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Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity
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Microbial Pathogenesis xxx (2014) 1e6
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Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity Q3
Abderrahmen Meghrni a, *, Mouna Ben Nejma a, Hajer Hentati b, Aouni Mahjoub a, Maha Mastouri a, c a Laboratoire des Maladies Transmissible et substances biologiquement actives « LR99ES27», Faculté de Pharmacie, Avenue Avicenne, 5000 de Monastir, Tunisia b Service de Médecine et chirurgie buccales, Clinique hospitalo-universitaire d’Odontologie, Monastir, Tunisia c Laboratoire de Microbiologie, CHU Fatouma Bourguiba, de Monastir, Tunisia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 February 2014 Received in revised form 2 May 2014 Accepted 5 May 2014 Available online xxx
Staphylococcus aureus is one of prominent bacterial pathogen that occurs in oral region. In this study, 21 strains of S. aureus isolated from the oral cavity of Tunisian patients were investigated for slime production using Congo red agar method (CRA) and adherence assay. Biofilm formation of oral isolates on orthodontic biomaterials (Bis-GMA and PMMA) was also evaluated by MTT reduction assay. In addition, the production of hydrolytic enzymes by S. aureus strains was analyzed and the presence of protease, lipase and b-hemolysin genes (sspA, sspB, geh, hlb) was achieved by polymerase chain reaction (PCR). Qualitative biofilm production tested on CRA revealed that 91% of strains were slime producers. The result of OD570 showed that five strains isolated from the oral cavity were highly biofilm positive. The metabolic activity of S. aureus biofilm formed on Bis-GMA and PMMA did not differ between tested strains. The atomic force micrographs demonstrated that biofilm formed by S. aureus strains was organized in typical cocci cells attached to each other through production of exopolymeric substances. The production of hydrolytic enzymes showed that all S. aureus strains were protease positive. Lipase (77%) and beta hemolytic (59%) activities were also detected. Among the tested strains, 17 were positive for sspA, sspB and hlb genes. While only ten S. aureus strains harbor the geh gene (48%). These data highlight the importance of evaluation of biofilm formation and exoenzyme production in oral S. aureus isolates to investigate the role of this pathogen and its impact in oral pathology. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Staphylococcus aureus Hydrolytic enzymes Biofilm Orthodontic biomaterials
1. Introduction The human oral cavity contains more than 500 species of bacteria that interact among themselves and with their host tissues [1,2]. Oral cavity is considered as a reservoir of opportunistic pathogens that played a role in the onset of oral diseases [3]. Staphylococcus aureus is known as the leading cause of nosocomial infections. It is responsible for a wide range of human diseases, including endocarditis, food poisoning, septicemia and skin infections [4]. Furthermore, several studies underlined the prevalence of S. aureus in the oral cavity [5]. In fact, it was isolated from root carious lesions [6], periodontal pockets [7], dental abscesses
* Corresponding author. Tel.: þ216 73 466959; fax: þ216 73 465754. E-mail address: [email protected] (A. Meghrni).
[8], as well as from saliva and supragingival plaque of healthy adults [9]. To survive and colonize the oral cavity, S. aureus should develop many strategies such as biofilm formation and extracellular enzyme secretion. Indeed, this pathogen has the capacity to form multicellular communities that grow embedded in a self-produced extracellular matrix, referred to as biofilms. The biofilm matrix, composed of exopolysaccharides, proteins, nucleic acids and lipids, plays a well-known role as a defense structure, protecting bacteria from the host immune system and antimicrobial therapy [10]. Adhesion ability is an important factor in bacterial pathogenicity since it precedes penetration of the microorganisms in the host tissues promoted by the production of toxins [11]. S. aureus can form biofilms on many host tissues and implanted medical devices often causing chronic infections [12,13]. The adherence of this bacterium to an either biotic or abiotic surface is the critical first event in the establishment of S. aureus infection. Indeed, it harbors
http://dx.doi.org/10.1016/j.micpath.2014.05.002 0882-4010/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Meghrni A, et al., Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.05.002
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a variety of proteins compounds that mediate attachment to a multitude of host factors, such as extracellular matrix and plasma proteins and human host cells, or intercellular adhesion, which is essential for biofilm accumulation [14]. This bacterium produces a wide array of cell surface and extracellular proteins involved in virulence [15]. It secreted enzymes that degrade host components and hemolysins that damage the host tissue [16]. Two major extracellular proteases, namly serine protease (V8 protease; SspA), and cysteine protease (SspB) are produces by S. aureus and encoded within the same operon [17]. Moreover, this pathogen secreted lipase enzyme, which encoded by geh gene [15] enable it to invade and destroy host tissue. In addition to enzymes production, S. aureus secretes multiple toxins acting in the immediate area of infection, such as alpha, beta, gamma, and delta toxins [18]. To date, few studies have analyzed the prevalence of S. aureus carriage on human oral mucosa [3]. Therefore, the role of S. aureus in several diseases of the oral cavity merits further investigation, and the characterization of a potential S. aureus virulence factor became a necessity to understand their subsistence in oral cavity. The aim of this study was to evaluate the slime production of oral S. aureus strains and their ability to adhere to polystyrene and orthodontic biomaterials and to investigate their potency to secrete exoenzymes and hemolytic factor. 2. Materials and methods 2.1. Patients and bacterial strains 2.1.1. Patients The study was done on 105 patients from the dental clinic of Monastir, Tunisia. The subjects were 50 males and 55 females, with dental caries, pyogenic granulomas or abscesses. The mean age was 45.84 15.82. Ethical clearance was taken prior to the commencement of study. Written informed consent was obtained from all participants. All clinical procedures were approved by the Ethical Committee of the Faculty of Medicine, University of Monastir, Tunisia. Medical data and dental history were obtained from each patient. The criteria for inclusion were: no antibiotic treatment during sampling, no use of mouth rinses or any other preventive measure that might involve exposure to antimicrobial agents. 2.1.2. Media and growth conditions Samples were taken from dental abscess, caries and saliva of each patient with a sterile swab. After incubation in brain heart infusion (BHI) medium during 24 h, the swab was plated aerobically on sheep blood agar plates containing 4% NaCl, for 24 h at 37 C. Suspected colonies of S. aureus were confirmed by their positive Gram stain, catalase and DNAse positive reaction and the presence of free plasma coagulase using rabbit plasma (Bio-Merieux, France). Specie identification was performed using API 20 Staph strips (BioMerieux, France) according to the manufacturer’s recommendation and the results were read using an automated microbiological mini-API (Bio-Merieux, France). 2.2. Phenotypic characterization of bacteria-producing slime Qualitative detection of slime producer strains was studied by culturing the isolates on Congo red agar (CRA) plate made by mixing 36 g saccharose (Sigma Chemical Company, St. Louis, MO) with 0.8 g Congo red in 1 L of brain heart infusion agar (Biorad, USA) as previously described [19]. The strains were incubated at 37 C for 24 h under aerobic conditions. Slime-producing strains gave black colonies with a rough surface against red colonies with a smooth
surface for non-producing strains. Variable phenotype strains gave colonies with a black center and red outline, or red center and black outline were considered as positive slime producers [20]. 2.3. Semi-quantitative adherence assay Biofilm production by S. aureus strains grown in BHI (Biorad, France) was determined using a semi-quantitative adherence assay on 96-well tissue culture plates (Nunc, Roskilde, Denmark) as described previously [21,22]. Adherent bacteria were fixed with 95% ethanol and stained with 100 mL of 1% crystal violet (Merck, France) for 5 min. The microplates were air-dried and the optical density of each well was measured at 570 nm (OD570) using an automated Multiskan reader (GIO. DE VITA E C, ome, Italy). Biofilm formation was interpreted as highly positive (OD570 1), low grade positive (0.1 OD570 < 1), or negative (OD570 < 0.1). 2.4. Biofilm formation on biomaterials 2.4.1. Preparation of strips In the present study, the denture base materials used is the bisphenol A glycidyl methacrylate (Bis-GMA) composite resins and the polymethyl methacrylate acrylic (PMMA). The Bis-GMA and PMMA strips were made according to the method used in the laboratory of Biomaterials and Biotechnology in the Faculty of Dentistry of Monastir (Tunisia). All strips were cut into 1 cm2 squares. They were disinfected by dipping in 70% alcohol for 30 min and washed with sterile distilled water. They were then ultrasonicated for 20 min to remove any contaminants and artifacts from the surfaces, washed again in sterile distilled water, dried and used for the biofilm assay. 2.4.2. MTT metabolic assay The composite disks were placed in a 24-well plate, inoculated with 1.5 mL of the inoculation medium, and cultured for 24 h. Each disk was transferred to a new 24-well plate for the MTT (3-(4,5dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay. It is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan [23]. This kind of indirect viability assay is based on the formation of insoluble purple formazan due to the reduction of MTT by (respiratory) reductases of living staphylococcal cells [24]. Briefly, 1 mL of MTT dye (0.5 mg/ml MTT in PBS) was added to each well and incubated at 37 C for 1 h. After 1 h, the disks were transferred to a new 24-well plate, 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals, and the plate was incubated for 20 min with gentle mixing at room temperature in the dark. A total of 200 mL of the DMSO solution from each well was transferred to a 96-well plate, and the absorbance at 540 nm (optical density OD540) was measured via a microplate reader (GIO. DE VITA E C, ome, Italy). A higher absorbance indicates a higher formazan concentration, which in turn indicates more metabolic activity in the biofilm on the composite. 2.4.3. Biofilm visualization by atomic force microscopy (AFM) To visualize the biofilm formed on Bis-GMA and PMMA surfaces and to study the morphological changes in the cells during biofilm production, S. aureus ATCC 6538 was used as a positive control. After biofilm formation on strips, the surfaces were fixed on the round cover slide and were examined by AFM [25]. 2.5. Characterization of the enzymatic activity The activities of various enzymes were determined after inoculation of cultures onto TSA-1 to which the following substrates had been added: 1% [wt/vol] skim milk for caseinase, 1% [wt/vol]
Please cite this article in press as: Meghrni A, et al., Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.05.002
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formation were examined by the Friedman test. P-values of <0.05 were considered significant.
Table 1 Primers used in this study. Primer
Sequence (50 / 30 )
Product size (pb)
Annealing T C
sspA-F sspA-R sspB-F sspB-R geh-F geh-R hlb-F hlb-R
GAC AAC AGC GAC ACT TGT GA AGT ATC TTT ACC TAC AAC TAC A TGA AGA AGA TGG CAA AGT TAG TTG AGA TAC ACT TTG TGC AAG GCACAAGCCTCGG GACGGGGGTGTAG GGTGCACTTACTGAC CGCATATACATCCCATGGC
292
45
493
47
473
40
854
47
3. Results 3.1. Phenotypic determination of slime production From all samples included in our study 21 S. aureus were isolated, among them 19 strains (90.5%) were slime producers developing positive and variable phenotype on the CRA plate. The two other strains showed a negative phenotype (9.5%) (Table 2). 3.2. Polystyrene adherence assay
gelatin for gelatinase, 1% Tween 80 for lipase, 5% [vol/vol] egg yolk for phospholipase (lecithinase). Elastase activity was evaluated by using 1% (w/v) soluble elastin (Fluka Chemical, France) in Columbia agar base as described by Williams et al. [26]. The hemolytic activity was evaluated by plating isolated strains on bacteriological agar supplemented with 5% sheep’s blood for alpha and beta-hemolysin production [27].
The result of OD570 presented in Table 2, showed that five strains (9P, 9S, 9D37, 9L37, C54) isolated from oral infections were highly biofilm positive (OD570 1), the other strains showed a low grade biofilm formation (0.1 OD570 < 1). 3.3. Biofilm formation on biomaterials
2.6. Detection of sspA, sspB, geh, and hlb genes
The metabolic activity of S. aureus biofilms was determined by the ability of the attached cells to reduce the MTT to the formazan dye. We found that there are no significant differences between biofilm formation on Bis-GMA and on PMMA surfaces (P > 005) (Table 2). Biofilm visualization was performed by AFM. Atomic force micrographs revealed the presence of cocci cells arranged in clusters which are a typical morphology of S. aureus (Fig. 1a,b and c,d). The bacterial surface was rough in texture from the both strips. Furthermore, cells have indistinct borders on both surfaces (BisGMA and PMMA) indicating the presence of extracellular polysaccharidic materials (Fig. 1).
Genomic DNA used for polymerase chain reaction (PCR) was extracted by using a standard phenolechloroform technique. The presence of protease, lipase and b-hemolysin genes in S. aureus strains was detected by PCR using the primers listed in Table 1. Amplifications were performed according the following cycle conditions: for all genes an initial denaturation at 94 C for 10 min was followed by 30 cycles of denaturation at 94 C for 1 min, annealing for 1 min at temperature determined for each gene as described in Table 1 and elongation at 72 C for 1 min, followed by 10 min of final extension at 72 C.
3.4. Production of hydrolytic enzymes 2.7. Statistical analysis In this study, all S. aureus tested strains were positive for Caseinase Gelatinase and Elastase. Lecithinase activity was detected in 20 strains (95%) and we noted that among the 22 S. aureus
Statistical analysis was performed using the SPSS 17.0 statistics package for Windows. The differences in the degree of biofilm
Table 2 Slime production, adherence assay and biofilm formation capacity of oral S. aureus strains. Strains
Clinical details
Biofilm phenotype (CRA)
OD570 SD
Adherence state
Biofilm formation (MTT reduction SD) Bis-GMA
ATCC 6538 9P 9S L36 D36 L37 45S Pg Cjj Cjb PPb PPj C54 C59 C60 C87 C91 C92 C98 99F 99P C102 a
_ Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dental caries Dental caries Dental caries Dental caries Dental caries Dental caries Dental caries Dental fistula Dental fistula Dental caries
a
abscess abscess abscess abscess abscess abscess abscess abscess abscess abscess abscess
Variable phenotype Variable phenotype Variable phenotype Negative phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Negative phenotype Positive phenotype Positive phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype
2.90 2.47 1.69 0.68 1.41 2.02 0.65 0.97 0.68 0.84 0.37 0.18 1.54 0.51 0.23 0.76 0.76 0.19 0.17 0.22 0.26 0.42
0.055 0.020 0.086 0.015 0.053 0.096 0.009 0.065 0.020 0.040 0.013 0.05 0.008 0.057 0.063 0.071 0.026 0.031 0.017 0.099 0.014 0.053
Highly positive Highly positive Highly positive Low grade positive Highly positive Highly positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive Highly positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive
0.249 0.292 0.299 0.262 0.275 0.276 0.220 0.217 0.209 0.217 0.218 0.211 0.247 0.211 0.249 0.205 0.240 0.245 0.265 0.233 0.233 0.264
0.0124 0.0065 0.0091 0.0024 0.0082 0.0256 0.0059 0.0060 0.0017 0.0159 0.0058 0.0033 0.0065 0.0004 0.0072 0.0033 0.0063 0.035 0.0093 0.0056 0.0033 0.0060
PMMA 0.358 0.210 0.305 0.256 0.265 0.245 0.204 0.208 0.232 0.237 0.242 0.180 0.210 0.206 0.209 0.238 0.235 0.2561 0.262 0.237 0.245 0.225
0.0233 0.0024 0.0058 0.0045 0.0044 0.0133 0.0124 0.0025 0.0020 0.0158 0.0053 0.0085 0.0053 0.0063 0.0060 0.0051 0.0032 0.0194 0.0177 0.0149 0.0192 0.0100
Strains presenting a variable phenotype were considered as positive slime producers.
Please cite this article in press as: Meghrni A, et al., Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.05.002
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Fig. 1. AFM 2D topography (left panel) and the corresponding 3D reconstructions (right panel) images of S. aureus cells adhered on Bis-GMA (a and b, scan area: 3 mm 3 mm) and PMMA surfaces (c and d, scan area: 3 mm 3 mm).
strains, 17 were phospholipase producers (77%). 12 out of 22 tested strains were beta hemolytic (59%) (Table 3). 3.5. Detection by PCR of sspA, sspB, geh and hlb genes Among the tested strains, 17 (81%) were positive for sspA and sspB. Our result revealed that 10 S. aureus isolated from oral infections harbor the geh gene (48%). The hlb gene encoding the btoxin was detected in 17 strains (81%) (Table 3). 4. Discussion This work represents the first attempt to study the adhesion ability and the enzymatic activity of oral S. aureus strains isolated from patients attending the Dental Hospital of Monastir, Tunisia during years 2012 and 2013. In this investigation we noted that patients suffering from oral infections were diabetics (18%), smokers (39%), or demonstrated poor oral hygiene (53%). Numerous risk factors have been reported that make patients with diabetes more susceptible to oral disease, especially those with
poor oral hygiene and who are smokers [28]. The potential role of the oral cavity as a reservoir of staphylococci has been underlined by previous studies [3]. Our study revealed the detection of 21 S. aureus strains isolated from the oral cavity of 105 Tunisian patients suffering from oral infections. The detection of this bacterium was in agreement with the result reported previously by Smith et al. [29] who showed the presence of S. aureus in saliva and from gingival swabs of patients with a range of oral diseases. Furthermore, in other reports methicillin-resistant S. aureus (MRSA) strains have been isolated from oral cavity [30,31]. In addition, S. aureus has been reported to be isolated from 24 to 36% of healthy oral cavities [32,33]. And more recently, Ohara et al. [9] showed the occurrence of this bacterium (46.4%) in plaque and saliva from healthy adults. Therefore, the presence of S. aureus in the oral cavity may be more frequent than previously thought. Once S. aureus adheres and forms biofilms to host tissues or prosthetic materials, it is able to grow and persist in various ways [34]. Slime production might be a virulence factor and simultaneously worsens the response to infection, protecting bacterial cells from the host’s natural defense mechanisms and from antibiotics
Please cite this article in press as: Meghrni A, et al., Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.05.002
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Table 3 Hydrolytic enzymes production and presence of S. aureus genes. Strains
ATCC 6538 9P 9S L36 D36 L37 45S Pg Cjj Cjb PP C54 C59 C60 C87 C91 C92 C98 99F 99P C102 % expression
Exoenzymes expression
Presence of genes
Lecithinase
Lipase
Caseinase
Gelatinase
Elastase
Hemolysin
geh
sspA
sspB
hlb
þ þ þ þ þ þ þ þ þ þ þ e þ þ þ þ þ þ þ þ þ 95
þ þ þ þ þ þ e e þ þ e e þ þ þ þ þ þ þ þ þ 77
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ 100
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ 100
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ 100
b b b b b b a a a a a a b b b b a b a a b
gehþ geh gehþ geh geh geh gehþ gehþ geh geh geh geh gehþ gehþ gehþ gehþ geh geh gehþ gehþ geh 48
sspAþ sspAþ sspAþ sspA sspAþ sspAþ sspAþ sspA sspAþ sspAþ sspAþ sspA sspAþ sspAþ sspAþ sspAþ sspA sspAþ sspAþ sspAþ sspAþ 81
sspB sspBþ sspBþ sspB sspBþ sspBþ sspBþ sspBþ sspBþ sspBþ sspB sspB sspBþ sspBþ sspBþ sspBþ sspBþ sspBþ sspBþ sspBþ sspBþ 81
hlbþ hlbþ hlbþ hlbþ hlbþ hlbþ hlb hlbþ hlb hlbþ hlbþ hlbþ hlb hlbþ hlbþ hlb hlbþ hlbþ hlbþ hlbþ hlbþ 81
activities [35]. In our study we found that 20 out of 22 strains of S. aureus (90.9%) were slime producers. In a recent study, Kouidhi et al. [36] demonstrated that 11 of 22 S. aureus associated to dental caries were slime positive. Furthermore, Zmantar et al. [37] found that S. aureus strains isolated from patients with an auricular infection were slime producers. Semi-quantitative adherence of S. aureus tested on polystyrene microplate showed that five strains isolated from oral infections were highly biofilm positive. The remaining strains showed low-grade biofilm formation. These results suggest a difference in adhesion ability between tested strains. In fact, variations in biofilm forming capacities of S. aureus could be explained by surface associated proteins and regulatory genes involved in biofilm production [38]. We noted a positive correlation between the slime-producing ability on CRA plates and the adhesion ability developed on polystyrene material. Likewise, previous study showed a high consistency between CRA screening and biofilm biomass crystal violet staining [39,40]. Nevertheless, other rapports showed that no correlation between slime-producing MRSA and methicillin susceptible S. aureus (MSSA) isolates and an enhanced tendency of biofilm formation [41,42]. In our study, we also determined the adhesion capacity of the oral S. aureus strains to orthodontic biomaterials Bis-GMA and PMMA. It was the first investigation in Tunisia to study biofilm formation of this bacterium to abiotic surfaces commonly used in dentistry practices. Our results demonstrated that the metabolic activity of S. aureus biofilm formed on Bis-GMA and PMMA surfaces did not differ among tested strains (P > 0.05). Otherwise, the capacity of S. aureus to adhere to indwelling medical devices was revealed in several studies [43]. S. aureus is one of the prominent gram positive human pathogen secretes many surface and secretary proteins including various enzymes and pathogenic factors that favor the successful colonization and infection of host tissue [44]. The production of S. aureus enzymes such as proteases, lipases, nucleases, and collagenases convert tissue components into nutrients, facilitating bacterial growth and invasion [45]. Toxins and enterotoxins have other effects such as superantigenicity, pyrogenicity and direct damage to endothelial [46]. The determination of hydrolytic enzymes production revealed that 95% of S. aureus strains were lecithinase positive, and 75% were lipase producer. Barretti et al. [35] found
59(b); 41(a)
that 97.1% of S. aureus isolated from peritonitis were both lecithinase and lipase positive, while Saising et al. [47] suggest that only 65.6% of the strains isolates from acne lesions were lipase positive. During infection, S. aureus produces numerous enzymes that enable it to invade and destroy host tissues and metastasize to other sites. In our work all tested strains have been found producers of caseinase gelatinase and elastase. Other studies reported that of S. aureus implicated in skin infections exhibited protease activity [48]. Furthermore, Wu et al. [49] showed that S. aureus isolated from corneal ulcers produced caseinase (100%), gelatinase (80%) and elastase (70%). Moreover, this bacterium is known to produce tow enzymes serine protease (V8 protease; SspA), and cysteine protease (SspB), which are implicated in virulence. In our study we showed that 81% of isolated strains are sspA and sspB positive. Other study revealed that S. aureus isolated from food samples harboring sspA and sspB genes [50]. Thus, the presence of extracellular protease encoding genes might sign the infectious abilities of these strains in humans. The extracellular proteases could help the invasion of bacteria through tissues during infection process [51], enable it to interact with the host defense mechanisms. In addition to enzyme production, S. aureus secrets a b-toxin as a neutral sphingomyelinase that lyses erythrocytes in order to evade the host immune system as well as scavenge nutrients [52]. This hemolysin is secreted by certain strains of S. aureus especially strains isolated from corneal infection [53]. While, in our study we demonstrated that this type of toxin is produced by 59% of oral S. aureus strains. 81% of our isolates are hlb positive which harbor the beta toxin gene but its expression may be influenced by regulatory genes.
5. Conclusion In conclusion, several reports highlight the presence of S. aureus in oral cavity, however this study is one of few reports dealing with the characterization of virulence factors in such isolates. According to our finding, oral S. aureus strains exhibits a considerable adhesion capacity to biomaterials and were producers of a variety of hydrolytic enzymes. All these factors contribute to the colonization of oral cavity by this bacterium.
Please cite this article in press as: Meghrni A, et al., Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.05.002
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Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity Q3
Abderrahmen Meghrni a, *, Mouna Ben Nejma a, Hajer Hentati b, Aouni Mahjoub a, Maha Mastouri a, c a Laboratoire des Maladies Transmissible et substances biologiquement actives « LR99ES27», Faculté de Pharmacie, Avenue Avicenne, 5000 de Monastir, Tunisia b Service de Médecine et chirurgie buccales, Clinique hospitalo-universitaire d’Odontologie, Monastir, Tunisia c Laboratoire de Microbiologie, CHU Fatouma Bourguiba, de Monastir, Tunisia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 February 2014 Received in revised form 2 May 2014 Accepted 5 May 2014 Available online xxx
Staphylococcus aureus is one of prominent bacterial pathogen that occurs in oral region. In this study, 21 strains of S. aureus isolated from the oral cavity of Tunisian patients were investigated for slime production using Congo red agar method (CRA) and adherence assay. Biofilm formation of oral isolates on orthodontic biomaterials (Bis-GMA and PMMA) was also evaluated by MTT reduction assay. In addition, the production of hydrolytic enzymes by S. aureus strains was analyzed and the presence of protease, lipase and b-hemolysin genes (sspA, sspB, geh, hlb) was achieved by polymerase chain reaction (PCR). Qualitative biofilm production tested on CRA revealed that 91% of strains were slime producers. The result of OD570 showed that five strains isolated from the oral cavity were highly biofilm positive. The metabolic activity of S. aureus biofilm formed on Bis-GMA and PMMA did not differ between tested strains. The atomic force micrographs demonstrated that biofilm formed by S. aureus strains was organized in typical cocci cells attached to each other through production of exopolymeric substances. The production of hydrolytic enzymes showed that all S. aureus strains were protease positive. Lipase (77%) and beta hemolytic (59%) activities were also detected. Among the tested strains, 17 were positive for sspA, sspB and hlb genes. While only ten S. aureus strains harbor the geh gene (48%). These data highlight the importance of evaluation of biofilm formation and exoenzyme production in oral S. aureus isolates to investigate the role of this pathogen and its impact in oral pathology. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Staphylococcus aureus Hydrolytic enzymes Biofilm Orthodontic biomaterials
1. Introduction The human oral cavity contains more than 500 species of bacteria that interact among themselves and with their host tissues [1,2]. Oral cavity is considered as a reservoir of opportunistic pathogens that played a role in the onset of oral diseases [3]. Staphylococcus aureus is known as the leading cause of nosocomial infections. It is responsible for a wide range of human diseases, including endocarditis, food poisoning, septicemia and skin infections [4]. Furthermore, several studies underlined the prevalence of S. aureus in the oral cavity [5]. In fact, it was isolated from root carious lesions [6], periodontal pockets [7], dental abscesses
* Corresponding author. Tel.: þ216 73 466959; fax: þ216 73 465754. E-mail address: [email protected] (A. Meghrni).
[8], as well as from saliva and supragingival plaque of healthy adults [9]. To survive and colonize the oral cavity, S. aureus should develop many strategies such as biofilm formation and extracellular enzyme secretion. Indeed, this pathogen has the capacity to form multicellular communities that grow embedded in a self-produced extracellular matrix, referred to as biofilms. The biofilm matrix, composed of exopolysaccharides, proteins, nucleic acids and lipids, plays a well-known role as a defense structure, protecting bacteria from the host immune system and antimicrobial therapy [10]. Adhesion ability is an important factor in bacterial pathogenicity since it precedes penetration of the microorganisms in the host tissues promoted by the production of toxins [11]. S. aureus can form biofilms on many host tissues and implanted medical devices often causing chronic infections [12,13]. The adherence of this bacterium to an either biotic or abiotic surface is the critical first event in the establishment of S. aureus infection. Indeed, it harbors
http://dx.doi.org/10.1016/j.micpath.2014.05.002 0882-4010/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Meghrni A, et al., Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.05.002
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a variety of proteins compounds that mediate attachment to a multitude of host factors, such as extracellular matrix and plasma proteins and human host cells, or intercellular adhesion, which is essential for biofilm accumulation [14]. This bacterium produces a wide array of cell surface and extracellular proteins involved in virulence [15]. It secreted enzymes that degrade host components and hemolysins that damage the host tissue [16]. Two major extracellular proteases, namly serine protease (V8 protease; SspA), and cysteine protease (SspB) are produces by S. aureus and encoded within the same operon [17]. Moreover, this pathogen secreted lipase enzyme, which encoded by geh gene [15] enable it to invade and destroy host tissue. In addition to enzymes production, S. aureus secretes multiple toxins acting in the immediate area of infection, such as alpha, beta, gamma, and delta toxins [18]. To date, few studies have analyzed the prevalence of S. aureus carriage on human oral mucosa [3]. Therefore, the role of S. aureus in several diseases of the oral cavity merits further investigation, and the characterization of a potential S. aureus virulence factor became a necessity to understand their subsistence in oral cavity. The aim of this study was to evaluate the slime production of oral S. aureus strains and their ability to adhere to polystyrene and orthodontic biomaterials and to investigate their potency to secrete exoenzymes and hemolytic factor. 2. Materials and methods 2.1. Patients and bacterial strains 2.1.1. Patients The study was done on 105 patients from the dental clinic of Monastir, Tunisia. The subjects were 50 males and 55 females, with dental caries, pyogenic granulomas or abscesses. The mean age was 45.84 15.82. Ethical clearance was taken prior to the commencement of study. Written informed consent was obtained from all participants. All clinical procedures were approved by the Ethical Committee of the Faculty of Medicine, University of Monastir, Tunisia. Medical data and dental history were obtained from each patient. The criteria for inclusion were: no antibiotic treatment during sampling, no use of mouth rinses or any other preventive measure that might involve exposure to antimicrobial agents. 2.1.2. Media and growth conditions Samples were taken from dental abscess, caries and saliva of each patient with a sterile swab. After incubation in brain heart infusion (BHI) medium during 24 h, the swab was plated aerobically on sheep blood agar plates containing 4% NaCl, for 24 h at 37 C. Suspected colonies of S. aureus were confirmed by their positive Gram stain, catalase and DNAse positive reaction and the presence of free plasma coagulase using rabbit plasma (Bio-Merieux, France). Specie identification was performed using API 20 Staph strips (BioMerieux, France) according to the manufacturer’s recommendation and the results were read using an automated microbiological mini-API (Bio-Merieux, France). 2.2. Phenotypic characterization of bacteria-producing slime Qualitative detection of slime producer strains was studied by culturing the isolates on Congo red agar (CRA) plate made by mixing 36 g saccharose (Sigma Chemical Company, St. Louis, MO) with 0.8 g Congo red in 1 L of brain heart infusion agar (Biorad, USA) as previously described [19]. The strains were incubated at 37 C for 24 h under aerobic conditions. Slime-producing strains gave black colonies with a rough surface against red colonies with a smooth
surface for non-producing strains. Variable phenotype strains gave colonies with a black center and red outline, or red center and black outline were considered as positive slime producers [20]. 2.3. Semi-quantitative adherence assay Biofilm production by S. aureus strains grown in BHI (Biorad, France) was determined using a semi-quantitative adherence assay on 96-well tissue culture plates (Nunc, Roskilde, Denmark) as described previously [21,22]. Adherent bacteria were fixed with 95% ethanol and stained with 100 mL of 1% crystal violet (Merck, France) for 5 min. The microplates were air-dried and the optical density of each well was measured at 570 nm (OD570) using an automated Multiskan reader (GIO. DE VITA E C, ome, Italy). Biofilm formation was interpreted as highly positive (OD570 1), low grade positive (0.1 OD570 < 1), or negative (OD570 < 0.1). 2.4. Biofilm formation on biomaterials 2.4.1. Preparation of strips In the present study, the denture base materials used is the bisphenol A glycidyl methacrylate (Bis-GMA) composite resins and the polymethyl methacrylate acrylic (PMMA). The Bis-GMA and PMMA strips were made according to the method used in the laboratory of Biomaterials and Biotechnology in the Faculty of Dentistry of Monastir (Tunisia). All strips were cut into 1 cm2 squares. They were disinfected by dipping in 70% alcohol for 30 min and washed with sterile distilled water. They were then ultrasonicated for 20 min to remove any contaminants and artifacts from the surfaces, washed again in sterile distilled water, dried and used for the biofilm assay. 2.4.2. MTT metabolic assay The composite disks were placed in a 24-well plate, inoculated with 1.5 mL of the inoculation medium, and cultured for 24 h. Each disk was transferred to a new 24-well plate for the MTT (3-(4,5dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay. It is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan [23]. This kind of indirect viability assay is based on the formation of insoluble purple formazan due to the reduction of MTT by (respiratory) reductases of living staphylococcal cells [24]. Briefly, 1 mL of MTT dye (0.5 mg/ml MTT in PBS) was added to each well and incubated at 37 C for 1 h. After 1 h, the disks were transferred to a new 24-well plate, 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals, and the plate was incubated for 20 min with gentle mixing at room temperature in the dark. A total of 200 mL of the DMSO solution from each well was transferred to a 96-well plate, and the absorbance at 540 nm (optical density OD540) was measured via a microplate reader (GIO. DE VITA E C, ome, Italy). A higher absorbance indicates a higher formazan concentration, which in turn indicates more metabolic activity in the biofilm on the composite. 2.4.3. Biofilm visualization by atomic force microscopy (AFM) To visualize the biofilm formed on Bis-GMA and PMMA surfaces and to study the morphological changes in the cells during biofilm production, S. aureus ATCC 6538 was used as a positive control. After biofilm formation on strips, the surfaces were fixed on the round cover slide and were examined by AFM [25]. 2.5. Characterization of the enzymatic activity The activities of various enzymes were determined after inoculation of cultures onto TSA-1 to which the following substrates had been added: 1% [wt/vol] skim milk for caseinase, 1% [wt/vol]
Please cite this article in press as: Meghrni A, et al., Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.05.002
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formation were examined by the Friedman test. P-values of <0.05 were considered significant.
Table 1 Primers used in this study. Primer
Sequence (50 / 30 )
Product size (pb)
Annealing T C
sspA-F sspA-R sspB-F sspB-R geh-F geh-R hlb-F hlb-R
GAC AAC AGC GAC ACT TGT GA AGT ATC TTT ACC TAC AAC TAC A TGA AGA AGA TGG CAA AGT TAG TTG AGA TAC ACT TTG TGC AAG GCACAAGCCTCGG GACGGGGGTGTAG GGTGCACTTACTGAC CGCATATACATCCCATGGC
292
45
493
47
473
40
854
47
3. Results 3.1. Phenotypic determination of slime production From all samples included in our study 21 S. aureus were isolated, among them 19 strains (90.5%) were slime producers developing positive and variable phenotype on the CRA plate. The two other strains showed a negative phenotype (9.5%) (Table 2). 3.2. Polystyrene adherence assay
gelatin for gelatinase, 1% Tween 80 for lipase, 5% [vol/vol] egg yolk for phospholipase (lecithinase). Elastase activity was evaluated by using 1% (w/v) soluble elastin (Fluka Chemical, France) in Columbia agar base as described by Williams et al. [26]. The hemolytic activity was evaluated by plating isolated strains on bacteriological agar supplemented with 5% sheep’s blood for alpha and beta-hemolysin production [27].
The result of OD570 presented in Table 2, showed that five strains (9P, 9S, 9D37, 9L37, C54) isolated from oral infections were highly biofilm positive (OD570 1), the other strains showed a low grade biofilm formation (0.1 OD570 < 1). 3.3. Biofilm formation on biomaterials
2.6. Detection of sspA, sspB, geh, and hlb genes
The metabolic activity of S. aureus biofilms was determined by the ability of the attached cells to reduce the MTT to the formazan dye. We found that there are no significant differences between biofilm formation on Bis-GMA and on PMMA surfaces (P > 005) (Table 2). Biofilm visualization was performed by AFM. Atomic force micrographs revealed the presence of cocci cells arranged in clusters which are a typical morphology of S. aureus (Fig. 1a,b and c,d). The bacterial surface was rough in texture from the both strips. Furthermore, cells have indistinct borders on both surfaces (BisGMA and PMMA) indicating the presence of extracellular polysaccharidic materials (Fig. 1).
Genomic DNA used for polymerase chain reaction (PCR) was extracted by using a standard phenolechloroform technique. The presence of protease, lipase and b-hemolysin genes in S. aureus strains was detected by PCR using the primers listed in Table 1. Amplifications were performed according the following cycle conditions: for all genes an initial denaturation at 94 C for 10 min was followed by 30 cycles of denaturation at 94 C for 1 min, annealing for 1 min at temperature determined for each gene as described in Table 1 and elongation at 72 C for 1 min, followed by 10 min of final extension at 72 C.
3.4. Production of hydrolytic enzymes 2.7. Statistical analysis In this study, all S. aureus tested strains were positive for Caseinase Gelatinase and Elastase. Lecithinase activity was detected in 20 strains (95%) and we noted that among the 22 S. aureus
Statistical analysis was performed using the SPSS 17.0 statistics package for Windows. The differences in the degree of biofilm
Table 2 Slime production, adherence assay and biofilm formation capacity of oral S. aureus strains. Strains
Clinical details
Biofilm phenotype (CRA)
OD570 SD
Adherence state
Biofilm formation (MTT reduction SD) Bis-GMA
ATCC 6538 9P 9S L36 D36 L37 45S Pg Cjj Cjb PPb PPj C54 C59 C60 C87 C91 C92 C98 99F 99P C102 a
_ Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dento-alveolar Dental caries Dental caries Dental caries Dental caries Dental caries Dental caries Dental caries Dental fistula Dental fistula Dental caries
a
abscess abscess abscess abscess abscess abscess abscess abscess abscess abscess abscess
Variable phenotype Variable phenotype Variable phenotype Negative phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Negative phenotype Positive phenotype Positive phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype Variable phenotype
2.90 2.47 1.69 0.68 1.41 2.02 0.65 0.97 0.68 0.84 0.37 0.18 1.54 0.51 0.23 0.76 0.76 0.19 0.17 0.22 0.26 0.42
0.055 0.020 0.086 0.015 0.053 0.096 0.009 0.065 0.020 0.040 0.013 0.05 0.008 0.057 0.063 0.071 0.026 0.031 0.017 0.099 0.014 0.053
Highly positive Highly positive Highly positive Low grade positive Highly positive Highly positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive Highly positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive Low grade positive
0.249 0.292 0.299 0.262 0.275 0.276 0.220 0.217 0.209 0.217 0.218 0.211 0.247 0.211 0.249 0.205 0.240 0.245 0.265 0.233 0.233 0.264
0.0124 0.0065 0.0091 0.0024 0.0082 0.0256 0.0059 0.0060 0.0017 0.0159 0.0058 0.0033 0.0065 0.0004 0.0072 0.0033 0.0063 0.035 0.0093 0.0056 0.0033 0.0060
PMMA 0.358 0.210 0.305 0.256 0.265 0.245 0.204 0.208 0.232 0.237 0.242 0.180 0.210 0.206 0.209 0.238 0.235 0.2561 0.262 0.237 0.245 0.225
0.0233 0.0024 0.0058 0.0045 0.0044 0.0133 0.0124 0.0025 0.0020 0.0158 0.0053 0.0085 0.0053 0.0063 0.0060 0.0051 0.0032 0.0194 0.0177 0.0149 0.0192 0.0100
Strains presenting a variable phenotype were considered as positive slime producers.
Please cite this article in press as: Meghrni A, et al., Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.05.002
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Fig. 1. AFM 2D topography (left panel) and the corresponding 3D reconstructions (right panel) images of S. aureus cells adhered on Bis-GMA (a and b, scan area: 3 mm 3 mm) and PMMA surfaces (c and d, scan area: 3 mm 3 mm).
strains, 17 were phospholipase producers (77%). 12 out of 22 tested strains were beta hemolytic (59%) (Table 3). 3.5. Detection by PCR of sspA, sspB, geh and hlb genes Among the tested strains, 17 (81%) were positive for sspA and sspB. Our result revealed that 10 S. aureus isolated from oral infections harbor the geh gene (48%). The hlb gene encoding the btoxin was detected in 17 strains (81%) (Table 3). 4. Discussion This work represents the first attempt to study the adhesion ability and the enzymatic activity of oral S. aureus strains isolated from patients attending the Dental Hospital of Monastir, Tunisia during years 2012 and 2013. In this investigation we noted that patients suffering from oral infections were diabetics (18%), smokers (39%), or demonstrated poor oral hygiene (53%). Numerous risk factors have been reported that make patients with diabetes more susceptible to oral disease, especially those with
poor oral hygiene and who are smokers [28]. The potential role of the oral cavity as a reservoir of staphylococci has been underlined by previous studies [3]. Our study revealed the detection of 21 S. aureus strains isolated from the oral cavity of 105 Tunisian patients suffering from oral infections. The detection of this bacterium was in agreement with the result reported previously by Smith et al. [29] who showed the presence of S. aureus in saliva and from gingival swabs of patients with a range of oral diseases. Furthermore, in other reports methicillin-resistant S. aureus (MRSA) strains have been isolated from oral cavity [30,31]. In addition, S. aureus has been reported to be isolated from 24 to 36% of healthy oral cavities [32,33]. And more recently, Ohara et al. [9] showed the occurrence of this bacterium (46.4%) in plaque and saliva from healthy adults. Therefore, the presence of S. aureus in the oral cavity may be more frequent than previously thought. Once S. aureus adheres and forms biofilms to host tissues or prosthetic materials, it is able to grow and persist in various ways [34]. Slime production might be a virulence factor and simultaneously worsens the response to infection, protecting bacterial cells from the host’s natural defense mechanisms and from antibiotics
Please cite this article in press as: Meghrni A, et al., Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.05.002
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Table 3 Hydrolytic enzymes production and presence of S. aureus genes. Strains
ATCC 6538 9P 9S L36 D36 L37 45S Pg Cjj Cjb PP C54 C59 C60 C87 C91 C92 C98 99F 99P C102 % expression
Exoenzymes expression
Presence of genes
Lecithinase
Lipase
Caseinase
Gelatinase
Elastase
Hemolysin
geh
sspA
sspB
hlb
þ þ þ þ þ þ þ þ þ þ þ e þ þ þ þ þ þ þ þ þ 95
þ þ þ þ þ þ e e þ þ e e þ þ þ þ þ þ þ þ þ 77
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ 100
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ 100
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ 100
b b b b b b a a a a a a b b b b a b a a b
gehþ geh gehþ geh geh geh gehþ gehþ geh geh geh geh gehþ gehþ gehþ gehþ geh geh gehþ gehþ geh 48
sspAþ sspAþ sspAþ sspA sspAþ sspAþ sspAþ sspA sspAþ sspAþ sspAþ sspA sspAþ sspAþ sspAþ sspAþ sspA sspAþ sspAþ sspAþ sspAþ 81
sspB sspBþ sspBþ sspB sspBþ sspBþ sspBþ sspBþ sspBþ sspBþ sspB sspB sspBþ sspBþ sspBþ sspBþ sspBþ sspBþ sspBþ sspBþ sspBþ 81
hlbþ hlbþ hlbþ hlbþ hlbþ hlbþ hlb hlbþ hlb hlbþ hlbþ hlbþ hlb hlbþ hlbþ hlb hlbþ hlbþ hlbþ hlbþ hlbþ 81
activities [35]. In our study we found that 20 out of 22 strains of S. aureus (90.9%) were slime producers. In a recent study, Kouidhi et al. [36] demonstrated that 11 of 22 S. aureus associated to dental caries were slime positive. Furthermore, Zmantar et al. [37] found that S. aureus strains isolated from patients with an auricular infection were slime producers. Semi-quantitative adherence of S. aureus tested on polystyrene microplate showed that five strains isolated from oral infections were highly biofilm positive. The remaining strains showed low-grade biofilm formation. These results suggest a difference in adhesion ability between tested strains. In fact, variations in biofilm forming capacities of S. aureus could be explained by surface associated proteins and regulatory genes involved in biofilm production [38]. We noted a positive correlation between the slime-producing ability on CRA plates and the adhesion ability developed on polystyrene material. Likewise, previous study showed a high consistency between CRA screening and biofilm biomass crystal violet staining [39,40]. Nevertheless, other rapports showed that no correlation between slime-producing MRSA and methicillin susceptible S. aureus (MSSA) isolates and an enhanced tendency of biofilm formation [41,42]. In our study, we also determined the adhesion capacity of the oral S. aureus strains to orthodontic biomaterials Bis-GMA and PMMA. It was the first investigation in Tunisia to study biofilm formation of this bacterium to abiotic surfaces commonly used in dentistry practices. Our results demonstrated that the metabolic activity of S. aureus biofilm formed on Bis-GMA and PMMA surfaces did not differ among tested strains (P > 0.05). Otherwise, the capacity of S. aureus to adhere to indwelling medical devices was revealed in several studies [43]. S. aureus is one of the prominent gram positive human pathogen secretes many surface and secretary proteins including various enzymes and pathogenic factors that favor the successful colonization and infection of host tissue [44]. The production of S. aureus enzymes such as proteases, lipases, nucleases, and collagenases convert tissue components into nutrients, facilitating bacterial growth and invasion [45]. Toxins and enterotoxins have other effects such as superantigenicity, pyrogenicity and direct damage to endothelial [46]. The determination of hydrolytic enzymes production revealed that 95% of S. aureus strains were lecithinase positive, and 75% were lipase producer. Barretti et al. [35] found
59(b); 41(a)
that 97.1% of S. aureus isolated from peritonitis were both lecithinase and lipase positive, while Saising et al. [47] suggest that only 65.6% of the strains isolates from acne lesions were lipase positive. During infection, S. aureus produces numerous enzymes that enable it to invade and destroy host tissues and metastasize to other sites. In our work all tested strains have been found producers of caseinase gelatinase and elastase. Other studies reported that of S. aureus implicated in skin infections exhibited protease activity [48]. Furthermore, Wu et al. [49] showed that S. aureus isolated from corneal ulcers produced caseinase (100%), gelatinase (80%) and elastase (70%). Moreover, this bacterium is known to produce tow enzymes serine protease (V8 protease; SspA), and cysteine protease (SspB), which are implicated in virulence. In our study we showed that 81% of isolated strains are sspA and sspB positive. Other study revealed that S. aureus isolated from food samples harboring sspA and sspB genes [50]. Thus, the presence of extracellular protease encoding genes might sign the infectious abilities of these strains in humans. The extracellular proteases could help the invasion of bacteria through tissues during infection process [51], enable it to interact with the host defense mechanisms. In addition to enzyme production, S. aureus secrets a b-toxin as a neutral sphingomyelinase that lyses erythrocytes in order to evade the host immune system as well as scavenge nutrients [52]. This hemolysin is secreted by certain strains of S. aureus especially strains isolated from corneal infection [53]. While, in our study we demonstrated that this type of toxin is produced by 59% of oral S. aureus strains. 81% of our isolates are hlb positive which harbor the beta toxin gene but its expression may be influenced by regulatory genes.
5. Conclusion In conclusion, several reports highlight the presence of S. aureus in oral cavity, however this study is one of few reports dealing with the characterization of virulence factors in such isolates. According to our finding, oral S. aureus strains exhibits a considerable adhesion capacity to biomaterials and were producers of a variety of hydrolytic enzymes. All these factors contribute to the colonization of oral cavity by this bacterium.
Please cite this article in press as: Meghrni A, et al., Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.05.002
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Please cite this article in press as: Meghrni A, et al., Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity, Microbial Pathogenesis (2014), http://dx.doi.org/10.1016/j.micpath.2014.05.002
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