Identification of a Novel Lytic Bacteriophage Obtained from Clinical MRSA Isolates and Evaluation of Its Antibacterial Activity

Abstract

Multidrug-resistant bacteria particularly MRSA is well known as a worldwide problem. Since the rate of development of novel antimicrobial agents has been slowed down during the last years, there have been a need for the exploration of alternative solutions for the treatment of resistant bacterial infections. Treatment of infections by bacteriophages (phages) that specifically kill the infecting pathogen, i.e. by the process known as phage therapy, is considered as a possible approach to treat multidrug resistant bacteria. Phage treatment has also been considered to treat Staphylococcus aureus infections. This study was aimed to evaluate the antibacterial and cytotoxic activities of a new lytic phage obtained from clinical MRSA strains. This lytic phage named as f LizAnk was obtained during the phage infectivity studies performed with 13 lysogenic phages against MRSA strains. The antibacterial activity of the f LizAnk phage was determined in vitro in BHI (Brain Heart Infusion) and LB (Leuria Bertani) broths and the in vivo antibacterial activity against MRSA strains and possible cytotoxic effect against mammalian cells were tested on fibroblastic cell cultures (3T3). This study was conducted using 20 MRSA strains isolated from hospitalized patients. Identification of the isolates was performed by conventional methods and methicillin resistance was detected with oxacillin disk diffusion test and mecA gene detection by PCR. The method described by Kaneko et al. [Biosci Biotechnol Biochem 1997; 61(11): 1960-2] was used with some modifications, for induction and isolation of the phages. In vitro studies indicated that this phage killed the six different MRSA strains (in 107 cfu/ml concentrations) in 8 hours, and this powerful lytic effect was similar in both of the liquid media. In vivo studies were performed by using cell cultures prepared in microplates, and the wells have been inoculated with only phage, phage + MRSA mixture, and only MRSA. The cells were then evaluated microscopically as well as by MTT assay which detected alive cells colorimetrically, at 2nd and 24th hours. In our study, the f LizAnk phage did not cause any toxic effect on fibroblast cell cultures, in addition it was observed that the antibacterial effect of the phage against MRSA has proceeded in the cell culture. In conclusion, since the fLizAnk phage described in this study exhibited strong antibacterial activity against MRSA strains and no cytotoxic effect was detected against mammalian cells, it might be safely used alone or in a phage cocktail to treat skin infection caused by MRSA.

Safety and efficacy of topical bacteriophage and ethylenediaminetetraacetic acid treatment of Staphylococcus aureus infection in a sheep model of sinusitis

“…Efficacy of both phage and EDTA therapies in the context of treating bacterial infections have been studied thus far mainly in the laboratory and animal models. Phage have previously been noted to be effective in vitro against S. aureus infection and biofilm, and in vivo against S. aureus planktonic infection . However, 2 studies assessing phage therapy against S. aureus biofilms contrast to our work, in that biofilm load was not reduced in the phage‐treated animals .…”

Bacteriophage Procurement for Therapeutic Purposes

“…Isolation of phages that are active against S. aureus turned out to be the most difficult. However, e.g., Kwiatek et al (2012) and Sahin et al (2013) obtained phages active against S. aureus clinical MRSA strains.…”

Application of the Resazurin Cell Viability Assay to Monitor Escherichia coli and Salmonella Typhimurium Inactivation Mediated by Phages

“…A great variety of methods to evaluate the efficiency of bacterial inactivation are already available and include viable plate count methods, turbidity measurements, bioluminescence assays and colorimetric test systems [ 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. However, the evaluation of the efficiency of bacterial inactivation by phages is currently, and has been for decades, mainly performed by monitoring bacterial concentration during treatment time using the standard colony-forming units method [ 15 ].…”

“…It consists of the spectrophotometric quantification of the intense purple-blue color of formazan, an enzymatic reduction of the lightly colored tetrazolium salt. This colorimetric assay has already been used to determine phage screening [ 22 ], assess bacterial viability after phage treatment [ 19 , 20 , 21 ] and monitor biofilm inactivation by phages [ 23 ]. However, the absorption wavelength of tetrazolium salt reduction products is between 500 and 600 nm [ 41 ], which coincides with the wavelength at which the bacterial optical density is read.…”