Staphylococcus epidermidis uses distinct mechanisms of biofilm formation to interfere with phagocytosis and activation of mouse macrophage-like cells 774A.1

Assembly of adherent biofilms is the key mechanism involved in Staphylococcus epidermidis virulence during device-associated infections. Aside from polysaccharide intercellular adhesin (PIA), the accumulation-associated protein Aap and the extracellular matrix binding protein Embp act as intercellular adhesins, mediating S. epidermidis cell aggregation and biofilm accumulation. The aim of this study was to investigate structural features of PIA-, Aap-, and Embp-mediated S. epidermidis biofilms in more detail and to evaluate their specific contributions to biofilm-related S. epidermidis immune escape. PIA-, Embp-, and Aap-mediated biofilms exhibited substantial morphological differences. Basically, PIA synthesis induced formation of macroscopically visible, rough cell clusters, whereas Aap- and Embp-dependent biofilms preferentially displayed a smooth layer of aggregated bacteria. On the microscopic level, PIA was found to form a string-like organized extracellular matrix connecting the bacteria, while Embp produced small deposits of intercellular matrix and Aap was strictly localized to the bacterial surface. Despite marked differences, S. epidermidis strains using PIA, Aap, or Embp for biofilm formation were protected from uptake by J774A.1 macrophages, with similarly efficiencies. In addition, compared to biofilm-negative S. epidermidis strains, isogenic biofilm-forming S. epidermidis induced only a diminished inflammatory J774A.1 macrophage response, leading to significantly (88.2 to 88.7%) reduced NF-κB activation and 68.8 to 83% reduced interleukin-1β (IL-1β) production. Mechanical biofilm dispersal partially restored induction of NF-κB activation, although bacterial cell surfaces remained decorated with the respective intercellular adhesins. Our results demonstrate that distinct S. epidermidis biofilm morphotypes are similarly effective at protecting S. epidermidis from phagocytic uptake and at counteracting macrophage activation, providing novel insights into mechanisms that could contribute to the chronic and persistent course of biofilm-related S. epidermidis foreign material infections.     

Detection of Staphylococcus aureus and Staphylococcus epidermidis in Clinical Samples by 16S rRNA-Directed In Situ Hybridization

Staphylococcus epidermidis and Staphylococcus aureus are the most common causes of medical device-associated infections, including septicemic loosenings of orthopedic implants. Frequently, the microbiological diagnosis of these infections remains ambiguous, since at least some staphylococci have the capacity to reduce their growth rate considerably. These strains exhibit a small-colony phenotype, and often they are not detectable by conventional microbiological techniques. Moreover, clinical isolates of S. aureus and S. epidermidis adhere to polymer and metal surfaces by the generation of thick, multilayered biofilms consisting of bacteria and extracellular polysaccharides. This study reports improved detection and identification of S. aureus and S. epidermidis by an in situ hybridization method with fluorescence-labeled oligonucleotide probes specific for staphylococcal 16S rRNA. The technique has proven to be suitable for the in situ detection of staphylococci, which is illustrated by the identification of S. epidermidis in a connective tissue sample obtained from a patient with septicemic loosening of a hip arthroplasty. We also show that this technique allows the detection of intracellularly persisting bacteria, including small-colony variants of S. aureus, and the differentiation of S. epidermidis from other clinically relevant staphylococci even when they are embedded in biofilms. These results suggest that the 16S rRNA in situ hybridization technique could represent a powerful diagnostic tool for the detection and differentiation of many other fastidious microorganisms.     

Mechanisms of biofilm formation in Staphylococcus epidermidis and Staphylococcus aureus: functional molecules, regulatory circuits, and adaptive responses

Biomaterial-associated infections, most frequently caused by Staphylococcus epidermidis and Staphylococcus aureus, are of increasing importance in modern medicine. Regularly, antimicrobial therapy fails without removal of the implanted device. The most important factor in the pathogenesis of biomaterial-associated staphylococcal infections is the formation of adherent, multilayered bacterial biofilms. In this review, recent insights regarding factors functional in biofilm formation of S. epidermidis, their role in pathogenesis, and regulation of their expression are presented. Similarly, in S. aureus the biofilm mode of growth affects gene expression and the overall metabolic status. Experimental approaches for analysis of differential expression of genes involved in these adaptive responses and evolving patterns of gene expression are discussed.     

Extracellular carbohydrate-containing polymers of a model biofilm-producing strain, Staphylococcus epidermidis RP62A

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. It has been established that clinical isolates often produce a biofilm, which is involved in adherence to biomaterials and provides enhanced resistance of bacteria against host defenses and antibiotic treatments. It has been thought that the staphylococcal biofilm contains two polysaccharides, one responsible for primary cell adherence to biomaterials (polysaccharide/adhesin [PS/A]) and an antigen that mediates bacterial aggregation (polysaccharide intercellular adhesin [PIA]). In the present paper we present an improved procedure for preparation of PIA that conserves its labile substituents and avoids contamination with by-products. Based on structural analysis of the polysaccharide antigens and a thorough overview of the previously published data, we concluded that PIA from S. epidermidis is structurally identical to the recently described poly-beta-(1-->6)-N-acetylglucosamine from PS/A-overproducing strain S. aureus MN8m. We also show that another carbohydrate-containing polymer, extracellular teichoic acid (EC TA), is an essential component of S. epidermidis RP62A biofilms. We demonstrate that the relative amounts of extracellular PIA and EC TA produced depend on the growth conditions. Moderate shaking or static culture in tryptic soy broth favors PIA production, while more EC TA is produced in brain heart infusion medium.     

Isolation and characterization of biofilm formation-defective mutants of Staphylococcus aureus

Staphylococcus aureus produces biofilm and this mode of colonization facilitates infections that are often difficult to treat and engender high morbidity and mortality. We have exploited bacteriophage Mu transposition methods to create an insertional mutant library in a highly biofilm-forming S. aureus clinical isolate. Our screen identified 38 insertions in 23 distinct genes together with one intergenic region that significantly reduced biofilm formation. Nineteen insertions were mapped in loci not previously known to affect biofilm in this organism. These include insertions in codY, srrA, mgrA, and fmtA, a putative DEAD-box helicase, two members of the zinc-metallo-beta lactamase/beta-CASP family, and a hypothetical protein with a GGDEF motif. Fifteen insertions occurred in the icaADBC operon, which produces intercellular adhesion antigen (PIA) and is important for biofilm formation in many strains of S. aureus and Staphylococcus epidermidis. Obtaining a high proportion of independent Em-Mu disruptions in icaADBC demonstrated both the importance of PIA for biofilm formation in this clinical strain and the strong validation of the screening procedure that concomitantly uncovered additional mutants. All non-ica mutants were further analyzed by immunoblotting and biochemical fractionation for perturbation of PIA and wall teichoic acid. PIA levels were diminished in the majority of non-ica insertional mutants. Three mutant strains were chosen and were functionally complemented for restored biofilm formation by transformation with plasmids carrying the cloned wild-type gene under the control of a xylose-inducible promoter. This is a comprehensive collection of biofilm-defective mutants that underscores the multifactorial genetic program underlying the establishment of biofilm in this insidious pathogen.     

Ica-expression and gentamicin susceptibility of Staphylococcus epidermidis biofilm on orthopedic implant biomaterials

Ica-expression by Staphylococcus epidermidis and slime production depends on environmental conditions such as implant material and presence of antibiotics. Here, we evaluate biofilm formation and ica-expression of S. epidermidis strains on biomaterials involved in total hip- and knee arthroplasty [polyethylene (PE), polymethylmethacrylate (PMMA), stainless steel (SS)]. Ica-expression, assayed using real-time RT-PCR, was highest on PE as confirmed using confocal laser scanning microscopy. Yet biofilm formation by S. epidermidis was most extensive on SS, with less slime production. Ica-expression and slime production were minimal on PMMA. After 3 h of continued growth of 24 h old biofilms in the presence of gentamicin, biofilms on PE showed lower susceptibility to gentamicin, relative to the other materials, presumably as a result of the stronger ica-expression. A higher gentamicin concentration further decreased metabolic activity on all biomaterials. It is concluded that the level of biomaterial-induced ica-expression does not correlate with the amount of biofilm formed, but initially aids bacteria in surviving antibiotic attacks. Once antibiotic treatment has started however, also the antibiotic itself induces slime production and only if its concentration is high enough, killing results. Results suggest that biomaterial-associated infections in orthopedics by S. epidermidis on PE may be more difficult to eradicate than on PMMA or SS.     

The Intercellular Adhesion (ica) Locus Is Present in Staphylococcus aureus and Is Required for Biofilm Formation

Nosocomial infections that result in the formation of biofilms on the surfaces of biomedical implants are a leading cause of sepsis and are often associated with colonization of the implants by Staphylococcus epidermidis. Biofilm formation is thought to require two sequential steps: adhesion of cells to a solid substrate followed by cell-cell adhesion, creating multiple layers of cells. Intercellular adhesion requires the polysaccharide intercellular adhesin (PIA), which is composed of linear beta-1,6-linked glucosaminylglycans and can be synthesized in vitro from UDP-N-acetylglucosamine by products of the intercellular adhesion (ica) locus. We have investigated a variety of Staphylococcus aureus strains and find that all strains tested contain the ica locus and that several can form biofilms in vitro. Sequence comparison with the S. epidermidis ica genes revealed 59 to 78% amino acid identity. Deletion of the ica locus results in a loss of the ability to form biofilms, produce PIA, or mediate N-acetylglucosaminyltransferase activity in vitro. Cross-species hybridization experiments revealed the presence of icaA in several other Staphylococcus species, suggesting that cell-cell adhesion and the potential to form biofilms is conserved within this genus.     

Disruption of Staphylococcus epidermidis biofilm formation using a targeted cationic peptide

This study reports the use of a targeted cationic peptide with the ability to disrupt Staphylococcus epidermidis biofilm formation. Complications due to nosocomial infections of implanted medical devices pose a significant health risk to patients, with Staphylococcus epidermidis often implicated in the case of blood-contacting biomaterials. S. epidermidis virulence relies mainly on its ability to form a biofilm, the main component of which is polysaccharide intercellular adhesin (PIA). We utilized the synthetic β6-20 peptide, known to specifically bind S. epidermidis, in order to deliver a cationic polylysine peptide (G(3)K(6)) to the bacterial surface and disrupt the charge-charge interactions needed for PIA retention and biofilm stability. The effects of the β6-20-G(3)K(6) peptide on biofilm formation were assessed using optical density, fluorescently labeled wheat germ agglutinin, nucleic acid stain (SYTO 9), and a metabolic assay (XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt). Biofilms formed in the presence of β6-20-G(3)K(6) peptide (100 μM) resulted in a 37.9% reduction in PIA content and a 17.5% reduction of adherent bacteria relative to biofilms grown in the absence of peptide. These studies demonstrate the targeting ability of the β6-20 peptide towards biomaterial-adherent S. epidermidis, and highlight the potential for disrupting the early stages of biofilm formation.     

Poly-N-acetylglucosamine is not a major component of the extracellular matrix in biofilms formed by icaADBC-positive Staphylococcus lugdunensis isolates

Staphylococcus lugdunensis is a pathogen of heightened virulence that causes infections resembling those caused by Staphylococcus aureus rather than those caused by its coagulase-negative staphylococcal counterparts. Many types of S. lugdunensis infection, including native valve endocarditis, prosthetic joint infection, and intravascular catheter-related infection, are associated with biofilm etiology. Poly-N-acetylglucosamine (PNAG), a polysaccharide synthesized by products of the icaADBC locus, is a common mechanism of intercellular adhesion in staphylococcal biofilms. Here we report the characterization of ica homologues and the in vitro biofilm formation properties of a collection of S. lugdunensis clinical isolates. Isolates formed biofilms in microtiter wells to various degrees. Biofilm formation by most isolates was enhanced with glucose but diminished by sodium chloride or ethanol. icaADBC homologues were found in all S. lugdunensis isolates tested, although the locus organization differed substantially from that of other staphylococcal ica loci. icaR was not detected in S. lugdunensis, but a novel open reading frame with putative glycosyl hydrolase function is located upstream of the ica locus. icaADBC sequence heterogeneity did not explain the variability in biofilm formation among isolates. PNAG was not detected in S. lugdunensis extracts by immunoblotting with an anti-deacetylated PNAG antibody or wheat germ agglutinin. Confocal microscopy with fluorescently labeled wheat germ agglutinin showed a paucity of PNAG in S. lugdunensis biofilms, but abundant extracellular protein was visualized with SYPRO Ruby staining. Biofilms were resistant to detachment by dispersin B and sodium metaperiodate but were susceptible to detachment by proteases. Despite the genetic presence of icaADBC homologues in S. lugdunensis isolates, PNAG is not a major component of the extracellular matrix of in vitro biofilms formed by this species. Our data suggest that the S. lugdunensis biofilm matrix contains proteinaceous factors.     

Mixed species biofilms of Candida albicans and Staphylococcus epidermidis

A simple catheter disk model system was used to study the development in vitro of mixed species biofilms of Candida albicans and Staphylococcus epidermidis, two organisms commonly found in catheter-associated infections. Two strains of S. epidermidis were used: a slime-producing wild type (strain RP62A) and a slime-negative mutant (strain M7). In mixed fungal-bacterial biofilms, both staphylococcal strains showed extensive interactions with C. albicans. The susceptibility of 48-h biofilms to fluconazole, vancomycin and mixtures of the drugs was determined colorimetrically. The results indicated that the extracellular polymer produced by S. epidermidis RP62A could inhibit fluconazole penetration in mixed fungal-bacterial biofilms. Conversely, the presence of C. albicans in a biofilm appeared to protect the slime-negative staphylococcus against vancomycin. Overall, the findings suggest that fungal cells can modulate the action of antibiotics, and that bacteria can affect antifungal activity in mixed fungal-bacterial biofilms.     

Molecular characterization of an early invasive Staphylococcus epidermidis prosthetic joint infection

Historically regarded as a skin commensal, Staphylococcus epidermidis has been increasingly implicated in invasive foreign body infections such as catheter-related bloodstream infections, indwelling device infections, and prosthetic joint infections. We report a case of an aggressive, difficult-to-eradicate, invasive prosthetic hip infection occurring early after hardware implant and associated with a high-grade bacteremia and assess its salient molecular characteristics. The clinical and molecular characteristics of this isolate mirror the pathogenesis and persistence commonly seen with invasive methicillin-resistant S. aureus and may be attributed to the combination of resistance genes (SCCmec type IV), putative virulence factors (arcA and opp3a), cytolytic peptide production (α-type phenol-soluble modulins), and biofilm adhesion, interaction, and maturation (bhp, aap, and β-type phenol-soluble modulins).     

Anaerobic conditions induce expression of polysaccharide intercellular adhesin in Staphylococcus aureus and Staphylococcus epidermidis

Products of the intercellular adhesion (ica) operon in Staphylococcus aureus and Staphylococcus epidermidis synthesize a linear beta-1,6-linked glucosaminylglycan. This extracellular polysaccharide mediates bacterial cell-cell adhesion and is required for biofilm formation, which is thought to increase the virulence of both pathogens in association with prosthetic biomedical implants. The environmental signal(s) that triggers ica gene product and polysaccharide expression is unknown. Here we demonstrate that anaerobic in vitro growth conditions lead to increased polysaccharide expression in both S. aureus and S. epidermidis, although the regulation is less stringent in S. epidermidis. Anaerobiosis also dramatically stimulates ica-specific mRNA expression in ica- and polysaccharide-positive strains of both S. aureus and S. epidermidis. These data suggest a mechanism whereby ica gene expression and polysaccharide production may act as a virulence factor in an anaerobic environment in vivo.     

Formation of Propionibacterium acnes biofilms on orthopaedic biomaterials and their susceptibility to antimicrobials

Failure to treat and eradicate prosthetic hip infection with systemic antibiotic regimens is usually due to the fact that the infection is associated with biofilm formation and that bacterial cells growing within a biofilm exhibit increased resistance to antimicrobial agents. In this in vitro study, we investigated the susceptibility of prosthetic hip Propionibacterium acnes and Staphylococcus spp. isolates growing within biofilms on polymethylmethacrylate (PMMA) bone cement to a range of antibiotics. All P. acnes isolates in the biofilm mode of growth demonstrated considerably greater resistance to cefamandole, ciprofloxacin and vancomycin. In contrast, only four of the eight P. acnes isolates demonstrated an increase in resistance to gentamicin. All ten Staphylococcus spp. isolates in the biofilm mode of growth exhibited large increases in resistance to gentamicin and cefamandole with eight of the ten isolates also exhibiting an increase in resistance to vancomycin. However, only three of the ten Staphylococcus spp. isolates exhibited an increase in resistance to ciprofloxacin. Biofilms were also formed on three different titanium alloys and on PMMA bone cement using P. acnes, Staphylococcus epidermidis and Staphylococcus aureus strains to determine if the underlying biomaterial surface had an effect on biofilm formation and the antimicrobial susceptibility of the bacteria growing within biofilms. Although differences in the rate at which the three strains adhered to the different biomaterials were apparent, no differences in biofilm antibiotic resistance between the biomaterials were observed. In the light of these results, it is important that the efficacy of other antibiotics against P. acnes and Staphylococcus spp. prosthetic hip isolates growing within biofilms on orthopaedic biomaterials be determined to ensure optimal treatment of orthopaedic implant infection.     

Accumulation-Associated Protein Enhances Staphylococcus epidermidis Biofilm Formation under Dynamic Conditions and Is Required for Infection in a Rat Catheter Model

Biofilm formation is the primary virulence factor of Staphylococcus epidermidis. S. epidermidis biofilms preferentially form on abiotic surfaces and may contain multiple matrix components, including proteins such as accumulation-associated protein (Aap). Following proteolytic cleavage of the A domain, which has been shown to enhance binding to host cells, B domain homotypic interactions support cell accumulation and biofilm formation. To further define the contribution of Aap to biofilm formation and infection, we constructed an aap allelic replacement mutant and an icaADBC aap double mutant. When subjected to fluid shear, strains deficient in Aap production produced significantly less biofilm than Aap-positive strains. To examine the in vivo relevance of our findings, we modified our previously described rat jugular catheter model and validated the importance of immunosuppression and the presence of a foreign body to the establishment of infection. The use of our allelic replacement mutants in the model revealed a significant decrease in bacterial recovery from the catheter and the blood in the absence of Aap, regardless of the production of polysaccharide intercellular adhesin (PIA), a well-characterized, robust matrix molecule. Complementation of the aap mutant with full-length Aap (containing the A domain), but not the B domain alone, increased initial attachment to microtiter plates, as did in trans expression of the A domain in adhesion-deficient Staphylococcus carnosus. These results demonstrate Aap contributes to S. epidermidis infection, which may in part be due to A domain-mediated attachment to abiotic surfaces.     

Rapid identification of staphylococci from prosthetic joint infections using MALDI-TOF mass-spectrometry

Hospital-acquired infections associated with implanted medical devices are most commonly caused by staphylococci. Current methods of species identification are slow, costly, and sometimes unreliable. We evaluated the ability of a Bruker Daltonics Microflex MALDI-TOF/MS in conjunction with MALDI Biotyper software to identify 158 characterized staphylococcal isolates from prosthetic joint infections, including 36 Staphylococcus aureus, 100 Staphylococcus epidermidis, 10 Staphylococcus capitis, 8 Staphylococcus lugdunensis, 2 Staphylococcus warneri, and 2 Staphylococcus haemolyticus isolates using the extraction method recommended by Bruker Daltonics. The suggested species identification by the MALDI Biotyper software was correct for all isolates, indicating reliable differentiation between S. aureus and coagulase-negative staphylococci. Applying the recommended criteria of the MALDI Biotyper software all 158 isolates gave scores ≥2.0, implying secure genus and probable species identification for all isolates. 34/36 S. aureus, 36/100 S. epidermidis, 5/10 S. capitis, 6/8 S. lugdunensis, 2/2 S. haemolyticus, 0/2 S. warneri displayed scores ≥2.3 implying highly probable species identification. For S. epidermidis 25/100 additional isolates had a score close to 2.3. It appears that additional clinically relevant staphylococcal isolates in the data base might aid in identification at scores implying highly probable species identification. The ability of the MALDI Biotyper software to recognize clonally-related strains within a species group (i.e. sub-typing) was investigated, and showed great potential. In conclusion, the MALDI-TOF/MS MALDI Biotyper system provides a promising rapid and reliable method of identifying clinical isolates from prosthetic joint infections to the species level, and has potential for sub-typing.     

Correlation between biofilm formation and production of polysaccharide intercellular adhesin in clinical isolates of coagulase-negative staphylococci

The ability to form a biofilm seems to play an essential role in the virulence of coagulase-negative staphylococci (CoNS) by permitting them to cause persistent prosthetic device-related infections. The most clearly characterized component of staphylococcal biofilms is the polysaccharide intercellular adhesin (PIA) encoded by the icaADBC operon. In the present paper, we assess the link between the ability to form a biofilm (Bf+/-), to synthesize PIA (PIA+/-) and the presence of the ica locus (ica+/-). For this purpose, 66 CoNS strains were tested in vitro. Seventy three percent of all strains revealed presence of the ica locus (ica+), and therefore were potentially able to produce PIA and to form a biofilm. However, the characteristics observed indicated that 15% of all strains were biofilm forming without PIA production (Bf+, PIA-, ica+/-) while 8% were PIA producers without biofilm formation (Bf-, PIA+, ica+). On the basis of the obtained data we conclude that (i) PIA synthesis alone is not sufficient to produce a biofilm and (ii) staphylococci can also form a biofilm without producing PIA.     

Staphylococcus biofilm components as targets for vaccines and drugs

Staphylococci have become the most common cause of nosocomial infections, especially in patients with predisposing factors such as indwelling or implanted foreign polymer bodies. The pathogenesis of foreign-body associated infections with S.aureus and S. epidermidis is mainly related to the ability of these bacteria to form thick, adherent multilayered biofilms. In a biofilm, staphylococci are protected against antibiotic treatment and attack from the immune system, thus making eradication of the infections problematic. This necessitates the discovery of novel prophylactic and therapeutic strategies to treat these infections. In this review, we provide an overview of staphylococcal biofilm components and discuss new possible approaches to controlling these persistent biofilm-dwelling bacteria.     

Biofilm extracellular-DNA in 55 Staphylococcus epidermidis clinical isolates from implant infections

Biofilm formation is broadly recognized as an important virulence factor in many bacterial species implicated in implant-related opportunistic infections. In spite of a long history of research and many investigative efforts aimed at elucidating their chemical composition, structure, and function, the nature of bacterial biofilms still remains only partly revealed. Over the years, different extracellular polymeric substances (EPS) have been described that contribute functionally and structurally to the organization of biofilms. Recently extracellular DNA (eDNA) has emerged as a quantitatively conspicuous and potentially relevant structural component of microbial biofilms of many microbial species, Staphylococcus aureus and S. epidermidis among them. The present study aims at comparatively investigating the amount of eDNA present in the biofilm of 55 clinical isolates of S. epidermidis from postsurgical and biomaterial-related orthopedic infections. Quantification of eDNA was performed by a non-destructive method directly on bacterial biofilms formed under static conditions on the plastic surface of 96-well plates.