene locus [72] have been studied in vitro. The proteins encoded by two genes, icaA and icaD, were shown to be able to synthesize an N-acetylglucosamine oligomer up to 20 residues in length from UPD-N-acetylglucosamine precursors. A third gene in the ica locus, icaC, was necessary to obtain N-acetylglucosamine polymers reactive with antisera raised to purified PIA. In spite of the clear evidence that the icaADC genes encode proteins that can synthesize a β-1,6-linked N-acetylglucosamine polymer in vitro [72], it has still not been shown that these genes encode the biosynthetic proteins for production of the PIA polymer in vivo. The presence of a 140-kDa adhesion-associated protein (AAP) from different clinical and laboratory CNS isolates is significantly associated with accumulative growth. However, a few biofilm-positive but 140-kDa antigen-negative strains were also observed. AAP may serve as an anchor molecule on the staphylococcal surface and bind to a complex polymeric substance, such as PIA or PS/A, through multiple interactive sites on the carbohydrate. The 140-kDa antigen-negative, biofilmproducing strains may possess similar proteins which do not share antigenicity with the 140-kDa protein, or they may provide other mechanisms to compensate for the apparent lack of the 140-kDa protein. Although much remains to be learned about the properties of PS/A and the proteins encoded by genes in the ica locus that synthesize PS/A, the findings reported here clarify some important aspects of the biology of coagulase-negative staphylococci. PS/A is a highmolecular-weight, variably (65
100%) N-succinylated, β-1-6-linked polyglycosamine synthesized by proteins encoded in the ica locus of S. epidermidis. PS/A production is correlated with adherence of S. epidermidis to biomaterials, accumulation into cellular aggregates, encapsulation, resistance to phagocytosis [77], and virulence in animal models [78]. Biofilm-producing, PS/Apositive S. epidermidis clearly make up the majority of significant clinical CNS isolates [64]. Another property of PS/A, and of bacterial capsules in general, is the ability to confer resistance to antibodyindependent, complement-mediated opsonic killing [79,80]. McKenney et al. [69] studied the susceptibility to complement-mediated killing of S. carnosus and found that 73.5% of cells carrying the control plasmid pCA44 were killed in 90 minutes by human peripheral blood leukocytes, while a strain transformed with pCN27, which contains ica, strongly resisted killing. This is comparable with results reported previously for isogenic PS/A-positive and PS/A-negative strains [81]. The antigen produced by the genes encoded by the ica locus, therefore, promotes resistance to complement-mediated phagocytic killing, as does PS/A.

BIOFILM FORMATION BY COAGULASENEGATIVE STAPHYLOCOCCI Staphylococcal cells become embedded in a secreted matrix of polysaccharide, protein and extracellular DNA (eDNA) forming a biofilm structure. For CNS this is a principal characteristic contributing to their ability to colonize the skin and mucosal surfaces and cause infections related to medical devices. On the skin surface this complex structure affords the cells protection against the hostile environment, whilst during episodes of infection the secreted matrix interferes with the innate immune responses, impedes phagocytes and offers resistance to antimicrobial agents. From a clinical aspect, biofilms formed by CNS are linked with significant levels of morbidity and mortality, typically being associated with nosocomial infections of indwelling medical devices, including central venous catheters, prosthetic joints and artificial heart valves. CNS biofilms can also form on tissue surfaces, leading to native valve endocarditis, with S. lugdunensis being a notable cause [82], and surgical site infections [83]. Unlike infections linked with the closely related S. aureus, CNS-associated disease is less severe, but is often difficult to treat primarily due to the involvement of a biofilm mode of growth leading to the development of chronic infection [84]. In addition to antimicrobial therapy, treatment will often require the removal of the infected device leading to further medical implications. Though S. epidermidis is the most commonly isolated CNS, reflecting the prevalence of the organism in the commensal flora, it is not the only member of the group to have been linked to biofilm-related infections [85]. In addition to causing native valve endocarditis, S. lugdunensis has also been implicated in intravenous catheter and other device-related infections, S. haemolyticus is particularly associated with infections of neonates and severely immunocompromised individuals [86], S. capitis with prosthetic valve endocarditis and pacemaker infection [87], and S. warneri and S. caprae have been reported to cause orthopaedic infections [88,89].

CNS Biofilm Formation CNS biofilm formation takes place in three phases: initial attachment of the staphylococcal cells to a surface, cell proliferation and accumulation leading to maturation of the biofilm and cellular detachment and dispersal. These stages of development lead to the formation of a multicellular layered structure with characteristic fluid channels embedded within a

Chapter | 43 Coagulase-Negative Staphylococci and Their Role in Infection

secreted polysaccharide matrix. It is only with recent technical advances that the complex, highly orchestrated system of genetic and molecular events that underlies staphylococcal biofilm formation is beginning to be understood.

Initial Attachment of Staphylococcal Cells to a Surface The staphylococcal cells may initially attach to either abiotic or biotic surfaces. Attachment to abiotic surfaces, such the plastics and metals used in medical devices, is reliant on physicochemical properties; hydrogen bonding, van der Waals forces, ionic and hydrophobic interactions that mediate reversible attachment to the unconditioned (native) surface. These non-specific interactions are facilitated principally by the bifunctional cell surface-bound autolysin (AltE), adhesin (Aae) and highly negatively charged cell envelope components, wall teichoic acid (WTA) and lipoteichoic acid (LTA). The 148-kDa AltE is the most predominant peptidoglycan hydrolase within the cell wall [68,90], whilst the smaller 35-kDa Aae displays both bacteriolytic and adhesive properties [91]. The teichoic acid polymers of staphylococci are typically alternating phosphate and ribitol groups (WTA) or glycerol groups (LTA) substituted with D-alanine and N-acetylglycosamine that are linked to the peptidoglycan (WTA) or the cytoplasmic membrane via glycolipid (LTA) and protrude through the cell wall into the extracellular milieu. Chemical analysis of the teichoic acids of the archetypally biofilmpositive S. epidermidis RP62A (ATCC 35984) strain revealed a (1-3)-linked poly-(glycerol phosphate) structure substituted with glucose, galactose and N-acetylated aminosugars, as well as D-alanyl, L-lysyl or acetyl residues [92]. These non-specific interactions with the abiotic surface are largely due to physicochemical properties of the device and the bacterial cell, unlike biofilm formation which takes place on biotic or conditioned indwelling device surfaces. Almost immediately on insertion into the bloodstream or soft tissues, indwelling devices are coated in host proteins, a deposit known as a conditioning layer or conditioning substratum. The biomaterial becomes layered in human serum and extracellular matrix proteins, including vitronectin, fibronectin, fibrinogen and collagen. These host proteins act as ligands, mediating irreversible attachment of the staphylococci through specific interactions with adherence receptors located on the bacterial cell surface. The specific interaction between a staphylococcal receptor and host ligand is an important initial step in biofilm formation on either conditioned biomaterials or host tissues. Although not as numerous on the surface of

801

S. epidermidis as on S. aureus, a number of cell surface protein receptors showing some capacity to be involved in CNS biofilm formation have been identified. For others their function remains indefinable at present. To date it has been shown that S. epidermidis can bind to vitronectin, fibronectin, fibrinogen, laminin and collagen, with descriptions of the cell wall protein receptors that facilitate attachment to fibrinogen (SdrG), fibronectin (Embp), collagen (GehD and SdrF) and vitronectin (Aae and AltE) being published [93]. CNS have a number of adhesive factors, known collectively as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), covalently attached through the catalytic activity of sortase to the peptidoglycan layer. The structural characteristics of MSCRAMMs are an N-terminal leader sequence of approximately 40 amino acids, a C-terminus that possesses a proline-rich wall-spanning region, a conserved amino acid motif LPXTG responsible for tethering the protein to the peptidoglycan layer, a hydrophobic transmembrane region and a positively charged cytoplasmic tail. Sequencing of the S. epidermidis ATCC 12228 genome has predicted the presence of nine LPXTG proteins, including SdrG and SdrF [7]. In a subsequent genome analysis of S. epidermidis RP62A, eight ORFs were found in common between the two strains; SdrF, SdrG, Aap, SesA, SesB, SesC, SesE and SesH, whilst Bhp, SesG and SesI were only present in the genome of S. epidermidis RP62A [94]. Within the MSCRAMM family there is a subgroup belonging to the serine-aspartate repeat (Sdr) proteins; SdrF, SdrG, SdrH. Studies on the related protein ClfA of S. aureus [95] suggest that the repetitive serine-aspartate region is a membrane-spanning region essential for the correct presentation of the ligand binding A domain located at the N-terminus on the cell surface. Evidence exists, however, to show that SdrF mediates attachment to collagen via the B domain [96,97] and SdrG (also referred to as Fbe [98]) binds to the Bβ chain of human fibrinogen [99]. Homologous proteins have been identified in other CNS, including the fibrinogen-binding protein Fbl of S. lugdunensis [100] and SdrX of S. capitis, which has been implicated in attachment to collagen VI [101]. Other adhesins include the lipase enzyme GehD [102], which has dual functionality in that it also expresses collagen I, II and IV specificity [103] and the extremely large 1-MDa extracellular matrix binding protein (Embp), which mediates attachment to fibronectin. The absence of the classic LPXTG motif suggests the protein is secured to the staphylococcal cell surface through non-covalent interactions. Current theory suggests that Embp is initially important for attachment to fibronectin-rich surfaces and subsequently promotes

802 PART | 6 Disseminating Bacterial Infections

intercellular adhesion. Unrelated to cell wall turnover and in contrast to the non-specific binding of unconditioned surfaces, the non-covalently attached autolysins AltE and Aae also facilitate specific binding to vitronectin [68]. In other CNS species proteins with demonstrated homology to AltE have been described, e.g. Aas in S. saprophyticus [104] and AtlC in S. caprae [105] and interactions with host extracellular matrix proteins demonstrated. Through these specific interactions nonreversible binding predisposes the colonizing population to undergo multiplication and intercellular aggregation leading to the development of a biofilm.

PROLIFERATION AND ACCUMULATION (MATURATION) Polysaccharide Intercellular Adhesion (PIA)Dependent Mechanisms of Biofilm Formation After the initial stage of bacterial attachment, the cells undergo a phase of proliferation and accumulation facilitated by intercellular interactions binding the cells to each other and leading to the formation of a mature biofilm. It is this stage of biofilm development that is characterized by a multilayered structure with cells deeply embedded within a secreted extracellular polysaccharide ‘slime’ layer apparent as a fibrous net-like structure enveloping the cells. Initial attempts to analyse this secreted substance were hindered by technical problems [106], but eventually a unique polysaccharide was purified, polysaccharide intercellular adhesin (PIA). PIA is also referred to as poly-N-acetylglucosamine (PNAG) due to its chemical composition, β-1,6linked N-acetylglucosaminoglycan [70]. Despite notable similarities between the exocellular PNAG substance secreted by different staphylococci, the failure of the S. epidermidis PNAG-degrading enzyme dispersin B (secreted by the biofilm forming oral commensal Aggregatibacter actinomycetemcomitans typically associated with periodontitis) to detach biofilms formed by S. lugdunensis illustrates fundamental differences in the macromolecular structure of biofilms between species within the Staphylococcus genus [107,108]. PIA is synthesized by the ica operon composed of icaADBC (biosynthesis) and icaR (regulatory) genes [71,72]. Initially, the transmembrane protein IcaA, which shares some homology to N-acetyl-glucosaminyltransferases, adds N-acetylglucosamine residues to the extending oligo-N-acetylglucosamine chain which will ultimately form PIA. IcaC assists in chain extension and translocation across the membrane, being essential for the formation of fully extended chains. In the absence of

IcaC, the chain length will not exceed 20 residues [72]. Cell surface-associated IcaB is responsible for the deacetylation of the poly-N-acetylglucosamine molecule, since 15
20% of the N-acetylglucosamine residues are non-Nacetylated [70]. Deacetylation generates a positive charge, which is unusual among bacterial exopolysaccharides, yet it is essential for attachment of the polymer to the surface of cells which are negatively charged [109,110]. Whilst WTA contribute significantly to the overall net negative charge of the bacterial cell envelope and their presence is known not to be essential for the binding of PNAG to the cell surface [111], it is highly likely that WTA contributes together with LTA. The role of IcaD is not clearly defined, but it is thought to assist with optimal gene activity. Expression of icaADBC is tightly controlled in staphylococci, though it is apparent that there are significant differences in the regulatory mechanisms not only between species but among strains [112]. Transcriptional regulation is complex with certain regulatory factors exerting direct control over the operon (IcaR and SarA), whilst others act at a global level and impact indirectly on icaADBC (SigmaB (σB) and SarA). Gene activity is directly coordinated by IcaR, a transcriptional repressor divergently transcribed from the icaADBC operon and belonging to the tetR family of transcriptional regulators, that binds in the region of icaA [113]. Whilst the ica operon and PIA synthesis have been extensively studied in S. epidermidis, only limited investigation of other CNS (including S. capitis, S. haemolyticus and S. lugdunensis) has been undertaken [86,89,108]. Acting with IcaR, TcaR, a protein that belongs to the MarR (multiple antibiotic resistance regulator) family, is also a weak negative regulator of the operon [114,115]. The Sar (staphylococcal accessory regulator) family encompasses a number of proteins including SarA and SarX. These small global regulatory proteins (molecular mass around 15 kDa) function by binding to the DNA at multiple sites of the icaR-icaA promotor region and are known, in contrast to IcaR and TcaR, to positively control expression of the icaADBC operon [116,117]. Deletion of sarA has a profound effect on transcription of icaA, but not icaR, and results in decreased PIA production. A similar pattern of ica-dependent biofilm regulatory activity has been demonstrated for sarX and sarZ [118,119]. Expression of PIA in S. epidermidis is also subject to phase variation due to the reversible insertion of IS256 into the icaADBC operon [120,121]. This ability to rapidly switch between phenotypes allows the staphylococcal cells to adapt to changing environmental conditions. This flexibility in being able to alternate between a biofilm and planktonic phases of growth may assist immune evasion and aid the establishment of persistent infection.

Chapter | 43 Coagulase-Negative Staphylococci and Their Role in Infection

Polysaccharide Intercellular Adhesion (PIA)Independent Mechanisms of Biofilm Formation PIA was previously considered to be essential for staphylococcal biofilm formation, but the characterization of ica-negative biofilm-forming S. epidermidis strains has cast doubt on this assumption [122]. A number of studies have screened clinical strain collections for ica genes and PIA production and found the operon to be absent in a substantial proportion of isolates; 55
62% of S. epidermidis recovered from prosthetic joint infections were icaADBC negative [5,123], as were 45% of strains isolated from infected CVC [124] and 26% of isolates cultured from pacemaker-related infections [123,124]. In the absence of PIA, specific proteins appear to assume the role of binding cells, Aap and Embp being two of note [75,125]. In addition to a direct role in surface attachment, the presence of Embp is sufficient to ensure the adequate cell-to-cell adhesion within a biofilm in the absence of any other recognized mechanisms. Similarly, Aap, a thin fibrillar LPXTG-type protein (molecular mass 240 kDa) composed of two domains (A and B) that extends 120 nm from the cell surface in localized clumps, has been implicated in intercellular interactions essential to mature biofilm formation in the absence of PIA [125,126]. Proteolytic cleavage of the A region of Aap occurs prior to the binding of Zn21 to repeat regions in the B domain (G5 region) which allows dimerization to occur and cell-to-cell interactions to form [127,128]. In vitro expression of aap has been observed during the accumulation phase of biofilm formation on a biomaterial, thus providing further evidence for the role of the protein during biofilm maturation [129]. The role of Bhp, a homologue of the S. aureus Bap protein, is less certain. Unlike Aap, expression has not been found to increase under conditions conducive to proteinaceous biofilm formation [130]. In addition to the PIA-dependent and PIA-independent mechanisms discussed, the non-specific electrostatic interactions that take place between the surface-located WTA and other surface components are also known to facilitate cell-to-cell interactions contributing to the structural integrity of the biofilm. There is also an increasing awareness of the potential contribution of eDNA, released through autolytic activity of AltE and cell lysis, to biofilm formation. Loss of SarA triggers a switch to proteinaceous and eDNA-mediated biofilms, possibly due to SarAinduced expression of the metalloprotease SepA which increases the activation of AltE, thus promoting autoinduced eDNA. eDNA is certainly present in the extracellular milieu of S. aureus biofilms [131] and to a lesser extent in S. epidermidis biofilms [107]. The negative charge of eDNA may act in a fashion comparable to WTA and assist intercellular linkage. However, DNase I

803

studies have indicated a relative lack of susceptibility by the biofilm to the enzyme suggesting that eDNA may only be of only minor structural importance [107]. Conversely, the susceptibility of S. epidermidis biofilms to human DNase I, which is freely available in blood plasma, suggests that eDNA is unlikely to have an important role in those disease-related biofilms formed in vivo [132]. It is highly likely that both exocellular polysaccharide and surface-bound proteins contribute to the establishment of robust biofilms, which are further influenced by nonspecific physical interactions. This shift between mechanisms of bacterial aggregation and biofilm maturation is clearly a direct response to the environmental conditions encountered by the CNS and their ability to rapidly respond to the environment underlies their success as opportunistic pathogens. The presence of differing mechanisms of biofilm formation also highlights the difficulties which will be encountered in the development of therapies targeting biofilm formation; impair one mechanism and another will likely compensate for the loss.

Environmental Stress and Biofilm Formation A switch between PIA-dependent and PIA-independent proteinaceous biofilm formation has been clearly demonstrated by Hennig et al. [130]. Exposure to ethanol resulted in a switch to a proteinaceous matrix and loss of the characteristic fibrous net of PIA [130], and, as previously noted, other environmental stimuli representative of conditions encountered during both invasive disease and colonization (serum and sodium chloride) are known to influence the switch to protein-mediated intercellular binding. The alternative sigma factor σB plays a pivotal role in the response of the staphylococci to environmental stimuli, including increased osmolarity, anaerobiosis, elevated temperature, extremes of pH and exposure to harmful agents such as antibiotics, disinfectants and ethanol [133
135]. The role of σB in biofilm regulation remains unclear. It is known to influence icaADBC, sarA and agr activity, but it is also known to regulate PIA/biofilm formation independent of SarA or icaADBC [117,136]. In the absence of conclusive evidence the mechanism of PIA regulation by σB is presently considered to be independent of icaADBC and mediated by factors that currently remain unknown.

Disaggregation of the Biofilm and Cell Dispersal As the biofilm matures, individual staphylococcal cells are dispersed from the surface. The clinical implications of this are clearly apparent; the staphylococcal cells shed from one site of infection are often disseminated into the

804 PART | 6 Disseminating Bacterial Infections

bloodstream or lymphatic system and are able to initiate secondary infections at distal sites. For example, cells adherent to the lumen of a central venous catheter enter the bloodstream and gain access to other artificial medical devices including heart valves, leading to infective endocarditis, or prosthetic hips, leading to loosening and failure of the joint. Unlike other biofilm-forming pathogens, such as A. actinomycetemcomitans, there is no evidence to suggest that staphylococci produce an enzyme with the capacity to degrade the PIA matrix. Similarly, the evidence to indicate that cell detachment is achieved through proteolytic activity is limited, though the serine protease Esp secreted by S. epidermidis has been demonstrated to inhibit both biofilm formation and nasal colonization by S. aureus [137,138]. Recent evidence suggests that Esp has the ability to degrade both adhesive and cohesive protein-based interactions fundamental to the biofilm structure [138]. An alternative supposition is the possibility that dispersal of the biofilm is achieved through the cessation rather than degradation of those factors responsible for biofilm integrity, namely PIA and adhesins. The staphylococcal agr locus encodes a quorum sensing system that controls the expression of many virulence factors, including numerous MSCRAMMs, via a twocomponent signalling pathway. Being essential in the initial stages of biofilm formation, expression of these adhesins is induced at low bacterial cell concentrations, with repression reducing expression in the mature biofilm structure when attachment becomes less important. Inhibition of MSCRAMMs under the control of agr in itself does not appear to lead to disaggregation of the biofilm structure. Correspondingly, repression of components that mediate cell-to-cell interaction (PIA) is not sufficient to facilitate detachment of cells. Quorum sensing does, however, appear to have a role as mutants defective in agr produce a particularly thick biofilm and are reportedly more often associated with biofilm-associated infections [139,140]. Furthermore, there is evidence for the differential expression of agr at various levels within the biofilm which might orchestrate biofilm architecture, including channel formation and dispersal of cells from the surface, through different factors [140]. Rather, degradative molecules such as phenol-soluble modulins (PSMs) under control of the Agr system have been proposed [141]. PSMs are small (approximately 20
45 amino acids in length) secreted peptides regulated by agr; the agr response regulator protein AgrA binds directly to the psm operon promotor controlling expression of PSMs [142]. Whilst a variety of functions have been attributed to PSMs, it is the surfactant activity due to their amphipathic alpha-helical structure that is assumed to inhibit the noncovalent interactions between the bacterial cell and the biofilm. Identified in virtually all Staphylococcus species,

it is the beta-type PSM (PSMβ) which is believed to be the primary facilitator of cell dispersal from S. epidermidis biofilms [143]. Wang et al. [143] revealed the capacity of PSMβ to influence the structure of a S. epidermidis biofilm on a catheter surface and also demonstrated dissemination both in vitro and in vivo. The observed differential agr activity in the surface layers of biofilms linked with the regulatory control that the quorum sensing system exerts over PSM activity lends credence to the suggestion that PSMs are responsible for the threedimensional sculpting of the biofilm and cell dispersal from the matrix surface in vivo [140].

ROLE OF VIRULENCE FACTORS IN CLINICAL INFECTION CAUSED BY COAGULASE-NEGATIVE STAPHYLOCOCCI A number of studies have attempted to define the role of several of the aforementioned virulence factors in clinical infection. For example, in an analysis of 112 episodes of S. epidermidis bacteraemia, 35% grew S. epidermidis from multiple cultures, while the remainder were positive for S. epidermidis in only one blood culture [144]. One hundred and nine isolates were examined for their ability to produce biofilm and possession of the ica operon; 83.5% carried the ica operon but only 54% produced biofilm. No specific clinical features were associated with ica carriage. Another study suggested that strains of S. epidermidis carrying the ica operon have a competitive advantage for colonization of indwelling medical devices, especially long-term catheters [145]. An experimental animal model was used to assess the importance of biofilm production, which is mediated by PIA, in the pathogenesis of a biomaterial-based infection [146]. Mice were inoculated along the length of a subcutaneously implanted intravenous catheter with either wildtype S. epidermidis or its isogenic PIA-negative mutant. The wild-type strain was significantly more likely to cause a subcutaneous abscess than the mutant strain (P , 0.01) and was significantly less likely to be eradicated from the inoculation site by host defence (P , 0.05). In addition, the wild-type strain was found to adhere to the implanted catheters more abundantly than the PLA/HA-negative mutant (P , 0.05). Binding of the wild-type strain and its isogenic mutant in vitro to a fibronectin-coated surface was similar. These results confirm the importance of biofilm production, mediated by PIA, in the pathogenesis of S. epidermidis experimental foreign body infection. PIA is also thought to protect the producer organism from phagocytosis and the action of antimicrobial peptides. A related extracellular polysaccharide, 20-kDa PS, also appears to influence phagocytosis by macrophages in the absence of complement.

Chapter | 43 Coagulase-Negative Staphylococci and Their Role in Infection

PS-positive strains of S. epidermidis displayed 30% less susceptibility to phagocytosis as compared to PS-negative strains. Addition of increasing amounts of 20-kDa PS to a non-PS producing strain decreased susceptibility to phagocytosis. Specific antibody to the 20-kDa PS enhanced phagocytosis of a PS-positive strain of S. epidermidis. The 20-kDa PS does not appear to be produced by other CNS or S. aureus. Lambe et al. [147] tested the ability of six strains of S. lugdunensis to induce abscess formation in a mouse model of foreign-body infection. Pieces of silicone rubber catheter pre-adhered with each test strain were subcutaneously implanted in mice that subsequently received a subcutaneous injection with the test strain. After 7 days, the six S. lugdunensis strains collectively induced abscess formation in 76% of mice. S. lugdunensis was cultured from 97% of the explanted foreign bodies and/or surrounding tissues. The results obtained with S. lugdunensis (766 19% abscess formation) did not statistically vary from the collective results obtained from nine S. epidermidis strains (916 13% abscess formation) in the same model, suggesting that the ability of S. lugdunensis to infect foreign bodies is similar to that of S. epidermidis. To further investigate the relationship of the foreign body to S. lugdunensis virulence, Ferguson et al. used the same mouse model described above but compared abscess formation with and without the presence of the foreign body [148]. Abscess formation and positive culture of the surrounding tissues were statistically higher when a foreign body was present for four out of five S. lugdunensis strains tested. Similar results were obtained with S. epidermidis; S. epidermidis induced abscess formation in the absence of a foreign body at a rate of 11
88%, whereas abscess formation in the absence of a foreign body occurred in 8
46% of infections induced by the five S. lugdunensis strains, attesting to the ability of S. lugdunensis to cause diseases on a par with those of other pathogenic CNS. Virulence was enhanced with most tested strains when a foreign body was involved, suggesting that biofilm formation on implanted foreign devices is a virulence mechanism used by S. lugdunensis.

CONCLUSIONS The association of some species of coagulase-negative staphylococci with infection is so well established that attention should be focused not so much on their isolation and identification but on their ability to cause infection. CNS resemble in many ways clinical isolates of S. aureus, including their ability to elaborate many of its toxins and enzymes. In addition, CNS have evolved mechanisms by which they can adhere and accumulate as a biofilm on a variety of prosthetic devices. They therefore live in an

805

TABLE 43.6 Stages in Pathogenesis of Biomaterial Associated Infection with S. epidermidis Step

Virulence Factor

Adhesion

Polysaccharide adhesin (PS/A)

Accumulation

Slime associated antigen (SAA)

Evasion of host defences

Extracellular slime substance (PS/A and SAA)

Protection from antibiotics

Extracellular slime substance (ESS)

Degradation of host nutrients

Lipase/esterase, DNase

Damage to endothelial cells of host

δ-haemolysin, protease

Damage to inflammatory cells of host

δ-haemolysin

intimate relationship with their host, producing only a moderate inflammatory response, which allows them to develop a long-term relationship in situations such as infected joint replacements and prosthetic valve endocarditis. This chapter has attempted to place the various virulence attributes of CNS into some perspective and to suggest how colonization can progress to infection (Table 43.6). The fact that these organisms of low pathogenic potential are able to cause chronic infections associated, in particular, with indwelling medical devices is exclusively due to their ability to form a robust biofilm that is recalcitrant to treatment with antimicrobials. The mechanisms underlying biofilm formation by the CNS are clearly complex and much remains to be determined. However, what is apparent is the multifarious nature of the regulatory systems that control the various stages of biofilm formation by this opportunistic pathogen.

REFERENCES [1] Quinn EL, Cox F, Fisher M. The problem of associating coagulasenegative staphylococci with disease. Ann NY Acad Sci 1965;128:428
43. [2] Nord CE, Holta-Oie S, Ljungh A, Wadstro¨m T. Characterization of coagulase-negative staphylococci species from human infections. In: Jeljaszewicz J, editor. Staphylococci and Staphylococcal Infections. Stuttgart: Gustav Fischer Verlag; 1976. p. 105
11. [3] Smith HBH, Farkas-Himsley H. The relationship of pathogenic coagulase-negative staphylococci to Staphylococcus aureus. Can J Microbiol 1969;15:879
90. [4] Kloos WE, Schleifer KH. Simplified scheme for routine identification of human Staphylococcus species. J Clin Microbiol 1975;1:82
8.

806 PART | 6 Disseminating Bacterial Infections

[5] Frank KL, Hanssen AD, Patel R. icaA is not a useful diagnostic marker for prosthetic joint infection. J Clin Microbiol 2004;42:4846
9. [6] Gill SR, Fouts DE, Archer GL, et al. Insights on the evolution of virulence and resistance from the complete genome analysis of an early methicillin resistant S. aureus strain and a biofilmproducing methicillin-sensitive strain of S. epidermidis. J Bacteriol 2005;187:2426
38. [7] Zhang YQ, Ren SX, Li HL, Wang YX, Fu G, Yang J, et al. Genome-based analysis of virulence genes in a non-biofilmforming Staphylococcus epidermidis strain (ATCC 12228). Mol Microbiol 2003;49:1577
93. [8] Rogers KJ, Fey FD, Rupp MR. Coagulase-negative staphylococcal infections. Inf Dis Clin North Amer 2009;23:73
98. [9] Kokianova S, Vuong C, Yao Y, Voyich JM, Fisher ER, DeLeo FR. Key role of poly-γ-DL-glutamic acid in immune evasion and virulence of Staphylococcus epidermidis. J Clin Invest 2005;115:688
94. [10] Miragaia M, Thomas JC, Couto I, Enright MC, de Lencastre H. Inferring a population structure for Staphylococcus epidermidis from multilocus sequence typing data. J Bacteriol 2007;189:2540
52. [11] Raymond EA, Traub WH. Identification of staphylococci isolated from clinical material. Appl Microbiol 1970;19:919
22. [12] Kocur M, Precechtel F, Martinec T. Haemolysins in coagulasenegative staphylococci. J Pathol Bacteriol 1966;92:331
6. [13] Zierdt CH, Golde DW. Deoxyribonuclcase-positive Staphylococcus epidermidis strains. Appl Microbiol 1970;20:54
7. [14] Males BM, Rogers WA, Parisi JT. Virulence factors of biotypes of Staphylococcus epidermidis from clinical sources. J Clin Microbiol 1975;1:256
61. [15] Grigorova M, Bailjosov D. Enzyme activity and phage sensitivity of Staphylococcus epidermidis. In: Jeljaszewicz J, editor. Staphylococci and Staphylococcal Infections. Stuttgart: Gustav Fischer Verlag; 1976. p. 175
7. [16] Gemmell CG. Extracellular toxins and enzymes of coagulasenegative staphylococci. In: Easmon CSF, Adlam C, editors. Staphylococci and Staphylococcal Infections, vol. 2. London: Academic Press; 1983. p. 809
27. [17] Gemmell CG, Roberts CE. Toxins and enzymes of coagulasenegative staphylococci isolated from human infections. J Hyg Epidemiol Microbiol Immunol 1973;18:261
6. [18] Dornbusch K, Nord CE, Olsson B, Wadstro¨m T. Some properties of coagulase-negative deoxyribonuclease producing strains of staphylococci from human infections. Med Microbiol Immunol 1976;162:143
52. [19] Abramson C, Friedman H. Enzymatic activity of primary isolates of staphylococci in relation to antibiotic resistance and phage type. J Infect Dis 1967;117:242
8. [20] Elek SD, Levy E. Distribution of haemolysins in pathogenic and non-pathogenic staphylococci. J Pathol Bacteriol 1950;62: 541
5. [21] Heczko PB, Jeljaszewicz J, Pulverer G. Classification of Micrococcaceae isolated from clinical sources. Zbl Bakteriol Hyg I 1974;229:171
7. [22] Baird-Parker AC. The basis for the present classification of staphylococci and micrococci. Ann NY Acad Sci 1974;236:7
13. [23] Kleck JL, Donahue JH. Production of thermostable haemolysin by cultures of Staphylococcus epidermidis. J Infect Dis 1968;18:317
23.

[24] Gemmell CG, Thelestam M. Toxinogenicity of clinical isolates of coagulase-negative staphylococci towards various animal cells. Acta Pathol Microbiol Scand B 1981;89:417
21. [25] Marks J, Yaughan ACT. Staphylococcal δ-haemolysin. J Pathol Bacteriol 1950;62:597
615. [26] McLeod JW. Thermostable staphylococcal toxin. J Pathol Bacteriol 1963;86:35
53. [27] Brun Y, Forey F, Fleurette J, Gonthier B. Pouvoir pathogene des staphylocoques coagulase negatifs. Lyon: Proc Soc Franc¸aise de Microbiologie; 1971. [28] Wadstro¨m T, Kjellgren M, Ljungh A. Extracellular proteins from different Staphylococcus species: a preliminary study. In: Jeljaszewicz J, editor. Staphylococci and Staphylococcal Infections. Stuttgart: Gustav Fischer Verlag; 1976. p. 623
34. [29] Turner WH, Pickard DI. Immunological relationship between delta-hemolysins of Staphylococcus aureus and coagulasenegative strains of staphylococci. Infect Immun 1979;23:910
1. [30] Thelestam M, Mo¨lby R. Determination of toxin induced leakage of different-size nucleotides through the plasma membrane of human diploid fibroblasts. Infect Immun 1974;11:640
8. [31] Gemmell CG, Thelestam M, Wadstro¨m T. Toxinogenicity of coagulase-negative staphylococci. In: Jeljaszewicz J, editor. Staphylococci and Staphylococcal Infections. Stuttgart: Gustav Fischer Verlag; 1976. p. 133
6. [32] Vandenesch F, Storrs MJ, Poitevin-Later F, Etienne J, Courvalin P, Fleurette J. Delta-like haemolysin produced by Staphylococcus epidermidis. FEMS Microbiol Lett 1991;62:65
8. [33] Donvito B, Etienne J, Deneroy L, Greenland T, Benito Y, Vandenesch F. Synergistic hemolytic activity of Staphylococcus lugdunensis is mediated by three peptides encoded by a non-agr genetic locus. Infect Immun 1999;65:95
100. [34] McKevitt AL, Bjornson GL, Mauracher CA, Scheifele DW. Amino acid sequence of a delta-like toxin from Staphylococcus epidermidis. Infect Immun 1990;58:1473
5. [35] Tegmark K, Morfeldt E, Arvidson S. Regulation of agr-dependent virulence genes in Staphylococcus aureus by RNAIII from coagulase-negative staphylococci. J Bacteriol 1998;180:3181
6. [36] Otto M, Sussmuth R, Vuong C, Jung G, Gotz F. Inhibition of virulence factor expression in Staphylococcus aureus by the Staphylococcus epidermidis agr pheromone and derivatives. FEBS Lett 1999;450:257
62. [37] Otto M. Staphylococccus aureus and Staphylococcus epidermidis peptide pheromones produced by the accessory gene regulator agr system. Peptides 2001;22:1603
8. [38] Laird W, Groman N. Rapid direct tissue culture test for toxinogenicity of Corynebacterium diphtheriae. Appl Microbiol 1973;25:709
12. [39] Menzies RE. Comparison of coagulase deoxyribonuclease (DNase) and heat-stable nuclease tests for identification of Staphylococcus aureus. J Clin Microbiol 1977;30:606
8. [40] Arvidson SO. Extracellular enzymes from Staphylococcus aureus. In: Easmon CSF, Adlam C, editors. Staphylococci and Staphylococcal Infections, vol. 2. London: Academic Press; 1983. p. 745
808. [41] Zhang S, Maddox CW. Cytotoxic activity of coagulase-negative staphylococci in bovine mastitis. Infect Immun 2000;68:1102
8. [42] Jeljaszewicz J. Studies on staphylococcal coagulases. VI. Appearance of various toxic and biochemical factors among

Chapter | 43 Coagulase-Negative Staphylococci and Their Role in Infection

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

coagulase-negative and coagulase-positive strains of staphylococci. Med Dosw Mikrobiol 1960;12:33
41. Brunner H, Gemmell CG, Huser H, Fehrenbach FJ. Chemical and biological properties of Staphylococcus aureus lipase. In: Jeljaszewicz J, editor. Staphylococci and Staphylococcal Infections. Stuttgart: Gustav Fischer Verlag; 1981. p. 329
33. Vandenesch F, Storrs MJ, Poitevin-Later F, Etienne. J, Novick RP. Agr-related sequences in Staphylococcus lugdunensis. FEMS Microbiol Lett 1993;111:115
22. Freney J, Brun J, Bes M, Meugnier H, Grimont F, Grimont PAD, et al. Staphylococcus lugdunensis sp.nov. and Staphylococcus schleiferi sp.nov., two species from human clinical specimens. Int J Syst Bacteriol 1988;38:168
72. Hogt AH, Dankert J, de Vries TA, Feijen J. Adhesion of coagulase-negative staphylococci to biomaterials. J Gen Microbiol 1983;129:2959
68. Baddour LM, Christensen CD, Hester MG, Bisno AL. Production of experimental endocarditis by staphylococci. Variability in species virulence. J Infect Dis 1984;150:721
6. Marrie TJ, Costerton JW. Scanning and transmission electron microscopy of in situ bacterial colonization of intravenous and intraarterial catheters. J. Clin Microbiol 1984;19:687
93. Wilkinson BJ. Staphylococcal capsules and slime. In: Easmon CSF, Adlam C, editors. Staphylococci and Staphylococcal Infections, vol. 2. London: Academic Press; 1983. p. 481
524. Drewry DT, Galbraith L, Wilkinson BJ, Wilkinson SG. Staphylococcal slime: a cautionary tale. J Clin Microbiol 1990;28:1292
6. Anderson TC, Craven N. Recruitment of neutrophils by an encapsulated coagulase-negative strain of Staphylococcus simulans in the mammary gland of the mouse. J Leukoc Biol 1984;36:633
45. Peters G, Schumacher-Perdreau F, Jansen B, Bey M, Pulverer G. Biology of S. epidermidis extracellular slime. Zbl Bakteriol Hyg I 1987;16:15
32. Tojo M, Yamashita N, Goldmann DA, Pier GB. Isolation and characterisation of a capsular polysaccharide adhesin from Staphylococcus epidermidis. J Infect Dis 1988;157:713
22. Hussain MC, Hastings JGM, White PJ. Comparison of cell wall teichoic acid with high-molecular-weight extracellular slime material from S. epidermidis. J Med Microbiol 1992;37:368
75. Mack D, Nedelmann M, Krototsch A, Schwarzkopf-Hessemann J, Laufs R. Characterization of transposon mutants of biofilmproducing Staphylococcus epidermidis impaired in the accumulation phase of biofilm production; genetic identification of a hexosamine-containing polysaccharide intercellular adhesin. Infect Immun 1994;62:3244
53. Christensen GD, Barker LP, Mawhinney TP, Baddour LM, Simpson WA. Identification of an antigenic marker of slime production for Staphylococcus epidermidis. Infect Immun 1990;58:2906
11. Baldassarri L, Doneili G, Gelosia A, Voglino MC, Simpson AW, Christensen GD. Purification and characterization of the staphylococcal slime-associated antigen and its occurrence among Staphylococcus epidermidis clinical isolates. Infect Immun 1996;64:3410
5. Staat RH, Langley SD, Doyle RJ. Streptococcus mutans adherence: presumptive evidence for protein-mediated attachment followed by glucan-dependent cellular accumulation. Infect Immun 1980;27:675
81.

807

[59] Olson ME, Ruseka I, Costerton JW. Colonization of n-butyl-2cyanoacrylate tissue adhesive by Staphylococcus epidermidis. J Biomed Mater Res 1988;22:485
95. [60] Ishak MA, Groschel DHM, Mandell GL, Wenzel RP. Association of slime with pathogenicity of coagulase-negative staphylococci causing nosocomial septicaemia. J Clin Microbiol 1985;22:1025
9. [61] Davenport DS, Massanari RM, Pfaller MT, Bal MJ, Streed SA, Hierholzer WJ. Usefulness of a test for slime production as a marker for clinically significant infections with coagulasenegative staphylococci. J Infect Dis 1986;153:332
9. [62] Buret A, Ward KH, Olsen ME, Costerton JW. An in vivo model to study the pathobiology of infectious biofilms on biomedical surfaces. J Biomed Mater Res 1991;25:865
74. [63] Kojima Y, Tojo M, Goldmann DA, Tosteson TD, Pier GB. Antibody to the capsular polysaccharide/adhesin protects rabbits against catheter related bacteraemia due to coagulase-negative staphylococci. J Infect Dis 1990;162:435
41. [64] Muller E, Hubner J, Gutierrez N, Takeda S, Goldmann DA, Pier GB. Isolation and characterization of transposon mutants of Staphylococcus epidermidis deficient in capsular polysaccharide/ adhesin and slime. Infect Immun 1993;61:551
8. [65] Cramton SE, Gerke C, Schnell NF, Nichols WW, Gotz F. The intercellular adhesin (ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infect Immun 1999;67:5427
33. [66] Kloos WE. Systematics and the natural history of staphylococci. Soc Appl Bacteriol Symp Series 1990;19:25S
37S. [67] Schleifer KH, Kroppenstedt RM. Chemical and molecular classification of staphylococci. Soc Appl Bacteriol Symp Series 1990;19:9S
24S. [68] Heilmann C, Hussain M, Peters G, Gotz F. Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Mol Microbiol 1997;24:1013
24. [69] McKenney D, Hubner J, Muller E, Wang Y, Goldman DA, Pier GB. The ica locus of Staphylococcus epidermidis encodes production of the capsular polysaccharide/adhesin. Infect Immun 1998;66:4711
20. [70] Mack D, Fischer W, Krokotsch A, Leopold K, Hartmann R, Egge H, et al. The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear β-1,6-linked glucosaminoglycan: purification and structural analysis. J Bacteriol 1996;178:175
83. [71] Heilmann C, Schweitzer O, Gerke C, Vanittanakom N, Mack D, Gotz F. Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis. Mol Microbiol 1996;20:1083
91. [72] Gerke C, Kraft A, Sussmuth R, Schweitzer O, Gotz F. Characterization of the N-acetylglucosaminyltransferase activity involved in the biosynthesis of the Staphylococcus epidermidis polysaccharide intercellular adhesin. J Biol Chem 1998;273:18586
93. [73] Mack D, Siemssen N, Laufs R. Parallel induction by glucose of adherence and a polysaccharide antigen specific for plastic adherent Staphylococcus epidermidis
evidence for functional relationship to intercellular adhesion. Infect Immun 1992;60:2048
57. [74] Timmerman CP, Fleer A, Besnier JM, De Graaf L, Cremers F, Verhoef J. Characterization of a proteinaceous adhesin of Staphylococcus epidermidis which mediates attachment to polystyrene. Infect Immun 1991;59:4187
92.

808 PART | 6 Disseminating Bacterial Infections

[75] Hussain M, Herrmann M, von Eiff C, Perdreau-Remington F, Peters G. A 140-kilodalton extracellular protein is essential for the accumulation of Staphylococcus epidermidis strains on surfaces. Infect Immun 1997;65:519
24. [76] Ziebuhr W, Heilmann C, Gotz F, Meyer P, Wilms K, Straube E, et al. Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates. Infect Immun 1997;65:890
6. [77] Kristinsson KG, Hastings JG, Spencer RC. The role of extracellular slime in opsonophagocytosis of Staphylococcus epidermidis. J Med Microbiol 1988;27:207
13. [78] Stout RD, Li Y, Miller AR, Lambe DW. Staphylococcal glycocalyx activates macrophage prostaglandin E2 and interleukin I production and modulates tumour necrosis factor alpha and nitric oxide production. Infect Immun 1994;62:4160
6. [79] Gray ED, Peters G, Verstegen M, Regelmann WE. Effect of extracellular slime substance from Staphylococcus epidermidis on the human cellular immune response. Lancet 1984;I:365
7. [80] Johnson GM, Lee PL, Regelmann WE, Gray ED, Peters G, Quie PG. Interference with granulocyte function by Staphylococcus epidermidis slime. Infect Immun 1986;54:13
20. [81] Shiro H, Meluleni G, Groll A, et al. The pathogenic role of Staphylococcus epidermidis capsular polysaccharide/adhesin in a low-inoculum rabbit model of prosthetic valve endocarditis. Circulation 1995;92:2715
22. [82] Van Hoovels L, De Munter P, Colaert J, Surmont I, Van Wijnggaerden E, Peetermans WE, et al. Three cases of destructive native valve endocarditis caused by Staphylococcus lugdunensis. Eur J Clin Microbiol Infect Dis 2005;24:149
52. [83] Soderquist B. Surgical site infections in cardiac surgery: microbiology. APMIS 2007;115:1008
11. [84] McCann MT, Gilmore BF, Gorman SP. Staphylococcus epidermidis device-related infections: pathogenesis and clinical management. J Pharm Pharmacol 2008;60:1551
71. [85] Vuong C, Otto M. Staphylococcus epidermidis infections. Microbes Infect 2002;4:481
9. [86] Fredheim EG, Klingenberg C, Rohde H, Frankenberger S, Gaustad P, Flaegstad T, et al. Biofilm formation by Staphylococcus haemolyticus. J Clin Microbiol 2009; 47:1172
80. [87] Nalmas S, Bishburg E, Meurillio J, Khoobiar S, Cohen M. Staphylococcus capitis prosthetic valve endocarditis: report of two rare cases and review of literature. Heart Lung 2008; 37:380
4. [88] Campoccia D, Montanaro L, Visai L, Corazzari T, Poggio C, Pegreffi F, et al. Characterization of 26 Staphylococcus warneri isolates from orthopedic infections. Int J Artif Organs 2010;33:575
81. [89] Allignet J, Aubert S, Dyke KG, El Solh N. Staphylococcus caprae strains carry determinants known to be involved in pathogenicity: a gene encoding an autolysin-binding fibronectin and the ica operon involved in biofilm formation. Infect Immun 2001;69:712
8. [90] Biswas R, Voggu L, Simon UK, Hentschel P, Thumm G, Gotz F. Activity of the major staphylococcal autolysin Atl. FEMS Microbiol Lett 2006;259:260
8. [91] Heilmann C, Thumm G, Chatwal GS, Hartleib J, Uekotter A, Peters G. Identification and characterization of a novel autolysin

[92]

[93] [94]

[95]

[96]

[97]

[98]

[99]

[100]

[101]

[102]

[103]

[104]

[105]

[106] [107]

(Aae) with adhesive properties from Staphylococcus epidermidis. Microbiology 2003;149:2769
78. Sadovskaya I, Vinogradov E, Li J, Jabbouri S. Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus epidermidis RP62A, a reference biofilm-positive strain. Carbohydr Res 2004;339:1467
73. Otto M. Staphylococcus epidermidis
the ‘accidental’ pathogen. Nat Rev Microbiol 2009;7:555
67. Bowden MG, Chen W, Singvall J, Xu Y, Peacock SJ, Valtulina V, et al. Identification and preliminary characterization of cellwall-anchored proteins of Staphylococcus epidermidis. Microbiology 2005;151:1453
64. Hartford O, Francois P, Vaudaux P, Foster TJ. The dipeptide repeat region of the fibrinogen-binding protein (clumping factor) is required for functional expression of the fibrinogen-binding domain on the Staphylococcus aureus cell surface. Mol Microbiol 1997;25:1065
76. Arrecubieta C, Lee MH, Macey A, Foster TJ, Lowy FD. SdrF, a Staphylococcus epidermidis surface protein, binds type I collagen. J Biol Chem 2007;282:18767
76. Arrecubieta C, Toba FA, Von Bayern M, AkashI H, Deng MC, Naka Y, et al. SdrF, a Staphylococcus epidermidis surface protein, contributes to the initiation of ventricular assist device driveline-related infections. PLoS Pathog 2009;5:e1000411. Nilsson M, Frykberg L, Flock JI, Pei L, Lindberg M, Guss B. A fibrinogen-binding protein of Staphylococcus epidermidis. Infect Immun 1998;66:2666
73. Ponnuraj K, Xu Y, Moore D, Deivanayagam CC, Boque L, Hook M, et al. Crystallization and preliminary X-ray crystallographic analysis of Ace: a collagen-binding MSCRAMM from Enterococcus faecalis. Biochim Biophys Acta 2002;1596:173
6. Mitchell J, Tristan A, Foster TJ. Characterization of the fibrinogen-binding surface protein Fbl of Staphylococcus lugdunensis. Microbiology 2004;150:3831
41. Liu Y, Ames B, Gorovits E, Prater BD, Syribeys P, Vernachio JH, et al. SdrX, a serine-aspartate repeat protein expressed by Staphylococcus capitis with collagen VI binding activity. Infect Immun 2004;72:6237
44. Longshaw CM, Farrell AM, Wright JD, Holland KT. Identification of a second lipase gene, gehD, in Staphylococcus epidermidis: comparison of sequence with those of other staphylococcal lipases. Microbiology 2000;146(Pt 6):1419
27. Bowden MG, Visai L, Longshaw CM, Holland KT, Speziale P, Hook M. Is the GehD lipase from Staphylococcus epidermidis a collagen binding adhesin? J Biol Chem 2002;277:43017
23. Hell W, Meyer HG, Gatermann SG. Cloning of aas, a gene encoding a Staphylococcus saprophyticus surface protein with adhesive and autolytic properties. Mol Microbiol 1998;29:871
81. Allignet J, Galdbart JO, Morvan A, Dyke KG, Vaudaux P, Aubert S, et al. Tracking adhesion factors in Staphylococcus caprae strains responsible for human bone infections following implantation of orthopaedic material. Microbiology 1999;145 (Pt 8):2033
42. Gotz F. Staphylococcus and biofilms. Mol Microbiol 2002;43:1367
78. Izano EA, Amarante MA, Kher WB, Kaplan JB. Differential roles of poly-N-acetylglucosamine surface polysaccharide and

Chapter | 43 Coagulase-Negative Staphylococci and Their Role in Infection

[108]

[109]

[110]

[111]

[112]

[113]

[114]

[115]

[116]

[117]

[118]

[119]

[120]

[121]

extracellular DNA in Staphylococcus aureus and Staphylococcus epidermidis biofilms. Appl Environ Microbiol 2008;74:470
6. Frank KL, Patel R. Poly-N-acetylglucosamine is not a major component of the extracellular matrix in biofilms formed by icaADBC-positive Staphylococcus lugdunensis isolates. Infect Immun 2007;75:4728
42. Joyce JG, Abeygunawardana C, Xu Q, Cook JC, Hepler R, Przysiecki CT, et al. Isolation, structural characterization, and immunological evaluation of a high-molecular-weight exopolysaccharide from Staphylococcus aureus. Carbohydr Res 2003;338:903
22. Vuong C, Kocianova S, Voyich JM, Yao Y, Fischer ER, Deleo FR, et al. A crucial role for exopolysaccharide modification in bacterial biofilm formation, immune evasion, and virulence. J Biol Chem 2004;279:54881
6. Vergara-Irigaray M, Maira-Litran T, Merino N, Pier GB, Penades JR, Lasa I. Wall teichoic acids are dispensable for anchoring the PNAG exopolysaccharide to the Staphylococcus aureus cell surface. Microbiology 2008;154:865
77. Cue D, Lei MG, Lee CY. Genetic regulation of the intercellular adhesion locus in staphylococci. Front Cell Infect Microbiol 2012;2:38. Jefferson KK, Cramton SE, Gotz F, Pier GB. Identification of a 5-nucleotide sequence that controls expression of the ica locus in Staphylococcus aureus and characterization of the DNA-binding properties of IcaR. Mol Microbiol 2003;48:889
99. Jefferson KK, Pier DB, Goldmann DA, Pier GB. The teicoplanin-associated locus regulator (TcaR) and the intercellular adhesin locus regulator (IcaR) are transcriptional inhibitors of the ica locus in Staphylococcus aureus. J Bacteriol 2004;186:2449
56. Chang YM, Chen CK, Chang YC, Jeng WY, Hou MH, Wang AH. Functional studies of ssDNA binding ability of MarR family protein TcaR from Staphylococcus epidermidis. PLoS One 2012;7:e45665. Tormo MA, Marti M, Valle J, Manna AC, Cheung AL, Lasa I, et al. SarA is an essential positive regulator of Staphylococcus epidermidis biofilm development. J Bacteriol 2005;187:2348
56. Handke LD, Slater SR, Conlon KM, O’DonnelL ST, Olson ME, Bryant KA, et al. σB and SarA independently regulate polysaccharide intercellular adhesin production in Staphylococcus epidermidis. Can J Microbiol 2007;53:82
91. Wang L, Li M, Dong D, Bach TH, Sturdevant DE, Vuong C, et al. SarZ is a key regulator of biofilm formation and virulence in Staphylococcus epidermidis. J Infect Dis 2008; 197:1254
62. Rowe SE, Mahon V, Smith SG, O’Gara JP. A novel role for SarX in Staphylococcus epidermidis biofilm regulation. Microbiology 2011;157:1042
9. Zeibuhr W, Krimmer V, Rachi S, Lossner I, Gotz F, Hacker J. A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. Mol Microbiol 1999;32:345
56. Conlon KM, Humphreys H, O’Gara JP. Inactivations of rsbU and sarA by IS256 represent novel mechanisms of biofilm

[122]

[123]

[124]

[125]

[126]

[127]

[128]

[129]

[130]

[131]

[132]

[133]

[134]

[135]

809

phenotypic variation in Staphylococcus epidermidis. J Bacteriol 2004;186:6208
19. Qin Z, Yang X, Yang L, Jiang J, Ou Y, Molin S, et al. Formation and properties of in vitro biofilms of ica-negative Staphylococcus epidermidis clinical isolates. J Med Microbiol 2007;56:83
93. Rohde H, Burandt EC, Siemssen N, Frommelt L, Burdelski C, Wurster S, et al. Polysaccharide intercellular adhesin or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infections. Biomaterials 2007;28:1711
20. Cafiso V, Bertuccio T, Santagati M, Campanile F, Amicosante G, Perilli MG, et al. Presence of the ica operon in clinical isolates of Staphylococcus epidermidis and its role in biofilm production. Clin Microbiol Infect 2004;10:1081
8. Rohde H, Burdelski C, Bartscht K, Hussain M, Buck F, Horstkotte MA, et al. Induction of Staphylococcus epidermidis biofilm formation via proteolytic processing of the accumulation-associated protein by staphylococcal and host proteases. Mol Microbiol 2005;55:1883
95. Banner MA, Cunniffe JG, Macintosh RL, Foster TJ, Rohde H, Mack D, et al. Localized tufts of fibrils on Staphylococcus epidermidis NCTC 11047 are comprised of the accumulationassociated protein. J Bacteriol 2007;189:2793
804. Conrady DG, Brescia CC, Horii K, Weiss AA, Hassett DJ, Herr AB. A zinc-dependent adhesion module is responsible for intercellular adhesion in staphylococcal biofilms. Proc Natl Acad Sci USA 2008;105:19456
61. Macintosh RL, Brittan JL, Bhattacharya R, Jenkinson HF, Derrick J, Upton M, et al. The terminal A domain of the fibrillar accumulation-associated protein (Aap) of Staphylococcus epidermidis mediates adhesion to human corneocytes. J Bacteriol 2009;191:7007
16. Patel JD, Colton E, Ebert M, Anderson JM. Gene expression during S. epidermidis biofilm formation on biomaterials. J Biomed Mater Res A 2012;100:2863
9. Hennig S, Wai NS, Ziebuhr W. Spontaneous switch to PIAindependent biofilm formation in an ica-positive Staphylococcus epidermidis isolate. Int J Med Microbiol 2007;297:117
22. Rice KC, Mann EE, Endres JL, Weiss EC, Cassat JE, Smeltzer MS, et al. The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus. Proc Natl Acad Sci USA 2007;104:8113
8. Kaplan JB, Lovetri K, Cardona ST, Madhyastha S, Sadovskaya I, Jabbouri S, et al. Recombinant human DNase I decreases biofilm and increases antimicrobial susceptibility in staphylococci. J Antibiot (Tokyo) 2012;65:73
7. Cotter JJ, O’Gara JP, Mack D, Casey E. Oxygen-mediated regulation of biofilm development is controlled by the alternative sigma factor σB in Staphylococcus epidermidis. Appl Environ Microbiol 2009;75:261
4. Knobloch JK, Jager S, Horstkotte MA, Rohde H, Mack D. RsbU-dependent regulation of Staphylococcus epidermidis biofilm formation is mediated via the alternative sigma factor σB by repression of the negative regulator gene icaR. Infect Immun 2004;72:3838
48. Rachid S, Cho S, Ohlsen K, Hacker J, Ziebuhr W. Induction of Staphylococcus epidermidis biofilm formation by environmental

810 PART | 6 Disseminating Bacterial Infections

[136]

[137]

[138]

[139]

[140]

[141] [142]

[143]

factors: the possible involvement of the alternative transcription factor sigB. Adv Exp Med Biol 2000;485:159
66. Pintens V, Massonet C, Merckx R, Vandecasteele S, Peetermans WE, Knobloch JK, et al. The role of σB in persistence of Staphylococcus epidermidis foreign body infection. Microbiology 2008;154:2827
36. Iwase T, Uehara Y, Shinji H, Tajima A, Seo H, Takada K, et al. Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization. Nature 2010;465:346
9. Sugimoto S, IwamotO T, Takada K, OkudA K, Tajima A, Iwase T, et al. Staphylococcus epidermidis Esp degrades specific proteins associated with Staphylococcus aureus biofilm formation and host-pathogen interaction. J Bacteriol 2013;195:1645
55. Vuong C, Gerke C, Somerville GA, Fischer ER, Otto M. Quorum-sensing control of biofilm factors in Staphylococcus epidermidis. J Infect Dis 2003;188:706
18. Vuong C, Kocianova S, Yao Y, Carmody AB, Otto M. Increased colonization of indwelling medical devices by quorum-sensing mutants of Staphylococcus epidermidis in vivo. J Infect Dis 2004;190:1498
505. Otto M. Molecular basis of Staphylococcus epidermidis infections. Semin Immunopathol 2012;34:201
14. Periasamy S, Joo HS, Duong AC, Bach TH, Tan VY, Chatterjee SS, et al. How Staphylococcus aureus biofilms develop their characteristic structure. Proc Natl Acad Sci USA 2012;109:1281
6. Wang R, Khan BA, Cheung GY, Bach TH, Jameson-Lee M, Kong KF, et al. Staphylococcus epidermidis surfactant

[144]

[145]

[146]

[147]

[148]

peptides promote biofilm maturation and dissemination of biofilm-associated infection in mice. J Clin Invest 2011;121:238
48. Ninin E, Caroff N, Espaze E, Maraillac J, Lepelletier D, Milpied N, et al. Assessment of ica operon carriage and biofilm production in Staphylococcus epidermidis isolates causing bacteraemia in bone marrow transplants. J Clin Microbiol Infect 2006;12:446
52. Vandencasteele SJ, Peetermans WER, Merckx J, Rinders BJ, Van Eldere J. Reliability of the ica, aap and attE genes in the discrimination between invasive, colonising and contaminating Staphylococcus epidermidis isolates in the diagnosis of catheterrelated infections. J Clin Microbiol Infect 2003;9:114
9. Rupp ME, Ulphani IS, Fey PD, Bartsch K, Mack D. Characterization of the importance of polysaccharide intercellular adhesin/hemagglutinin of Staphylococcus epidermidis in the pathogenesis of biomatcrial-bascd infection in a mouse foreign body infection model. Infect Immun 1999;67:2627
32. Lambe DW, Ferguson KP, Keplinger JL, Gemmell CG, Kalbfleisch JH. Pathogenicity of Staphylococcus lugdunensis, Staphylococcus schleiferi and three other coagulase-negative staphylococci in a mouse model and possible virulence factors. Can J Microbiol 1990;36:455
63. Ferguson KP, Lambe DW, Keplinger JL, Kalbfleisch JH. Comparison of the pathogenicity of three species of coagulasenegative Staphylococcus in a mouse model with and without a foreign body. Can J Microbiol 1991;37:722
4.