Biofilm Formation by Staphylococcus Aureus Isolation from Atopic Dermatitis Patients on Defined Lewis Types Saliva

Bacteria adherence and biofilm formation vary depending on strain of microorganism as well as the substrate .Here ,we evaluated the ability of different isolates of Staphylococcus aureus (S. aureus) ,isolated. from atopic dermatitis patients skin and stool to form biofilm on different Lewis types saliva ,including Le (a) , (b) ,(c) and Le a+b+ saliva. S. aureus isolates were cultured on tryptose soy broth .The bacteria were used to form biofilm on 96well flat bottom Microtitration plate that was coated with the respective , optimally diluted,processed saliva of Lewis blood groups (a),(b),(c) and Le a+b+ .The intensity of biofilm formation was assessed by measuring the biofilm – associated optical density reading of bacterial bound dye. The results indicated a superior ability of biofilm formation on Lewis(a) saliva type .The highest binding gave a mean of 1.25 ± 0.39 compared to biofilm formation on non – coated wells that showed a mean of 0.23 ± 0.016(P=0.01 , CL= 0.390 – 1.635). On the other hand however, a variable degree of binding to Le(b) saliva was demonstrated for the isolates. The isolates didn’t demonstrate binding on Lea-b-(Lec) and Le a+b+ -(Led) saliva. We conclude that biofilm formation on Lewis types saliva of the atopic dermatitis bacterial isolates requires Le(a) in a ligand – receptor based system for biofilm establishment.


-Introduction
Biofilm is a microbial structure that causes numerous issues in restorative field, in nour-ishment industry and even to nature.Along these lines, biofilm could cause numerous many public health problems.In reality, almost 80% of constant bacterial infections are related with the presence of biofilm [1].
Staphylococcus aureus (S. aureus) is regular pathogen related with different diseases and biofilm of the bacterium was observed to be associated with an assortment of microbial infection and to expand harmfulness [2] [3] [4]. S. aureus biofilm is made out of three noteworthy segments,the polysaccharide poly-N-acetylglucosamine (PNAG), otherwise called polysaccharide intercellular attachment (PIA), cell surface and emitted bacterial proteins, and extracel-lular nucleic acid, which shape extracellular polymeric substances [5].Atopic dermatitis (AD) is an interminable fiery skin illness typically beginning in early youth with a revealed lifetime commonness of 15 -30% in youngsters, while the com-paring figure for adults is 2-10%.[6].Staphylococcus aureus is accounted for to rule in AD injuries and reports have uncovered the nearness of Staphylo-coccal biofilms.These infections add to irritation of the eczema [7].Nonetheless, the part of S. aureus in the pathogenesis of AD remains ineffectively poorly understood [8].
Biofilm formation on synthetic saliva was demonstrated for mixed Candida albicans and Streptococcus gordonii [9], S. aureus predominate in the skin of atopic dermatitis patients [10].
Lewis blood group molecules expressed in a variety of surfaces [11] and their role in bacterial colonization and biofilm formation is well de-fined [12] [13] [14].In this work it was tried to explore the role of Lewis blood groups determinants carried on salivary mucins on biofilm formation of S. aureus isolates from atopic dermatitis patients.

-Bacterial isolates
The isolates used in this study included (9) isolates from skin and one isolate from stool of atopic dermatitis patients.They were maintained on Tryptose soy slants .Bacteria were grown on tryptose soy broth at 37 • C for 24 hours and used in biofilm formation.

-Saliva coating
Saliva of typed Lewis blood groups were used .Lewis typing was performed as described [15] (Ahmed et al.,2009).The saliva obtained were diluted 1:2 with saline and boiled at 100 • C for 10minutes , centrifuged at 4000 r.p.m and the supernatant was used as a source of saliva .Saliva were stored at -20 • C until used.
To find optimal dilution of saliva coat ,experiment was set using serially diluted saliva in 0.05M bicarbonate buffer pH 8.5.Optimal dilution of saliva was used to coat wells of 95well polystyrene microtitration plate .100µl of saliva in bicarbonate buffer was used to coat wells.Uncoated wells included and served as controls.
The culture was diluted to 1:20 in medium and 200 µl of this suspension was used to inoculate 96-well polystyrene plate .The plate was incubated at 37 • C for 24 hours, washed twice with 200 µl sterile saline ,air dried then stained with 0.1% safranin for 30 second .The O.D of the adhered bacteria was measured at 490 nm using a microplate ELISA reader.Each assay was performed in triplicates .

-Biostatistical analysis
Significant differences between mean were evaluated using student t-test using a computer program of epidemiological statistics.p≤0.05 were regarded significant.

-Saliva optimal coating
Saliva of Le(a) and Le(b) serially diluted and used to coat 96-wells of polystyrene plate.As seen in (Figure 1).There was a dose response in biofilm formation that depends on the coating concentration .The intensity of biofilm for a selected isolate of the bacterium was performed .Accordingly an optimal (1:8) coating dilution was used in subsequent experiments.

-Biofilm formation by the isolates
A total of (10) isolates from atopic dermatitis patients were assayed for biofilm formation on a defined set of Le(a) ,(b), (c) saliva .The majority of isolates were potent biofilm for-mers on Le(a) saliva compared to binding on non saliva -coated wells(Figure 2).Nonethe-less, however ,the degree of biofilm formation vary between isolates .Some were strong biofilm formation as seen isolates No.4 (Binding mean was 1.17 ±0.14) in Le(a) coated wells compared to 0.27±0.04 in non-coated wells.(P=3.1× 10 −4 , CL=0.74-1.203).This gave a percent increase of 334.07 in binding (Table 1).Other isolates showed a high degree of biofilm formation also.

Control
Biofilm formation on Le(b) saliva was studied also.As depicted in (Figure 3) ,the ability to form biofilm on this substrate was high also ,but a bit less compared to that seen with Le(a) saliva(Table 1).

Figure 3. Binding of S. aureus and biofilm formation of isolates from atopic dermatitis patients to Lewis ( b ) saliva coating compared to non-coated wells
Interestingly ,biofilm formation on Le (a+b+) and Le (a-b-) was mostly negative on these substrates.
The finding presented in this report demonstrated clearly that S. aureus colonizing atopic dermatitis patients required a defined ligand for adherence .
As show here Le(a) is a strong presumptive ligand for the bacteria as this ligand which is represented in the salivary mucin [17] was involved in biofilm formation besides Le (b) moiety in mucin was also involved.
What strengthen then this finding is that neither Le (a+b+) nor Le (a-b-) acted as having ligands ,a notion emphasizes that a defined structural specificity is preferred among Lewis molecular types that are structurally well defined [18].[19], that are used to bind substrate in biofilm formation .In atopic dermatitis, the most suspected bacteria involved in the pathogenesis of this disease was shown to be S. aureus [20].

S.aureus processes adhesins
The novel findings presented here ,uncover a peculiar system for S. aureus binding that might initiate colonization in skin of affected individules.

Figure 1 .
Figure 1 .Titration of saliva of Lewis (a) and (b) coating for optimal biofim formation by S. aureus.

Figure 2 .
Figure 2 .Binding of S .aureus and biofilm formation of isolates from atopic dermatitis patients to saliva of Lewis (a) coating compared to non-coated wells