The Ciliate Protist Tetrahymena pyriformis as a Cellular Adhesion Model for the Pathogenic Bacterium Staphylococcus aureus

Staphylococcus aureus is one of the main pathogenic agents responsible for nosocomial and community-acquired bacterial infections. The pathogenicity of this Gram-positive bacterium is ensured by its different adhesion factors. Collagen and the extracellular glycoprotein adhesin are among the Staphylococcus most important virulence factors. It has been shown that most of the S. aureus strains carry the ica operon, responsible for biofilm production. However, the coexpression of the icaA and the icaD genes is necessary for complete biofilm synthesis. The aim of our study was to study a collection of 15 clinical strains of S. aureus from different sources for the presence of cna and icaD genes coding intercellular adhesion proteins. We also intended to estimate the strains’ ability to form biofilms by the red Cong method and to test the adhesion ability of S. aureus to the ciliated protist Tetrahymena pyriformis, which we used as a novel cellular adhesion model. Finally, we checked the adhesion’s inhibition capacity of some plants extracts. The molecular detection of adhesion genes revealed that 80% of strains are cna positive, and 73% are icaD positive. Qualitative biofilm production of S. aureus revealed that 66.6% of strains were slime producers. The adhesion test revealed that 20% of strains are strongly adhering to T. pyriformis and that the Clematis cirrhosa extract has an anti-adhering effect of S. aureus to the ciliate T. pyriformis.


Introduction
Staphylococcus (from the Greek: σταφυλή, staphylē, "grape" and κόκκος, kókkos, "granule") is a genus of Gram-positive bacteria in shells shape, without flagellum, which has a cellular envelope made of a unique plasma membrane, surrounded by a relatively thick cell wall [1] [2].Staphylococcus strains are able to grow in aerobic or anaerobic conditions.They are ubiquitous species: having the ability to live in soil, water or diverse animal tissues [3] [4].
Among the Staphylococci, there are three main human pathogenic species: S. aureus, S. epidermidis and S. saprophyticus.Staphylococcus aureus is usually called golden Staphylococcus because of the yellow colored colonies that it forms on the agar.As described in 1881 by Alexander Ogston, it is a Gram-positive spherical bacterium, optional anaerobic, immobile and making regular clusters like bunch of grapes of 0.5 to 1.5 µm.It has the most important pathogenicity potential among the Staphylococci, and is the only strain able to produce coagulase, an exoenzyme able to coagulate blood plasma, which allows Staphylococcus fast and easy identification.It is also one of the main causes of nosocomial and community-acquired infections.Most often, these infections involve biofilms, which are multicellular communities omnipresent in natural, industrial and medical areas and can affect human health [3] [4].
S. aureus infection pathogenicity is related to its ability to colonize host tissues, to proliferate and to evade the immune defense system of the host thanks to specific virulence factors (adhesion factors, exoenzymes and toxin production) [5] [6].
Bacterial adhesion to host cells or surfaces is often an essential first stage in pathogenic mechanisms.S. aureus has a large number of surface proteins called adhesins, belonging to the MSCRAMMs class (Microbial Surface Components Recognizing Adhesive Matrix Molecules) [7] [8] that enable specific adhesion to components of the host tissue, namely, the fibrinogen binding protein (Clumping factor A or ClfA), the fibronectin binding proteins FnBPA and FnBPB (fibronectin-binding protein A and B) [9] [10], the collagen binding protein (collagen adhesin or Cna) and the elastin binding protein (EbpS) [8].Other surface proteins also described in S. aureus, such as Eap (extracellular adherence protein) [11], Ebh (extracellular matrix binding homologue protein) [12] and Emp (extracellular matrix binding protein) [13] may have a role in colonization.
Biofilms formation is the outcome of a set of physical, chemical and biological processes.According to the model suggested by Mack et al. [14], it consists of two phases: initial attachment and accumulation.The first phase is the initial attachment or adhesion of cells to a solid support, this support can either be the host tissue (skin, epithelium…), materials used in medical field (catheters, prosthesis•••) or a food industry support (cutting surface, floors, walls…).The initial adhesion is the result of non covalent physicochemical interactions between support and bacteria such as Van der Waals forces, electrostatic forces, Lewis acid-base properties and hydrophobic/hydrophilic properties [15] [16].Adhe- sion is also affected by components of the bacterial cell wall such as teichoic acids and surface proteins (adhesins and autolysins identified in S. aureus and S. epidermidis) [17] [18].
The second phase is the intercellular aggregation, which leads to micro colonies formation.This step includes cell division and exopolysaccharides production and leads to a mature biofilm establishment.Among the responsible factors for intercellular aggregation, there are three main surface compounds such as: polysaccharide intercellular adhesion (PIA) described in S. epidermidis, S. aureus and S. caprae [14] [19] [20], Accumulation Associated Protein (AAP) described in S. epidermidis [21] and the Biofilm Associated Protein (BAP) originally described in bovine S. aureus strains [22].Biofilm maturation is sometimes followed by a detachment phase that allows bacteria to colonize other sites.This phenomenon has been slightly studied in Staphylococci.In S. epidermidis, this phase has been studied using electric currents or enzymes that would reverse the establishment of biofilms on catheters, causing thereby the detachment [23].In S. aureus, the biofilm viscoelasticity allows resistance to detachment caused by mechanical stress or surrounding flux [24].In addition, this viscoelasticity leads to rolling phenomenon in micro-colonies that allows them to migrate to other sites.Stoodley et al. showed that cells spontaneous removal is divided into two processes: erosion and sloughing [25].Erosion is a continual detachment of single cells and small cell aggregates while the sloughing is the rapid and massive loss of biofilm.Erosion occurs during the whole biofilm maturation period whereas sloughing occurs after a nutritional deficiency [26] [27].The detachment step seems to be genetically programmed, and would facilitates the propagation of the infection and/or the colonization of other sites [28] [29] [30] [31].
In the current study, we screened the presence of cna and icaD genes coding for adhesion proteins in 15 clinical S. aureus strains and assessed their ability to produce biofilms.We also tested, for the first time, the adhesion ability of S. aureus to ciliated protist Tetrahymena pyriformis, a well-known model organism in biomedical research with a cellular architecture similar to human cell [32], and finally evaluated the anti-adhesion effect of some plant extracts.

Staphylococcus aureus
Fifteen Staphylococcus aureus bacterial strains of different origins have been studied, including a resistant strain to methicillin (SARM).These strains were provided by the molecular bacteriology laboratory of the Pasteur Institute of Morocco (IPM) and the bacteriology department of the Ibn Rochd hospital of Casablanca (Morocco).
Identification of the 15 strains of S. aureus was performed using primers specific for the AF gene, which is specific to the identification of Staphylococcus aureus.Advances in Bioscience and Biotechnology The reference strain Staphylococcus aureus ATCC 25923, which was provided by the Laboratory of Analysis, Treatment and Valorization of Environmental and Products Pollutants, Faculty of Pharmacy, Monastir (Tunisia) was used as a control.
Culture of Staphylococcus aureus was performed on Nutrient Broth (NB) medium, and/or brain heart infusion broth (BHI) and incubated at 37˚C.

2) Plant Extracts
Plant extracts were prepared from the aerial part (stem + leaf) of Pisenlit (Taraxacum officinale), Clematis cirrhosa, Mesembryanthemum crystallinum and Rubia pergrina, and stored at 4˚C in the National Institute of Medicinal and Aromatic Plants, Taounate, Morocco.
3) Bacterial DNA Extraction Colonies of S. aureus were scraped using an inoculation loop and mixed with 200 μl of distilled water in a conical centrifuge tube under sterile conditions.Tubes were placed in a water bath for 10 min at 100˚C, then immediately placed on ice for 5 min (heat shock), and centrifuged at 12,000 rpm for 10 min at 4˚C.The supernatant representing the DNA was collected in sterile Eppendorf tubes of 1.5 ml which were stored at −20˚C.

4) Polymerase Chain Reaction
Molecular detection of AF, icaD and can genes was performed with PCR using the primers shown in Table 1.For all the PCR experiments, reaction mixtures contained 10 µM of each forward and reverse primer, 25 mM of MgCl 2 , 5× flexi buffer Promega, 10 µM of dNTPs and 5 U/μl of Taq DNA polymerase.The PCR program used for strains identification was: 1 cycle of 3 min at 96˚C, 30 cycles (30 sec at 95˚C, 30 sec at 52˚C and 1 min at 72˚C) and a final elongation step of 10 min at 7˚C.For cna and icaD genes detection, we used the following PCR program: 1 cycle of 5 minutes at 94˚C, 30 cycles (30 sec at 94˚C, 30 sec at 57˚C (for the cna gene, 55˚C for the icaD gene) and 1 min at 72˚C) and a final elongation step of 10 min at 72˚C.Using the following protocol, we aimed to study the adhesion capacity of Staphylococcus aureus to the ciliated protist T. pyriformis cells.Seventeen cover slips were prepared: The first one served as a control in which the protist cells were incubated alone, in the second one T. pyriformis cells were incubated with Pseudomonas syringae and for the 15 remaining cover slips, the protist cells were incubated with the 15 strains of Staphylococcus aureus.Then, each cover slip was placed in a sterile Petri box and 100 μl of an exponential phase culture of T. pyriformis were added.Afterward, using a toothpick, 30 colonies of each bacterial culture were took from the gelose medium, spread over the entire cover slip and incubated for 2 h at 28˚C.Subsequently, the cover slips were fixed with 1 ml of methanol for 20 min, stained with 1 ml of Giemsa solution for 20 min and washed 2 -3 times with 1 ml of PBS.After drying, cover slips were placed on slides.Finally, a drop of immersion oil was added and preparations were observed on light microscope.The experiment was carried out in triplicate.

Study of the antibacterial effect of plant extracts
The antibacterial effect of Pisenlit, Clematis cirrhosa, Mesembryenthemum hallinum and Rubia peregrina plant extracts on S. aureus was determined by the well diffusion technique on solid medium.The antimicrobial activity is determined in terms of the inhibition zone diameter generated around the wells.
Three boxes were used, the first one contained only the culture medium (negative control), the second one contained the bacterial culture without plant extracts (positive control) and the third box contained bacterial suspension incubated with plant extracts.The experiment was carried out in triplicate for each plant extract.

Effects of plant extracts on bacterial adhesion
To study the anti-adhesion effect, 30 colonies of bacteria were incubated with T. pyriformis cells and plant extracts, as described above (Section 6. Adhesion test of Staphylococcus aureus to Tetrahymena pyriformis cells).50 μl of one of the analyzed plant extracts (Pisenlit, Clematis cirrhosa, Mesembryentemum hallinum, and Rubia pergrina) were added to each coverslip before 2 h of incubation.The same steps of adhesion test were followed and the result was observed using a light microscope.The experiment was carried out in triplicate for each plant extract.

Results and Discussion
Molecular identification of S. aureus strains In this study, all the fifteen clinical bacterial strains collected were identified by PCR using the AF gene.The results were revealed by electrophoresis on 1% agarose gel.The Figure 1 is a demonstrative profile representing the result of a set of selected clinical strains.
Thereby as shown in Figure 1, all strains presented the expected DNA fragment of 108 bp, which is in line with the microbiological identification (results not shown), indicating that all clinical strains belong to the Staphylococcus aureus species.
Molecular detection of cna and icaD genes The cna gene PCR amplification of the cna gene encoding the collagen-binding adhesin protein was performed for all Staphylococcus aureus clinical strains using genomic DNA as a template, and the results were revealed on 1% agarose gels.Figure 2 shows an example of some selected strains.
Based on the results of PCR amplifications, out of the fifteen clinical strains available, twelve Staphylococcus aureus strains (80%) carried the cna gene as indicated by the amplified DNA fragment of 192 bp (Figure 2), while only three strains did not possess this gene.
According to literature, the staphylococcal collagen-binding adhesion has been described as one of the most important virulence factor proteins in the infection  pathogenesis of this bacterium [33].[34] showed that cna gene is carried by 56.5% of the tested S. aureus strains while Nizami Duran et al. [35] found cna gene presence in 78.4% of the staphylococcal strains.However, Peacock et al. [36] underlined that the prevalence of cna-positive strains was 52% while Aricola et al. [37] showed that cna was carried by 46% of S. aureus strains.Furthermore, Tristan et al. [38] and Rohdet et al. [39] described the presence of cna gene in 36% and 22% of the strains respectively.
The icaD gene PCR amplification of the icaD gene, which is a member of the ica operon encoding the proteins that synthesize the polysaccharide intercellular adhesin (PIA), was obtained for most tested Staphylococcus aureus clinical strains (Figure 3).
Eleven among fifteen Staphylococcus aureus strains (73%) presented the icaD gene, as revealed by the amplified band on agarose gel which corresponds to a DNA fragment of 198 bp (Figure 3).The remaining four strains did not show the highlighted gene.

Phenotypic determination of biofilm production
Slime production by Staphylococcus aureus clinical strains was assessed using Congo red agar technique; the results were interpreted according to what has been reported in the literature [37] [40].Strains forming biofilms presented crystalline dry colonies, in black, dark black or slightly black color.While those not forming biofilm developed pink, red or bordeaux colonies and sometimes can present red colonies with a black dot in the middle known as "bulls eye".
We found that ten of the Staphylococcus aureus strains analyzed in this study (66.6%; including reference strain) were biofilm producing strains, giving dry crystalline black, slightly black or dark black colonies (Figure 4), this difference in coloration intensity can be attributed to the different metabolic pathways incurred by S. aureus for biofilm formation [41].
These strains demonstrated an intense biofilm production, indicating a high enzymatic activity of polysaccharide intercellular adhesin (PIA), while the remaining five clinical strains (33.3%) were classified as non-biofilm forming strains, and developed pink, red or bordeaux colored colonies (Figure 4).
Our results are close to those obtained by Arciola et al. [42] who reported 61% of icaA and icaD genes holders among S. aureus strains.While Tarek Zmantar et al. [43] found that 78.26% of the tested strains were icaA and icaD positive.
We also found that nine icaD positive strains (including the reference strain) were characterized as slime forming, whereas two strains were positive for this gene but were not producing biofilm.These results are inconsistent with those described by Arciola et al. [42] who found that icaA and icaD genes were only detected in slime forming strains.These differences may be caused by environmental conditions [44] [45].In the case of many biofilm-forming bacteria, differentiation from planktonic state to sessile state is associated to environmental stress factors [29].However, biofilm formation by Staphylococcus is subject to complex regulation influenced by a number of environ- mental factors including osmolarity, glucose, anaerobiosis and temperature [46] [47].
On the other hand, Gundogan et al. [48] found that 58 out of 110 (52.7%) S. aureus strains were slime producing; furthermore, Vasudevan et al. [49] underlined that 32 out of 35 (91.42%) S. aureus strains were slime positive after 24 -48 h of incubation, indicating that S. aureus biofilm production depends on the incubation time.
Staphylococcus aureus is able to adhere and form biofilms and therefore cause severe infections [6].This pathogen has the ability to produce a number of exoenzymes, some of them are involved in virulence [50].PIA production is responsible for the staphylococcal biofilm development [43].Biofilm formation is considered as a two-step process that requires the adhesion of bacteria to a surface or substrate followed by a cell-cell adhesion, forming the multiple layers of the biofilm [18].It was shown by several authors that most of S. aureus strains have the entire ica operon [20] [51].Thereby, the single icaA expression induces only low enzyme activity, whereas the co-expression of icaA and icaD leads to a significant increase of activity and is related to the phenotypic expression of capsular polysaccharide PIA [52].

S. aureus Adhesion Test to Tetrahymena pyriformis cells
Adhesion is an essential step in the development of the infectious process.It is recognized that, in order to adhere to human tissue surfaces, a bacterium must first adhere to appropriate host cells or to their extracellular matrix, in order to withstand the various mechanisms that may eliminate it [53].Advances in Bioscience and Biotechnology Different human cell lines were used to study the phenomenon of bacterial adhesion but most of them are very expensive and are likely to be contaminated during subcultures.For this reason, we chose to use the ciliated protist Tetrahymena pyriformis as a more suitable host to which S. aureus may adhere.
Comparative analysis of the obtained results showed that among the fifteen bacterial strains studied, three of them {Sa67, Sa99 and Sa114} (20%) showed a strong adhesion to protist cells, six {Sa65, Sa116, Sa122, SARM, Ria and the reference strain ATCC25923} (40%) had a moderate adhesion capacity, and the remaining six strains (40%) presented poor adhesion to the protist cells (Figure 5).
The capacity of S. aureus to adhere to host cells is considered the first step towards colonization and then cell infection.It is generally accepted that the ability of the bacteria to adhere to the host cells surface is an important factor in the initial interaction between S. aureus and its host [54] [55] [56].

Anti-adhesion effect of some plant extracts
The antibacterial effect of a number of plant extracts on S. aureus was first studied in order to search for the optimal concentration of each extract for which bacterial adhesion is inhibited without stressing the protist.For this purpose, the well diffusion technique was used.The obtained results are shown in Table 2.The well diffusion technique revealed that all the tested plant extracts (Pisenlit, C. cirrhosa, M. crystallinum and R. peregrina) exhibit an antibacterial effect.Interestingly, the R. peregrina extract produced a growth inhibition zone of 1 cm and showed the strongest effect, followed by C. cirrhosa and M. crystallinum with an inhibition zone of 0.8 cm.Thereby, R. peregrina and C. cirrhosa extracts were selected for further tests.
Our results are consistent with a study on the antibacterial effect of R. peregrina, which showed that the ethyl acetate and chloroform fractions of the ex- tract are effective against S. aureus and Escherichia coli, respectively [57].The anti-adhesion effects of R. peregrina and C. cirrhosa extracts were assessed and the results are shown in Figure 6.
Our results clearly show that both plant extracts exhibit an anti-adhesion effect.However, it seems that C. cirrhosa features a better effect on S. aureus adhesion-inhibition without stressing T. pyriformis cells.
The results obtained with the S. aureus strains on the relationships between the presence of genes encoding adhesion proteins, biofilm formation phenotypes and adhesion tests with Tetrahymena pyriformis are summarized in Table 3.

Conclusions
In conclusion, our study confirms variations in the presence and expression of genes encoding important adhesion proteins among clinical S. aureus strains.
Slime production is also subjected to strain selection and can be affected by the environmental conditions.
In addition, we suggest that our study on S. aureus adhesion to Tetrahymena pyriformis cells may be considered as a first step in understanding the estab- lishment of different adhesion mechanisms in the pathogenesis of medical device-associated staphylococcal infections.Furthermore, the anti-adhesion effect of plant extracts could be relevant in the development of new preventive and therapeutic approaches against staphylococcal infections.

Figure 5 .
Figure 5. Adhesion test of Staphylococcus aureus to Tetrahymena pyriformis cells observed under light microscope (magnification, ×100).(a) Control; (b) S. aureus strains with low adhesion capacity; (c) Strains with moderate adhesion capacity; (d) and (e) Strains with strong adhesion capacity.

Table 1 .
Primers sequences and PCR conditions.

Table 2 .
Antibacterial effect of plant extracts on the S. aureus strain "114" in solid medium.The antibacterial effect was determined by measuring the diameter of the growth inhibition zone.50 µl of plant extract were poured on the wells.

Table 3 .
Summary of S. aureus strains features showing the relationships between presence of cna and icaD genes, biofilm formation and adhesion tests with Tetrahymena pyriformis.