Detection of ica genes and slime production in a collection of Staphylococcus epidermidis strains from catheter-related infections in neutropenic patients

Slime production, principal virulence factor of Staphylococcus epidermidis associated with catheter-related infections is mediated by icaADBC operon wich expression is subject to phase variation. Reversible transposition of IS256 element into this operon is one of the most important mechanisms of biofilm phenotypic variation. Our study compared 28 S. epidermidis strains from catheter-related infection to 28 strains from nasal carriage concerning slime production on Congo red agar plate and ica genes and IS256 presence by PCR. ica operon was present among all slime-producing strains, and was absent among slime-negative strains. Only 79% of ica-positive strains were slime producers and no insertion of IS256 element was detected inside ica genes. A significative difference was found between catheter-related infections strains and commensal ones in terms of oxacillin (67,8 versus 35,7%) and ofloxacin resistance (75 versus 35,7%), slime production (64,2 versus 28,5%), phase variability (46,4 versus 7,1%) and ica genes presence (82,1 versus 35,7%). Our study demonstrates the role of ica genes, of phenotypic variability of slime production and antibiotic multiresistance as virulence factors of S. epidermidis associated with catheter-related infections; it confirms also the complexity and the diversity of regulation mechanisms implicated in biofilm formation.     

Collagen binding, elastase production, and slime production associated with coagulase-negative staphylococci isolated from bovine intramammary infections.

Collagen binding, elastase production, slime production, and associated somatic cell counts were determined with 160 strains of coagulase-negative staphylococci isolated from bovine intramammary infections. Mean binding values for type I and II collagen with Staphylococcus epidermidis, S. chromogenes, and S. hyicus strains were 5.8, 6.6, and 7.4 and 4.3, 4.2, and 4.9%, respectively. Eleven of 28 (39.3%) S. epidermidis, 1 of 38 (2.6%) S. chromogenes, and 1 of 94 (1.1%) S. hyicus strains were elastase positive. Slime production was noted with 12 (42.9%) S. epidermidis, 1 (2.6%) S. chromogenes, and 11 (11.7%) S. hyicus strains. No differences in somatic cell counts were observed with type I or type II collagen binding, elastase production, or slime production with S. epidermidis or S. chromogenes. However, somatic cell counts associated with S. hyicus strains with collagen type I binding affinities of greater than 5 and type II binding affinities of greater than 3 were 320.7 x 10(3) compared with 163.9 x 10(3) for strains with lower binding affinities.     

Regulation of slime production in Staphylococcus epidermidis by iron limitation.

Slime production by most strains of Staphylococcus epidermidis was enhanced by conditions of iron limitation produced by the addition of ethylenediamine-di-o-hydroxyphenol acetic acid to the growth medium. The density of the biofilm which formed on the base of microtiter plates was dependent on the degree of iron limitation, the stage of the growth cycle, and the nutritional state of the initial inoculum. One repeatedly slime-negative S. epidermidis strain, passaged in tryptic soya broth containing ethylenediamine-di-o-hydroxyphenol acetic acid, expressed high levels of slime after two passages. These observations suggest that iron limitation is one factor that regulates slime production by S. epidermidis. These findings could explain inconsistencies between the in vivo observation that biofilms invariably form on implanted catheters and the in vitro finding that some isolates from catheter-associated infection fail to produce slime.     

Species distribution and antibiotic sensitivity pattern of Coagulase negative staphylococci isolated from various clinical specimens

A study of 192 strains of Coagulase negative staphylococcus (CONS) showed that Staphylococcus epidermidis was the most common species, 158 (82.29%) isolated from all clinical specimens followed by S. saprophyticus (30, 15.62%) isolated mainly from urine. Slime production was exhibited by 77 (48.7%) strains of S. epidermidis and 8 (26.6%) of S. saprophyticus and the difference in the slime producing activity was statistically significant (p< 0.005). Antibiotic susceptibility testing against 15 commonly used antibiotics showed multidrug resistance with more than 90% resistance to penicillin, more than 50% to cephalexin and ciprofloxacin and more than 20% to methicillin, thus, highlighting the importance of species identification and antibiotic susceptibility testing for clinical isolates of CONS.     

Phase variation of slime production in Staphylococcus aureus: implications in colonization and virulence.

Two methods commonly used for slime detection in coagulase-negative staphylococci (tube biofilm formation and colony morphology in Congo red agar) were used to study 144 ruminant mastitis Staphylococcus aureus strains. Slime production was detected in 21 strains. A majority of cells (85%) in slime-producing (SP) strains and a minority of cells (5%) in non-slime-producing (NSP) strains showed a condensed exopolysaccharide matrix (slime) surrounding the bacterial cell wall, as revealed by electron microscopy and immunofluorescence. In vivo slime production was also detected immunohistochemically after experimental infection of the mammary gland in sheep. Upon repeated subcultures in Congo red agar, NSP variants were obtained from four ovine and four bovine SP strains at a frequency ranging from 0.5 x 10(-4) to 10(-4). Because SP variants could not be obtained from NSP strains within this range or at a higher frequency, they were obtained by the tube biofilm formation (requiring repeated subculturing of NSP strains in tryptic soy broth containing 2% glucose for subsequent recovery of colonies adherent to the walls of the culture tubes). In experimental challenge, the SP variant showed a significantly higher colonization capacity than did the NSP variant of the same strain used (P < 0.001). However, the NSP variant had a higher virulence than did the SP variant (P < 0.001). These results may help to explain the different roles of S. aureus slime production cell types (SP and NSP) coexisting in disease.     

Association of slime with pathogenicity of coagulase-negative staphylococci causing nosocomial septicemia.

To assess the role of slime in the pathogenesis of nosocomial bloodstream infections caused by coagulase-negative staphylococci, we compared the characteristics of 27 nosocomial bloodstream isolates with those of 27 skin isolates from non-hospital personnel. Of 27 bloodstream isolates, 14 were judged to be significant by a clinical index, and 13 were contaminants. Slime production was observed in 13 of 14 significant isolates but in only 3 of 13 contaminants (P = 0.0003) and 4 of 27 skin isolates (P = 0.0001). The 14 pathogens were identified as Staphylococcus epidermidis. Only 7 of 13 contaminants and 9 of 27 skin isolates belonged to the same species (P less than 0.006). Slime-producing strains of S. epidermidis represented 13 of 14 pathogens but only 2 of 13 contaminants (P less than 0.0003). Neither adherence to Teflon catheters nor phagocytosis and killing of coagulase-negative staphylococci by polymorphonuclear leukocytes was significantly influenced by slime production. Nevertheless, the identity of the organism and the slime production test predicted the clinical significance of blood isolates of coagulase-negative staphylococci with an overall accuracy of 89%.     

Furanones as potential anti-bacterial coatings on biomaterials

A major barrier to the long-term use of medical devices is development of infection. Staphylococcus epidermidis is one of the most common bacterial isolates from these infections with biofilm formation being their main virulence factor. Currently, antibiotics are used as the main form of therapy. However with the emergence of staphylococcal resistance, this form of therapy is fast becoming ineffective. In this study, the ability of a novel furanone antimicrobial compound to inhibit S. epidermidis adhesion and slime production on biomaterials was assessed. Furanones were physically adsorbed to various biomaterials and bacterial load determined using radioactivity. Slime production was assessed using a colorimetric method. Additionally, the effect of the furanone coating on material surface characteristics such as hydrophobicity and surface roughness was also investigated. The results of this study indicated that there was no significant change in the material characteristics after furanone coating. Bacterial load on all furanone-coated materials was significantly reduced (p<0.001) as was slime production (p<0.001). There is a potential for furanone-coated biomaterials to be used to reduce medical device-associated infections.     

A rapid PCR method for the detection of slime-producing strains of Staphylococcus epidermidis and S. aureus in periprosthesis infections.

In periprosthesis tissues, Staphylococcus epidermidis produces extracellular polysaccharide slime. Recently it has been shown that S. aureus also produces slime and that both S. epidermidis and S. aureus contain the ica operon responsible for slime production. In the operon, icaA encodes for N-acetylglutaminyltransferase, the enzyme for polysaccharide synthesis. However, co-expression of icaA and icaD is required for full slime synthesis. The slime-producing strains of both S. epidermidis and S. aureus are more virulent and are responsible for severe postsurgical or periprosthesis infections. The authors describe a simple, rapid, and reliable polymerase chain reaction method to detect icaA and icaD. The method was applied to the detection of ica genes on two reference strains, 15 strains each of S. epidermidis and S. aureus from periprosthesis infections and 10 strains from the skin and mucosa of healthy volunteers. icaA and icaD were detectable only in slime-producing strains (tested for slime production on Congo Red agar), and never in nonslime-producing ones. This method is a straightforward way of detecting the slime-producing ability by S. epidermidis and S. aureus. In clinical specimens this polymerase chain reaction method enables rapid diagnosis of virulent slime-producing strains with respect to the traditional culture method on Congo Red agar, which requires much more time. Rapid identification of the virulent properties of the bacterial strain responsible for a staphylococcal infection is crucial for deciding treatment.     

The effect of antibiotics on bacterial colonisation of vascular cannulae in a novel in-vitro model

An in-vitro model for studying semi-quantitatively the bacterial colonisation of the external and internal surfaces of peripheral intravascular cannulae is described. Using this model, we studied the effect of ciprofloxacin, teicoplanin and fusidic acid on cannula colonisation by Staphylococcus epidermidis. Exposure of colonised cannulae to sub-MICs of ciprofloxacin and fusidic acid reduced bacterial attachment, whereas sub-MIC levels of teicoplanin had little effect. Pre-exposure of S. epidermidis to sub-MICs of ciprofloxacin and fusidic acid also reduced slime production and colonisation. In comparison, pre-exposure of S. epidermidis to teicoplanin 1.0 mg/L did not influence colonisation, whereas at 0.1 mg/L it was reduced. The model allowed investigation of bacterial colonisation of cannulae and offers a screening system for the assessment of potential agents for the prophylaxis and treatment of these infections.     

Slime Production by Staphylococcus aureus and Staphylococcus epidermidis Strains Isolated from Patients with Diabetic Foot Ulcers

Slime production is a very important factor related to biofilm formation. The objective of the present study was to determine the frequency of slime production by Staphylococcus aureus and Staphylococcus epidermidis strains recovered from 50 patients with diabetic foot ulcers. Slime production was determined using the Congo red agar (CRA) method and compared with immunocytochemistry for the production of polysaccharide intercellular adhesin (PIA). Out of 55 S. aureus strains, 69% produced slime as shown by the CRA method. Of them, 84.2% also produced PIA. Of 17 CRA-negative strains, 70.6% produced PIA. Out of 20 S. epidermidis strains, 75% were CRA positive and 93.3% produced PIA. All CRA-negative S. epidermidis produced PIA. In conclusion, PIA production is a very common trait of S. aureus and S. epidermidis isolates obtained from diabetic foot ulcer patients.     

Adherence of staphylococci to intravascular catheters

Adherence of seven strains of Staphylococcus epidermidis and three strains of S. aureus to three types of intravascular catheters was assessed by ATP bioluminescence, by culture after ultrasonication and by scanning electronmicroscopy. The catheter materials studied were silicone elastomer, thermoplastic polyurethane and polyurethane coated with Hydromer, a coating which absorbs water and provides a hydrophilic sheath around the catheter. The adherence assays were performed in phosphate-buffered saline on a rotary shaker at 37 degrees C, with the catheters precoated with serum and uncoated, and the results were correlated with bacterial hydrophobicity. There was wide strain-to-strain variation in bacterial adherence; S. aureus and slime-producing S. epidermidis strains adhered better than did non-slime-producing strains. Overall, there was less bacterial adherence to Hydromer-coated catheters than to polyurethane and silicone catheters but it was unrelated to bacterial hydrophobicity. Serum coating of catheters resulted in marked reduction of bacterial adherence.     

Presence of icaA and icaD genes and slime production in a collection of staphylococcal strains from catheter-associated infections

Both Staphylococcus epidermidis and Staphylococcus aureus are important causes of infections associated with catheters and other medical devices. It has recently been shown that not only S. epidermidis but also S. aureus can produce slime and carries the ica operon responsible for slime production. In the operon, coexpression of icaA and icaD is required for full slime synthesis. In this study, the presence of icaA and icaD was determined in a collection of 91 staphylococcal (68 S. epidermidis and 23 S. aureus) strains from intravenous catheter-associated infections, in 10 strains from the skin and mucosa of healthy volunteers, and in two reference strains by a PCR method. Slime-forming ability was tested on Congo red agar plates; 49% of S. epidermidis strains from catheters and, surprisingly, 61% of S. aureus strains were icaA and icaD positive and slime forming. All the saprophytic strains turned out to be negative for both icaA and icaD and also non-slime forming. Two S. aureus and one S. epidermidis strain from catheters, detected as icaA and icaD positive by PCR analysis and as slime forming (black colonies) at 24 h on Congo red agar, at 48 h exhibited tiny red spikes at the center of black colonies. The onset of these variants could not be ascribed to a mutagenic potential of Congo red, which, in the Ames test, was devoid of mutagenicity. PCR analysis showed that these red variants were negative for both icaA and icaD and even lacking the entire icaADBC operon. The data reported indicate an important role of ica genes as a virulence marker in staphylococcal infections from intravenous catheters.     

Staphylococcal slime: a cautionary tale.

Slime production by Staphylococcus epidermidis may be important in the adherence to and colonization of biomedical devices, and slime has been proposed to have various effects on the immune system. Attempts were made to isolate, purify, and chemically characterize slime from S. epidermidis cultivated under fluid on tryptic soy broth-agar medium. "Crude slime" from slime-producing strain RP-12 was characterized by a high galactose content. Similar materials in similar yields were isolated from slime-producing strain Kaplan, a non-slime-producing mutant, Kaplan-6A, and sterile medium controls, suggesting that crude slime was derived mainly from the medium. The occurrence of D- and L-galactose and pyruvate and sulfate residues and methylation analysis of these crude slime preparations, monitored by gas-liquid chromatography and mass spectrometry, showed that the agar was the main source of crude slime, suggesting that the preparation was largely an artifact of the growth and isolation procedures. Similar high-galactose-content preparations from both S. epidermidis and Staphylococcus aureus, assumed to be bacterial products and with a variety of biological activities, have been described by other investigators. Growth attached to a solid surface appears to be important for slime production. An accumulation of turned-over cell surface molecules and released macromolecules such as DNA may contribute to slime production. Avoidance of agar and development of a chemically defined medium for slime production are recommended for further studies.     

Role of the Staphylococcus epidermidis slime layer in experimental tunnel tract infections.

An experimental animal model was used to assess the slime layer of Staphylococcus epidermidis as a pathogenic factor in tunnel tract infections. Mice were inoculated with high-slime-producing or non-slime-producing strains of S. epidermidis, either along the length of a subcutaneous catheter or in the area where a catheter had been placed and immediately removed (controls). Among the catheter-bearing mice, the phenotypically distinct staphylococci produced similar, high frequencies of abscess formation (72% [44 of 61] versus 81% [31 of 38]; P = 0.29). In controls, the non-slime-producing organisms were significantly more pathogenic (87% [40 of 46] versus 57% [25 of 44] abscess formation; P = 0.001). No consistent difference was detected between blood isolates obtained from patients with central venous catheter bacteremia and those from neonates with bacteremia in the absence of a prosthetic medical device. Quantitative culture of removed catheters showed greater adherence by the slime-producing isolates (P = 0.014). In this mouse model, slime production by S. epidermidis did not increase the risk of catheter tunnel tract infection, despite the greater catheter adherence of the slime-producing organisms. These findings suggest that traumatized tissue may be a sufficient condition for the development of S. epidermidis catheter-associated infections.     

Experimental foreign body infections in mice challenged with slime-producing Staphylococcus epidermidis.

The virulence of two previously described Staphylococcus epidermidis strains was examined in an experimental model of foreign body infection in mice. Animals challenged with the slime-producing strain developed three times as many infections as animals challenged with the strain that did not produce slime (P less than 0.001). Bacterial isolates recovered from the infected sites retained the characteristics of the inoculated strain. Animals without foreign bodies but challenged in a similar manner with either staphylococcal strain did not become infected. Thus, the presence of a foreign body predisposed the animals to S. epidermidis infection. These results indicate that the production of slime by S. epidermidis is a stable characteristic retained after animal passage and may be important in the pathogenesis of these infections.     

In catheter infections by Staphylococcus epidermidis the intercellular adhesion (ica) locus is a molecular marker of the virulent slime-producing strains.

Recently, it has been shown that S. epidermidis includes the ica operon responsible for slime production. In the operon, coexpression of icaA and icaD genes is required for full slime synthesis. In this study, the presence of icaA and icaD genes was searched for in a collection of 100 Staphylococcus epidermidis strains from catheter-associated infections by an original PCR method. Another 51 strains of S. epidermidis isolated from the skin or mucosa of healthy volunteers (26 of which derived from the hospital staff) were also investigated. Slime-forming ability was phenotypically tested on Congo red agar plates. Sixty-one percent of the strains isolated from catheters were icaA- icaD-positive and produced slime. The results indicate that detection of ica genes by a PCR method is a useful tool for prompt identification of S. epidermidis slime-forming strains isolated from catheter-related infections. Also, three saprophytic strains from the hospital staff were positive for slime synthesis and presence of ica genes, suggesting a potential diffusion of slime-forming strains in hospital personnel.     

Identification of an antigenic marker of slime production for Staphylococcus epidermidis.

The pathogenic Staphylococcus epidermidis strain RP62A (ATCC 35984) adheres to smooth surfaces by forming a tenacious bacterial film known as slime. The mechanism of slime production is not known; however, workers in the laboratory of G. Pier (Harvard Medical School, Boston, Mass.) have isolated from RP62A a galactose-rich capsular polysaccharide adhesin (CPA) which mediates the attachment of the organism to smooth surfaces. We have obtained two daughter strains from RP62A that no longer produce slime. One daughter strain, H4A, was obtained by selection for a spontaneous variant; the other strain, HAM892, was obtained by treating growing cultures of RP62A with acriflavin. Using an antiserum generated against whole cells of RP62A, we have examined lysozyme-lysostaphin digests of RP62A, H4A, and HAM892 by double immunodiffusion. The two strains that no longer produced slime no longer produced a particular antigen, which we refer to as the slime-associated antigen (SAA). SAA was also produced by unrelated strains of slime-producing S. epidermidis. SAA was heat and protease stable, had a molecular weight of greater than 50,000, and could be partially purified by chromatographing trypsin-digested material over a Sephadex G-200 column. Chemical analysis of partially purified SAA by gas-liquid chromatography found SAA to be glucose rich (59%) and galactose poor (1.4%). This analysis chemically distinguished SAA from CPA. When tested together by double immunodiffusion with anti-RP62A and anti-CPA antisera, partially purified SAA did not cross-react with CPA. Kinetic studies suggested that SAA is a marker for surface accumulation whereas CPA mediates initial adherence.     

Slime production by clinical isolates of <Emphasis Type="Italic">Blastoschizomyces capitatus</Emphasis> from patients with hematological malignancies and catheter-related fungemia

In order to expand the present knowledge of the pathogenic potential of Blastoschizomyces capitatus in central venous catheter (CVC)-related bloodstream infections, six strains of the organism recovered from three leukemic patients with CVC-related fungemia in different years were investigated. Isolates and control strains were tested for their genetic relatedness and for their ability to produce slime in glucose-containing solutions. DNA restriction enzyme analysis revealed that all strains of B. capitatus were identical, whereas slime production assays and examination of ex vivo material showed that they were able to produce large amounts of slime. Slime production may therefore play a relevant pathogenic role in cases of CVC-related fungemia caused by B. capitatus.     

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.     

Genotypic variation and slime production among blood and catheter isolates of Candida parapsilosis.

Candida parapsilosis is an important nosocomial pathogen that can proliferate in high concentrations of glucose and form biofilms on prosthetic materials. We investigated the genotypic diversity and slime production among 31 isolates of C. parapsilosis from individual patients with bloodstream or catheter infections. DNA subtyping was performed by using electrophoretic karyotyping plus restriction endonuclease analysis with BssHII followed by pulsed-field gel electrophoresis. Slime production was evaluated by growing organisms in Sabouraud broth with 8% glucose and examining the walls of the tubes for the presence of an adherent slime layer. Overall there were 14 DNA subtypes among the 31 isolates. Eighty percent of the isolates produced slime; 67% of the isolates were moderately to strongly positive, 13% were weakly positive, and 20% were not slime producers. The ability of isolates of a given DNA type to produce slime under these conditions was variable. The results of these studies indicate moderate genotypic variation among clinical isolates of C. parapsilosis. The propensity of these isolates to form slime in glucose-containing solutions suggests that this phenotypic characteristic may contribute to the ability of C. parapsilosis to adhere to plastic catheters and cause infections.