Binding of Extracellular Matrix Molecules by Staphylococci from Wild Herbivores

· tyr iak I . , A. Lauková, Å. Ljungh: Binding of Extracellular Matrix Molecules by Staphylococci from Wild Herbivores. Acta Vet. Brno 2002, 71: 369-374. Nine strains of staphylococci were examined for their antibiotic resistance and for binding of seven extracellular matrix (ECM) molecules (bovine mucin, porcine mucin, porcine fibronectin, bovine fibrinogen, fetuin, bovine lactoferrin and heparin) immobilized on latex beads in the particle agglutination assay. All nine strains were resistant to bacitracin, and most of them were multiresistant. Four strains displayed resistance to 5 of 13 antibiotics tested. Different binding capability between individual strains was observed. There were strains binding several ECM molecules (as S. warneri SW6 and S. epidermidis SE14) as well as strains which bound only one (S. aureus SA11) or no ECM molecule (S. saprophyticus SS16) of seven tested. While some ECM molecules, e.g. porcine fibronectin and heparin, were bound by most of strains, others (e.g. mucins) were bound only by 1 or 2 strains. Some strains bound these substrates weakly, however, other strains displayed strong binding especially of heparin and porcine fibronectin. The preincubation of bacteria for 1 hour at room temperature with a protein used subsequently in the PAA completely prevented the agglutination reaction, thus indicating the specificity of the assay. ECM binding, mucin, fibronectin, fibrinogen, fetuin, heparin, lactoferrin, antibiotic resistance, Bacterial pathogens commonly adhere to host tissues as a first step in the pathogenic process of many infectious diseases. Following epithelial trauma, these pathogens may interact with various subepithelial extracellular matrix (ECM) structures (Ljungh and Wadström 1995). This bacterial adherence is mediated primarily by adhesins on the bacterial surface which bind specifically to complimentary ligands (Höök et al. 1990). The ECM is composed of glycoproteins and glycosaminoglycans which form a network by means of a number of specific interactions between different ECM components (Ljungh et al. 1996). Since most ECM are covered by epithelial or endothelial cells, they are not available for bacterial binding. However, damaged tissue surface may expose ECM and allow microbial colonization and infection (Ljungh and Wadström 1995). In recent years, binding of several ECM proteins by many Gram positive cocci (including staphylococci) has been investigated. Despite the fact that only a few bacterial species have been proven to use the ability to bind an ECM protein as a virulence determinant (Lowrance et al. 1990; Pat t i et al. 1994; Hienz et al. 1996), this property may be involved in the pathogenicity of several bacterial species (Naidu et al. 1988; Ljungh and Wadström 1995). While some staphylococci display high virulence, others may have a commensal relationship with their host. Generally, staphylococci form an important component of the bacterial community in the rumen content as well as on the rumen wall of domestic and wild ruminants (Wallace et al. 1979; Fonty et al. 1984; KmeÈ et al. 1990; Lauková 1994). Despite the fact that staphylococci are known also as lactic acid producers and belong ACTA VET. BRNO 2002, 71: 369–374 Address for correspondence: Dr. Igor ·tyriak, Ph. D. Institute of Animal Physiology SAS ·oltésovej 4-6 040 01 Ko‰ice, Slovakia Phone: +421 55 633 6251 Fax: +421 55 678 2162 E-mail:[email protected] http://www.vfu.cz/acta-vet/actavet.htm together with lactobacilli and enterococci to the first bacterial groups colonizing the rumen of young ruminants (Lauková 1994), they may be important in causing infection under the appropriate predisposing conditions similar to those described by Jet t et al. (1994) for enterococci. The aim of the present study was to determine the ability of 9 selected staphylococcal strains originally isolated from rumen content of mouflons and European bison to bind some ECM molecules immobilised on latex beads by rapid PAA (particle agglutination assay), and to compare results obtained with previously investigated animal staphylococci (· ty r iak et al. 1999a). The extension of our results is in the examination of bacterial resistance to 13 antibiotics. This fact is interesting because especially multiresistant strains could complicate a usual antibiotic therapy in case of infection. Materials and Methods Sources and cul t ivat ion of s t ra ins Nine strains of staphylococci from the bacterial collection of Dr. Lauková originally isolated from rumen content of wild herbivores (seven isolates from rumen of mouflons and two from European bison, ZOO Ko‰ice) were used together with 5 control strains from the collection of the Medical Microbiology Department at Lund University in our assay. The strain Helicobacter pylori NCTC 11637 was used as positive control for fetuin and mucin binding (Lelwala–Guruge et al. 1993) and 4 staphylococcal strains (Staphylococcus aureus Cowan1, S. saprophyticus TW111, S. haemolyticus SM 131 and Staphylococcus sp. LS1) as controls for the binding of the other ECM molecules. Staphylococci were cultivated using blood agar plates (Difco, Detroit, MI) with 5% horse erythrocytes overnight at 37 °C and H.pylori NCTC 11637 on GAB-CAMP agar (blood agar supplemented with 5% saponin lysed equine erythrocytes) under microaerophilic conditions (5% O2, 10% CO2, 85% N2) at 37 °C for 2 to 3 d. Ruminal staphylococci were phenotypically allotted by Becton-Dickinson identification system (Cockeysville, USA) to following species: Staphylococcus warneri, S. saprophyticus, S. epidermidis, S. xylosus, S. haemolyticus and S. aureus (Table 1). They are lactic acid producing and ureolytic bacteria (Lauková 1993). Chemicals Human serum albumin (HSA), bovine lactoferrin, fetuin, mucin type III (partially purified) from porcine stomach, piperazine N-N-bis-2-ethanolsulfonic acid, and EDAC (1-Ethyl 3-3-dimethylamino propyl carbodiimide hydrochloride) were purchased from Sigma Chemicals Co., St Louis (Missouri, USA). Citric acid and glycine were from Merck AG (Darmstadt, Germany). Heparin sodium salt was supplied from Fluka (Buchs, Switzerland), porcine plasma fibronectin was from BioInvent International AB (Lund, Sweden), bovine serum fibrinogen from Behring Diagnostics (La Jolla, CA, USA), mucin from bovine submaxillary glands from ICN Biomedicals Inc. (Aurora, Ohio, USA), merthiolate from Kabi AB (Stockholm, Sweden), latex beads (0.81 μm diameter) from Difco Laboratories, Detroit, (MI, USA), and carboxylated modified latex beads (0.55 μm diameter) from Seradyn Inc. Particle Technol. Div., Indianapolis (Indiana, USA). All buffers and chemicals were of analytical grade. Antibiot ic res is tance Staphylococcal resistance to 13 antibiotics was tested by agar diffusion method using the following antibiotic disks (Becton and Dickinson, Cockeysville, USA): ampicillin 10 μg (Amp), erythromycin 15 μg (Eryth), fosfomycin 50 μg (Fosf) gentamicin 10 μg (Gent), chloramphenicol 30 μg (CHC), bacitracin 2 units (Bac), kanamycin 30 μg (Kan), lincomycin 10 μg (Linc), methicilin 5 μg (Met), novobiocin 30 μg (NB), tetracycline 30 μg (TTC), vancomycin 30 μg (Vanc), and rifampicin 30 μg (Rif). Testing was performed on Columbia blood agar (Becton and Dickinson). Staphylococcus aureus CB44 (University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic) was used as a control organism. Adsorpt ion of ECM molecules to la tex beads The ECM molecules were adsorbed to the latex beads (0.81 μm diameter) by electrostatic and hydrophobic interactions according to the method described earlier (Naidu et al. 1988; · tyr iak et al. 1999b). Covalent coupl ing of fe tuin and mucins to carboxylated modif ied la tex beads Hundred μl of carboxylated modified latex (CML) beads were washed twice with 0.1 M phosphate buffer (pH 8.1) and were resuspended in 1 ml of phosphate buffer (pH 8.1) containing 2 mg/ml EDAC (1-ethyl 3-3dimethylamino propyl carbodiimide hydrochloride). This solution was added to beads drop by drop and the mixtures were incubated overnight at 4 °C on a rotatory mixer. The suspensions were then centrifuged at 12 000 g for 10 min, suspended in 1 ml of phosphate buffer containing 1 mg/ml protein, and then incubated overnight at 4 °C on a rotatory mixer. Latex beads were subsequently centrifuged at 12 000 g for 10 min, and the unbound protein 370