Detection of Biofilm Producing Staphylococci among Different Clinical Isolates and Its Relation to Methicillin Susceptibility

ID Design Press, Skopje, Republic of Macedonia Open Access Macedonian Journal of Medical Sciences. 2018 Aug 20; 6(8):1335-1341. https://doi.org/10.3889/oamjms.2018.246 eISSN: 1857-9655 Basic Science Detection of Biofilm Producing Staphylococci among Different Clinical Isolates and Relation to Methicillin Susceptibility * Rania M. Abdel Halim , Nevine N. Kassem, Basma S. Mahmoud Clinical Pathology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt Abstract Citation: Abdel Halim RM, Kassem NN, Mahmoud BS. Detection of Biofilm Producing Staphylococci among Different Clinical Isolates and Relation to Methicillin Susceptibility. Open Access Maced J Med Sci. 2018 Aug 20; 6(8):1335-1341. https://doi.org/10.3889/oamjms.2018.246 Keywords: Biofilm; Staphylococci; Methicillin; TCP; TM; CRA *Correspondence: Rania M Abdel Halim. Clinical Pathology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt. E-mail: Received: 31-Dec-2017; Revised: 12-Feb-2018; Accepted: 25-Jun-2018; Online first: 05-Aug-2018 Copyright: © 2018 Rania M. Abdel Halim, Nevine N. Kassem, Basma S. Mahmoud. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0) Funding: This research did not receive any financial support Competing Interests: The authors have declared that no competing interests exist AIMS: To evaluate three in vitro phenotypic methods; tissue culture plate, tube method, and Congo red agar for detection of biofilm formation in staphylococci and assess the relation of biofilm formation with methicillin resistance and anti-microbial resistance. METHODS: The study included 150 staphylococcal isolates. Biofilm detection in staphylococci was performed using tissue culture plate, tube method, and Congo red agar. RESULTS: Tissue culture plate, tube method, and Congo red agar detected 74%, 42.7%, and 1.3% biofilm producing staphylococci respectively. S. aureus isolates were more common biofilm producers (53.2%) than CONS (46.8%). Biofilm production in CONS species was highest in S. hemolyticus (57.7%). Tube method was 51.4% sensitive, 82.1% specific. As for Congo red agar, sensitivity was very low (0.9%), but specificity was 97.4%. Biofilm producers were mostly; isolated from blood specimens (82.6%) and detected in methicillinresistant strains 96/111 (86.5%). They were resistant to most antibiotics except vancomycin and linezolid. CONCLUSIONS: Tissue culture plate is a more quantitative and reliable method for detection of biofilm producing staphylococci compared to tube method and Congo red agar. Hence, it can still be used as a screening method for biofilm detection. Vancomycin and Linezolid are the most sensitive antibiotics among biofilm producing staphylococci. Introduction Staphylococcus aureus is a virulent organism that is resistant to most of the conventionally available antibiotics. This is attributed to the fact that they are capable of biofilms formation [1]. Biofilm consists of multilayered cell clusters embedded in a matrix of extracellular polysaccharide, which facilitate the adherence of microorganism [2]. The interior of the bacterial biofilms presents greater resistance to the opsonisation by antibodies and phagocytosis. This explains the chronic character of these infections such as endocarditis, osteomyelitis and especially those infections associated with implanted medical devices that are difficult to be treated [1]. Coagulase-negative staphylococci especially S. epidermidis is the most frequent cause of hospital-acquired infections. Most S. epidermidis infections are subacute or chronic and occur mainly in immunocompromised individuals or patients with indwelling medical devices. Biofilm formation on the surface of indwelling devices is often involved in the pathogenesis [3]. Biofilms can resist antibiotic concentration 1010,000 folds higher than those required to inhibit the growth of free-floating Staphylococci. Biofilm producing staphylococci have also been isolated from various clinical samples like blood, urine, pus, skin surface etc. The differentiation of staphylococci concerning its biofilm phenotype might help in their diagnosis and thereby, prevention of infections [4]. Biofilm is an increasing cause of morbidity and mortality associated with chronic and nosocomial _______________________________________________________________________________________________________________________________ Open Access Maced J Med Sci. 2018 Aug 20; 6(8):1335-1341. 1335 Basic Science _______________________________________________________________________________________________________________________________ infections, so a greater understanding of the nature of intracellular bacterial communities in infections, their early detection and management will aid in the development of new and more effective treatments [5]. A number of tests are available to detect slime production by staphylococci; which include quantitative methods such as tissue culture plate (TCP), which is considered as the gold-standard method for biofilm detection [6], and qualitative methods such as tube method (TM) [7], and Congo red agar (CRA) [8]. freshly prepared sodium acetate (2%) was added to each well (for biofilm fixation) for 10 minutes and then discarded. This was followed by adding 200 l crystal violet (0.1%) to each well for biofilm staining. The Plates was kept at room temperature for 30 minutes, and then the stain was discarded. The washing step was repeated once more. Finally, the plate was left to dry at room temperature for one hour, after which, the absorbance was read on a spectrophotometer at 620 nm OD (Figure 1). Materials and Methods This study was conducted on 150 staphylococcal isolates randomly selected from different clinical specimens submitted to the Microbiology Laboratory of Ain Shams University Hospitals. They were isolated from different specimens; 30 pus, 46 blood, nine (9) wound, 15 urine, 22 sputa, 17 central line, five body fluids and six others (two ear swabs, two throat swabs, one bile drain and one radivac). All the isolates were identified morphologically by Gram stain, colonial morphology on culture, catalase test to differentiate it from Streptococcus species and DNase test to differentiate S. aureus from coagulase- negative staphylococci (CONS). Identification of CONS species and antibiotic susceptibility testing for all isolates were made using a n automated identification system (Vitek 2, bioMérieux, France) according to CLSI guidelines 2015 [9]. Biofilm detection was performed using TCP [6], TM [7] and CRA [8]. S. aureus (ATCC 25923) was used as negative control. Tissue culture plate method was performed as described by Christensen et al., 1985 [6] for quantitative measurement of biofilm production in Staphylococcus spp. Using a microtiter assay. A single colony from each subcultured plate on blood agar was inoculated in a glass tube co (...truncated)