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Staphylococcus epidermidis as a cause of bacteremia Sharon Kleinschmidt*,1,2, Flavia Huygens1, Joan Faoagali1, Irani U Rathnayake1 & Louise M Hafner1
Staphylococcus epidermidis is a biofilm-producing commensal organism found ubiquitously on human skin and mucous membranes, as well as on animals and in the environment. Biofilm formation enables this organism to evade the host immune system. Colonization of percutaneous devices or implanted medical devices allows bacteria access to the bloodstream. Isolation of this organism from blood cultures may represent either contamination during the blood collection procedure or true bacteremia. Staphylococcus epidermidis bloodstream infections may be indolent compared with other bacteria. Isolation of S. epidermidis from a blood culture may present a management quandary for clinicians. Over-treatment may lead to patient harm and increases in healthcare costs. There are numerous reports indicating the difficulty of predicting clinical infection in patients with positive blood cultures with this organism. No reliable phenotypic or genotypic algorithms currently exist to predict the pathogenicity of a S. epidermidis bloodstream infection. This review will discuss the latest advances in identification methods, global population structure, pathogenicity, biofilm formation, antimicrobial resistance and clinical significance of the detection of S. epidermidis in blood cultures. Previous studies that have attempted to discriminate between invasive and contaminating strains of S. epidermidis in blood cultures will be analyzed. Staphylococcus epidermidis is one of the most prevalent species of bacteria found universally on the human skin and mucous membranes, and is generally regarded as a commensal organism [1] . They belong to the coagulase-negative group of staphylococci (CoNS) and due to their paucity of virulence factors are less invasive than their coagulase-positive relation, Staphylococcus aureus. However, in recent years S. epidermidis has become a frequent and important nosocomial pathogen, particularly in immunocompromised patients [2,3] . Many strains of S. epidermidis are able to produce biofilms and readily colonize implanted medical devices, in particular intravascular devices, cerebrospinal fluid shunts, intraocular lenses, prosthetic joints and heart valve replacements. Colonization of such medical devices may progress to infections that manifest as subacute or chronic in nature. S. epidermidis bacteremia is predominantly caused by entry of the bacteria through colonized intravascular medical devices and removal of the device is recommended as an integral part of patient treatment [4] . The high prevalence (70–85%) of multi-resistance in nosocomial strains of S. epidermidis means that vancomycin infusion is one of a limited range of current therapeutic options. In view of the fact that vancomycin has limited biocidal properties, requiring therapeutic drug monitoring and may cause nephrotoxicity and/or ototoxicity in selected patient groups [5,6] it is prudent to confirm that culture recovery of S. epidermidis from patient specimens is indeed due to infection and not sample contamination. 1 School of Biomedical Sciences, Institute of Health & Biomedical Innovation, Faculty of Health, Queensland University of Technology, Brisbane, QLD, Australia 2 Microbiology Department, Pathology Queensland, Princess Alexandra Hospital, Woolloongabba, QLD, Australia *Author for correspondence: [email protected]
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Keywords
• bacteremia • biofilm • Staphylococcus epidermidis • virulence
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ISSN 1746-0913
Review Kleinschmidt, Huygens, Faoagali, Rathnayake & Hafner There is a preponderance of literature on neonatal sepsis especially with reference to CoNS bacteremia in preterm neonates. The most prevalent CoNS species causing bacteremia in neonates and other patient groups is S. epidermidis [7–9] . Neonates are colonized by S. epidermidis within a few days of birth and generally the commensal role of these bacteria limits the colonization of more virulent bacteria such as S. aureus. Unfortunately, neonates requiring intensive care may become colonized with strains previously exposed to the selective pressure of the neonatal intensive care unit (ICU) that is, multi-resistant strains of S. epidermidis. Intensive care is associated with the use of invasive devices for therapy in these patients. Such devices may include indwelling foreign body devices and colonization of these devices with CoNS may lead to invasive infections in these hosts. Preterm neonates have immature immune systems and their mucosal barriers are more permeable, permitting microbial access to body fluids [10] . Investigations have demonstrated that many preterm neonates are not only colonized with multi-resistant strains of S. epidermidis but will succumb to infections with the same colonizing strains [7,10–11] . Moreover, antimicrobial treatment of these infections does not always preclude continued colonization with these multi-resistant strains [10] . Bacteremia caused by CoNS is difficult to eradicate and is associated with increased hospitalization times, increased healthcare costs, morbidity and infrequent mortality [12] . Juthani-Mehta and colleagues audited 137 episodes of CoNS bacteremia in a USA cancer hospital and calculated the average cost per episode at US$7594 [12] . Identification S. epidermidis strains are facultative anaerobic, Gram-positive cocci belonging to the Micrococcaceae family. There are more than 20 species of coagulase-negative bacteria in the Staphylococcus genus and these may be phenotypically differentiated by using a battery of biochemical tests [13] that may available in a card or plate format and are mostly processed using an automated instrument-based platform. Specified dilutions of test isolates are used to inoculate identification cards and utilization of each chromogenic or fluorometric substrate is assessed using an automated optics system. In general, an incubation period of between 2h and 8 h is required for identification of CoNS using
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phenotypic automated platforms. The optical reading center of the instrument generates a biochemical profile and this is fed through a computer interface and compared with a database of reference isolates [14] . The most common automated platforms used by Australian clinical microbiology laboratories include the Vitek 2 systems (bioMérieux), Phoenix automated systems (BectonDickinson Diagnostic System) and the Microscan systems (Siemens Healthcare). A comparative analysis of the performance of the three previously mentioned rapid automated systems using 27 Gram-positive clinical isolates showed correct identification at 100%, 96% and 81.5% using the Vitek 2, Phoenix and Microscan systems, respectively [15] . Chatzigeorgiou and colleagues [16] analyzed 147 staphylococcal strains (clinical and reference strains) using the Phoenix, Vitek 2 and the tuf gene as the reference method. These researchers found the Vitek 2 system more accurate than the Phoenix system with correct species identification at 96.8% versus 87.4%, respectively. Specifically, 50 clinical strains of S. epidermidis were identified correctly, 100% by the Vitek 2 system and 92% by the Phoenix system. The chief factor limiting the accuracy of phenotypic identification of CoNS and other bacteria is that it is based upon variable expression of phenotypic properties. The quality of the database library is an important factor affecting the efficiency of genotypic and phenotypic methods. The extent of the number of species and replicates of each species together with timely software updates, incorporating new species is of paramount importance in all identification methods. In recent years, matrix-assisted laser desorption/ionization mass spectrometry (MALDITOF MS) systems have transformed identification methods used in clinical microbiology laboratories because this technology provides rapid, accurate and cost-efficient results [17] . Minute quantities of test organism with MALDI matrix are exposed to a laser beam, producing ions that are captured to generate a mass/charge spectrum [18] . The test spectra are compared with peptide databases from reference strains and test results are expressed as an absolute score or percentage match. Two of the most commonly employed MALDI-TOF MS instruments used in clinical microbiology laboratories are the Bruker MALDI-TOF MS systems (Bruker Daltonics) and the Shimadzu Accuspot/Vitek MS MALDI-TOF systems (bioMérieux) [19] .
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Staphylococcus epidermidis as a cause of bacteremia Jamal and colleagues [20] evaluated the identification of 70 clinical CoNS isolates using the Vitek MALDI-TOF MS system and the Bruker MALDI-TOF MS system in parallel with the phenotypic automated Vitek 2 system, resolving discrepant results using 16S rRNA gene or other specific gene amplification and sequencing. The conventional Vitek 2 system and the Vitek MS system misidentified one S. epidermidis strain, the 16S rRNA method misidentified two strains and the Bruker MS system identified all S. epidermidis strains correctly. Both MALDITOF MS methods required less than 20 min to identify colonies compared with more than 24 h using the phenotypic Vitek 2 system, demonstrating the clinical value of MALDI-TOF MS identification systems. The major limitation of this study was that only seven different CoNS species were represented among the 70 clinical strains. Dupont and colleagues [21] compared the identifications of 234 clinical CoNS using the Bruker MALDI-TOF MS (Autoflex, Bruker Diagnostics) with flex control software (Bruker Daltronics), the BD Phoenix automated system (Becton Dickinson Diagnostic Systems) and the Vitek 2 system (bioMérieux). Twenty different species were represented among the 234 clinical CoNS isolates. Using sodA gene sequencing as the reference method, the Bruker MALDITOF MS platform proved most accurate with 93.2% agreement with the reference method. Species identification using the BD Phoenix automated system showed 75.6% agreement with the reference method and the Vitek 2 system, 75.2%. With regard to S. epidermidis, the Bruker MALDI-TOF MS system provided 100% concordance, followed by 92.9% with the Vitek MALDI-TOF and 89.3% with the Phoenix instrument. The Vitek MALDI-TOF MS system (bioMérieux) was assessed for CoNS species identification by comparing results against a reference method using 16S rRNA sequencing and sodA and rpoB partial gene sequencing [22] . Two hundred and forty nine CoNS isolates representing eight different species yielded 96% concordance with the sequencing methods, with 98% agreement for S. epidermidis. The chief limiting factor of MALDI-TOF MS identification is that the method requires bacterial growth of colonies on solid agar plates prior to analysis. Recently, however, a number of researchers directly identified CoNS
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(predominantly S. epidermidis) from patient fluids using various approaches that included extra steps prior to processing in the MALDI-TOF instrument, for example, lysis, protein extraction, differential centrifugation and short incubation agar plates [23–26] . These investigations provided correct identification to species level ranging from 20% to 90% for CoNS depending on the MALDI-TOF MS preceding step. Short incubation cultures on solid media with a protein extraction step prior to MALDI-TOF MS analysis identified CoNS to the correct species in 15 of 35 CoNS cultures [23] . Moussaoui and colleagues used differential centrifugation and protein extraction prior to loading an extract in the Bruker MALDI-TOF MS [25] . In total, 152 CoNS isolates were processed and results yielded 136 correct identifications, with 97.5% of 118 representing correct identification for S. epidermidis. MALDI-TOF MS has also been used for strain differentiation of many bacterial species for epidemiologic purposes [27] . Dubois and colleagues [28] correctly identified 99.3% of 152 clinical and environmental isolates of staphylococci using the Ultraflex II MALDI-TOF MS system (Bruker). Score-oriented dendrogram analysis of S. epidermidis strains separated the clinical strains into two different clusters with one singleton strain and the environmental strains into a separate cluster with one singleton. The authors propose that MALDI-TOF MS analysis provides adequate discriminatory power beyond species level for epidemiological studies. Variation in the sequences of specific gene targets, including tuf, sodA, rpoB and gap genes has been exploited to identify CoNS to species level and beyond [29–32] . The tuf gene and 16S rRNA sequencing were used in two separate studies to analyze 47 and 96 clinical CoNS isolates, respectively [30,33] . Both research groups concluded that tuf gene amplification and sequencing was more discriminating than 16S rRNA sequencing. The increasing importance of CoNS as a cause of hospital-acquired infections means that correct identification to the species level is necessary for optimal patient and infection control management. The earlier identification methods in clinical microbiology laboratories relied upon phenotypic properties which have proven to be less accurate, costly and time consuming. The availability of PCR, next-generation sequencing and MALDI-TOF MS has transformed identification methods with reduced costs, rapid
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Review Kleinschmidt, Huygens, Faoagali, Rathnayake & Hafner turn-around-times and high specificity. The low cost, simplicity and rapid processing time required for MALDI-TOF identification has resulted in this method becoming the methodof-choice in most modern clinical microbiology laboratories. Further application development of MALDI-TOF MS methods promise to deliver direct specimen identification, strain discrimination and antimicrobial susceptibility information. Population structure Genotyping methods compare bacterial genetic material and are therefore the methods of choice for epidemiological studies [34] . Whole-genome analysis (WGA) will ultimately provide a discriminatory epidemiologic tool but presently the three main typing methods are pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and multiple-locus variable number of tandem DNA repeat (MLVA). PFGE genotyping methods use more than 90% of the bacterial chromosome for comparative analysis, therefore, it is highly discriminatory and is considered the gold standard for S. epidermidis [35,36] . Macrorestriction of bacterial DNA by restriction endonucleases and the separation of the resultant fragments by electrophoresis produces distinct PFGE patterns for unrelated strains [35] . However, there are limitations associated with PFGE: it is time consuming and laborious; inter-laboratory reproducibility is problematic; and interpretation of bands across a number of isolates is subjective [34] . MLVA genotyping is based upon the numbers of short tandem repeats in defined coding and non-coding genes within bacterial genomes. Strains are classified according to the number of repeats at defined loci [34] . This genotyping method was used in two studies: one investigating clonal lineage within 30 multi-drug resistant clinical strains of S. epidermidis and the other study characterizing a set of 65 clinical isolates with 21 previously characterized isolates. Both research groups concluded that MLVA was rapid, robust and provided similar discriminatory capacity to PFGE [37,38] . Limitations of this method include the lack of validated interpretative criteria, the absence of standard nomenclature and that the discriminatory power of MLVA has not yet been proven for S. epidermidis [34] . MLST methods for S. epidermidis genotyping are based on comparison of DNA sequences of internal fragments of seven highly conserved
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housekeeping genes. Variations in nucleotide sequences of each gene fragment are assigned unique allele numbers and a sequence type (ST) is assigned for each set of alleles for each of the seven gene fragments [39,40] . These data are stored on a central, curated database [41] allowing any user to compare test allelic profiles with reference sets and together with the eBURST software allows the user to make population structure inferences [34,36,42] . This focus on conserved housekeeping genes means that MLST typing is more suited for analyzing long-term evolutionary changes as well as across different geographical locations [36] . Several studies have revealed that the S. epidermidis global population appears epidemic exhibiting a minimum of nine clonal lineages found worldwide. Clonal Complex2 (CC2) accounts for almost 74% of global strains and ST2 accounts for 31% of global isolates (Figure 1) [42–45] . Figure 1 depicts a global population snapshot of S. epidermidis showing the primary founder, ST2 and several subgroup founders. Wisplinghoff and colleagues [45] investigated the clonal association of staphylococcal chromosomal cassette mec (SCCmec) types on 54 clinical strains of methicillin-resistant S. epidermidis (MRSE) using MLST and SCCmec typing. MLST data separated 52 of the 54 MRSE strains into three related clonal clusters. Three methicillinsensitive S. epidermidis (MSSE) strains isolated from bacteremia cases from the 1970s belonged to the same clonal cluster as the MRSE strains harboring type IV SCCmec. The investigators suggest that S. epidermidis provides a reservoir of resistance genes that may be transferred to S. aureus. [45] . Thomas and colleagues [47] interrogated the S. epidermidis MLST database for genetic clusters using a Bayesian cluster program. This analysis revealed six major genetic clusters (GCs) with GC2 having a high prevalence of commensal markers. Strains belonging to GC5 harbored multiple antibiotic resistance genes and virulence markers demonstrating adaptations consistent with a nosocomial lifestyle. Molecular typing of hospital-acquired S. epidermidis strains by the methods explained above have shown the considerable diversity within the S. epidermidis population. This was observed in studies involving isolates from diverse geographic locations, clinical origins and collections which originated from the same hospital and even in a single intensive care unit [42] . However, there
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Staphylococcus epidermidis as a cause of bacteremia
Review
Figure 1. Global population snapshot of Staphylococcus epidermidis showing clusters of linked and unlinked sequence types. The largest blue dot shows the primary founder, ST2 representing almost 31% of global isolates (March 2015, created from [46]). ST: Sequence type; Black dots: Other STs; Blue dots: Primary founders; Yellow dots: Subgroup founders.
are limitations with existing typing methods such as, they are time consuming, expensive and complex. Therefore there is a need to develop and apply new robust, rapid and cost-effective techniques that are likely to yield more definitive results in the routine monitoring and population structure analysis of S. epidermidis. Pathogenicity & biofilm formation Despite the absence of typical virulence factors, CoNS are able to colonize, evade the immune system, and cause disease [48] . Two well-known reference strains of S. epidermidis have been fully sequenced with the intent of revealing virulence factors, S. epidermidis ATCC 12228 [49] and S. epidermidis ATCC 35984 (also known as RP62A) [50] . Zhang and colleagues [49] revealed a ∼2.5 Mb genome with paucity of known staphylococcal toxin genes with the exception of two hemolysin genes, however, several adhesin genes were identified. Gill and colleagues [50] fully sequenced S. epidermidis ATCC 35984 revealing a ∼2.6 Mb genome and compared this genome to four S. aureus strains previously characterized. Both staphylococcal species share a core set of
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1681 open reading frames (ORFs), however the key virulence factors found in S. aureus (enterotoxins, exotoxins, leucotoxins and leucocidins) were not identified in S. epidermidis. A novel genome island was identified in the two reference strains and this was found to encode for phenol-soluble modulins (PSMs), manifesting as small cytokine-stimulating amphipathic peptides that are postulated to play important roles in the virulence of S. epidermidis strains. Cheung and co-authors [51] investigated the ability of S. epidermidis and S. aureus PSMs to lyse human erythrocytes, finding that PSMs are capable of cell lysis in both organisms and concluding that S. epidermidis PSM δ peptides play an important role in virulence in BSIs in particular. PSMs produced by S. epidermidis have also been investigated in a murine model by Wang and colleagues [52] . These researchers vaccinated test mice with β-PSM antibodies and implanted test and control mice with catheter sections impregnated with heavy suspensions of S. epidermidis. Results indicated that catheter infections in the vaccinated group were less severe and more localized than catheter infections in the control
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Review Kleinschmidt, Huygens, Faoagali, Rathnayake & Hafner group which had progressed to systemic infection. The authors propose the roles of β-PSMs in S. epidermidis are to promote biofilm production and facilitate detachment of staphylococcal cells from the biofilm which is necessary for the establishment of disseminated infections. ●●Biofilm formation
Biofilm formation and the consequent ability of bacterial strains to escape the host immune defense are regarded as the chief mechanisms of S. epidermidis disease [2,48,53] . A biofilm results from the interaction of single bacterial cells acting together using quorum sensing to produce a protective layer of polysaccharide, protein and nucleic acids to effectively inhibit host defence mechanisms and antibiotic therapy [54] . S. epidermidis infections require access through the epidermal barrier and this breach is facilitated by implanted intravascular devices in the host. The most important mechanisms for virulence are the adherence of S. epidermidis to indwelling medical devices and the accumulation of biofilm, facilitating evasion of host defence and antimicrobial treatment [55–57] . The discovery of the ica operon in S. epidermidis by Heilmann and colleagues was published in 1996 [58] . This paper demonstrated that the intercellular adhesin (icaABC) genes are responsible for the production of polysaccharide intercellular adhesin (PIA). Sequencing of the S. epidermidis icaABC gene cluster led to suggestions that three genes make up the operon and are co-transcribed from the icaA promoter. The authors inserted a transposon into the icaABC gene cluster and observed the loss of biofilm production, cell aggregation and PIA production. This mutant was able to revert to producing biofilm after transformation with the icaABCcarrying plasmid pCN27. Attachment to the polymer surface of biomedical devices is essential for the first stage of biofilm formation and is mediated by surface-associated and cell wall-anchored proteins together with electrostatic and hydrophobic interactions [48] . After implantation of medical devices, the surfaces are rapidly coated with a conditioning film consisting of host human serum proteins: fibronectin; fibrinogen; vitronectin; elastin and collagen. The affinity of staphylococcal adherence factors for these serum proteins facilitates binding. These adherence factors are termed ‘microbial surface components recognizing adhesive matrix molecules’ (MSCRAMMs) [2] .
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The MSCRAMM, Embp (extracellular matrixbinding protein) is another bifunctional protein, serving as an adhesin by binding fibronectin and mediating biofilm accumulation [59] . Christner and colleagues [59] used transposon mutagenesis and phage transduction to produce aap, ica and embp mutant strains of S. epidermidis and observed these gene mutation effects on biofilm production using microtiter plates and cell culture flasks. The investigators concluded that Embp is an important multi-functional protein with a central role in biofilm formation in both the adherence and accumulation phases and may act as a surrogate for icaADBC and aap-negative S. epidermidis strains. Confocal laser scanning microscopy of anti-staphylococcal antibody treated murine macrophages demonstrated that Embp-mediated biofilm protects S. epidermidis cells from phagocytosis. Bowden and colleagues [60] used labeled collagen and monoclonal antibody probes to demonstrate that the S. epidermidis extracellular lipase, GehD is bifunctional, acting as a cell surface associated collagen adhesin as well as a lipase. GehD antibodies provided to the rat model caused a significant reduction of bacterial attachment to collagen. SesC is another cell wall-anchored protein of S. epidermidis, attaching specifically to fibrinogen-coated surfaces. Shahrooei and colleagues [61] observed increased SesC levels of bacteria grown in biofilms compared with planktonic cultures. Furthermore, antibodies to SesC provided to a rat model with a catheter infection demonstrated a reduction in adherence of S. epidermidis to fibrinogen [61,62] . Another MSCRAMM, SdrF was demonstrated to bind host collagen and adhere directly to the hydrophobic polymeric outer coating on the drive lines of ventricular assist devices (VADs) [63] . Arrecubieta and colleagues [63] used anti-SdrF antibodies in a mouse model drive line infection to demonstrate marked reduction of S. epidermidis adherence to the VAD drivelines. The protease ClpP was observed to play an integral role in initial adherence and PIA/PNAG production in a study using an isogenic clpP mutant S. epidermidis strain in a rat model [64] . Wang and colleagues [64] observed a significant reduction in adherence of the clpP S. epidermidis mutant strain to the polystyrene surface and biofilm production in a catheter-implanted rat model.
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Staphylococcus epidermidis as a cause of bacteremia The S. epidermidis specific protease, SepA is postulated to degrade antimicrobial peptides (AMPs) and inhibit neutrophil killing [65] . Li and colleagues [66] discovered an antimicrobial peptide sensor system, aps in S. epidermidis that upregulates AMP-protective mechanism by increasing the positive charge of the bacterial cell surface thereby increasing repulsion against cationic AMPs. Surface proteins produced by S. epidermidis have been demonstrated to adhere to conditioned abiotic surfaces and these include the bifunctional adhesins/autolysins AtlE and Aae. An additional important effect of AtlE is its ability to hydrolyze peptidoglycan present in bacterial cells, thereby releasing extracellular DNA (eDNA) which promotes surface attachment to other cells and contributes to the biofilm aggregate [67,68] . Non-proteinaceous adhesins include PIA (also known as Poly-N-acetyl glucosamine [PNAG]) in addition to teichoic and lipoteichoic acids. PIA facilitates biofilm formation by attachment to the cell surface, surface colonization, phagocyte evasion and this PIAassociated virulence was verified using a murine model [69,70] . The fibrinogen-binding protein (Fbe) also called SdrG is one such molecule and in one investigation, deletion of the sdrG gene resulted in significant reduction of adherence to fibrinogen-coated polyethylene catheter surfaces [71] . The accumulation-associated protein (Aap) is an important bifunctional, cell wall anchored protein mediating both adherence and biofilm production and is extruded by the staphylococcal cell in fibrillar tufts [72] . Aap is prevalent in clinical strains of S. epidermidis and investigations have demonstrated that Aap is responsible for mediating biofilm production in a PIA-independent mode [72–74] . The second and third phases of biofilm formation are co-dependent and involve accumulation and maturation. The multi-functional PIA is a well-researched biofilm polymer and is encoded by the icaADBC operon of S. epidermidis [56] . The roles of both the icaA and icaD genes were demonstrated in biofilm producing CoNS by a number of researchers [75–78] . Regulation of the icaACBD operon in S. epidermidis has been thoroughly investigated and factors controlling its expression include SarA, SarZ, LuxS, ClpP, σB and the tricarboxylic acid cycle [64,79–83] . PIA is integral for the formation of cellular aggregates in most S. epidermidis biofilms. The positively charged PIA functions as a mechanical barrier
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against peptides and phagocytes, and by the electrostatic repulsion of predominantly cationic antibacterial peptides. Thus, by inhibiting major mechanisms of the human innate immune defence, PIA may significantly contribute to the success of S. epidermidis in chronic infections [48] . Interestingly clinically significant nonPIA producing S. epidermidis strains have been isolated and this suggests alternative mechanisms of biofilm production in these strains. Aap and Bap homolog protein (Bhp) are two surrogate proteins that play important roles in the biofilm accumulation stage [72,84–85] . Bhp was shown to be responsible for biofilm accumulation in PIA-negative strains when mutant S. epidermidis strains formed significantly reduced biofilm using a polystyrene microtiter plate method compared with wild strains of S. epidermidis [85] . Wang and colleagues [81] demonstrated the importance of SarZ as a regulator of biofilm formation and virulence in S. epidermidis by controlling the expression of virulence genes producing lipases and proteases, hemolysis and the human antimicrobial peptide, human beta defensin3 (hBD3). Abolishment in the production of SarZ corresponded with reduced virulence of S. epidermidis in the murine model [81] . Alteration of luxS, σB and the transcriptional regulator, icaR causes reduced transcription of icaADBC and resultant PIA synthesis [82,83] . Rohde and colleagues showed ica-negative strains of biofilm producing S. epidermidis were protease susceptible but resistant to polysaccharide degradation enzymes illustrating the role of surrogate accumulation proteins Embp, Aap and Bhp [73] . Quorum sensing is an essential means of cell-to-cell communication in biofilm accumulation and the effects of the signaling autoinducer, AI-2 was recently investigated by Xue and colleagues [86] . Biofilm assays using tissue culture plates were used to assess the effect of AI-2 upon biofilm formation on S. epidermidis RP62A cultures. A significant increase in transcript levels of bhp and ica was observed and it was determined that AI-2 functions at the intercellular adhesion stage, confirming that AI-2 plays an important role in quorum sensing dependent process of biofilm formation. Poly-γ-glutamic acid (PGA) is a virulence factor synthesized by S. epidermidis, inhibiting phagocytosis and the production of AMPs as well as protecting the cell against the high salt concentration environment found in human skin [87,88] . Kocianova and colleagues [88]
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Review Kleinschmidt, Huygens, Faoagali, Rathnayake & Hafner demonstrated that the secretion of PGA by S. epidermidis contributes to the protection, survival and persistence in device-associated infections in a murine model. Protection is required against increased salt concentrations and osmotic pressure normally found on the skin and the capsule inhibits phagocytosis by neutrophils as well as repelling antibacterial peptides found in human epithelial cells [88] . The accumulation and maturation of biofilm enhances the physical barrier protecting bacterial cells from the host defence system and antimicrobials [89,90] . The final stage of biofilm production is dispersal, essential for causing invasive disease because viable bacteria must detach from biomedical devices to cause bacteremia. S. epidermidis produces α-type PSMs (PSMα, PSMδ, PSMɛ, PSM-mec and PSMγ [also known as δ-toxins]) and β-type PSMs that are responsible for initiating detachment and are postulated to play an important role in invasive diseases caused by this bacterium [91,92] . The synthesis of PSMs is controlled by the agr system and S. epidermidis agr mutants demonstrated a reduction in dispersal and subsequent infection in the rabbit model [93] . PSMs act as surfactants, effectively reducing bonding between individual cells making up a biofilm [2] . This reduction in cell-to-cell bonding facilitates the production of channels permitting transport of nutrients and waste products [2,92] . The breakdown of biofilm produced by the protein biofilm factors, Bhp, Aap and EmBp is mediated by proteases whereas the breakdown of PIA-induced biofilm is mediated by sugar hydrolases [2] . Quorum sensing is a form of communication among bacteria resulting from changes in concentration of autoinducing peptide signals [4] . Activation of the staphylococcal agr-quorum sensing system may increase the dispersal of planktonic bacteria from biofilms by upregulating extracellular proteases. The various stages of biofilm formation leading to catheter-related bloodstream infections are shown in Figure 2 . Fey and Olson [2] have suggested that all mechanisms of biofilm production have not yet been fully elucidated because of the identification of a number of clinically significant S. epidermidis strains that do not harbor the recognized genes associated with biofilms, icaADBC, aap, embp and bhp. Wang and colleagues [94] discovered the novel gene, ygs in S. epidermidis. The researchers challenged both a mutant ygs strain and a wild strain
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of S. epidermidis to produce infection in mouse and rat models. The mutant S. epidermidis strain was unable to establish a catheter site infection under environmental pressure in contrast to the wild strain of S. epidermidis. The authors postulate that ygs plays an integral role in biofilm production under extreme pH and osmolarity pressures. The various genes that are associated with the ability of S. epidermidis strains to produce and regulate biofilms have been identified and discussed. Given the central role that biofilm production plays in the ability of S. epidermidis to cause disease it would appear that the presence of such markers could determine the virulence of certain strains. This association will be analyzed in several studies in the following sections. ●●Antimicrobial resistance
Antimicrobial resistance plays an important role in the success of S. epidermidis as a nosocomial pathogen and it is recognized that resistance in CoNS is associated with transposons and exposure to the selective pressure of hospital environments. Mack and colleagues [95] used transposon mutagenesis to demonstrate that the expression of methicillin resistance in S. epidermidis is associated with PIA-mediated biofilm production. Koksal and colleagues [96] analyzed 200 CoNS clinical isolates and demonstrated methicillin resistance in 81% of biofilm producing isolates compared with 57% methicillin resistance in biofilm negative isolates. Pinheiro and colleagues [97] analyzed 107 strains of S. epidermidis from bacteremia cases finding 81.3% were resistant to methicillin and out of those, 75.9% demonstrated increases in the minimum inhibitory concentration to vancomycin. These findings suggest an association between biofilm production, decreased susceptibility to vancomycin and methicillin resistance. More recently, a Japanese investigation reported that complete resistance to 1000 μg/ mL vancomycin in a biofilm-forming strain of S. epidermidis (RP62A) was expressed 4–8 h after adhesion of the bacterium to a metal surface [98] . Linezolid is an important antimicrobial agent that may be used to treat staphylococcal infections that are recalcitrant to vancomycin therapy [99,100] . Unfortunately a number of bacteremia cases caused by linezolid-resistant CoNS have recently been reported worldwide [101–105] . Russo and colleagues [103] compared the clinical features of 38 BSIs caused by linezolid-resistant
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1a. Direct attachment to device: • Electrostatic/hydrophobic interactions • ClpP, Bhp, wall teichoic acids
Epidermis
1b. Attachment to device via conditioning film: • MSCRAMMs – SdrG/Fbe, SdrF, SesC, Aap • AtlE, Aae, Embp, GehD
Commensal CoNS strains may be replaced by nosocomial strains
Blood vessel
3. Detachment and dispersal: • Microemboli • Extracellular enzymes: glycosyl hydrolases, proteases, nucleases • PSMs
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Conditional film: • Fibrinogen • Fibronectin • Vitronectin • Collagen • Elastin
2. Biofilm accumulation and maturation: Intercellular adherence via • Polysaccharides: PIA, PNAG • Proteins: Aap, Bhp, PSMs, eDNA, AtIE
Figure 2. The stages of biofilm formation leading to catheter-related bloodstream infections. Blue dots represent staphylococcal bacteria. Red layer on internal section of intravascular line depicts conditioning film.
strains of CoNS against 40 patients having linezolid-susceptible CoNS BSIs and 90 patients without infections. The authors found that linezolid-resistant CoNS causing bacteremia were strongly associated with poor clinical outcomes including increased 30-day mortality rates. Additionally, independent risk factors for bacteremia with these resistant staphylococci included previous linezolid therapy, antibiotic therapy in the previous 30 days, antibiotic therapy for more than 14 days and hospitalization in the preceding 90 days. BSIs caused by S. epidermidis are more prevalent in patients with long-term indwelling or implanted medical devices. This group of patients often require care in high dependency clinical units where commensal and environmental bacteria are exposed to the selective pressures of a high-use antimicrobial environment. The prevalence of antimicrobial resistance genes in S. epidermidis strains isolated from bacteremia cases would be expected to be higher than in patients without previous hospital admissions. Unfortunately, antimicrobial multi-resistance does not help distinguish invasive from contaminating strains of S. epidermidis because the
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contaminating commensal or environmental strains are just as likely to harbor antimicrobial resistance genes. It appears that those factors that allow S. epidermidis to remain a successful commensal, the ability to evade the host immune defense system and the ability to produce biofilms are the factors that have enabled it to transform into a successful opportunistic pathogen in accessible hosts [2] . Difficulties in the diagnosis of true CoNS bacteremia There are conflicting published studies on the criteria for separating true and contaminant bacteremias with CoNS (106 , 107 ) and these will be discussed in detail below. Bloodstream infections are major causes of morbidity and mortality in hospitalized patients worldwide [108] . The diagnosis of sepsis is made clinically, with or without the detection of a positive blood culture. Loonen and colleagues [109] consider that culture-based methods are the ‘gold standard’ because they allow identification as well as antimicrobial susceptibility testing that is specific for the organism isolated.
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Review Kleinschmidt, Huygens, Faoagali, Rathnayake & Hafner Studies have shown that up to 40% of organisms isolated from blood cultures may represent contamination [110–112] . CoNS make up the majority of these contaminants; with S. epidermidis the most frequently implicated species in both contaminated blood cultures and in true cases of bacteremia [113–117] . Previously we have emphasized that S. epidermidis is a normal component of the commensal flora of human skin but isolation of S. epidermidis from blood cultures is problematic. Growth from blood cultures may indicate a false-positive bacteremia resulting from skin contamination of the culture or more importantly the organism may represent a clinically significant bloodstream infection. Patients with intravenous devices or previous open operations and insertion of prosthetic medical devices provide not only bloodstream access for this organism but also provide a surface conducive to its attachment and subsequent biofilm formation following organism access. These devices may include prosthetic heart valves, stents and orthopedic prostheses [118] . The virulence section of this review described the production of various factors by S. epidermidis facilitating adherence to materials in medical devices, production of biofilm and the dispersal of globules of biofilm parts or planktonic bacteria from this biofilm into the bloodstream [2,56,78,115,119] . Therefore, isolation of S. epidermidis from the blood cultures of patients with indwelling medical devices presents a quandary for the treating clinician and the testing medical laboratory. There have been a number of indicators and algorithms postulated as criteria to assist with the clinical diagnosis of sepsis. The true diagnosis of sepsis is based on evidence of infection and the systemic inflammatory response syndrome (SIRS) [106] . The guidelines developed by Bone et al., [106] classify sepsis on a clinical basis, in other words, sepsis, severe sepsis, septic shock and this is dependent upon the proinflammatory phase of sepsis which does not take into account the immune status of the patient [120] . The clinical path of S. epidermidis blood stream infections is more indolent than those infections caused by typical BSI pathogens. Specific definitions for diagnosis of catheter-related primary bloodstream infection (CR-BSI) have been developed by the US Centers for Disease Control and Prevention (CDC) [107] . In summary, CDC criterion 1 states the patient has a recognized pathogen cultured from one or more blood cultures and the pathogen cultured from
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the bloodstream is not related to an infection at another site. This first criterion excludes the following skin contaminants: CoNS; diphtheroids; Bacillus species; Propionibacterium species; and micrococci. Criterion 2 defines a CR-BSI: the patient has one or more symptoms (fever, chills or hypotension) and symptoms and laboratory results are not related to infection at another site and a common skin contaminant has been cultured from two or more blood samples obtained on separate occasions [121] . Tokars [107] from the CDC developed a mathematical model to assist in clinical decisionmaking with regard to blood cultures positive for CoNS, taking into account both the number of blood cultures positive, the total number of collections and the method of collection, in other peripheral blood or a vascular line. Enlisting 540 hospitals, Tokars revealed a true bacteremia prevalence of between 27 and 38% and a contamination rate of between 0.9 and 5.1%. The positive predictive value (PPV) of a true bacteremia with positive CoNS cultures from two of two vascular line collections was 50% whereas the PPV for one vascular line collection and one peripheral blood collection was 96%. The PPV increased to 98% for two positive peripheral blood collections. Interestingly, the PPV for one positive vascular line collection from only one collection decreased to 24%. The results using this model emphasize the importance of collection of not only more than one blood culture sample but that peripheral blood culture collection is more valuable than line cultures in the diagnosis of CR-BSI. Beekmann and colleagues [122] analyzed the clinical symptoms of 405 patients with positive blood cultures with a CoNS and found 89 patients (22%) had true BSIs compared with 316 contaminant cases. The authors noted that patients with BSI were significantly more likely to be neutropenic and exhibit clinical signs of sepsis syndrome than contaminant cases. The best algorithm for BSI prediction provided a sensitivity of 62% and specificity of 91% and the criteria for BSI included two or more positive blood cultures within 5 days together with one or more clinical signs of infection. A 3-year retrospective study was performed by Favre and colleagues to evaluate the clinical significance of a single blood culture growing a CoNS [123] . Four hundred and eleven of these cases were reviewed with 234 cases exhibiting signs of sepsis. It was found that the associated
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Staphylococcus epidermidis as a cause of bacteremia mortality was similar whether the CoNS was isolated from one blood culture or more than two blood cultures. The authors concluded that CoNS bacteremia is associated with significant mortality and a single positive blood culture from a patient with clinical signs of sepsis should be considered relevant. Al Wohoush and colleagues [124] compared the CDC clinical criteria with species identification to determine the accuracy of the diagnosis of CoNS bacteremia. The CDC clinical criteria requires the detection of CoNS from two or more blood cultures within 48 h and the patient’s symptoms must include fever, chills or hypotension. A total of 101 retrospective cases and the associated CoNS strains were analyzed. A true bacteremia was defined as fitting the clinical criteria and two or more blood cultures yielding identical species and strains. All CoNS were identified to species level and clinical data collected and compared with the assigned reference standard of genotype identification (using DiversiLab repetitive-PCR genotyping system, Spectral Genomics, TX, USA). The PPV of the CDC criteria alone was 74% with a sensitivity of 91% and a specificity of only 11%. The sensitivity and PPV for species identification was 100 and 84% with a higher specificity of 48%. This is perhaps not surprising because species identification is intrinsically linked to genotype, the defined reference standard. The reduced specificity of the CDC criteria was attributed to the different genotypes isolated from the same patient case. By combining the CDC criteria with species identification, the specificity of the diagnosis of CoNS bacteremia increased to 52% with a PPV of 84% and sensitivity of 91%. Genotyping and comparison of CoNS strains provides the optimum diagnostic tool, however the cost, time and labor requirements make this method impracticable for hospital laboratories. The researchers [124] concluded that the CDC criteria alone is only acceptable as a screening tool, however, in combination with species identification provides a more sensitive and specific tool for diagnosis of CoNS bacteremia. Simple clinical findings were assessed for their ability to distinguish true BSIs from contaminant cases in 471 patients with CoNS isolated from their blood cultures [125] . Abnormal heart rate, body temperature and systolic blood pressure did not significantly correlate with true BSIs. Conversely, Elzi and colleagues [126] examined 654 patients with CoNS cultured from
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Review
blood cultures and observed significant correlation between clinical indicators of SIRS and true BSI. The most significant clinical criteria assessed were fever or hypothermia, tachycardia, tachypnea, leukocytosis or leukopenia and each additional SIRS criterion increased the probability of true BSI. The most likely combination of factors predictive of BSI was three SIRS criteria alone or two SIRS criteria with a central line in-situ at blood culture collection. Clearly, there is no simple clinical algorithm for distinguishing true BSI from contaminated blood collections when CoNS are grown from patient blood cultures. Compounding the dilemma of treating or not treating the patient is the inherent resistance of most S. epidermidis strains to antibiotics, often resulting in vancomycin as the only perceived useful treatment [99] . Previous studies have shown that up to 30% of patients were unnecessarily commenced on vancomycin due to contaminated blood cultures [125] . There is disagreement among studies attempting to find reliable predictive clinical symptoms and this may result from investigating different hospital populations or from a complex plethora of host factors. Putative virulence markers used to predict true bacteremia caused by S. epidermidis Review of the relevant literature reveals a number of putative markers that have been postulated to contribute to the virulence of S. epidermidis strains and have been employed in research studies to discriminate true bacteriemia from contaminated blood cultures. Table 1 provides an overview of studies that have linked putative virulence factors with pathogenicity. Both phenotypic and genotypic methods, singly or together have been used in many CoNS bacteremia studies. A further approach to identifying true bacteremia is the analysis of blood culture data. These approaches are reviewed in separate subsections. ●●Blood culture bottle data
In a Brisbane hospital study, an investigation into the link between time to positive (TTP) of blood culture bottle incubation and clinical parameters assessing organ function was performed retrospectively on 569 positive CoNS blood culture cases [136] . The authors found that TTP 20 h were associated with low bacterial counts of
Staphylococcus epidermidis as a cause of bacteremia Sharon Kleinschmidt*,1,2, Flavia Huygens1, Joan Faoagali1, Irani U Rathnayake1 & Louise M Hafner1
Staphylococcus epidermidis is a biofilm-producing commensal organism found ubiquitously on human skin and mucous membranes, as well as on animals and in the environment. Biofilm formation enables this organism to evade the host immune system. Colonization of percutaneous devices or implanted medical devices allows bacteria access to the bloodstream. Isolation of this organism from blood cultures may represent either contamination during the blood collection procedure or true bacteremia. Staphylococcus epidermidis bloodstream infections may be indolent compared with other bacteria. Isolation of S. epidermidis from a blood culture may present a management quandary for clinicians. Over-treatment may lead to patient harm and increases in healthcare costs. There are numerous reports indicating the difficulty of predicting clinical infection in patients with positive blood cultures with this organism. No reliable phenotypic or genotypic algorithms currently exist to predict the pathogenicity of a S. epidermidis bloodstream infection. This review will discuss the latest advances in identification methods, global population structure, pathogenicity, biofilm formation, antimicrobial resistance and clinical significance of the detection of S. epidermidis in blood cultures. Previous studies that have attempted to discriminate between invasive and contaminating strains of S. epidermidis in blood cultures will be analyzed. Staphylococcus epidermidis is one of the most prevalent species of bacteria found universally on the human skin and mucous membranes, and is generally regarded as a commensal organism [1] . They belong to the coagulase-negative group of staphylococci (CoNS) and due to their paucity of virulence factors are less invasive than their coagulase-positive relation, Staphylococcus aureus. However, in recent years S. epidermidis has become a frequent and important nosocomial pathogen, particularly in immunocompromised patients [2,3] . Many strains of S. epidermidis are able to produce biofilms and readily colonize implanted medical devices, in particular intravascular devices, cerebrospinal fluid shunts, intraocular lenses, prosthetic joints and heart valve replacements. Colonization of such medical devices may progress to infections that manifest as subacute or chronic in nature. S. epidermidis bacteremia is predominantly caused by entry of the bacteria through colonized intravascular medical devices and removal of the device is recommended as an integral part of patient treatment [4] . The high prevalence (70–85%) of multi-resistance in nosocomial strains of S. epidermidis means that vancomycin infusion is one of a limited range of current therapeutic options. In view of the fact that vancomycin has limited biocidal properties, requiring therapeutic drug monitoring and may cause nephrotoxicity and/or ototoxicity in selected patient groups [5,6] it is prudent to confirm that culture recovery of S. epidermidis from patient specimens is indeed due to infection and not sample contamination. 1 School of Biomedical Sciences, Institute of Health & Biomedical Innovation, Faculty of Health, Queensland University of Technology, Brisbane, QLD, Australia 2 Microbiology Department, Pathology Queensland, Princess Alexandra Hospital, Woolloongabba, QLD, Australia *Author for correspondence: [email protected]
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Keywords
• bacteremia • biofilm • Staphylococcus epidermidis • virulence
part of
ISSN 1746-0913
Review Kleinschmidt, Huygens, Faoagali, Rathnayake & Hafner There is a preponderance of literature on neonatal sepsis especially with reference to CoNS bacteremia in preterm neonates. The most prevalent CoNS species causing bacteremia in neonates and other patient groups is S. epidermidis [7–9] . Neonates are colonized by S. epidermidis within a few days of birth and generally the commensal role of these bacteria limits the colonization of more virulent bacteria such as S. aureus. Unfortunately, neonates requiring intensive care may become colonized with strains previously exposed to the selective pressure of the neonatal intensive care unit (ICU) that is, multi-resistant strains of S. epidermidis. Intensive care is associated with the use of invasive devices for therapy in these patients. Such devices may include indwelling foreign body devices and colonization of these devices with CoNS may lead to invasive infections in these hosts. Preterm neonates have immature immune systems and their mucosal barriers are more permeable, permitting microbial access to body fluids [10] . Investigations have demonstrated that many preterm neonates are not only colonized with multi-resistant strains of S. epidermidis but will succumb to infections with the same colonizing strains [7,10–11] . Moreover, antimicrobial treatment of these infections does not always preclude continued colonization with these multi-resistant strains [10] . Bacteremia caused by CoNS is difficult to eradicate and is associated with increased hospitalization times, increased healthcare costs, morbidity and infrequent mortality [12] . Juthani-Mehta and colleagues audited 137 episodes of CoNS bacteremia in a USA cancer hospital and calculated the average cost per episode at US$7594 [12] . Identification S. epidermidis strains are facultative anaerobic, Gram-positive cocci belonging to the Micrococcaceae family. There are more than 20 species of coagulase-negative bacteria in the Staphylococcus genus and these may be phenotypically differentiated by using a battery of biochemical tests [13] that may available in a card or plate format and are mostly processed using an automated instrument-based platform. Specified dilutions of test isolates are used to inoculate identification cards and utilization of each chromogenic or fluorometric substrate is assessed using an automated optics system. In general, an incubation period of between 2h and 8 h is required for identification of CoNS using
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phenotypic automated platforms. The optical reading center of the instrument generates a biochemical profile and this is fed through a computer interface and compared with a database of reference isolates [14] . The most common automated platforms used by Australian clinical microbiology laboratories include the Vitek 2 systems (bioMérieux), Phoenix automated systems (BectonDickinson Diagnostic System) and the Microscan systems (Siemens Healthcare). A comparative analysis of the performance of the three previously mentioned rapid automated systems using 27 Gram-positive clinical isolates showed correct identification at 100%, 96% and 81.5% using the Vitek 2, Phoenix and Microscan systems, respectively [15] . Chatzigeorgiou and colleagues [16] analyzed 147 staphylococcal strains (clinical and reference strains) using the Phoenix, Vitek 2 and the tuf gene as the reference method. These researchers found the Vitek 2 system more accurate than the Phoenix system with correct species identification at 96.8% versus 87.4%, respectively. Specifically, 50 clinical strains of S. epidermidis were identified correctly, 100% by the Vitek 2 system and 92% by the Phoenix system. The chief factor limiting the accuracy of phenotypic identification of CoNS and other bacteria is that it is based upon variable expression of phenotypic properties. The quality of the database library is an important factor affecting the efficiency of genotypic and phenotypic methods. The extent of the number of species and replicates of each species together with timely software updates, incorporating new species is of paramount importance in all identification methods. In recent years, matrix-assisted laser desorption/ionization mass spectrometry (MALDITOF MS) systems have transformed identification methods used in clinical microbiology laboratories because this technology provides rapid, accurate and cost-efficient results [17] . Minute quantities of test organism with MALDI matrix are exposed to a laser beam, producing ions that are captured to generate a mass/charge spectrum [18] . The test spectra are compared with peptide databases from reference strains and test results are expressed as an absolute score or percentage match. Two of the most commonly employed MALDI-TOF MS instruments used in clinical microbiology laboratories are the Bruker MALDI-TOF MS systems (Bruker Daltonics) and the Shimadzu Accuspot/Vitek MS MALDI-TOF systems (bioMérieux) [19] .
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Staphylococcus epidermidis as a cause of bacteremia Jamal and colleagues [20] evaluated the identification of 70 clinical CoNS isolates using the Vitek MALDI-TOF MS system and the Bruker MALDI-TOF MS system in parallel with the phenotypic automated Vitek 2 system, resolving discrepant results using 16S rRNA gene or other specific gene amplification and sequencing. The conventional Vitek 2 system and the Vitek MS system misidentified one S. epidermidis strain, the 16S rRNA method misidentified two strains and the Bruker MS system identified all S. epidermidis strains correctly. Both MALDITOF MS methods required less than 20 min to identify colonies compared with more than 24 h using the phenotypic Vitek 2 system, demonstrating the clinical value of MALDI-TOF MS identification systems. The major limitation of this study was that only seven different CoNS species were represented among the 70 clinical strains. Dupont and colleagues [21] compared the identifications of 234 clinical CoNS using the Bruker MALDI-TOF MS (Autoflex, Bruker Diagnostics) with flex control software (Bruker Daltronics), the BD Phoenix automated system (Becton Dickinson Diagnostic Systems) and the Vitek 2 system (bioMérieux). Twenty different species were represented among the 234 clinical CoNS isolates. Using sodA gene sequencing as the reference method, the Bruker MALDITOF MS platform proved most accurate with 93.2% agreement with the reference method. Species identification using the BD Phoenix automated system showed 75.6% agreement with the reference method and the Vitek 2 system, 75.2%. With regard to S. epidermidis, the Bruker MALDI-TOF MS system provided 100% concordance, followed by 92.9% with the Vitek MALDI-TOF and 89.3% with the Phoenix instrument. The Vitek MALDI-TOF MS system (bioMérieux) was assessed for CoNS species identification by comparing results against a reference method using 16S rRNA sequencing and sodA and rpoB partial gene sequencing [22] . Two hundred and forty nine CoNS isolates representing eight different species yielded 96% concordance with the sequencing methods, with 98% agreement for S. epidermidis. The chief limiting factor of MALDI-TOF MS identification is that the method requires bacterial growth of colonies on solid agar plates prior to analysis. Recently, however, a number of researchers directly identified CoNS
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(predominantly S. epidermidis) from patient fluids using various approaches that included extra steps prior to processing in the MALDI-TOF instrument, for example, lysis, protein extraction, differential centrifugation and short incubation agar plates [23–26] . These investigations provided correct identification to species level ranging from 20% to 90% for CoNS depending on the MALDI-TOF MS preceding step. Short incubation cultures on solid media with a protein extraction step prior to MALDI-TOF MS analysis identified CoNS to the correct species in 15 of 35 CoNS cultures [23] . Moussaoui and colleagues used differential centrifugation and protein extraction prior to loading an extract in the Bruker MALDI-TOF MS [25] . In total, 152 CoNS isolates were processed and results yielded 136 correct identifications, with 97.5% of 118 representing correct identification for S. epidermidis. MALDI-TOF MS has also been used for strain differentiation of many bacterial species for epidemiologic purposes [27] . Dubois and colleagues [28] correctly identified 99.3% of 152 clinical and environmental isolates of staphylococci using the Ultraflex II MALDI-TOF MS system (Bruker). Score-oriented dendrogram analysis of S. epidermidis strains separated the clinical strains into two different clusters with one singleton strain and the environmental strains into a separate cluster with one singleton. The authors propose that MALDI-TOF MS analysis provides adequate discriminatory power beyond species level for epidemiological studies. Variation in the sequences of specific gene targets, including tuf, sodA, rpoB and gap genes has been exploited to identify CoNS to species level and beyond [29–32] . The tuf gene and 16S rRNA sequencing were used in two separate studies to analyze 47 and 96 clinical CoNS isolates, respectively [30,33] . Both research groups concluded that tuf gene amplification and sequencing was more discriminating than 16S rRNA sequencing. The increasing importance of CoNS as a cause of hospital-acquired infections means that correct identification to the species level is necessary for optimal patient and infection control management. The earlier identification methods in clinical microbiology laboratories relied upon phenotypic properties which have proven to be less accurate, costly and time consuming. The availability of PCR, next-generation sequencing and MALDI-TOF MS has transformed identification methods with reduced costs, rapid
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Review Kleinschmidt, Huygens, Faoagali, Rathnayake & Hafner turn-around-times and high specificity. The low cost, simplicity and rapid processing time required for MALDI-TOF identification has resulted in this method becoming the methodof-choice in most modern clinical microbiology laboratories. Further application development of MALDI-TOF MS methods promise to deliver direct specimen identification, strain discrimination and antimicrobial susceptibility information. Population structure Genotyping methods compare bacterial genetic material and are therefore the methods of choice for epidemiological studies [34] . Whole-genome analysis (WGA) will ultimately provide a discriminatory epidemiologic tool but presently the three main typing methods are pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and multiple-locus variable number of tandem DNA repeat (MLVA). PFGE genotyping methods use more than 90% of the bacterial chromosome for comparative analysis, therefore, it is highly discriminatory and is considered the gold standard for S. epidermidis [35,36] . Macrorestriction of bacterial DNA by restriction endonucleases and the separation of the resultant fragments by electrophoresis produces distinct PFGE patterns for unrelated strains [35] . However, there are limitations associated with PFGE: it is time consuming and laborious; inter-laboratory reproducibility is problematic; and interpretation of bands across a number of isolates is subjective [34] . MLVA genotyping is based upon the numbers of short tandem repeats in defined coding and non-coding genes within bacterial genomes. Strains are classified according to the number of repeats at defined loci [34] . This genotyping method was used in two studies: one investigating clonal lineage within 30 multi-drug resistant clinical strains of S. epidermidis and the other study characterizing a set of 65 clinical isolates with 21 previously characterized isolates. Both research groups concluded that MLVA was rapid, robust and provided similar discriminatory capacity to PFGE [37,38] . Limitations of this method include the lack of validated interpretative criteria, the absence of standard nomenclature and that the discriminatory power of MLVA has not yet been proven for S. epidermidis [34] . MLST methods for S. epidermidis genotyping are based on comparison of DNA sequences of internal fragments of seven highly conserved
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housekeeping genes. Variations in nucleotide sequences of each gene fragment are assigned unique allele numbers and a sequence type (ST) is assigned for each set of alleles for each of the seven gene fragments [39,40] . These data are stored on a central, curated database [41] allowing any user to compare test allelic profiles with reference sets and together with the eBURST software allows the user to make population structure inferences [34,36,42] . This focus on conserved housekeeping genes means that MLST typing is more suited for analyzing long-term evolutionary changes as well as across different geographical locations [36] . Several studies have revealed that the S. epidermidis global population appears epidemic exhibiting a minimum of nine clonal lineages found worldwide. Clonal Complex2 (CC2) accounts for almost 74% of global strains and ST2 accounts for 31% of global isolates (Figure 1) [42–45] . Figure 1 depicts a global population snapshot of S. epidermidis showing the primary founder, ST2 and several subgroup founders. Wisplinghoff and colleagues [45] investigated the clonal association of staphylococcal chromosomal cassette mec (SCCmec) types on 54 clinical strains of methicillin-resistant S. epidermidis (MRSE) using MLST and SCCmec typing. MLST data separated 52 of the 54 MRSE strains into three related clonal clusters. Three methicillinsensitive S. epidermidis (MSSE) strains isolated from bacteremia cases from the 1970s belonged to the same clonal cluster as the MRSE strains harboring type IV SCCmec. The investigators suggest that S. epidermidis provides a reservoir of resistance genes that may be transferred to S. aureus. [45] . Thomas and colleagues [47] interrogated the S. epidermidis MLST database for genetic clusters using a Bayesian cluster program. This analysis revealed six major genetic clusters (GCs) with GC2 having a high prevalence of commensal markers. Strains belonging to GC5 harbored multiple antibiotic resistance genes and virulence markers demonstrating adaptations consistent with a nosocomial lifestyle. Molecular typing of hospital-acquired S. epidermidis strains by the methods explained above have shown the considerable diversity within the S. epidermidis population. This was observed in studies involving isolates from diverse geographic locations, clinical origins and collections which originated from the same hospital and even in a single intensive care unit [42] . However, there
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Review
Figure 1. Global population snapshot of Staphylococcus epidermidis showing clusters of linked and unlinked sequence types. The largest blue dot shows the primary founder, ST2 representing almost 31% of global isolates (March 2015, created from [46]). ST: Sequence type; Black dots: Other STs; Blue dots: Primary founders; Yellow dots: Subgroup founders.
are limitations with existing typing methods such as, they are time consuming, expensive and complex. Therefore there is a need to develop and apply new robust, rapid and cost-effective techniques that are likely to yield more definitive results in the routine monitoring and population structure analysis of S. epidermidis. Pathogenicity & biofilm formation Despite the absence of typical virulence factors, CoNS are able to colonize, evade the immune system, and cause disease [48] . Two well-known reference strains of S. epidermidis have been fully sequenced with the intent of revealing virulence factors, S. epidermidis ATCC 12228 [49] and S. epidermidis ATCC 35984 (also known as RP62A) [50] . Zhang and colleagues [49] revealed a ∼2.5 Mb genome with paucity of known staphylococcal toxin genes with the exception of two hemolysin genes, however, several adhesin genes were identified. Gill and colleagues [50] fully sequenced S. epidermidis ATCC 35984 revealing a ∼2.6 Mb genome and compared this genome to four S. aureus strains previously characterized. Both staphylococcal species share a core set of
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1681 open reading frames (ORFs), however the key virulence factors found in S. aureus (enterotoxins, exotoxins, leucotoxins and leucocidins) were not identified in S. epidermidis. A novel genome island was identified in the two reference strains and this was found to encode for phenol-soluble modulins (PSMs), manifesting as small cytokine-stimulating amphipathic peptides that are postulated to play important roles in the virulence of S. epidermidis strains. Cheung and co-authors [51] investigated the ability of S. epidermidis and S. aureus PSMs to lyse human erythrocytes, finding that PSMs are capable of cell lysis in both organisms and concluding that S. epidermidis PSM δ peptides play an important role in virulence in BSIs in particular. PSMs produced by S. epidermidis have also been investigated in a murine model by Wang and colleagues [52] . These researchers vaccinated test mice with β-PSM antibodies and implanted test and control mice with catheter sections impregnated with heavy suspensions of S. epidermidis. Results indicated that catheter infections in the vaccinated group were less severe and more localized than catheter infections in the control
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Review Kleinschmidt, Huygens, Faoagali, Rathnayake & Hafner group which had progressed to systemic infection. The authors propose the roles of β-PSMs in S. epidermidis are to promote biofilm production and facilitate detachment of staphylococcal cells from the biofilm which is necessary for the establishment of disseminated infections. ●●Biofilm formation
Biofilm formation and the consequent ability of bacterial strains to escape the host immune defense are regarded as the chief mechanisms of S. epidermidis disease [2,48,53] . A biofilm results from the interaction of single bacterial cells acting together using quorum sensing to produce a protective layer of polysaccharide, protein and nucleic acids to effectively inhibit host defence mechanisms and antibiotic therapy [54] . S. epidermidis infections require access through the epidermal barrier and this breach is facilitated by implanted intravascular devices in the host. The most important mechanisms for virulence are the adherence of S. epidermidis to indwelling medical devices and the accumulation of biofilm, facilitating evasion of host defence and antimicrobial treatment [55–57] . The discovery of the ica operon in S. epidermidis by Heilmann and colleagues was published in 1996 [58] . This paper demonstrated that the intercellular adhesin (icaABC) genes are responsible for the production of polysaccharide intercellular adhesin (PIA). Sequencing of the S. epidermidis icaABC gene cluster led to suggestions that three genes make up the operon and are co-transcribed from the icaA promoter. The authors inserted a transposon into the icaABC gene cluster and observed the loss of biofilm production, cell aggregation and PIA production. This mutant was able to revert to producing biofilm after transformation with the icaABCcarrying plasmid pCN27. Attachment to the polymer surface of biomedical devices is essential for the first stage of biofilm formation and is mediated by surface-associated and cell wall-anchored proteins together with electrostatic and hydrophobic interactions [48] . After implantation of medical devices, the surfaces are rapidly coated with a conditioning film consisting of host human serum proteins: fibronectin; fibrinogen; vitronectin; elastin and collagen. The affinity of staphylococcal adherence factors for these serum proteins facilitates binding. These adherence factors are termed ‘microbial surface components recognizing adhesive matrix molecules’ (MSCRAMMs) [2] .
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The MSCRAMM, Embp (extracellular matrixbinding protein) is another bifunctional protein, serving as an adhesin by binding fibronectin and mediating biofilm accumulation [59] . Christner and colleagues [59] used transposon mutagenesis and phage transduction to produce aap, ica and embp mutant strains of S. epidermidis and observed these gene mutation effects on biofilm production using microtiter plates and cell culture flasks. The investigators concluded that Embp is an important multi-functional protein with a central role in biofilm formation in both the adherence and accumulation phases and may act as a surrogate for icaADBC and aap-negative S. epidermidis strains. Confocal laser scanning microscopy of anti-staphylococcal antibody treated murine macrophages demonstrated that Embp-mediated biofilm protects S. epidermidis cells from phagocytosis. Bowden and colleagues [60] used labeled collagen and monoclonal antibody probes to demonstrate that the S. epidermidis extracellular lipase, GehD is bifunctional, acting as a cell surface associated collagen adhesin as well as a lipase. GehD antibodies provided to the rat model caused a significant reduction of bacterial attachment to collagen. SesC is another cell wall-anchored protein of S. epidermidis, attaching specifically to fibrinogen-coated surfaces. Shahrooei and colleagues [61] observed increased SesC levels of bacteria grown in biofilms compared with planktonic cultures. Furthermore, antibodies to SesC provided to a rat model with a catheter infection demonstrated a reduction in adherence of S. epidermidis to fibrinogen [61,62] . Another MSCRAMM, SdrF was demonstrated to bind host collagen and adhere directly to the hydrophobic polymeric outer coating on the drive lines of ventricular assist devices (VADs) [63] . Arrecubieta and colleagues [63] used anti-SdrF antibodies in a mouse model drive line infection to demonstrate marked reduction of S. epidermidis adherence to the VAD drivelines. The protease ClpP was observed to play an integral role in initial adherence and PIA/PNAG production in a study using an isogenic clpP mutant S. epidermidis strain in a rat model [64] . Wang and colleagues [64] observed a significant reduction in adherence of the clpP S. epidermidis mutant strain to the polystyrene surface and biofilm production in a catheter-implanted rat model.
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Staphylococcus epidermidis as a cause of bacteremia The S. epidermidis specific protease, SepA is postulated to degrade antimicrobial peptides (AMPs) and inhibit neutrophil killing [65] . Li and colleagues [66] discovered an antimicrobial peptide sensor system, aps in S. epidermidis that upregulates AMP-protective mechanism by increasing the positive charge of the bacterial cell surface thereby increasing repulsion against cationic AMPs. Surface proteins produced by S. epidermidis have been demonstrated to adhere to conditioned abiotic surfaces and these include the bifunctional adhesins/autolysins AtlE and Aae. An additional important effect of AtlE is its ability to hydrolyze peptidoglycan present in bacterial cells, thereby releasing extracellular DNA (eDNA) which promotes surface attachment to other cells and contributes to the biofilm aggregate [67,68] . Non-proteinaceous adhesins include PIA (also known as Poly-N-acetyl glucosamine [PNAG]) in addition to teichoic and lipoteichoic acids. PIA facilitates biofilm formation by attachment to the cell surface, surface colonization, phagocyte evasion and this PIAassociated virulence was verified using a murine model [69,70] . The fibrinogen-binding protein (Fbe) also called SdrG is one such molecule and in one investigation, deletion of the sdrG gene resulted in significant reduction of adherence to fibrinogen-coated polyethylene catheter surfaces [71] . The accumulation-associated protein (Aap) is an important bifunctional, cell wall anchored protein mediating both adherence and biofilm production and is extruded by the staphylococcal cell in fibrillar tufts [72] . Aap is prevalent in clinical strains of S. epidermidis and investigations have demonstrated that Aap is responsible for mediating biofilm production in a PIA-independent mode [72–74] . The second and third phases of biofilm formation are co-dependent and involve accumulation and maturation. The multi-functional PIA is a well-researched biofilm polymer and is encoded by the icaADBC operon of S. epidermidis [56] . The roles of both the icaA and icaD genes were demonstrated in biofilm producing CoNS by a number of researchers [75–78] . Regulation of the icaACBD operon in S. epidermidis has been thoroughly investigated and factors controlling its expression include SarA, SarZ, LuxS, ClpP, σB and the tricarboxylic acid cycle [64,79–83] . PIA is integral for the formation of cellular aggregates in most S. epidermidis biofilms. The positively charged PIA functions as a mechanical barrier
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against peptides and phagocytes, and by the electrostatic repulsion of predominantly cationic antibacterial peptides. Thus, by inhibiting major mechanisms of the human innate immune defence, PIA may significantly contribute to the success of S. epidermidis in chronic infections [48] . Interestingly clinically significant nonPIA producing S. epidermidis strains have been isolated and this suggests alternative mechanisms of biofilm production in these strains. Aap and Bap homolog protein (Bhp) are two surrogate proteins that play important roles in the biofilm accumulation stage [72,84–85] . Bhp was shown to be responsible for biofilm accumulation in PIA-negative strains when mutant S. epidermidis strains formed significantly reduced biofilm using a polystyrene microtiter plate method compared with wild strains of S. epidermidis [85] . Wang and colleagues [81] demonstrated the importance of SarZ as a regulator of biofilm formation and virulence in S. epidermidis by controlling the expression of virulence genes producing lipases and proteases, hemolysis and the human antimicrobial peptide, human beta defensin3 (hBD3). Abolishment in the production of SarZ corresponded with reduced virulence of S. epidermidis in the murine model [81] . Alteration of luxS, σB and the transcriptional regulator, icaR causes reduced transcription of icaADBC and resultant PIA synthesis [82,83] . Rohde and colleagues showed ica-negative strains of biofilm producing S. epidermidis were protease susceptible but resistant to polysaccharide degradation enzymes illustrating the role of surrogate accumulation proteins Embp, Aap and Bhp [73] . Quorum sensing is an essential means of cell-to-cell communication in biofilm accumulation and the effects of the signaling autoinducer, AI-2 was recently investigated by Xue and colleagues [86] . Biofilm assays using tissue culture plates were used to assess the effect of AI-2 upon biofilm formation on S. epidermidis RP62A cultures. A significant increase in transcript levels of bhp and ica was observed and it was determined that AI-2 functions at the intercellular adhesion stage, confirming that AI-2 plays an important role in quorum sensing dependent process of biofilm formation. Poly-γ-glutamic acid (PGA) is a virulence factor synthesized by S. epidermidis, inhibiting phagocytosis and the production of AMPs as well as protecting the cell against the high salt concentration environment found in human skin [87,88] . Kocianova and colleagues [88]
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Review Kleinschmidt, Huygens, Faoagali, Rathnayake & Hafner demonstrated that the secretion of PGA by S. epidermidis contributes to the protection, survival and persistence in device-associated infections in a murine model. Protection is required against increased salt concentrations and osmotic pressure normally found on the skin and the capsule inhibits phagocytosis by neutrophils as well as repelling antibacterial peptides found in human epithelial cells [88] . The accumulation and maturation of biofilm enhances the physical barrier protecting bacterial cells from the host defence system and antimicrobials [89,90] . The final stage of biofilm production is dispersal, essential for causing invasive disease because viable bacteria must detach from biomedical devices to cause bacteremia. S. epidermidis produces α-type PSMs (PSMα, PSMδ, PSMɛ, PSM-mec and PSMγ [also known as δ-toxins]) and β-type PSMs that are responsible for initiating detachment and are postulated to play an important role in invasive diseases caused by this bacterium [91,92] . The synthesis of PSMs is controlled by the agr system and S. epidermidis agr mutants demonstrated a reduction in dispersal and subsequent infection in the rabbit model [93] . PSMs act as surfactants, effectively reducing bonding between individual cells making up a biofilm [2] . This reduction in cell-to-cell bonding facilitates the production of channels permitting transport of nutrients and waste products [2,92] . The breakdown of biofilm produced by the protein biofilm factors, Bhp, Aap and EmBp is mediated by proteases whereas the breakdown of PIA-induced biofilm is mediated by sugar hydrolases [2] . Quorum sensing is a form of communication among bacteria resulting from changes in concentration of autoinducing peptide signals [4] . Activation of the staphylococcal agr-quorum sensing system may increase the dispersal of planktonic bacteria from biofilms by upregulating extracellular proteases. The various stages of biofilm formation leading to catheter-related bloodstream infections are shown in Figure 2 . Fey and Olson [2] have suggested that all mechanisms of biofilm production have not yet been fully elucidated because of the identification of a number of clinically significant S. epidermidis strains that do not harbor the recognized genes associated with biofilms, icaADBC, aap, embp and bhp. Wang and colleagues [94] discovered the novel gene, ygs in S. epidermidis. The researchers challenged both a mutant ygs strain and a wild strain
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of S. epidermidis to produce infection in mouse and rat models. The mutant S. epidermidis strain was unable to establish a catheter site infection under environmental pressure in contrast to the wild strain of S. epidermidis. The authors postulate that ygs plays an integral role in biofilm production under extreme pH and osmolarity pressures. The various genes that are associated with the ability of S. epidermidis strains to produce and regulate biofilms have been identified and discussed. Given the central role that biofilm production plays in the ability of S. epidermidis to cause disease it would appear that the presence of such markers could determine the virulence of certain strains. This association will be analyzed in several studies in the following sections. ●●Antimicrobial resistance
Antimicrobial resistance plays an important role in the success of S. epidermidis as a nosocomial pathogen and it is recognized that resistance in CoNS is associated with transposons and exposure to the selective pressure of hospital environments. Mack and colleagues [95] used transposon mutagenesis to demonstrate that the expression of methicillin resistance in S. epidermidis is associated with PIA-mediated biofilm production. Koksal and colleagues [96] analyzed 200 CoNS clinical isolates and demonstrated methicillin resistance in 81% of biofilm producing isolates compared with 57% methicillin resistance in biofilm negative isolates. Pinheiro and colleagues [97] analyzed 107 strains of S. epidermidis from bacteremia cases finding 81.3% were resistant to methicillin and out of those, 75.9% demonstrated increases in the minimum inhibitory concentration to vancomycin. These findings suggest an association between biofilm production, decreased susceptibility to vancomycin and methicillin resistance. More recently, a Japanese investigation reported that complete resistance to 1000 μg/ mL vancomycin in a biofilm-forming strain of S. epidermidis (RP62A) was expressed 4–8 h after adhesion of the bacterium to a metal surface [98] . Linezolid is an important antimicrobial agent that may be used to treat staphylococcal infections that are recalcitrant to vancomycin therapy [99,100] . Unfortunately a number of bacteremia cases caused by linezolid-resistant CoNS have recently been reported worldwide [101–105] . Russo and colleagues [103] compared the clinical features of 38 BSIs caused by linezolid-resistant
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Staphylococcus epidermidis as a cause of bacteremia
1a. Direct attachment to device: • Electrostatic/hydrophobic interactions • ClpP, Bhp, wall teichoic acids
Epidermis
1b. Attachment to device via conditioning film: • MSCRAMMs – SdrG/Fbe, SdrF, SesC, Aap • AtlE, Aae, Embp, GehD
Commensal CoNS strains may be replaced by nosocomial strains
Blood vessel
3. Detachment and dispersal: • Microemboli • Extracellular enzymes: glycosyl hydrolases, proteases, nucleases • PSMs
Review
Conditional film: • Fibrinogen • Fibronectin • Vitronectin • Collagen • Elastin
2. Biofilm accumulation and maturation: Intercellular adherence via • Polysaccharides: PIA, PNAG • Proteins: Aap, Bhp, PSMs, eDNA, AtIE
Figure 2. The stages of biofilm formation leading to catheter-related bloodstream infections. Blue dots represent staphylococcal bacteria. Red layer on internal section of intravascular line depicts conditioning film.
strains of CoNS against 40 patients having linezolid-susceptible CoNS BSIs and 90 patients without infections. The authors found that linezolid-resistant CoNS causing bacteremia were strongly associated with poor clinical outcomes including increased 30-day mortality rates. Additionally, independent risk factors for bacteremia with these resistant staphylococci included previous linezolid therapy, antibiotic therapy in the previous 30 days, antibiotic therapy for more than 14 days and hospitalization in the preceding 90 days. BSIs caused by S. epidermidis are more prevalent in patients with long-term indwelling or implanted medical devices. This group of patients often require care in high dependency clinical units where commensal and environmental bacteria are exposed to the selective pressures of a high-use antimicrobial environment. The prevalence of antimicrobial resistance genes in S. epidermidis strains isolated from bacteremia cases would be expected to be higher than in patients without previous hospital admissions. Unfortunately, antimicrobial multi-resistance does not help distinguish invasive from contaminating strains of S. epidermidis because the
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contaminating commensal or environmental strains are just as likely to harbor antimicrobial resistance genes. It appears that those factors that allow S. epidermidis to remain a successful commensal, the ability to evade the host immune defense system and the ability to produce biofilms are the factors that have enabled it to transform into a successful opportunistic pathogen in accessible hosts [2] . Difficulties in the diagnosis of true CoNS bacteremia There are conflicting published studies on the criteria for separating true and contaminant bacteremias with CoNS (106 , 107 ) and these will be discussed in detail below. Bloodstream infections are major causes of morbidity and mortality in hospitalized patients worldwide [108] . The diagnosis of sepsis is made clinically, with or without the detection of a positive blood culture. Loonen and colleagues [109] consider that culture-based methods are the ‘gold standard’ because they allow identification as well as antimicrobial susceptibility testing that is specific for the organism isolated.
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Review Kleinschmidt, Huygens, Faoagali, Rathnayake & Hafner Studies have shown that up to 40% of organisms isolated from blood cultures may represent contamination [110–112] . CoNS make up the majority of these contaminants; with S. epidermidis the most frequently implicated species in both contaminated blood cultures and in true cases of bacteremia [113–117] . Previously we have emphasized that S. epidermidis is a normal component of the commensal flora of human skin but isolation of S. epidermidis from blood cultures is problematic. Growth from blood cultures may indicate a false-positive bacteremia resulting from skin contamination of the culture or more importantly the organism may represent a clinically significant bloodstream infection. Patients with intravenous devices or previous open operations and insertion of prosthetic medical devices provide not only bloodstream access for this organism but also provide a surface conducive to its attachment and subsequent biofilm formation following organism access. These devices may include prosthetic heart valves, stents and orthopedic prostheses [118] . The virulence section of this review described the production of various factors by S. epidermidis facilitating adherence to materials in medical devices, production of biofilm and the dispersal of globules of biofilm parts or planktonic bacteria from this biofilm into the bloodstream [2,56,78,115,119] . Therefore, isolation of S. epidermidis from the blood cultures of patients with indwelling medical devices presents a quandary for the treating clinician and the testing medical laboratory. There have been a number of indicators and algorithms postulated as criteria to assist with the clinical diagnosis of sepsis. The true diagnosis of sepsis is based on evidence of infection and the systemic inflammatory response syndrome (SIRS) [106] . The guidelines developed by Bone et al., [106] classify sepsis on a clinical basis, in other words, sepsis, severe sepsis, septic shock and this is dependent upon the proinflammatory phase of sepsis which does not take into account the immune status of the patient [120] . The clinical path of S. epidermidis blood stream infections is more indolent than those infections caused by typical BSI pathogens. Specific definitions for diagnosis of catheter-related primary bloodstream infection (CR-BSI) have been developed by the US Centers for Disease Control and Prevention (CDC) [107] . In summary, CDC criterion 1 states the patient has a recognized pathogen cultured from one or more blood cultures and the pathogen cultured from
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the bloodstream is not related to an infection at another site. This first criterion excludes the following skin contaminants: CoNS; diphtheroids; Bacillus species; Propionibacterium species; and micrococci. Criterion 2 defines a CR-BSI: the patient has one or more symptoms (fever, chills or hypotension) and symptoms and laboratory results are not related to infection at another site and a common skin contaminant has been cultured from two or more blood samples obtained on separate occasions [121] . Tokars [107] from the CDC developed a mathematical model to assist in clinical decisionmaking with regard to blood cultures positive for CoNS, taking into account both the number of blood cultures positive, the total number of collections and the method of collection, in other peripheral blood or a vascular line. Enlisting 540 hospitals, Tokars revealed a true bacteremia prevalence of between 27 and 38% and a contamination rate of between 0.9 and 5.1%. The positive predictive value (PPV) of a true bacteremia with positive CoNS cultures from two of two vascular line collections was 50% whereas the PPV for one vascular line collection and one peripheral blood collection was 96%. The PPV increased to 98% for two positive peripheral blood collections. Interestingly, the PPV for one positive vascular line collection from only one collection decreased to 24%. The results using this model emphasize the importance of collection of not only more than one blood culture sample but that peripheral blood culture collection is more valuable than line cultures in the diagnosis of CR-BSI. Beekmann and colleagues [122] analyzed the clinical symptoms of 405 patients with positive blood cultures with a CoNS and found 89 patients (22%) had true BSIs compared with 316 contaminant cases. The authors noted that patients with BSI were significantly more likely to be neutropenic and exhibit clinical signs of sepsis syndrome than contaminant cases. The best algorithm for BSI prediction provided a sensitivity of 62% and specificity of 91% and the criteria for BSI included two or more positive blood cultures within 5 days together with one or more clinical signs of infection. A 3-year retrospective study was performed by Favre and colleagues to evaluate the clinical significance of a single blood culture growing a CoNS [123] . Four hundred and eleven of these cases were reviewed with 234 cases exhibiting signs of sepsis. It was found that the associated
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Staphylococcus epidermidis as a cause of bacteremia mortality was similar whether the CoNS was isolated from one blood culture or more than two blood cultures. The authors concluded that CoNS bacteremia is associated with significant mortality and a single positive blood culture from a patient with clinical signs of sepsis should be considered relevant. Al Wohoush and colleagues [124] compared the CDC clinical criteria with species identification to determine the accuracy of the diagnosis of CoNS bacteremia. The CDC clinical criteria requires the detection of CoNS from two or more blood cultures within 48 h and the patient’s symptoms must include fever, chills or hypotension. A total of 101 retrospective cases and the associated CoNS strains were analyzed. A true bacteremia was defined as fitting the clinical criteria and two or more blood cultures yielding identical species and strains. All CoNS were identified to species level and clinical data collected and compared with the assigned reference standard of genotype identification (using DiversiLab repetitive-PCR genotyping system, Spectral Genomics, TX, USA). The PPV of the CDC criteria alone was 74% with a sensitivity of 91% and a specificity of only 11%. The sensitivity and PPV for species identification was 100 and 84% with a higher specificity of 48%. This is perhaps not surprising because species identification is intrinsically linked to genotype, the defined reference standard. The reduced specificity of the CDC criteria was attributed to the different genotypes isolated from the same patient case. By combining the CDC criteria with species identification, the specificity of the diagnosis of CoNS bacteremia increased to 52% with a PPV of 84% and sensitivity of 91%. Genotyping and comparison of CoNS strains provides the optimum diagnostic tool, however the cost, time and labor requirements make this method impracticable for hospital laboratories. The researchers [124] concluded that the CDC criteria alone is only acceptable as a screening tool, however, in combination with species identification provides a more sensitive and specific tool for diagnosis of CoNS bacteremia. Simple clinical findings were assessed for their ability to distinguish true BSIs from contaminant cases in 471 patients with CoNS isolated from their blood cultures [125] . Abnormal heart rate, body temperature and systolic blood pressure did not significantly correlate with true BSIs. Conversely, Elzi and colleagues [126] examined 654 patients with CoNS cultured from
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blood cultures and observed significant correlation between clinical indicators of SIRS and true BSI. The most significant clinical criteria assessed were fever or hypothermia, tachycardia, tachypnea, leukocytosis or leukopenia and each additional SIRS criterion increased the probability of true BSI. The most likely combination of factors predictive of BSI was three SIRS criteria alone or two SIRS criteria with a central line in-situ at blood culture collection. Clearly, there is no simple clinical algorithm for distinguishing true BSI from contaminated blood collections when CoNS are grown from patient blood cultures. Compounding the dilemma of treating or not treating the patient is the inherent resistance of most S. epidermidis strains to antibiotics, often resulting in vancomycin as the only perceived useful treatment [99] . Previous studies have shown that up to 30% of patients were unnecessarily commenced on vancomycin due to contaminated blood cultures [125] . There is disagreement among studies attempting to find reliable predictive clinical symptoms and this may result from investigating different hospital populations or from a complex plethora of host factors. Putative virulence markers used to predict true bacteremia caused by S. epidermidis Review of the relevant literature reveals a number of putative markers that have been postulated to contribute to the virulence of S. epidermidis strains and have been employed in research studies to discriminate true bacteriemia from contaminated blood cultures. Table 1 provides an overview of studies that have linked putative virulence factors with pathogenicity. Both phenotypic and genotypic methods, singly or together have been used in many CoNS bacteremia studies. A further approach to identifying true bacteremia is the analysis of blood culture data. These approaches are reviewed in separate subsections. ●●Blood culture bottle data
In a Brisbane hospital study, an investigation into the link between time to positive (TTP) of blood culture bottle incubation and clinical parameters assessing organ function was performed retrospectively on 569 positive CoNS blood culture cases [136] . The authors found that TTP 20 h were associated with low bacterial counts of
