Microbiology Research 2020; volume 11:8356 Extremophilic actinomycetes isolated from soil in Introduction Correspondence: Azliyati Azizan, Nazarbayev Kazakhstan: Classification and Antibiotics, first time discovered in University School of Medicine (NUSOM), 5/1 1928 by Alexander Fleming,1 have become Kerey and Zhanibek Khans Street, Nur- antimicrobial activities excellent chemotherapeutic agents effective Sultan, 010000, Republic of Kazakhstan in treating human infections caused by E-mail: [email protected] Tel.: +7-7172- 69-4633 Ainur Zhulamanova,1,2 many dangerous pathogenic bacteria. Even though it was unexpectedly discovered, Zhanat Koshenov,1,2,3 Key words: Extremophiles; antibiotics; natu- antibiotics and search for newer more effec- Saikal Shamkeeva,1,4 ral products; ESKAPE pathogens. 1 tive antimicrobial agents brought science Zhannur Markhametova, and medicine to an extensive research in 1 Acknowledgements: The authors would like Diyora Abdukhakimova, this field. As a result, many more antibi- 5 to acknowledge the Research and Production Lyudmila Trenozhnikova, otics, such as vancomycin, tetracycline, Center for Microbiology and Virology, as well 1 Azliyati Azizan ampicillin and others were discovered with- as the Republican Diagnostic Center for sup- 2 1Nazarbayev University School of in a short period of time. porting us through this project. We also want Natural sources of antibiotics can be Medicine (NUSOM), Nur-Sultan, to thank Nazarbayev University for funding 2 bacteria and fungi. The most prevalent this research. Kazakhstan; National Laboratory antibiotic producers are actinomycetes Astana, (NLA), Nazarbayev University, species, which are gram-positive fungi-like Contributions: AZ performed microbiolgical 3 Nur-Sultan, Kazakhstan; Medical bacteria. Today 45% of all known antibi- part of the study; ZK performed chemical University of Graz, Gottfried Schatz otics are produced by these species which analysis on analytical HPLC, adjusted param- Research Center, Department of are generally found in soil and freshwater.3 eters, and designed a method for analysis; SS, Molecular Biology and Biochemistry, Most of the actinomycetes grow in neu- ZM, DA performed molecular analysis includ- ing primer selection, PCR, and sequencing; Graz, Austria; 4Leipzig University, trophilic and slightly alkaline conditions. LT provided the extremomophilic actino- Institute for Laboratory Medicine, However, there are also acidophilic species that grow in the pH range of 3.5 - 6.5 with mycetes from her collection, and guided the Clinical Chemistry and Molecular only salt concentrations that are above 0.2 mol/L team throughout the project; AA supervised Diagnostics, Lepzig, Germany; the project, provided assistance to all group NaCl.4 These microorganisms are usually 5 members, manuscript writing and checking. Research and Production Center for found in saline soil and are very promising Microbiology and Virology (RPCMV), targets for antimicrobial agents. The search Funding: the funding was provided by Almaty, Kazakhstan use for these organisms as producers of novel Nazarbayev University (NU), grant no. antibiotics is the subject of our current SOM2017001. research. Abstract We have found that there are actino- Conference presentations: Part of this paper mycetes species inhabiting soil with high was presented at the American Society for Extremophilic actinomycetes species salt and high alkaline concentration; these Microbiology Microbe 2018 Conference, are capable of surviving in extreme envi- species appeared to produce antibiotics Atlanta, Georgia, USA, in June 7-11, 2018. ronment and producing antibiotics. In this while growing in saline, alkaline, or neutral study, we hypothesize that extremophiles conditions.5 We continue our efforts in Conflict of interest: The authors declare no produce antimicrobial compounds that are researching extremophilic actinomycetes potential conflict of interests. potentially novel agent(s) effective against that can be isolated from these extreme drug resistant pathogens. The goal of this environmental growth conditions and Received for publication: 29 October 2019. study is to test inhibitory activity of the screen their activity against multi-drug Accepted for publication: 3 January 2020. extracts derived from extremophilic actino- resistant bacteria that can cause life-threat- This work is licensed under a Creative mycetes species against the most prevalent ening infections. drug-resistant bacteria in Kazakhstani hos- Commons Attribution NonCommercial 4.0 Bacterial ESKAPE pathogens, which License (CC BY-NC 4.0). pitals, and preliminarily analyzeNon-commercial chemical are Enterococcus faecium, Staphylococcus composition of the active extracts. aureus, Klebsiella pneumoniae, ©Copyright: the Author(s), 2020 Actinomycetes species isolated from the Acinetobacter baumannii, Pseudomonas Licensee PAGEPress, Italy soil of Kazakhstan were cultured in modi- aeruginosa, and Enterobacter spp., are rec- Microbiology Research 2020; 11:8356 fied media mimicking extreme environment ognized as Multi-Drug Resistant (MDR) doi:10.4081/mr.2020.8356 the species were isolated from. microorganisms. These ESKAPE pathogens Antimicrobial compound(s) extracted with are resistant to almost all known classes of organic solvent were tested against condi- antibiotics and can cause healthcare associ- diminishing treatment options. tionally pathogenic and multi-drug resistant ated infections (HAI).6 It is estimated that Even though most of the antibiotics pathogens Acinetobacter baumanni and by 2050 infections caused by HAI have been rediscovered, which led to the Pseudomonas aeruginosa. pathogens with Antimicrobial Resistance decline in drug discovery research activi- Our study generated promising results (AMR) will cause fatality in up to 10 mil- ties, there is still hope that nature has solu- regarding the potential discovery of novel lion people per year.7 Currently effective tions to the health problems that we have components effective against drug resistant treatment against these drug resistant today. Antibiotics from natural sources usu- pathogens. Future studies will focus on fur- pathogens is scarce or nonexistent in some ally are small compounds that are responsi- ther chemical analysis to identify the active cases, making the situation even more seri- ble for the inhibitory activity against other component within these extremophilic ous. Thus, the search for novel antibacterial bacteria. However, synthesis of these natu- extracts. compounds that are effective against these rally occurring compounds in the laboratory multi-drug resistant pathogenic bacteria is difficult because of the complexity of the becomes even more crucial in this era of compound structure. Chemical analysis is [Microbiology Research 2020; 11:8356] [page 11] Article required to elucidate the exact component which were then grown and extracted to grown in four different modified Bennett’s within the natural products that is responsi- obtain the source of the inhibitory antibiotic broth media, which were neutral, saline 1, ble for the activity against these pathogens. for testing against bacterial pathogens. This saline 2 and alkaline. Neutral medium was Most of the research on screening step was then followed by chemical charac- composed of Oatmeal flour (1.5g), CaCO3 antibiotic compounds from natural products terization of active components using (0.25 g), and Glucose (1.5 g) in a total target prokaryotic organisms from the phy- HPLC analysis, and finally molecular growth media volume of 100 ml per flask lum actinomycetes extracted from neutral analysis for identification of the strains. (strains grown in triplicate flasks/strain/ environment. Interestingly, actinomycetes medium). Saline 1 medium was prepared are also one of the most widespread Subject, sampling, recruitment similarly as neutral media but with the addi- extremophiles that can grow in harsh Soil samples from deserts, solonchaks tion of 2.5 g of salt, while Saline 2 medium growth conditions and they can maintain and forests of Ile-Balkhash and contained 5 g of salt, and alkaline medium favorable conditions for themselves. These Amankaragay regions in Kazakhstan were contained 0.5g of Na2CO3 solution. This microorganisms produce antibiotics natu- collected to obtain the 5 extremophilic growth culture condition was designed to rally as a defense mechanism in order to actinomycete strains number 9, 78, 334, mimic natural environment from where survive and thrive in their natural environ- 536, and 550 that are described in this study, these bacteria were obtained. Actinomy- ments. Thus, it is reasonable to investigate as potential producers of novel antibiotics cetes were grown for 5 days at 28°C with antibiotic producing potential of these (Figure 1). sufficient aeration (continuous shaking in a microorganisms by isolating and growing large 1000ml flask) for optimal growth, them in extreme laboratory culture condi- Isolation and maintenance of after which extraction was performed using tions that would mimic their natural growth organic solvent. This protocol was Actinomycetes 9 environment. Five strains of actinomycetes species described in our previous research; howev- We found that extremophilic actino- (strains #9, 78, 334, 536, and 550) were er, in this study n-butanol was used to mycetes that were isolated from the saline soil were able to inhibit growth of other bacteria including our tested MDR organ- isms.8 Moreover, they produce inhibitory only antibiotics only