International Journal of Science and Research (IJSR) ISSN (Online): 2319-7064 Index Copernicus Value (2013): 6.14 | Impact Factor (2015): 6.391 Volume 5 Issue 10, October 2016 www.ijsr.net Licensed Under Creative Commons Attribution CC BY Comparison of Three Methods for the Detection of Biofilm Formation by Clinical Isolates of Staphylococcus aureus Isolated in Casablanca Achmit Mohamed 1 , Ait Mhand Rajaa 2 , Zerouali Khalid 3 , Mellouki Fouad 4 , Rhallabi Naima 5 1, 2, 4, 5 Laboratory of Virology, Microbiology and Quality / Eco-toxicology and Biodiversity, Faculty of Sciences and Techniques, Mohammedia, Morocco 3 Laboratory of Microbiology, Ibn Rochd University Hospital, Faculty of Medicine and Pharmacy, Casablanca, Morocco Abstract: Biofilms are group of microorganisms encased in a matrix of extracellular polysaccharide (slime), called polysaccharide intercellular adhesin (PIA). They have been associated with a variety of chronic and persistent infections. Staphylococcus aureus is from bacteria which have high ability to form a biofilm. In this study, detection of biofilm production by Staphylococcus aureus was done by using three different methods: tube method (TM), congo red agar method (CRA) and tissue culture plate method (TCP), three repetitions were made for every method. Among of 117 strains of Staphylococcus spp isolated between October 2015 and January 2016 at the University Hospital Ibn Rochd in Casablanca, 74 were identified as Staphylococcus aureus. 58 isolates were detected as biofilm producer by TCP method, 49 by TM and 42 by CRA method. We can conclude from our study that the TCP method is a more quantitative and reliable method for the detection of biofilm formation by Staphylococcus aureus as compared to TM and CRA methods. Keywords: Biofilm, Staphylococcus aureus, Tube method (TM), Congo red agar method (CRA), Tissue culture plate method (TCP). 1. Introduction Staphylococccus aureus is the most frequent cause of nosocomial and community-acquired infections and is recognized as the most frequent causes of biofilm-associated infections. The ability of Staphylococcus aureus to form biofilms helps the bacteria to survive in hostile environments within the host and is considered to be responsible for chronic or persistent infections [1, 2]. Biofilm consists of multilayered cell clusters embedded in a matrix of extracellular polysaccharide (slime), called polysaccharide intercellular adhesin (PIA), which facilitates the adherence of these microorganisms to biomedical surfaces and protect them from host immune system and antimicrobial therapy [3]. The synthesis of PIA is mediated by the products of the chromosomal ica gene (intercellular adhesion), which are organized in an operon structure. This operon contains the icaADBC genes, in addition to the icaR gene which exerts a regulatory function and is transcribed in the opposite direction. Once this operon is activated, four proteins are transcribed, IcaA, IcaD, IcaB and IcaC, which are necessary for the synthesis of PIA [4-6]. The three methods broadly used for the phenotypic identification of biofilm-producing strains are Tube method (TM), Congo red agar method (CRA) and Tissue culture plate method (TCP) [1, 7- 8]. The Aim of this study is to compare three different methods for detection of biofilm formation in Staphylococcus aureus in order to determine the most reliable method. 2. Materiel and Methods A total of 117 strains of Staphylococcus spp were isolated between October 2015 and January 2016 from different samples from patients hospitalized in various services of the University Hospital Ibn Rochd in Casablanca. Isolates were initially identified by standard microbiological techniques including Gram stain, catalase test, coagulase test and mannitol fermentation [9]. API Staph gallery (Biomerieux, France) was performed for identification of different Staphylococcus strains then biofilm ability formation was detected for each isolate by Tube method (TM), Congo red agar method (CRA) and Tissue culture plate method (TCP). Tube method (TM) Biofilm production was investigated by the tube adherence test proposed by Christensen et al.. 10 ml of Trypticase soy broth with 1% glucose was inoculated with a loopfull of test organism from overnight culture on nutrient agar individually. Broths were incubated at 37 °C for 24 hours. The cultures were decanted and tubes were washed with phosphate buffer saline pH 7.3. The tubes were dried and stained with 0.1% crystal violet. Excess stain was washed with deionized water. Tubes were dried in inverted position and observed for biofilm formation. Biofilm Production was considered positive when a visible film lined the wall and bottom of the tube. Ring formation at the liquid interface was not indicative of biofilm formation. Tubes were examined and amount of biofilm formation was scored as 0- absent, 1-weak, 2-moderate, 3-strong [10, 11]. Congo red agar method (CRA) The medium composed of Brain heart infusion broth (37 mg/l), sucrose (5 mg/l), agar number 1 (10 mg/l) and Congo red dye (0.8 mg/l). Congo red stain was prepared as concentrated aqueous solution and autoclaved at 121°C for Paper ID: ART20162319 DOI: 10.21275/ART20162319 1156