Jul 11, 2013 - of Anatomy (AAK), Jawaharlal Nehru Medical College and. Hospital, Aligarh Muslim University, Aligarh, Institute of Microbial. Technology ...
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Indian Journal of Medical Microbiology, (2015) 33(1): 101-109
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Original Article
Anti-biofilm efficacy of silver nanoparticles against MRSA and MRSE isolated from wounds in a tertiary care hospital MA Ansari*, HM Khan, AA Khan, SS Cameotra, MA Alzohairy
Abstract Purpose: Different approaches have been used for preventing biofilm-related infections in health care settings. Many of these methods have their own de-merits, which include chemical-based complications; emergent antibiotic resistant strains, etc. The formation of biofilm is the hallmark characteristic of Staphylococcus aureus and S. epidermidis infection, which consists of multiple layers of bacteria encased within an exopolysachharide glycocalyx. Nanotechnology may provide the answer to penetrate such biofilms and reduce biofilm formation. Therefore, the aim of present study was to demonstrate the biofilm formation by methicillin resistance S. aureus (MRSA) and methicillin resistance S. epidermidis (MRSE) isolated from wounds by direct visualisation applying tissue culture plate, tube and Congo Red Agar methods. Materials and Methods: The anti-biofilm activity of AgNPs was investigated by Congo Red, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) techniques. Results: The minimum inhibitory concentration (MIC) was found to be in the range of 11.25-45 μg/ml. The AgNPs coated surfaces effectively restricted biofilm formation of the tested bacteria. Double fluorescent staining (propidium iodide staining to detect bacterial cells and fluorescein isothiocyanate concanavalin A (Con A-FITC) staining to detect the exopolysachharides matrix) technique using CLSM provides the visual evidence that AgNPs arrested the bacterial growth and prevent the glycocalyx formation. In our study, we could demonstrate the complete anti-biofilm activity AgNPs at a concentration as low as 50 μg/ml. Conclusions: Our findings suggested that AgNPs can be exploited towards the development of potential anti-bacterial coatings for various biomedical and environmental applications. In the near future, the AgNPs may play major role in the coating of medical devices and treatment of infections caused due to highly antibiotic resistant biofilm. Key words: Anti-biofilm, AgNPs, confocal laser scanning microscopy, exopolysachharide, scanning electron microscopy
Introduction Staphylococci are most often associated with chronic infections of implanted medical devices.[1] The use of indwelling medical devices is important in the treatment of critically and chronically ill patients, however, bacterial colonisation of implanted foreign material can cause major medical and economic sequel. The increased use *Corresponding author (email: ) Nanotechnology and Antimicrobial Drug Resistance Research Lab, Department of Microbiology (MAA, HMK), and Department of Anatomy (AAK), Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, Institute of Microbial Technology (IMTECH) (SSC), Sector 39-A, Chandigarh, Department of Medical Laboratories College of Applied Medical Science Buraydah Colleges (MAA), Buraydah 51452 Saudi Arabia Received: 11-07-2013 Accepted: 29-01-2014 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.148402
of indwelling medical devices has had considerable impact on the role of staphylococci in clinical medicine. The predominant species isolated in these infections are Staphylococcus epidermidis and S. aureus, their major pathogenic factor being ability to form biofilm on polymeric surfaces.[2] Biofilm producing staphylococci frequently colonise catheters and medical devices and may cause foreign body-related infections. They easily get attached to polymer surfaces.[3] Bacterial biofilms are a predominant challenge to wound healing.[4] The first recorded observation concerning biofilm was probably given by Henrici in 1933, who observed that water bacteria are not free floating but grow upon submerged surfaces.[5] Biofilm consists of multilayered cell clusters embedded in a matrix of extracellular polysaccharide (slime), which facilitates the adherence of these microorganisms to biomedical surfaces and protect them from host immune system and anti-microbial therapy.[6] Biofilm formation is regulated by expression of polysaccharide intracellular adhesin (PIA), which mediates cell to cell adhesion and is the gene product of icaADBC.[7] Various reports attest to the presence of icaADBC gene in S. aureus and S. epidermidis isolated from infections associated with indwelling medical devices.[8] It is now well documented that biofilms are notoriously difficult to eradicate and are often resistant to systemic
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antibiotic therapy and removal of infected device becomes necessary.[3,9] According to National Institute of Health, more than 60% of all infections are caused by biofilm.[10] Biofilm organisms have an inherent resistance to antibiotics, disinfectants and germicides. Unlike planktonic populations, bacterial cells embedded in biofilms exhibit intrinsic resistance to antibiotics due to several specific defense mechanisms conferred by the biofilm environment, including the inactivation of anti-microbial agents by exopolysachharide (EPS), over expression of stress-responsive genes, oxygen gradients within the biofilm matrix and differentiation of a subpopulation of biofilm cells into resistant dormant cells.[11,12] The intrinsic resistance of bacterial cells within biofilms to conventional anti-microbials has motivated new approaches for the treatment of biofilm-associated infections, including the use of silver preparations. Several silver-containing dressings are recommended for long-term de-contamination and wound healing based on silver’s broad-spectrum, high-level anti-microbial activity.[13] The difficulty in eradicating a chronic infection associated with biofilm formation lies in the fact that biofilm bacteria are able to resist higher antibiotic concentration than bacteria in suspension.[14] Nanotechnology may provide the answer to penetrate such biofilms and reduce biofilm formation. Silver nanotechnology chemistry can prevent the formation of life-threatening biofilms on medical devices. Silver is one of the oldest known anti-microbials. It has recently been demonstrated that AgNPs hydrogel hybrid with different sizes of AgNPs can be effectively employed as anti-bacterial agents.[15] Saxena et al. studied that propylene-based sutures immobilised AgNPs show anti-bacterial activity against S. aureus and Escherichia coli.[16] Due to the strong anti-bacterial properties and low toxicity towards mammalian cells, AgNPs have been applied in a wide range of areas including wound dressing, coatings on medical devices to reduce nosocomial infection rates,[17] protective clothing, anti-bacterial surfaces, water treatment, food preservation and cosmetics as biocidal and disinfecting agents.[18] Although the literature reports that some studies are related to the anti-bacterial activity of AgNPs, to the authors’ knowledge, there are very few studies concerning the effect of these particles against adhered cells and biofilms of methicillin resistance S. aureus (MRSA) and methicillin resistance S. epidermidis (MRSE). Thus, the aim of the present study was to evaluate the anti-biofilm potential of AgNPs against biofilms of MRSA and MRSE by Congo Red Agar (CRA), scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM).
Biochem, Inc. (Columbia, USA). The subsequent dilutions were made in autoclaved Milli Q water. The morphological features and particle size of the procured NPs were characterised by high-resolution transmission electron microscopy (HR-TEM, Technai, FEI, Electron Optics, USA) [Figure 1]. Bacterial strains, media and materials A total of 62 non-repetitive clinical isolates of Staphylococcus spp were recovered over a period of 6 months (September 2012 to February 2013) from skin lesion such as pus, wounds, burn, etc., were subjected to the study. In vitro antibiotic susceptibility test Individual isolates were tested, based on the recommendations of the Clinical and Laboratory Standards Institute (CLSI),[19] by the Kirby-Bauer disc diffusion method for susceptibility to the following antibiotics: Amikacin (AK, 30μg), Gentamycin (G, 10μg), Oxacillin (Ox, 1 μg), Ceftriaxone (Ci, 30 μg), Cefotaxime (Ce, 30 μg), Vancomycin (Va, 30 μg), Chloramphenicol (C, 30 μg), Tobramycin (Tb, 10 μg), Novobiocin (No, 30 μg), Levofloxacin (Le, 5 μg), Clindamycin (Cd, 1 μg), Erythromycin (E, 1 μg). Antibiotic discs used were procured by Hi-Media (Mumbai, India). Screening for methicillin resistance Resistance to oxacillin was determined in all S. aureus isolates using an oxacillin broth screening and disc diffusion test recommended by the British Society for Antimicrobial Chemotherapy (BSAC),[20] respectively. For the oxacillin broth screening test, isolated colonies from an 18 to 24 h sheep blood agar plate were used to prepare a direct inoculum equivalent to a 0.5 McFarland and suspended in 2 ml nutrient broth, supplemented with 2% NaCl. Breakpoints published by the CLSI were used: oxacillin susceptible (S)
