- Email: [email protected]
Phenotypic and genotypic markers of Staphylococcus epidermidis virulence
ORIGINAL AR TICLE
Phenotypic and genotypic markers of Staphylococcus epidermidis virulence A. Gelosia1, L. Baldassarri2, M. Deighton1 and T. van Nguyen1 1
Department of Applied Biology and Biotechnology, RMIT University, Melbourne, Australia and 2Laboratorio di Ultrastrutture, Istituto Superiore di Sanita, Rome, Italy
Objectives To analyze Staphylococcus epidermidis strains, previously tested for their virulence in a mouse model
of subcutaneous infection, for various phenotypic traits (biofilm density, extracellular polysaccharide, slimeassociated antigen (SAA)) and for the presence of the ica gene cluster, to determine which of these phenotypic and genotypic methods best correlates with virulence in the mouse model. Methods The quantitative biofilm assay was performed on 10 strains of S. epidermidis, comprising (1) RP62A
(ATCC 35984), (2) the strongest and weakest biofilm producers in our collection, (3) a pair of phenotypic variants, and (4) a strain whose biofilm density was enhanced in iron-limited media. Biofilm density was measured after growth at 37 8C and at ambient temperature, in trypticase soy broth (TSB) with and without glucose supplementation and using both chemical and heat fixation. Strains were assayed for SAA using a double immunodiffusion method. Extracellular polysaccharide was detected by transmission electron microscopy (TEM). A 546-base-pair segment of the ica gene cluster was amplified by PCR. Results Biofilm formation in TSB, glucose-enriched TSB, extracellular polysaccharide (observed by TEM),
expression of SAA and presence of the ica gene predicted virulence of nine, nine, nine, eight and eight of 10 strains, respectively. The phenotypic expression of biofilm and related properties was medium and temperature dependent. We encountered one ica-positive strain that failed to express biofilm in standard TSB at 37 8C, but was virulent in a mouse model, and another strain that lacked ica, produced biofilm and was virulent in the model. Conclusions Mouse virulence in our model can be predicted by any of the phenotypic or genotypic methods examined for 80% of strains. Medium and incubation conditions affect the expression of phenotypic markers by some strains. For the remaining strains, possible reasons for inconsistencies between the presence of the ica gene, phenotypic markers and mouse virulence include (1) dependence of biofilm on genes other than ica, (2) sequence differences in ica, (3) dependence of biofilm expression in vivo on strain characteristics and media used to prepare inocula for in vivo studies. Keywords
Staphylococcus epidermidis, biofilm, slime-associated antigen, mouse virulence, ica gene
Accepted 20 November 2000
Clin Microbiol Infect 2001; 7: 193–199
IN T R ODUC T ION Staphylococcus epidermidis is the most common cause of nosocomial sepsis associated with the use of implanted medical devices [1]. The initial stage of infection consists of attachment of the staphylococci to the plastic surface. This is followed by local multiplication of staphylococci, with the formation of an adherent surface growth (originally referred to as slime, now
Corresponding author and reprint requests: M. Deighton, Department of Applied Biology and Biotechnology, RMIT University, 124 Latrobe Street, 3000 Melbourne, Victoria, Australia Tel: þ61 39925 2482 Fax: þ61 39662 3421 E-mail: [email protected]
called biofilm) consisting of staphylococci enclosed in a protective layer of extracellular polysaccharide. Biofilm production by S. epidermidis is usually demonstrated by measuring the density of adherent growth on microtiter plates [2]. The original description of this method for quantitation of staphylococcal biofilm used Bouin’s fixative; however, fixation in air has been shown to be equally effective and does not involve the use of picric acid, which is explosive [3]. The presence of extracellular material around single cells may be observed by transmission electron microscopy (TEM). The amount of biofilm produced varies between strains of S. epidermidis, but is also influenced by environmental factors [4,5]. We and others have described phenotypic variants that differ in biofilm production as well as other characteristics [1,6–9]. Slime-associated antigen (SAA) has been demonstrated in 59% of biofilm-producing S. epidermidis strains and is involved
ß 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases
194 Clinical Microbiology and Infection, Volume 7 Number 4, April 2001
in the second (accumulation) stage of biofilm formation [10–12]. The main constituent of SAA is a polysaccharide composed of N-acetylglucosamine [11]. Two different polymers of Nacetylglucosamine, polysaccharide intercellular adhesin (PIA), described by Mack et al [13], and polysaccharide adhesin (PS/ A) [14], have been shown to be involved in the accumulation phase of biofilm formation. Preparations of SAA, PIA and PS/A apparently differ in molecular size, and substitutions on the glucosamine molecule [12–14]. PS/A and PIA are synthesized by a protein encoded by the icaADBC locus of S. epidermidis [14–16]. The product of icaA is an N-acetylglucosamine transferase, icaC and icaD encode membrane proteins, while icaB encodes a signal peptide that is secreted into the medium [15,16]. Two recent papers have demonstrated that significantly higher proportions of significant blood culture isolates than contaminants possess the ica gene cluster [17,18]. In a previous study [19], 10 strains of S. epidermidis (five biofilm positive and five biofilm negative) were assessed for virulence in a mouse model of subcutaneous infection. In three separate experiments using different starting cultures, sets of 40 mice were inoculated subcutaneously with 2 108 CFU of the test strain, suspended in sterile saline. Mice were killed on days 1, 3, 7 and 14 after inoculation. At autopsy, macroscopic evidence of inflammation and abscess formation at the inoculation sites was scored on a five-point scale ranging from no evidence of inflammation to the presence of an abscess 2–10 mm in diameter. Lesions were also scored histologically and microbiologically for evidence of infection. This information was amalgamated to allow categorization of mice as not infected, showing active infection (a macroscopic abscess at autopsy together with microbiological or histologic evidence of inflammation) or showing low-grade chronic infection (mild inflammatory response detected only by histologic or microbiological examination). With the use of these criteria, 73% of mice inoculated with the biofilm-positive strains developed
infections compared with 35% inoculated with the biofilmnegative strains. For each strain, the total number of mice (out of a possible 12) with any evidence of infection (acute or chronic) was recorded. This value ranged from eight to 10 for biofilmpositive strains and from two to seven for biofilm-negative strains. For the purposes of the present communication, we designated the number of infected mice (out of 12) the virulence index and arbitrarily defined a virulent strain as one with a virulence index of 8 ( 65% of mice infected). The aims of the present study were to extend our observations on the strains previously examined for virulence in the mouse model. We wished to examine the relationship between mouse virulence and the expression of various phenotypic traits (biofilm density, SAA, extracellular polysaccharide) and presence of the ica gene cluster. M AT ER I AL S AND ME T HOD S Staphylococcal strains Ten well-characterized strains from a large collection of clinical isolates of S. epidermidis ranging from minimal to heavy biofilm producers were chosen for this study. The strains had been assessed previously by the quantitative biofilm assay described by Christensen et al [2], after growth in TSB without enrichment and in iron-limited TSB, using Bouin’s reagent as a fixative [4], and for virulence in the mouse model described in the previous section [19]. The 10 strains were: (1) three of the heaviest biofilm producers in the collection (A285, A263, A285); (2) four of the weakest biofilm producers (A62, A211, A250, A315); (3) a pair of phenotypic variants isolated from valvular tissue of a patient with native valve endocarditis (A204 and A205) [6,7]; and (4) a blood culture isolate that produced significantly more biofilm under iron-limited conditions than in iron-replete conditions (A73) [4]. The reference
Table 1 Staphylococcal strains
S. epidermidis strain
Biofilm (TSB)
Biofilm (iron-limited TSB)
Virulence indexa
References
RP62A A285 A263 A205 A73 A62 A204 A211 A250 A315
þ þ þ þ ^ ^ ^ ^ ^ ^
þ þ þ þ þ ^ ^ ^ ^ ^
9 8 10 8 9 2 3 5 7 3
2, 19 19 19 6, 19 19 19 6, 19 19 19 19
a
In a mouse model of subcutaneous infection, mice were killed on days 1, 3, 7 and 14 after inoculation. At autopsy, macroscopic evidence of inflammation and abscess formation at the inoculation sites was scored on a five-point scale. Lesions were also scored histologically and microbiologically for evidence of infection. This information was amalgamated to allow the categorization of mice as not infected, showing active infection or showing low-grade chronic infection. For each strain, the total number of mice (out of a possible 12) with any evidence of infection was recorded.
ß 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 7, 193–199
Gelosia et al Phenotypic and genotypic markers of Staphylococcus epidermidis virulence
strain, S. epidermidis RP62A (ATCC 35984), was included as positive control. Strains A204 and A205 were defined as phenotypic variants on the basis of differences in expression of several phenotypic characteristics, including colony morphology on blood agar and Congo red agar [7], biofilm production and the expression of several enzymes, while having identical pulsed-field gel electrophoresis profiles [6,7]. We included strain A73 because we hypothesized that this strain might express strong biofilm under the iron-limited conditions encountered in vivo, although it is biofilm negative in the standard assay. The characteristics of these strains are listed in Table 1.
Biofilm assay Briefly, the biofilm-producing capacity of strains was investigated after growth of a standard inoculum in wells of microtiter plates. The biofilm was fixed with either Bouin’s fixative [2] or by drying in air for 1 h at 60 8C [3], and stained with Hucker crystal violet; the OD600 was measured spectrophotometrically (Dynatech ELISA reader MR 700). An OD600 of 0.12 was interpreted as negative, between 0.12 and 0.24 as weak and 0.24 as positive. The media and incubation conditions used were varied as shown in Table 2. Glucose-enriched TSB consisted of TSB (Oxoid, Heidelberg, Australia) with additional 1% filter-sterilized glucose.
Transmission electron microscopy (TEM) Strains were grown in glucose-enriched TSB and TEM was performed on overnight cultures fixed as described by Fassell et al [20]. Briefly, cells were prefixed for 20 min in 2.5% sodium cacodylate-buffered glutaraldehyde containing 0.075% ruthenium red (RR) and 75 mM lysine. Fixation in 2.5% glutaraldehyde þ 0.075% RR for 2 h was followed by postfixation in 1% OsO4 for 1 h. Samples were dehydrated through a graded series of ethanol solutions and embedded in Agar 100 resin (Agar Scientific Ltd, Cambridge, Essex, UK) and examined by transmission electron microscopy ( Jeol 100CX).
Slime-associated antigen SAA expression was assayed as previously described [11]. Briefly, bacteria from overnight cultures in glucose-enriched TSB were resuspended in phosphate-buffered saline and sonicated on ice for a total of 3 min. Bacterial cells and debris were removed by centrifugation, and the antigen-containing supernatant concentrated 10-fold by SpeedVac SC100 (Savant Instruments Inc., Farmingdale, NY, USA). SAA was detected by double immunodiffusion using an antiserum raised against whole cells of the biofilm-producing strain S. epidermidis ATCC 35984. SAA was purified as previously described [11], and
195
extracts from an SAA-negative, isogenic mutant of S. epidermidis ATCC 35984 (HAM 892) were used as positive and negative controls, respectively.
PCR of the ica gene A 546-base-pair segment of the ica gene was amplified using reaction mixtures, conditions and primers based on those described by Frebourg et al [18]. Briefly, DNA was extracted using the InstaGene Matrix kit (BioRad, Hercules, California, USA) and target DNA amplified with Hot Start Taq polymerase (Qiagen, Hilden, Germany) and the primers ica-F (50 -TTA TCA CCG CAG TTG TC-30 ) and ica-R (50 -GTT TAA CGC GAG TGA GCT AT-30 ). These primers amplify a segment of the ica gene locus that includes part of icaA icaB and the entire length of icaD. For an internal control, the universal primers 91E-F (50 -GGA ATT CAA A[T/G]G AAT TGA CGG GGG C-30 ) and 13B-R (50 -CGG GAT CCC AGC CCC GGG AAC GTA TTC AC-30 ) were used to amplify a 478-base-pair product of 16S rRNA [21]. PCR products were amplified by agarose gel electrophoresis, and gel images captured on a gel documentation system (GelDoc, BioRad). R E SULT S AND DI SC US S ION The type of fixative used in the biofilm assay (Bouin’s or in air at 60 8C) had no significant effect on the results. This is in agreement with previous findings [3]. The latter method should be adopted, since it avoids the potential hazards of using picric acid. Seven of the 10 strains examined by the biofilm assay generated similar results regardless of media and incubation conditions (Table 2, Figure 1). For the other three strains (A73, A205, A315), the biofilm density was dependent on incubation conditions and medium. Two strains (A73 and A315) were designated biofilm negative or weak producers on the basis of the conventional assay alone (growth at 35 8C in TSB), but produced biofilm in most other test situations. It is interesting to note that A73 and A315 showed a preference for incubation at ambient temperature rather than 35 8C. The most consistent results were obtained in glucose-enriched TSB, and these correlated with the presence of extracellular polysaccharide after growth in the same medium and SAA (except for strain A73). It is possible that strain A73 produces low levels of SAA that were not detected in our assay. The medium dependence of biofilm production by S. epidermidis is well known, but we are not aware of any reports describing strains with a preference for ambient temperature if grown in non-enriched TSB. The phenotypic variants (A204 and A205) gave opposite results in all phenotypic tests, including expression of SAA, but with the exception that both variants failed to produce biofilm in enriched TSB at ambient temperature.
ß 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 7, 193–199
þþ
þþþ
^^
^^
^^
^^
þþþ
þþþ
^^
^^
^^
^^
þþþ
-
þ
^
^
^
^
þ
A205
A73
A62
A204
A211
A250
A315
wþþ
þþ
^^
^^
^^
^^
þþþ
-wþ
þþþ
þþþ
þþw
Heat
þþ
^^
^^
^^
^w^
þþþ
^^
þþ
þþþ
þw
Bouin’s
Enriched TSB (1% glucose added) ambient temperature
w^ ^
^^
^^
^^
^^
^þ^
þþþ
þþþ
þþþ
þþw
Heat
TSB 35 8C
^^
^^
^^
^^
^^
^w^
www
þþþ
þþþ
þþw
Bouin’s
þþþ
^^
^^
^^
^^
þþ^
þþþ
þþþ
þþþ
þþþ
Heat
þwþ
^^
^^
^^
^w^
þþ^
þþþ
þþþ
þþþ
þþþ
Bouin’s
Enriched TSB (1% glucose added) 35 8C
Heat
þ
^
^
^
^
^
þ
þ
þ
þ
TEM
þ
^
^
^
^
þ
þ
þ
þ
þ
Bouin’s
Enriched TSB (1% glucose added) 35 8C
SAA
The biofilm-producing capacity of strains was investigated after growth of a standard inoculum in wells of microtiter plates.The biofilm was fixed with either Bouin’s fixative [2] or by drying in air for 1 h at 60 8C [3], and stained with Hucker crystal violet; the OD600 was measured with a spectrometer. An OD600 of 0.12 was interpreted as negative (^), between 0.12 and 0.24 as weak (w), and 0.24 as positive (þ). The three entries for the biofilm assay represent three replicate assays performed on different occasions. The other assays produced identical results on repeat testing.
a
þwþ
þþþ
þ
A263
þþþ
þþþ
þ
A285
þþþ
þþþ
Bouin’s
þ
Heat
TSB ambient temperature
ica gene Biofilm assaya
RP62A
Strain
Table 2 Expression of slime and slime-associated antigen under different environmental conditions
196 Clinical Microbiology and Infection, Volume 7 Number 4, April 2001
ß 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 7, 193–199
Gelosia et al Phenotypic and genotypic markers of Staphylococcus epidermidis virulence
197
Figure 1 Transmission electron micrograph of (a) S. epidermidis A205, showing loosely adherent extracellular material (slime), and (b) S. epidermidis A62 (slime negative). Bar: 0.5mm.
The ica gene cluster was detected in five of six strains that expressed some or all of the phenotypic markers that we looked for (RP62A, A285, A263, A73, A315), but not in four strains that were negative for all phenotypic markers (A62, A204, A211, A250) (Table 2, Figure 2). The only biofilm-positive strain that lacked ica was A205. As this strain is one of a pair of phenotypic variants, it is conceivable that a reversion to the biofilm-negative phenotype occurred in the preparation of
cultures for PCR. However, the biofilm-positive phenotype of A205 is relatively stable. Moreover, repeat testing of cultures that were clearly biofilm positive failed to detect the ica gene. Other possible explanations for our results are that A205 contains an altered ica gene cluster that was not detected in the PCR assay or that biofilm production by A205 is not determined by the ica gene cluster but by novel genes. Ziebuhr et al [17] also found a minority of biofilm-positive strains that
ß 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 7, 193–199
198 Clinical Microbiology and Infection, Volume 7 Number 4, April 2001
Figure 2 PCR amplification of ica and 16S rRNA gene fragments of S. epidermidis strains. Molecular weight marker (DNA MolecularWeight MarkerVIII, Roche Diagnostics, Mannhein, Germany), followed by S. epidermidis RP62A, A285, A283, A205, A73, A62, A204, A211, A250 and A315, each amplified with ica primers and 16S rRNA primers, respectively. The last lane is a negative control without DNA.
lacked the ica gene. It is interesting to note that biofilm produced by A205 is less tenacious and less granular than that of RP62A and other strong biofilm producers. In general, ica-positive strains were more virulent in our mouse model than strains lacking the ica gene cluster. The virulence indices of ica-positive and -negative strains were 9, 8, 10, 9 and 3 (mean 8, median 8) and 8, 2, 3, 5 and 7 (mean 5, median 5), respectively. These results are in general agreement with two recent studies that detected the ica gene cluster more commonly among clinically significant isolates than commensals or contaminants [17,18]. Ziebuhr et al [17], using Southern hybridization with a gene probe encoding the icaA, icaD and truncated icaB gene, showed that the ica gene was present in 85% (44 of 52) of clinical isolates compared with 6% (2 of 36) of commensals. In another study [18], icaADB was detected by PCR in 77% of blood culture isolates compared with 38% of commensals. Strain A315 (ica positive, virulence index 3) was originally selected for the virulence study in mice because it was the weakest biofilm producer in our collection, on the basis of quantitative biofilm assays in standard TSB and iron-limited TSB, both before and after mouse passage. However, in glucose-enriched TSB, we have demonstrated biofilm, SAA and extracellular polysaccharide. One possible explanation for these findings is that cultures of A315 contain a dominant biofilmnegative phenotype, with a variable but low proportion of cells belonging to the opposite (biofilm-positive) phenotype. In addition, since biofilm expression in A315 is strongly dependent on environmental conditions, the TSB culture used to prepare the inoculum for mice may have been dominated by cells that were not expressing extracellular polysaccharide. Regarding strain A205 (ica negative, biofilm positive, virulence index 8), it
is possible that biofilm is determined by a novel gene cluster or that there is an altered ica gene that was not detected by the PCR assay. In summary, we have confirmed the dependence on medium and incubation conditions for the phenotypic expression of biofilm and related characteristics. All of the phenotypic and genotypic methods examined in this study predicted the mouse virulence of 80% of strains. We have described an ica-positive strain that appears to express biofilm under conditions unlikely to be encountered in vivo and an ica-negative strain that produced biofilm, possibly via a novel mechanism or due to sequence differences in ica, and is virulent in our mouse model. ACK NOW L EDGME N T S This research was supported by grants from the Italian Ministry of Health, Project 1% ‘Interactions between opportunistic pathogens and biomaterials in the pathogenesis of prosthesis infections’ (ICS 080.1/RS 98.43) and RMIT University Competitive Grant Support Scheme, 1998. R EFER E NCE S 1. Christensen GD, Baldassarri L, Simpson WA. Colonization of medical devices by coagulase-negative staphylococci. In: Bisno AL, Waldvogel FA, eds. Infections associated with medical devices, 2nd edn. Washington DC: American Society for Microbiology, 1994: 45–78. 2. Christensen GD, Simpson WA, Younger JJ et al. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 1985; 22: 996–1006. 3. Baldassarri L, Simpson WA, Donelli G, Christensen GD. Variable fixation of staphylococcal slime by different histochemical fixatives. Eur J Clin Microbiol Infect Dis 1993; 12: 866–8.
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Gelosia et al Phenotypic and genotypic markers of Staphylococcus epidermidis virulence
4. Deighton MA, Borland R. Regulation of slime production in Staphylococcus epidermidis by iron. Infect Immun 1993; 61: 4473–9. 5. Barker LP, Simpson WA, Christensen GD. Differential production of slime under aerobic and anaerobic conditions. J Clin Microbiol 1990; 28: 2578–9. 6. Deighton M, Pearson S, Capstick J, Spelman D, Borland R. Phenotypic variation of Staphylococcus epidermidis isolated from a patient with native valve endocarditis. J Clin Microbiol 1992; 30: 2385–90. 7. Deighton MA, Capstick J, Borland R. A study of phenotypic variation of Staphylococcus epidermidis using Congo red agar. Epidemiol Infect 1992; 108: 423. 8. Christensen GD, Baddour LM, Simpson WA. Phenotypic variation of Staphylococcus epidermidis slime production in vitro and in vivo. Infect Immun 1987; 55: 2870–7. 9. Christensen GD, Baddour LM, Parisi JT et al. Colonial morphology of staphylococci on Memphis agar: phase variation of slime production, resistance to b-lactam antibiotics, and virulence. J Infect Dis 1990; 161: 1153–69. 10. Christensen GD, Barker LP, Mawhinney TP, Baddour LM, Simpson WA. Identification of an antigenic marker of slime production for Staphylococcus epidermidis. Infect Immun 1990; 58: 2906–11. 11. Baldassarri L, Donelli G, Gelosia A, Voglino MC, Simpson AW, Christensen GD. Purification and characterization of the staphylococcal slime-associated antigen and its occurrence among Staphylococcus epidermidis clinical isolates. Infect Immun 1996; 64: 3410–15. 12. Ammendolia MG, Di Rosa R, Montanaro L, Arciola CR, Baldassarri L. Slime production and expression of the slimeassociated antigen by staphylococcal clinical isolates. J Clin Microbiol 1999; 37(10): 3235–8. 13. Mack D, Nedelmann M, Krokotsch A, Schwarzkopf AM, Hessemann J, Laufs R. Characterisation of transposon mutants
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of biofilm-producing Staphylococcus epidermidis impaired in the accumulative phase of biofilm-production: genetic identification of a hexosamine-containing polysaccharide intracellular adhesin. Infect Immun 1994; 62: 3244–53. McKenney D, Hu¨ bner J, Muller E, Wang Y, Goldmann D, Pier G. The ica locus of Staphylococcus epidermidis encodes production of capsular polysaccharide/adhesin. Infect Immun 1998; 66: 4711–20. Heilmann C, Schweitzer O, Gerke C, Vanittanakom N, Mack D, Go¨ tz F. Molecular basis of intracellular adhesion in the biofilmforming Staphylococcus epidermidis. Mol Microbiol 1996; 20: 1083–91. Gerke C, Kraft A, Su¨ bmuth R, Schweitzer O, Go¨ tz F. Characterisation of the N-acetylglucosamine activity involved in the biosynthesis of the Staphylococcus epidermidis polysaccharide intracellular adhesin. J Biol Chem 1998; 273: 18586–93. Ziebuhr W, Heilmann C, Go¨ tz F et al. Detection of the intracellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates. Infect Immun 1997; 65: 890–6. Frebourg N, Lefebvre S, Baert S, Lumeland J-F. PCR-based assay for discrimination between invasive and contaminating Staphylococcus epidermidis strains. J Clin Microbiol 2000; 38: 877–80. Deighton MA, Borland R, Capstick J. Virulence of Staphylococcus epidermidis in a mouse model: significance of extracellular slime. Epidemiol Infect 1996; 117: 267–80. Fassell TA, Van Over JE, Hauser CC et al. Evaluation of bacterial glycocalyx preservation and staining by ruthenium red, ruthenium red-lysine and alcian blue for several methanotroph and staphylococcal species. Cells Materials 1992; 2: 37–48. Relman DA, Loutit JS, Schmidt TM, Falkow S, Tompkins LS. The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens. N Engl J Med 1990; 327: 293–301.
ß 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 7, 193–199
Phenotypic and genotypic markers of Staphylococcus epidermidis virulence A. Gelosia1, L. Baldassarri2, M. Deighton1 and T. van Nguyen1 1
Department of Applied Biology and Biotechnology, RMIT University, Melbourne, Australia and 2Laboratorio di Ultrastrutture, Istituto Superiore di Sanita, Rome, Italy
Objectives To analyze Staphylococcus epidermidis strains, previously tested for their virulence in a mouse model
of subcutaneous infection, for various phenotypic traits (biofilm density, extracellular polysaccharide, slimeassociated antigen (SAA)) and for the presence of the ica gene cluster, to determine which of these phenotypic and genotypic methods best correlates with virulence in the mouse model. Methods The quantitative biofilm assay was performed on 10 strains of S. epidermidis, comprising (1) RP62A
(ATCC 35984), (2) the strongest and weakest biofilm producers in our collection, (3) a pair of phenotypic variants, and (4) a strain whose biofilm density was enhanced in iron-limited media. Biofilm density was measured after growth at 37 8C and at ambient temperature, in trypticase soy broth (TSB) with and without glucose supplementation and using both chemical and heat fixation. Strains were assayed for SAA using a double immunodiffusion method. Extracellular polysaccharide was detected by transmission electron microscopy (TEM). A 546-base-pair segment of the ica gene cluster was amplified by PCR. Results Biofilm formation in TSB, glucose-enriched TSB, extracellular polysaccharide (observed by TEM),
expression of SAA and presence of the ica gene predicted virulence of nine, nine, nine, eight and eight of 10 strains, respectively. The phenotypic expression of biofilm and related properties was medium and temperature dependent. We encountered one ica-positive strain that failed to express biofilm in standard TSB at 37 8C, but was virulent in a mouse model, and another strain that lacked ica, produced biofilm and was virulent in the model. Conclusions Mouse virulence in our model can be predicted by any of the phenotypic or genotypic methods examined for 80% of strains. Medium and incubation conditions affect the expression of phenotypic markers by some strains. For the remaining strains, possible reasons for inconsistencies between the presence of the ica gene, phenotypic markers and mouse virulence include (1) dependence of biofilm on genes other than ica, (2) sequence differences in ica, (3) dependence of biofilm expression in vivo on strain characteristics and media used to prepare inocula for in vivo studies. Keywords
Staphylococcus epidermidis, biofilm, slime-associated antigen, mouse virulence, ica gene
Accepted 20 November 2000
Clin Microbiol Infect 2001; 7: 193–199
IN T R ODUC T ION Staphylococcus epidermidis is the most common cause of nosocomial sepsis associated with the use of implanted medical devices [1]. The initial stage of infection consists of attachment of the staphylococci to the plastic surface. This is followed by local multiplication of staphylococci, with the formation of an adherent surface growth (originally referred to as slime, now
Corresponding author and reprint requests: M. Deighton, Department of Applied Biology and Biotechnology, RMIT University, 124 Latrobe Street, 3000 Melbourne, Victoria, Australia Tel: þ61 39925 2482 Fax: þ61 39662 3421 E-mail: [email protected]
called biofilm) consisting of staphylococci enclosed in a protective layer of extracellular polysaccharide. Biofilm production by S. epidermidis is usually demonstrated by measuring the density of adherent growth on microtiter plates [2]. The original description of this method for quantitation of staphylococcal biofilm used Bouin’s fixative; however, fixation in air has been shown to be equally effective and does not involve the use of picric acid, which is explosive [3]. The presence of extracellular material around single cells may be observed by transmission electron microscopy (TEM). The amount of biofilm produced varies between strains of S. epidermidis, but is also influenced by environmental factors [4,5]. We and others have described phenotypic variants that differ in biofilm production as well as other characteristics [1,6–9]. Slime-associated antigen (SAA) has been demonstrated in 59% of biofilm-producing S. epidermidis strains and is involved
ß 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases
194 Clinical Microbiology and Infection, Volume 7 Number 4, April 2001
in the second (accumulation) stage of biofilm formation [10–12]. The main constituent of SAA is a polysaccharide composed of N-acetylglucosamine [11]. Two different polymers of Nacetylglucosamine, polysaccharide intercellular adhesin (PIA), described by Mack et al [13], and polysaccharide adhesin (PS/ A) [14], have been shown to be involved in the accumulation phase of biofilm formation. Preparations of SAA, PIA and PS/A apparently differ in molecular size, and substitutions on the glucosamine molecule [12–14]. PS/A and PIA are synthesized by a protein encoded by the icaADBC locus of S. epidermidis [14–16]. The product of icaA is an N-acetylglucosamine transferase, icaC and icaD encode membrane proteins, while icaB encodes a signal peptide that is secreted into the medium [15,16]. Two recent papers have demonstrated that significantly higher proportions of significant blood culture isolates than contaminants possess the ica gene cluster [17,18]. In a previous study [19], 10 strains of S. epidermidis (five biofilm positive and five biofilm negative) were assessed for virulence in a mouse model of subcutaneous infection. In three separate experiments using different starting cultures, sets of 40 mice were inoculated subcutaneously with 2 108 CFU of the test strain, suspended in sterile saline. Mice were killed on days 1, 3, 7 and 14 after inoculation. At autopsy, macroscopic evidence of inflammation and abscess formation at the inoculation sites was scored on a five-point scale ranging from no evidence of inflammation to the presence of an abscess 2–10 mm in diameter. Lesions were also scored histologically and microbiologically for evidence of infection. This information was amalgamated to allow categorization of mice as not infected, showing active infection (a macroscopic abscess at autopsy together with microbiological or histologic evidence of inflammation) or showing low-grade chronic infection (mild inflammatory response detected only by histologic or microbiological examination). With the use of these criteria, 73% of mice inoculated with the biofilm-positive strains developed
infections compared with 35% inoculated with the biofilmnegative strains. For each strain, the total number of mice (out of a possible 12) with any evidence of infection (acute or chronic) was recorded. This value ranged from eight to 10 for biofilmpositive strains and from two to seven for biofilm-negative strains. For the purposes of the present communication, we designated the number of infected mice (out of 12) the virulence index and arbitrarily defined a virulent strain as one with a virulence index of 8 ( 65% of mice infected). The aims of the present study were to extend our observations on the strains previously examined for virulence in the mouse model. We wished to examine the relationship between mouse virulence and the expression of various phenotypic traits (biofilm density, SAA, extracellular polysaccharide) and presence of the ica gene cluster. M AT ER I AL S AND ME T HOD S Staphylococcal strains Ten well-characterized strains from a large collection of clinical isolates of S. epidermidis ranging from minimal to heavy biofilm producers were chosen for this study. The strains had been assessed previously by the quantitative biofilm assay described by Christensen et al [2], after growth in TSB without enrichment and in iron-limited TSB, using Bouin’s reagent as a fixative [4], and for virulence in the mouse model described in the previous section [19]. The 10 strains were: (1) three of the heaviest biofilm producers in the collection (A285, A263, A285); (2) four of the weakest biofilm producers (A62, A211, A250, A315); (3) a pair of phenotypic variants isolated from valvular tissue of a patient with native valve endocarditis (A204 and A205) [6,7]; and (4) a blood culture isolate that produced significantly more biofilm under iron-limited conditions than in iron-replete conditions (A73) [4]. The reference
Table 1 Staphylococcal strains
S. epidermidis strain
Biofilm (TSB)
Biofilm (iron-limited TSB)
Virulence indexa
References
RP62A A285 A263 A205 A73 A62 A204 A211 A250 A315
þ þ þ þ ^ ^ ^ ^ ^ ^
þ þ þ þ þ ^ ^ ^ ^ ^
9 8 10 8 9 2 3 5 7 3
2, 19 19 19 6, 19 19 19 6, 19 19 19 19
a
In a mouse model of subcutaneous infection, mice were killed on days 1, 3, 7 and 14 after inoculation. At autopsy, macroscopic evidence of inflammation and abscess formation at the inoculation sites was scored on a five-point scale. Lesions were also scored histologically and microbiologically for evidence of infection. This information was amalgamated to allow the categorization of mice as not infected, showing active infection or showing low-grade chronic infection. For each strain, the total number of mice (out of a possible 12) with any evidence of infection was recorded.
ß 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 7, 193–199
Gelosia et al Phenotypic and genotypic markers of Staphylococcus epidermidis virulence
strain, S. epidermidis RP62A (ATCC 35984), was included as positive control. Strains A204 and A205 were defined as phenotypic variants on the basis of differences in expression of several phenotypic characteristics, including colony morphology on blood agar and Congo red agar [7], biofilm production and the expression of several enzymes, while having identical pulsed-field gel electrophoresis profiles [6,7]. We included strain A73 because we hypothesized that this strain might express strong biofilm under the iron-limited conditions encountered in vivo, although it is biofilm negative in the standard assay. The characteristics of these strains are listed in Table 1.
Biofilm assay Briefly, the biofilm-producing capacity of strains was investigated after growth of a standard inoculum in wells of microtiter plates. The biofilm was fixed with either Bouin’s fixative [2] or by drying in air for 1 h at 60 8C [3], and stained with Hucker crystal violet; the OD600 was measured spectrophotometrically (Dynatech ELISA reader MR 700). An OD600 of 0.12 was interpreted as negative, between 0.12 and 0.24 as weak and 0.24 as positive. The media and incubation conditions used were varied as shown in Table 2. Glucose-enriched TSB consisted of TSB (Oxoid, Heidelberg, Australia) with additional 1% filter-sterilized glucose.
Transmission electron microscopy (TEM) Strains were grown in glucose-enriched TSB and TEM was performed on overnight cultures fixed as described by Fassell et al [20]. Briefly, cells were prefixed for 20 min in 2.5% sodium cacodylate-buffered glutaraldehyde containing 0.075% ruthenium red (RR) and 75 mM lysine. Fixation in 2.5% glutaraldehyde þ 0.075% RR for 2 h was followed by postfixation in 1% OsO4 for 1 h. Samples were dehydrated through a graded series of ethanol solutions and embedded in Agar 100 resin (Agar Scientific Ltd, Cambridge, Essex, UK) and examined by transmission electron microscopy ( Jeol 100CX).
Slime-associated antigen SAA expression was assayed as previously described [11]. Briefly, bacteria from overnight cultures in glucose-enriched TSB were resuspended in phosphate-buffered saline and sonicated on ice for a total of 3 min. Bacterial cells and debris were removed by centrifugation, and the antigen-containing supernatant concentrated 10-fold by SpeedVac SC100 (Savant Instruments Inc., Farmingdale, NY, USA). SAA was detected by double immunodiffusion using an antiserum raised against whole cells of the biofilm-producing strain S. epidermidis ATCC 35984. SAA was purified as previously described [11], and
195
extracts from an SAA-negative, isogenic mutant of S. epidermidis ATCC 35984 (HAM 892) were used as positive and negative controls, respectively.
PCR of the ica gene A 546-base-pair segment of the ica gene was amplified using reaction mixtures, conditions and primers based on those described by Frebourg et al [18]. Briefly, DNA was extracted using the InstaGene Matrix kit (BioRad, Hercules, California, USA) and target DNA amplified with Hot Start Taq polymerase (Qiagen, Hilden, Germany) and the primers ica-F (50 -TTA TCA CCG CAG TTG TC-30 ) and ica-R (50 -GTT TAA CGC GAG TGA GCT AT-30 ). These primers amplify a segment of the ica gene locus that includes part of icaA icaB and the entire length of icaD. For an internal control, the universal primers 91E-F (50 -GGA ATT CAA A[T/G]G AAT TGA CGG GGG C-30 ) and 13B-R (50 -CGG GAT CCC AGC CCC GGG AAC GTA TTC AC-30 ) were used to amplify a 478-base-pair product of 16S rRNA [21]. PCR products were amplified by agarose gel electrophoresis, and gel images captured on a gel documentation system (GelDoc, BioRad). R E SULT S AND DI SC US S ION The type of fixative used in the biofilm assay (Bouin’s or in air at 60 8C) had no significant effect on the results. This is in agreement with previous findings [3]. The latter method should be adopted, since it avoids the potential hazards of using picric acid. Seven of the 10 strains examined by the biofilm assay generated similar results regardless of media and incubation conditions (Table 2, Figure 1). For the other three strains (A73, A205, A315), the biofilm density was dependent on incubation conditions and medium. Two strains (A73 and A315) were designated biofilm negative or weak producers on the basis of the conventional assay alone (growth at 35 8C in TSB), but produced biofilm in most other test situations. It is interesting to note that A73 and A315 showed a preference for incubation at ambient temperature rather than 35 8C. The most consistent results were obtained in glucose-enriched TSB, and these correlated with the presence of extracellular polysaccharide after growth in the same medium and SAA (except for strain A73). It is possible that strain A73 produces low levels of SAA that were not detected in our assay. The medium dependence of biofilm production by S. epidermidis is well known, but we are not aware of any reports describing strains with a preference for ambient temperature if grown in non-enriched TSB. The phenotypic variants (A204 and A205) gave opposite results in all phenotypic tests, including expression of SAA, but with the exception that both variants failed to produce biofilm in enriched TSB at ambient temperature.
ß 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 7, 193–199
þþ
þþþ
^^
^^
^^
^^
þþþ
þþþ
^^
^^
^^
^^
þþþ
-
þ
^
^
^
^
þ
A205
A73
A62
A204
A211
A250
A315
wþþ
þþ
^^
^^
^^
^^
þþþ
-wþ
þþþ
þþþ
þþw
Heat
þþ
^^
^^
^^
^w^
þþþ
^^
þþ
þþþ
þw
Bouin’s
Enriched TSB (1% glucose added) ambient temperature
w^ ^
^^
^^
^^
^^
^þ^
þþþ
þþþ
þþþ
þþw
Heat
TSB 35 8C
^^
^^
^^
^^
^^
^w^
www
þþþ
þþþ
þþw
Bouin’s
þþþ
^^
^^
^^
^^
þþ^
þþþ
þþþ
þþþ
þþþ
Heat
þwþ
^^
^^
^^
^w^
þþ^
þþþ
þþþ
þþþ
þþþ
Bouin’s
Enriched TSB (1% glucose added) 35 8C
Heat
þ
^
^
^
^
^
þ
þ
þ
þ
TEM
þ
^
^
^
^
þ
þ
þ
þ
þ
Bouin’s
Enriched TSB (1% glucose added) 35 8C
SAA
The biofilm-producing capacity of strains was investigated after growth of a standard inoculum in wells of microtiter plates.The biofilm was fixed with either Bouin’s fixative [2] or by drying in air for 1 h at 60 8C [3], and stained with Hucker crystal violet; the OD600 was measured with a spectrometer. An OD600 of 0.12 was interpreted as negative (^), between 0.12 and 0.24 as weak (w), and 0.24 as positive (þ). The three entries for the biofilm assay represent three replicate assays performed on different occasions. The other assays produced identical results on repeat testing.
a
þwþ
þþþ
þ
A263
þþþ
þþþ
þ
A285
þþþ
þþþ
Bouin’s
þ
Heat
TSB ambient temperature
ica gene Biofilm assaya
RP62A
Strain
Table 2 Expression of slime and slime-associated antigen under different environmental conditions
196 Clinical Microbiology and Infection, Volume 7 Number 4, April 2001
ß 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 7, 193–199
Gelosia et al Phenotypic and genotypic markers of Staphylococcus epidermidis virulence
197
Figure 1 Transmission electron micrograph of (a) S. epidermidis A205, showing loosely adherent extracellular material (slime), and (b) S. epidermidis A62 (slime negative). Bar: 0.5mm.
The ica gene cluster was detected in five of six strains that expressed some or all of the phenotypic markers that we looked for (RP62A, A285, A263, A73, A315), but not in four strains that were negative for all phenotypic markers (A62, A204, A211, A250) (Table 2, Figure 2). The only biofilm-positive strain that lacked ica was A205. As this strain is one of a pair of phenotypic variants, it is conceivable that a reversion to the biofilm-negative phenotype occurred in the preparation of
cultures for PCR. However, the biofilm-positive phenotype of A205 is relatively stable. Moreover, repeat testing of cultures that were clearly biofilm positive failed to detect the ica gene. Other possible explanations for our results are that A205 contains an altered ica gene cluster that was not detected in the PCR assay or that biofilm production by A205 is not determined by the ica gene cluster but by novel genes. Ziebuhr et al [17] also found a minority of biofilm-positive strains that
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198 Clinical Microbiology and Infection, Volume 7 Number 4, April 2001
Figure 2 PCR amplification of ica and 16S rRNA gene fragments of S. epidermidis strains. Molecular weight marker (DNA MolecularWeight MarkerVIII, Roche Diagnostics, Mannhein, Germany), followed by S. epidermidis RP62A, A285, A283, A205, A73, A62, A204, A211, A250 and A315, each amplified with ica primers and 16S rRNA primers, respectively. The last lane is a negative control without DNA.
lacked the ica gene. It is interesting to note that biofilm produced by A205 is less tenacious and less granular than that of RP62A and other strong biofilm producers. In general, ica-positive strains were more virulent in our mouse model than strains lacking the ica gene cluster. The virulence indices of ica-positive and -negative strains were 9, 8, 10, 9 and 3 (mean 8, median 8) and 8, 2, 3, 5 and 7 (mean 5, median 5), respectively. These results are in general agreement with two recent studies that detected the ica gene cluster more commonly among clinically significant isolates than commensals or contaminants [17,18]. Ziebuhr et al [17], using Southern hybridization with a gene probe encoding the icaA, icaD and truncated icaB gene, showed that the ica gene was present in 85% (44 of 52) of clinical isolates compared with 6% (2 of 36) of commensals. In another study [18], icaADB was detected by PCR in 77% of blood culture isolates compared with 38% of commensals. Strain A315 (ica positive, virulence index 3) was originally selected for the virulence study in mice because it was the weakest biofilm producer in our collection, on the basis of quantitative biofilm assays in standard TSB and iron-limited TSB, both before and after mouse passage. However, in glucose-enriched TSB, we have demonstrated biofilm, SAA and extracellular polysaccharide. One possible explanation for these findings is that cultures of A315 contain a dominant biofilmnegative phenotype, with a variable but low proportion of cells belonging to the opposite (biofilm-positive) phenotype. In addition, since biofilm expression in A315 is strongly dependent on environmental conditions, the TSB culture used to prepare the inoculum for mice may have been dominated by cells that were not expressing extracellular polysaccharide. Regarding strain A205 (ica negative, biofilm positive, virulence index 8), it
is possible that biofilm is determined by a novel gene cluster or that there is an altered ica gene that was not detected by the PCR assay. In summary, we have confirmed the dependence on medium and incubation conditions for the phenotypic expression of biofilm and related characteristics. All of the phenotypic and genotypic methods examined in this study predicted the mouse virulence of 80% of strains. We have described an ica-positive strain that appears to express biofilm under conditions unlikely to be encountered in vivo and an ica-negative strain that produced biofilm, possibly via a novel mechanism or due to sequence differences in ica, and is virulent in our mouse model. ACK NOW L EDGME N T S This research was supported by grants from the Italian Ministry of Health, Project 1% ‘Interactions between opportunistic pathogens and biomaterials in the pathogenesis of prosthesis infections’ (ICS 080.1/RS 98.43) and RMIT University Competitive Grant Support Scheme, 1998. R EFER E NCE S 1. Christensen GD, Baldassarri L, Simpson WA. Colonization of medical devices by coagulase-negative staphylococci. In: Bisno AL, Waldvogel FA, eds. Infections associated with medical devices, 2nd edn. Washington DC: American Society for Microbiology, 1994: 45–78. 2. Christensen GD, Simpson WA, Younger JJ et al. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 1985; 22: 996–1006. 3. Baldassarri L, Simpson WA, Donelli G, Christensen GD. Variable fixation of staphylococcal slime by different histochemical fixatives. Eur J Clin Microbiol Infect Dis 1993; 12: 866–8.
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