Molecular typing of dandruff pathogens and evaluation of antifungal activity of plant extracts

Abstract

Background : Dandruff is a common dermatological problem, dispersed flaking of the scalp and hair. Various intrinsic and environmental factors such as skin surface fungal colonization, individual susceptibility, are affecting.

The etiology of dandruff is not well understood. The study aims to verify the unequivocal understanding of the fungal relationship with dandruff by identification of filamentous fungi, Candida and survey of the bacterial companioning dandruff of hair samples and investigate the antifungal activity of water extracts of some medicinal plants in isolated fungi. A total of 280 hairs scalps samples,and hair swabs were collected from patients attenuated in Hilla hospitals and private clinics inthe Babylon province (n= 152 hair samples,n= 74 dandruff samples,n= 54 scalp swabs).Clinical samples were cultured on Sabouraud dextrose agar (SDA) medium with / without antibiotics based on stander cultured methods.Candida spp. was preliminary identified based on CHROMagar medium. Molecular typing of isolated yeasts via amplification ITS region and sequence analysis and multiple alignment was performed and constricted the phylogeny tree.

Keyword: Dandruff, Molecular typing, fungi,Plant extract activity.

Introduction

Scalp skin has unique predisposing factors like hair follicles density, moist ,dark and warm environment ,these factors make it susceptible to superficial mycotic infections such as dandruff, dermatitis and tinea captis [1].Dandruff is extremely common, affecting close to 50% of the world’s population [2].It's characterize white or gray flakes, occurred in patches or scattered on the scalp, usually accompanied by itching [1] [3] [4].The health care orientation, high costs. In USA the cost exceeded $1.4 billion in the United States in 2004 and causes patient's psychological suffering [5]. Many factors were suspected with occurring of dandruff, some of them are intrinsic and environmental factors, such as skin surface fungal colonization, individual susceptibility, genetic, patient immunity and interactions between these factors, led to the dandruff occurrence [6]. The yeasts Malassezia and Candida may aggravate atopic dermatitis due to an allergic reaction and led to damaging the surface of the hair, scalp may share in the development of dandruff [7].

The etiology of dandruff appears to be dependent upon many factors: scalp gland secretions, head lice, microfloral metabolism, individual susceptibility and their interactions [8-10], but the exact underlying cause of dandruff is unknown [11]. Fungi were one of the most suspected causative agents of scalp health problematic worldwide [12].

Unfortunately, most of previous studies consider the dandruff were given more attention to the Malassezia spp is the unique causes. A secreted lipase of Malassezia is an associated virulence factor of development of dandruff, and quite neglected others fungi [13-16].Faergemann [17] pointed to there is little information about the distribution and colonization of Candidaspp. of skin in patients with atopic dermatitis. The Deutromycete,Ascomycetes and Basidiomycete yeast are colonizing the human scalp and hairs and get their role in the development of dermatitis as primary or secondary pathogens [18].

The study aims to verify the unequivocal understanding of the fungiassociated with dandruff by identification of filamentous fungi, yeastsand bacteria companioning dandruff of hair samples, and investigate the antifungal activity of aqueous extracts of some medicinal plants in isolated fungi.

2. Material and Methods

Two hundred and eighty dandruff samples (152 hair samples and 54 swabs and 74 scraps) were collected from patients at different ages and gender, those were previously diagnosed by a physician for the presence of hair dandruff symptoms.

Clinical specimens (piece of hairs, flakes and swabs were cultured into two sets: first one was cultured on Sabouraud's dextrose agar medium(SDA)without cyclohexamide and incubated for 24-48h 30˚C. Second set with cyclohexamide and incubated at 28-30˚C for 1-3 weeks. After incubation periods, single yeast and fungal colonies were isolated in pure cultures, phenotypic identification was performed based on standard methods [19] [20]. The subcultures in each isolate were preserved in slant SDA media for future tests21.While the bacterial isolation, purification and identification on the blood agar base and manitol salt agar based on McFadden [22].

2.1.CHROMagar test

This test was used for preliminary identification most of Deutromycetes, Ascomycetes and Basidiomycetes yeast. Each single colony yeast with white –cream color were pick up and streaking on CHROMagar medium [23]. All the plates were cultured and incubated at 30˚C for 24-48h.While the red colonies were directly identified as Rhodotorulla.

2.2.Lipase and Phospholipase production assay

Candid a species were screened for the production of extracellular lipase and phospholipase activity by growing them on a substrate with SDA: tween 80 medium and egg yolk respectively and incubated at 37˚C for 48h, after which the colony diameter plus precipitation zone was measured for each isolate. Calculation of the zone of phospholipase activity was performed according to Price et.al. [24].

2.3.Plants crud extraction and preparation

Plant material included of Lawsonia inermis( leaves of Henna), Eugenia caryophyllus (floral buds of Clove),Cinnamomum verum (bark) ,Camellia sinenesis (leaves) and Eucalpytus globules(leaves) were collected from markets. They were identified by the taxonomist in the biology department. All women college for Science in Babylon University .The extracts were prepared based on Vijayakumar et al.[25]. These rial concentration (1%,4%,8%)were evaluated their antifungal activities against dermatophytes and yeasts.

2.4.Evaluate the antifungal activities

Brief description of the method, pour about 20 mlSDA to Petri dish, 0.2ml of cell suspension(1x106) was spread on the surface of SDA and left it to been absorption in the medium. Wells (0.5cm diameter) in SDA were performed by cork pore and filled each well of SDA with 100 µl of plant extract, incubate 28-30˚C for 24-48 h, the inhibition zone was measured by metric ruler. All tests were performed in triplicate [26].

2.5.PCR and sequencing analysis

The phenotypic identification of fungi under interested was confirmed by simple PCR by universal primer pair ITS5/ITS4.OneμLofDNA(20µg/ml)from each of 24 isolates were mixed with a PCR mixture (final reaction volume 25 uL) consisted of 12 uL of 20x Master Mix (Promega),2 uL of primers (10 pmole) and rest molecular-grade water. The PCR conditions and gel electrophoresis were performed based on Imran and Al- Asadi [27.

Representative 22PCR products off ungi were subjected to sequencing analysis in the Macro gene Lab. USA. Pairwise alignment sequences were compared with the BLAST database. The phylogenetic tree (UPGM) based on sequencing were constructed employing the Mega 6 software, multiple alignment sequences based on BioEdit software was performed [28].

2.4. Statistical analysis

All statistical analysis was undertaken using factorial experimental randomized block design. A P.value < 0.05 was considered significant.

3. Results

3.1. Fungal survey

A total of 2313 colonies (1591 yeast colonies, 722 filamentous fungi colonies)was isolated from clinical specimens (hair, flakes, scalp swabs).The percentage of filamentous fungi were summarized in table (1), the results showed that the Aspergillus flavus and Asp.fumegatus were the highest molds while the T. rubrum and M.caniswere the highest dermatophytes fungi. The survey of bacteria with dandruff cases showed a frequency percentage of S. aureus and S. epidermidis 35.1 % for each.

[Table is omitted from this preview]

Table (1): Summarized the appearance and frequency percentage of filamentous fungi isolated from clinical samples

Data on the growth of Candidaspp andRhodotorulla on hairs planted on SDA comparison with direct scalp's swab streaking and scattered flakes on SDA medium are presented in figure (1).Both yeast grew well in whole hairs. Candida spp and Rhodotorulla demonstrated more rapid and abundant growth of hairs than SDA medium after 24-48h,most frequent and vital colonies on hairs, may supported growth both Candida and Rhodotorulla compare with their growth on SDA (Figure 1).

[Figure is omitted from this preview]

Figure (1): Abundant of yeasts grown on clinical samples : A=scatter of flakes, B= Swab streak from dandruff cases, C= Hairs infected by dandruff.(Rh= Rhodotorulla, C=Candida, M=Mould, H=Hairs)

The percentage of appearance Candida spp. were summarized in table (2),the results showed C.parapsilosis was the highest yeast (28.75% (80/280), while the frequency of percentage of it was 25.14% (400/280). The appearance of R.mucitaginosa was 7.14% (20/280) and the frequency percentage was 8.79% (140/280).

[Table is omitted from this preview

Table (2): Summarized the Percentage of appearance and frequency values of yeasts and their colors on CHROMagar

3.2.Lipase and phospholipase production tests

Our results showed that C.albicans, C.tropicalis and C.parapsilosis gave positive results for their ability to produce lipase and Phospholipase in vitro. The singer of lipase and Phospholipase production is a precipitation around the colony after an incubation period of 72 h at 30˚C (Figure 2), other yeast showed positive tests (data not show on).

[Figure is omitted from this preview]

Figure (2): Lipase (A) and Phospholipase(B) production singe as precipitation around Candida colony at incubation for 72h,30C

3.3. Crude of plant extract activity

The activity of plant extract crude showed significant inhibition in the colony diameter of fungi. Figure (3) showed the variation of inhibition zones based on plant species: the aqueous extract of cloves was more effective (0.62 mm),followed by Cinnomumm, Eucalyptus, green tea ,henna (0.25,0.28,0.29,0.23) respectively.

[Figure is omitted from this preview]

Figure (3): Effect of the extract type in the inhibition zone of dermatophytes and yeast

The sensitivity of fungi to the effects of plant extracts were showed significant difference :T.rubrum more sensitive followed by M.canis, C.albicans, C.glabrata, C.parapsilosis, C.intermedia and R.mucitaginosa respectively ,(Figure 2).also the concentration 8% showed more effective than 2% and 4% respectively (data not show on).

[Figures are omitted from this preview]

Figure (2): Fungal sensitivity to the crude of plant extract determined based on the inhibition zone of dermatophytes and yeast growth

Antfactivitungal of five extracts were comparable with three reference antifungals (Econazole (ECN), Miconazole (MCL) and 5-fluorocytosine AFY), the extract of clove gave antifungal activity higher than that of references (ECN, MCL and AFY),while the other four plant extract gave lower antifungal activities than that reference antifungals (Figure 3).

[Figure is omitted from this preview]

Figure (3): Antifungal activities of five plants extracts: Cl= Cloves, E=Euclaptus, C= Cinnamomum, Ca= Camellia. H=hana, ECN, MCL, AFY=reference antifungals, A=culture T.rubrum, B= culture of C.intermedia and C=culture of R.mucitaginosa

3.4. PCR and sequencing assay

The results of amplification ITS1-5.8S –ITS2 and flanking of primer pairs ITS5/ITS4 showed variation in the amplicons sizes of 24 isolates dermatophytes and yeasts for each(Figure4,A&B). T.mentogrophytes isolates (680,650 bp). T.rubrum isolates (780,800bp).

[Figure is omitted from this preview]

Figure (4 ): Agarose gel electrophoresis of PCR products: A. Fordermatophytesspecies isolateamplified by pair primer ITS5/ITS4: Lane M= Molecular marker100 bp.; lane one T.mentogrophytes isolates (680 bp.). Lanes 2-4,6,8-14,17-21 T.rubrum isolates (800bp), Lanes 5 T.mentogrophytes isolates (650bp).Lanes 7,15,16,22-24 T.rubrum isolates (780bp).B. ForCandida species isolate. Lane M = Molecular marker 100 bp.; Lanes 1-2,4-8,11,18-19,21-24:680 bp.(C.tropicalis); Lanes 3,9,13,15-17,20 630 bp.(C.parapsilosis ); Lanes 14: 720 bp.(C.kruzea); Lanes 10,12 : 550 bp.(C.albicans)

3.5.Multiple alignment of 17 sequences of ITS region

[Figure is omitted from this preview]

Figure (5): The Multiple-alignment of Sequence analysis of ITS region amplified by ITS5/ITS4 for suspected fungal dandruff pathogens.

The results of the multiple alignment analysis based on BioEdit software performed for 22 isolates of yeast: C.albicans and Cryptococcusspp ,C.parapsilosis Aureobasidium iranianum,Issatchenkia orientalis, R.mucitaginosa and dermatophyte (Figure5). Each set of isolates of Candida spp was showed high similarity to leading sequences with some mutation or substitutions, these sequence variations were indicated to microevolution in each set of isolates.

3.6. Phylogeny tree

The Phylogenetic tree (UPGM) for 21 fungal species was constructed based on sequences of ITS region. The fungal species were isolated from dandruff samples showed closed related intra-isolates groups as in Figure (6), many clusters of closely related fungal species. The dermatophytes occurred in neighbored clusters, the same relationship between C.albicans, C.sake and C.parapsilosis were close to either. The Basidiomycetes yeasts separated their clusters of previous clusters depended on their sequences.

[Figure is omitted from this preview]

Figure (6): Phylogeny tree (UPGM) based on sequence of 21 fungalspecies suspected as dandruff pathogens occurred in five clusters (C1=dermatophyte,C2 and C4 =Basidiomycetes yeast,C2=Deutromycets yeast, C5= Ascomycetes yeast).

4. Discussion

Mycotic pathogens were one of the suspected on the scalp of dandruff patients, most of previous works considered Malassezia sppas the most common pathogen, others works reported to role of combined factors, but in the general mechanism of occurrence of the disease is not clearly understood [9] [10] [15] [16].

This study identified 12 fungal speciesis sociated with all clinical samples :(4 dermatophytes, 3 Candida, one Aureobasidium iranianum, Issatchenkia orientalis and R.mucitaginosa for each, 2Cryptococcus)based on molecular assays (Figures 4-6). Most of these dermatophytic fungi cause dermatitis, our results agree with the results of studies of Tan, [29]; Jain et al. [30]; Abastabar et al.[31].

Our study was aimed to evaluate antifungal activities of five plant extracts in vitro against suspected dandruff pathogens, Clove extract showed significantly antifungal activity more than others extracts, either than reference antifungals drugs such as ECN, MCL and AFY (figure 3).the microbial inhibition activity of Clove depended on its composion of flavenoides and cartenoides ,this finding agreed with [32], our results recommended to evaluate the clinical efficacy and safety of Clove extract as anti-Dandruff Shampoo based on their size of inhibition zone compare with reference drugs .

This study was verified that most of yeast: C.albicans , C.parapsilosis, C.tropicales and R.mucitaginosa had the ability to produced lipase and Phospholipase, this result was agree with recent studies were reported that lipase causing dermal inflammation and tissue damage and play a key role in the lifestyle of opportunistic yeast [28], [33-37] .The high frequent of dermatophyte fungi and yeast such as Microsporum, Trichophyton and Candidain this study may take their role in the attacked of scalp hair follicle and led to development dandruff, this explanation agreed with Herbert [38] and Zinkevicieneet al.[39],also Staph.aureus and Staph.epidermidis, were considered an opportunistic pathogen has the ability to colonize in different niches [40].

Our finding results, in particular based on the abundant growth of Candida and Rhodotorulla on hairs, scalp and flakes and the ability of these yeasts for lipase and Phospholipase production (Figure 1, 2).This result tends to confirm the virulence of Candida spp and Rhodotorulla are considered to be the most virulent compared with Malassezia spp. has been questioned by 13-17,who studied only Malassezia have virulence factor depended on lipase production.

Our finding highly frequent and abundant growth of Candida spp, R.mucitaginosa and Cryptococcus with absent of Malassezia spp.in all clinical samples under interest (Figure 1).This finding was supportedby the results of Golubev [37] and Zinkevicieneet al.[39],the found negative relationship between Candida spp and Malassezia spp.when they grew in the same niche, Both Malassezia and Candida were not found together in any of the samples due to different growth rate, Candida had an antagonist role against Malassezia spp. And Candida spp. have the ability to produce kill factors (mycocin as lethal substance) led to inhibition and killed Malassezia spp. The high growth rate of Candida more than Malassezia spp this property led to overgrowth of Candida against Malassezia spp. which has no special requirement for growthmedia [37] [39] [40].These justifications were explained the predominance of Candida spp, R.mucitaginosa and Cryptococcus spp.

Our conclusion, based on this results and review: We think that it was impossible to note that were considered both Candida and Rhodotorulla as harmless yeast with dandruff patients from all previous works. And we consider the dermatophytes, Candida and Rhodotorulla were important dandruff mycotic pathogens, and the current data refute their contention about the consideration the Malassezia spp as the main dandruff pathogen. More studies are required to conform our results about the pathogenic role of fungi act as exacerbating factors in dandruff.

Ethical approval

Author hereby declared that all the actions have been examined in the studies were approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.

References

[1] Elewski, B. E.(2005).Clinical Diagnosis of Common Scalp DisordersJ Investig Dermatol Symp Proc. 10:190 –193.

[2] Cardin, C.(1998). Isolated dandruff. In: Baran R, Maibach H (eds). Textbook of Cosmetic Dermatology. Malden, MA: Blackwell Science, p 193–200.

[3] Bulmer, A. ; Bulmer, G.(1999).The antifungal action of dandruff shampoos, Mycopathologia 147: 63- 65 .

[4] Bhushan, B.(2010).Introduction-Human Hair, Skin,and Hair Care Products , Biophysics of Human hair Structural, Nanomechanical, and Nanotribiological Studies:Chapter 1pp.1-19.Springer.

[5] Bickers, D.R.; Lim,H. W.; Margolis, D.; Weinstock, M. A.; Goodman, C.; Faulkner, E.; Gould, C.; Gemmen, E. and Dall,T.(2006) .The burden of skin diseases: a joint project of the American Academy of Dermatology Association and the Society for Investigative Dermatology, J Am Acad Dermatol. 55: 490-500 .

[6] Pierard-Franchimont, C., Xhauflaire-Uhoda, E. and Pierard, G.E. Revisiting dandruff. Int. J. Cosmet. Sci. 28:311–318 (2006).

[7] Savolainen, J.; P. Lintu, J.; Kosonen, O. Kortekangas-Savolainen, M.; Viander, J.; Pene, K.; Kalimo, E. ; Terho, O.and Bousquet,J.(2001). Pityrospo-rum and Candida specific and non-specific humoral, cellular and cytokineresponses in atopic dermatitis patients.Clin. Exp. Allergy 31:125–134.

[8] Chosidow ,O .(2000).Scabies and pediculosis. Lancet. 355:819–826.

[9] DeAngelis, Y.M.; Gemmer, C.M.; Kaczvinsky, J.R.; Kenneally, D.C.; Schwartz, J.R.; Dawson, T.L .(2005).Three etiologic facets of dandruff and seborrheic dermatitis: Malassezia fungi, sebaceous lipids, and individual sensitivity, J Investig Dermatol Symp Proc 10: 295-297.

[10] Ro, B.I.; Dawson, T.L. (2005).The role of sebaceous gland activity and scalp microfloral metabolism in the etiology of seborrheic dermatitis and dandruff, J Investig Dermatol Symp Proc. 10: 194-197.

[11] Ravichandran,G.; Shivaram Bharadwaj, V. and Kolhapure, S.A. ( 2004).Evaluation of the clinical efficacy and safety of “Anti-Dandruff shampoo” in the treatment of dandruff The Antiseptic 201(1): 5- 8.

[12] Cowen, L. E. (2008). The evolution of fungal drug resistance: modulating the trajectory from genotype to phenotype. Nat. Rev. Microbiol. 6 :187–198.

[13] Morishita, N., Sei,Y.; Takiuchi, I.and Sugita,T. (2005). Examination of the causative agent of pityriasis versicolor. Jpn. J. Med. Mycol. 46:169–170.

[14] Nakabayashi, A. (2002). Identification of causative species in Malassezia-associated dermatoses. Jpn. J. Med. Mycol. 43:65–68.

[15] Sei, Y. (2006). Malassezia related disease. Jpn. J. Med. Mycol. 47:75–80.

[16] Schwartz, J. R.; DeAngelis, Y.M. and Dawson, T.L.( 2016). Dandruff and Seborrheic Dermatitis: A Head Scratcher.www.com/files/pdf/.../TRI_book_chapter_Ch12_Dandruff.pdf

[17] Faergemann, J. (2002).Atopic Dermatitis and Fungi.CLIN.Microbiol.Rev. 545–563.

[18] O’Neil, C.E.; Horner , W.E.; Reed, M.A.; Lopez, M.; Lehrer, S.B. ( 1990). Evaluation of Basidiomycete and Deuteromycete (Funfi Imperfecti) extracts for shared allergenic determinants, Clin Exp Allergy 20:533-538.

[19] Chapin, K. and Murray, P. (1999). Stains.In Manual of Clinical Microbiology, pp. 1674–1686.Edited by P. R. Murray, E. J. Baron, M. A.Pfaller, F. C. Tenover & R. H. Yolken. Washington, DC: American Society for Microbiology.

[20] Astrid, l. (1999). Preparation of lactophenol cotton blue slide mounts 12(30):24-28.

[21] Sharma, M. and Sharma,M. (2010). Incidence of dermatophytes and other Keratinophilic fungi in the schools and college playground soils of Jaipur, India. African Journal of Microbiology Research . 4(24):2647-2654 .

[22] Macfaddin, J.E. (2000) .Individual Biochemical Tests. In: Test for Identification of Medical Bacteria . 3thed.Biochemical Lippincott Williams and Wilkins co,Blatimore. USA. 27- 439 .

[23] .Agarwal,S.; Manchanda,V.;Verma,N. and Bhalla, P. (2011). Yeast identification in routine clinical microbiology laboratory and its clinical relevance .Indian J Med Microbiol . 29:172-177.

[24] Price, M.; Wilkinson, ID; Gentry, L. (1982). Plate method for detection of phospholipase activity in Candida albicans , Sabouraudia 20: 7–14.

[25] Vijayakumar, M.; Priya, K.; Nancy, F.T.; Noorlidah ,A. and Ahmed, A.B. ( 2013). Biosynthesis, characterisation and anti-bacterial effect of plant mediated silver nanoparticles using Artemisia nilagirica. Indust.Crops Products, 41: 235-240.

[26] Perez , C. ; Pauli, M. and Bazerque , P. (1990) . An antibiotic assay by the agar – Well diffusion method . J. Actabiologiae . 15:113-115 .

[27] Imran ,Z. and Al- Asadi, Y. (2014). Multiple molecular markers for diagnosis of conjunctivitis caused by Candida spp. in Iraq, Afr. J. Microbiol. Res. 8(38): 3482-3488.

[28] Imran, Z.K. and Ali , E.K. (2015) . Molecular identification of Candida glabrata and C. parapsilosis based on sequencing analysis of rDNA . International Journal Of Medical Science and Clinical Inventions . 2:1490-1497 .

[29] Tan, H. H. (2008) . Superficial fungal infections seen at the National Skin Centre . Singapore. Jpn. J. Med . Mycol. 46: 77- 80 .

[30] Jain, N.; Sharma, M. and Saxena, V.N.(2008). Indian Journal of Dermatology,Venereology and Leprology.74 (3) : 274-75.

[31] Abastabar, M.; Rezaei-Matehkolaei, A.; Shidfar, M.R. & et al., (2013) . A molecular epidemiological survey of clinically important dermatophytes in Iran based on specific RFLP profiles of betatubulin gene . Iranian J Publ Health . 42(9):1049-1057 .

[32] Ravichandran,G. and Derm. M.(2004). Evaluation of the clinical efficacy and safety of “Anti-Dandruff Shampoo” in the treatment of dandruff.The Antiseptic 201(1): 5-8.

[33] Paraje , M.C. ; Correa, S.G.; Renna, M.S.; Theumer, M. and ElenaC.S.(2008) . Candidaalbicans –secreted lipase induces injury and steatosis in immune andparenchymal cells . Canadian Journal ofMicrobiology . 54(8) : 647-659.

[34] Neugnot , V.; Moulin, G.; Dubreng, E. and Bigey, F. (2002). The lipase/ acyltransferase fromCandidaparapilosis : Molecular cloning and characterization of purified recombinant enzymes . European Journal of Biochemistry. 269(6) : 1734-1745 .

[35] Imran, Z. K.(2015).Candida albicansSsp. dubliniensis stat.et comb. nov., a new combination for Candidadubliniensis based on genetic criteria Afr. J. Microbiol. Res.,9(17) :1205-1214.

[36] Schaller , M.; Borelli C.; Korting, H. C. and Hube,B. (2005) . Hydrolytic enzymes as virulence factors of Candidaalbicans.Mycoses . 48: 365-377 .

[37] Golubev W., ( 1998) .Mycocins (Killer toxins), In: The yeasts, a taxonomic study, Kurtzman C.P., Fell J.W., 4th edn. Amsterdam: Elsevier Science Publishing .

[38] Herbert, A.(1988).Tineacapitis. Arch Dermatol. 124:1554–1557.

[39] Zinkeviciene, A.;Vaiciulioniene , N.; Baranauskiene, I.;Kvedariene, V.; Emuzyte , R.; Citavicius , D.(2011).Cutaneous yeast microflora in patients with atopic dermatitis.Cent. Eur. J. Med. 6(6) : 713-719.

[40] Sharvari , S.A. and Chitra, P.G. (2012). Evaluation of different detection methods of biofilm formation in clinical isolates of Staphylococci.Int. J. Pharm. Biol. Sci7(6): 640-648.