Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Preparation of nature inspired indicator based agar for detection and identifcation of MRSA and MRSE Cagla Celik a,1 , Nilay Ildiz b,∗∗ , Pinar Sagiroglu c , M. Altay Atalay c , Cevat Yazici d , Ismail Ocsoy a,∗ a Department of Analytical Chemistry, Faculty of Pharmacy, Erciyes University, 38039, Kayseri, Turkey b Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Erciyes University, 38039, Kayseri, Turkey c Department of Medical Microbiology, School of Medicine, Erciyes University, 38039, Kayseri, Turkey d Department of Medical Biochemistry, School of Medicine, Erciyes University, 38039, Kayseri, Turkey ARTICLE INFO Keywords: Methicillin-resistant Staphylococcus aureus MRSA Methicillin-resistant Staphylococcus epidermidis (MRSE) Colorimetric sensor Detection Agar Natural indicator ABSTRACT Natural indicator, red cabbage extract (RCE) incorporated agars were developed, for the frst time, as colori- metric sensors for identifcation of MRSA and MRSE. These strains were diferentiated in RCE media with ad- dition of plasma due to coagulase positive property of MRSA, they were diferentiated by manipulating NaCl and introducing gelatin in RCE agar. RCE agar was examined based on concentration of NaCl and MRSA con- centrations and incubation time for detection of MRSA. RCE agar was prepared mixing 10g peptone, 1g beef extract, NaCl, 15 mg/mL agar and 25% RCE in distilled water and sterilized in autoclave at 121°C for 15 min. 4 μg/mL cefoxitin was added to mixture based on ex- periment. The color of RCE agar including 50 mg/mL NaCl was turned to pink dependent upon growth of MRSA, MRSE and MSSA, growth of E. coli was inhibited due to its salt intolerance property. Introducing 4 μg/mL cefoxitin, growth of MRSA was not observed. 1 CFU/mL, 10 CFU/mL, 100 CFU/mL and 1000 CFU/mL of MRSA inoculated on the RCE agar showed growth and led color change in 24 hrs. Additionally, slight pink spots on RCE agar and pale pink color on whole RCE agar were appeared in 8th hrs and 11th hrs of inoculation, respectively when 1000 CFU/mL of MRSA used. The RCE agar was successfully used for detection of MRSA and diferentiation of them. Finally, the RCE agar can be implemented in clinics and may alleviate incubation time and cost compared to the chromogenic agars. 1. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin- resistant Staphylococcus epidermidis (MRSE) known as nosocomial bac- teria developed intrinsic resistance to various antibiotics containing beta lactam groups. [1–5]. These are considered as community-ac- quired and nosocomial pathogens and cause several serious diseases including meningitis, septicemia and infammatory wounds. [5–7]. Danger of MRSA and MRSE infections is associated with mortality and even recent reports revealed that the increase in mortality caused by nosocomial infections is much higher than HIV/AIDS-related diseases in the United States. [8,9]. For eradication of these multidrug-resistant bacteria, essential treatment option is to use broad-spectrum antibiotics. Unfortunately, prescribing broad-spectrum antibiotics until diagnosis of infection and prolongation of treatment both adversely infuence patient's immune system, hospitalization cost and national economy as well. [2,10,11]. Additionally, wrong and unnecessary antibiotic use not only afect patient's health but also results in the development of new resistances. [12]. To detect MRSA and MRSE, a variety of genotypic and phenotypic methods have been actively used in clinics. For instance, PCR (Poly- merase Chain Reaction) is used as a gold standard method to identify the MRSA with excellent sensitivity and specifcity with 2 hrs. The identifcation mechanism is to beneft multiplexed PCR primers for screening of specifc genes of MRSA. [13–15]. Despite these unique features of PCR, it is much costly, less available in every hospital and requires specialized personnel compared to phenotypic methods. Cul- ture methods including liquid media and agar show the growth of bacteria based upon 18–24 hrs of incubation. Up to date, various bac- teria selected agars have been developed and in use for bacteria iden- tifcation. [5,16–18]. For instance, Wolk and co-workers reported sys- tematic comparison study on Bio-Rad’s MRSASelect agar and Becton https://doi.org/10.1016/j.talanta.2020.121292 Received 6 April 2020; Received in revised form 9 June 2020; Accepted 10 June 2020 ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: nilaygucluer@erciyes.edu.tr (N. Ildiz), ismailocsoy@erciyes.edu.tr (I. Ocsoy). 1 First author. Talanta 219 (2020) 121292 Available online 05 July 2020 0039-9140/ © 2020 Elsevier B.V. All rights reserved. T