Sahin Fikret, Karasartova Djursun, Ozsan T Murat, Gerçeker Devran, Kıyan Mehmet
Ankara University Faculty of Medicine, Department of Medical Microbiology, Ankara, Turkey.
Mikrobiyol Bul. 2013 Jan;47(1):27-34. doi: 10.5578/mb.3790.
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.
多重耐药菌，尤其是耐甲氧西林金黄色葡萄球菌（MRSA），是一个众所周知的全球性问题。由于近年来新型抗菌药物的研发速度放缓，因此需要探索治疗耐药菌感染的替代解决方案。利用能特异性杀死感染病原体的噬菌体（phages）进行治疗，即通过所谓的噬菌体疗法，被认为是治疗多重耐药菌的一种可能方法。噬菌体治疗也被考虑用于治疗金黄色葡萄球菌感染。本研究旨在评估一种从临床MRSA菌株中获得的新型裂解性噬菌体的抗菌和细胞毒性活性。这种名为f LizAnk的裂解性噬菌体是在对13种溶原性噬菌体针对MRSA菌株进行噬菌体感染性研究的过程中获得的。在体外，于脑心浸液（BHI）肉汤和吕氏肉汤（LB）中测定f LizAnk噬菌体的抗菌活性，并在成纤维细胞培养物（3T3）上测试其对MRSA菌株的体内抗菌活性以及对哺乳动物细胞可能的细胞毒性作用。本研究使用了从住院患者中分离出的20株MRSA菌株。通过常规方法对分离株进行鉴定，并通过苯唑西林纸片扩散试验和PCR检测mecA基因来检测耐甲氧西林情况。噬菌体的诱导和分离采用了金子等人[《生物科学、生物技术与生物化学》1997年；61(11):1960 - 2]描述的方法，并做了一些修改。体外研究表明，这种噬菌体在8小时内杀死了六种不同的MRSA菌株（浓度为107 cfu/ml），并且在两种液体培养基中这种强大的裂解效果相似。体内研究通过使用微孔板中制备的细胞培养物进行，孔中分别接种仅噬菌体、噬菌体 + MRSA混合物以及仅MRSA。然后在第2小时和第24小时通过显微镜以及MTT法对细胞进行评估，MTT法通过比色法检测活细胞。在我们的研究中，f LizAnk噬菌体对成纤维细胞培养物未产生任何毒性作用，此外还观察到噬菌体对MRSA的抗菌作用在细胞培养物中持续存在。总之，由于本研究中描述的fLizAnk噬菌体对MRSA菌株表现出强大的抗菌活性，且未检测到对哺乳动物细胞的细胞毒性作用，它可能单独或与噬菌体鸡尾酒联合安全地用于治疗由MRSA引起的皮肤感染。
