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Adherence of Abiotrophia defectiva and Granulicatella species to fibronectin: is there a link with endovascular infections?

Laurence Senn, José M. Entenza, Guy Prod'hom
DOI: http://dx.doi.org/10.1111/j.1574-695X.2006.00142.x 215-217 First published online: 1 November 2006

Abstract

During a 6-year period, we isolated three Abiotrophia defectiva, six Granulicatella adiacens and two G. ‘para-adiacens’ strains from clinical specimens. All A. defectiva strains were isolated from immunocompetent patients with endovascular infections, whereas the Granulicatella spp. strains were isolated from immunosuppressed patients with primary bacteremia. As the capacity of bacteria to adhere to the host extracellular matrix (ECM) has been implicated in the pathogenesis of endovascular infection, we investigated the ability of A. defectiva and Granulicatella spp. isolates to bind different ECM components immobilized in microtiter plates. Adherence tests showed a strong attachment of A. defectiva strains to fibronectin, whereas Granulicatella spp. strains were not adherent. The poor adherence of Granulicatella spp. strains to the ECM could be correlated with a lower propensity to induce endocarditis.

Keywords
  • Abiotrophia
  • Granulicatella
  • nutritionally variant streptococci
  • endocarditis
  • fibronectin adherence

Introduction

Abiotrophia and Granulicatella species form part of the normal flora of the oral cavity (Ohara-Nemoto et al. 1997; Sato et al. 1999; Mikkelsen et al. 2000), the genitourinary tract and the intestinal tract (Ruoff, 1991). Bacteremia and endocarditis are the more frequently reported clinical infections due to Abiotrophia and Granulicatella species (Christensen & Facklam, 2001) and account for from 4.3% to 6% of all ‘streptococcal’ endocarditis (Brouqui & Raoult, 2001). In a previous study (Senn et al. 2006), we reviewed the clinical manifestations of infections due to Abiotrophia defectiva and Granulicatella species that occurred at our institution, an 800-bed tertiary care center, over a 6-year period. Bloodstream and endovascular isolation of A. defectiva was associated with bacterial endocarditis or vascular infection in immunocompetent patients, whereas the isolation of Granulicatella spp. was associated with primary bacteremia in immunocompromised hosts. As it is well established that bacteria that cause endocarditis interact with proteins of the extracellular matrix (ECM) in the course of adhesion and colonization of damaged heart valves (Patti & Hook, 1994), we studied the ability of the Abiotrophia and Granulicatella isolates to bind fibrinogen, fibronectin, collagen and laminin in vitro, to evaluate whether variations in adhesive abilities could explain the different clinical behavior of these strains.

Material and methods

Bacterial strains

Eleven strains — three A. defectiva, six Granulicatella adiacens and two G. ‘para-adiacens’ — were tested. All strains were isolated from blood cultures or vascular graft specimens and were described previously (Senn et al. 2006). Reference strains included Staphylococcus aureus Cowan, S. aureus 5880 and S. aureus 5883 (Vaudaux et al. 1995; Que et al. 2001). Bacteria were grown at 37°C for 18 h in brain-infusion broth (BHI; Difco Laboratories, Detroit, MI), supplemented with 1% Vitox (Oxoid Ltd., Basingstoke, England) for Abiotrophia and Granulicatella strains. Bacteria were collected by centrifugation, washed and suspended in sterile phosphate buffer saline (PBS) pH 7.0.

Adherence tests

In vitro adherence to immobilized fibrinogen, fibronectin, laminin and collagen was assessed as described previously (Styriak et al. 1999). Briefly, 96-well flat-bottomed microtiter plates (MaxiSorp, Nunc, Roskilde, Denmark) were coated overnight at 4°C with twofold decreasing concentrations (from 50 to 0.5 µg mL−1) of purified human fibrinogen, fibronectin, laminin or collagen (Sigma, Buchs, Switzerland). The plates were washed three times with PBS and the wells were incubated with 2 mg mL−1 bovine serum albumin in PBS for 1 h to block unoccupied sites. After washing with PBS, 50 µL of bacterial cells, corresponding to about 5 × 108 CFUs, were added to each well and incubated at 37°C for 2 h. The plates were then washed three times with PBS to remove nonadherent bacteria. Adherent bacterial cells were fixed by warm air (60°C) for 30 min and stained with 0.5% (w/v) crystal violet for 45 min. Wells were washed six more times with PBS before a 200 µL well−1 of citrate buffer (pH 4.3) was added. After incubation for 45 min at room temperature, absorbances were read in a plate reader at 570 nm (A570 nm). Each batch of assays included at least one reference strain with known protein binding levels as well as blank wells coated with bovine serum albumin. Two independent series of experiments were performed. Strains were classified as strongly adherent (A570 nm>0.3), weakly adherent (0.1≤A570 nm≤0.3) or nonadherent (A570 nm≤0.1). At each concentration, absorbances of A. defectiva and Granulicatella spp. were compared by the unpaired t-test. Differences between A. defectiva and Granulicatella spp. were considered significant when P was <0.05.

Results and discussion

Binding activity to either fibrinogen, laminin or collagen was marginal or negative (A570 nm≤0.1) for all Abiotrophia and Granulicatella strains. In contrast (Fig. 1), the A. defectiva strains (isolates 1 3) were strongly adherent (A570 nm≥0.3) and exhibited an at least threefold increased attachment to fibronectin compared to that of the Granulicatella spp. (isolates 4 11) (P≤0.001). Adherence values between independent experiences were highly reproducible with less than 10% variation. None of the strains bound to blank wells. The control strains behaved as expected (Vaudaux et al. 1995; Que et al. 2001).

Figure 1

Binding of Abiotrophia and Granulicatella strains to fibronectin. Binding of Abiotrophia defectiva isolates was significantly higher than that of Granulicatella spp. isolates (P≤0.001 at each fibronectin concentration as determined by the unpaired t-test).

Our previous review of 11 clinical cases of A. defectiva and Granulicatella spp. infections had suggested that each genus was associated with a distinct clinical presentation: all three A. defectiva infections were seen in immunocompetent patients with definite endocarditis according to modified Duke criteria or vascular graft infection, whereas seven of eight Granulicatella spp. infections occurred in immunosuppressed, mainly febrile neutropenic patients with primary bacteremia, and only one Granulicatella spp. bacteremia was associated with possible endocarditis (Senn et al. 2006). These clinical observations hint that A. defectiva isolates have a higher tropism for endovascular tissue. This hypothesis is supported by the present study showing an increased ability of the A. defectiva strains to bind fibronectin in vitro compared to the Granulicatella strains.

Fibronectin is a major component of the ECM. The ability of bacteria to adhere to fibronectin is considered a virulence factor in endocarditis (Kuypers & Proctor, 1989; Lowrance et al. 1990; Que et al. 2005). Only a few studies have investigated the binding ability of Abiotrophia and Granulicatella to ECM components. In accordance with our results, Tart & van de Rijn (1991) reported that 13 of 15 A. defectiva strains, but only one of six G. adiacens strains, isolated from patients with endocarditis, bound to the ECM. In contrast, Okada et al. showed that adherence to fibronectin was maximal for G. adiacens, followed by A. defectiva, G. para-adiacens and G. elegans (Okada et al. 2000). Differences in experimental conditions, such as variation of pH and salt concentrations, may modify the binding of bacteria (Ito et al. 2003) and could explain the difference in these results. A selection bias depending on the clinical context of the isolated strains (primary bacteremia or endocarditis) was also possible.

Conclusion

In our study, the A. defectiva strains associated with endocarditis in humans showed a higher fibronectin-binding ability than the Granulicatella spp strains isolated from patients with primary bacteremia. Adherence to fibronectin may be an important factor involved in the pathogenesis of A. defectiva endovascular infections. Granulicatella spp. bacteremia were observed only when predisposing conditions such as mucositis and neutropenia were present, suggesting a low pathogenicity of the isolated strains. Their poor ability to bind to ECM components could be correlated with a lower propensity to induce endocarditis.

Footnotes

  • Editor: Alex van Belkum

References

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