Extremophilic Actinomycetes Isolated from Soil in Kazakhstan

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 including 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 organisms.8 Moreover, they produce inhibitory only antibiotics only in response to specific growth conditions. Thus, salt for example can be the trigger for actinomycetes to produce antimicrobial compound. use Identification of the compound associated with the inhibitory antibacterial activity was performed using High-Performance Liquid Chromatography (HPLC); this was approached by analyzing and comparing differential peaks between extracts from the same strain grown in different culture conditions. We hypothesize that extremophiles grown in different extreme growth conditions (either saline or alkaline) could produce antimicrobial compounds that have not been yet identified and investigated, and therefore are good candidates of microorganisms that could produce novel antibacterial compounds effective against MDR bacterial pathogens. Non-commercial

Materials and Methods Overall study design This study was a continuation of an ongoing research as we have previously reported.8 Our study found that actinomycetes species are capable of producing antimicrobial activity when these are grown in modified growth media. All the strains used in this study were isolated from extreme environments of Kazakhstan. The strains were isolated from environmental samples collected, which were soils, muds, Figure 1. Sampling sites for Actinomycetes strains #9 (a), 78 (b), 334 (c), 536 (d), and and rhizosphere samples from Northern and 550 (e). a. Ile-Balkhash region, clay desert, salty soil; b. Amankaragay pine forest, saline Southern Kazakhstan. Samples collected solonchak, a sample of the rhizosphere; c. Ile-Balkhash region, sandy desert, saline solonchak; d. Amankaragay pine forest, saline solonchak, a sample of the rhizosphere; e. Ile- were transferred to the laboratory for isola- Balkhash region, sandy desert, typical solonchak. tion of extremophilic microorganisms

[page 12] [Microbiology Research 2020; 11:8356] Article extract the antibiotic from the cultural liq- ings, we tested the inhibitory activity of activities of the extracts to enable us to uid of actinomycetes broth (instead of ethyl samples against conditionally pathogenic compare with the results of the disc diffu- acetate that was previously used as a sol- bacteria Staphylococcus aureus and sion assay. The advantage of this method vent). Culture media from 3 flasks of each Escherichia coli. For the third level of was that more quantity of the extracts could strain grown in same medium were com- screening we performed testing on hospital be applied to the wells (up to 100 μl, com- bined and filtered through the Büchner fun- strains of MDR pathogens, which included pared to 20 μl total volume possible for the nel and a cloth. By doing this biomass and Acinetobacter baumannii and Pseudomonas disc method). For the wells method, liquid culture liquid were divided and used sepa- aeruginosa. These hospital pathogens were nutrient agar media was seeded with a solu- rately for extraction. Volume of the culture provided by the Republican Diagnostic tion of 109 CFU (colony forming unit) of broth was measured and transferred to a Center in Nur-Sultan, Kazakhstan. the test microorganisms and this mixture separating funnel. N-butanol was added in a Inhibitory antibacterial activity was deter- was then poured into the petri dishes. After 1:5 ratio to each funnel with the culture liq- mined using the standard disc diffusion and solidification of the agar, four 7 mm-diame- uid and mixed well periodically during the wells assay methods. The standard disc dif- ter wells were created using a cork borer. day and left to stay overnight under the fusion assay was performed by applying 20 Finally, 0.1 ml of each sample was pipetted fume hood, so that two phases (organic and μL of the extract to the sterile disc and test- into these wells clockwise starting from inorganic) separate. Organic (top) phase ing this along with the control antibiotics neutral media, saline 1, saline 2, and alka- was collected to the clean vial and further discs. Solutions containing the appropriate line. This was done for extracts from both evaporated in the rotary evaporator at 14 amount of test organisms in growth media the filtrate and the biomass. Inoculated petri mmHg pressure at 40ºC. Finally, the evapo- were prepared according to 0.5 Mc Farland dishes were incubated for one day, after rated dry extracts were dissolved in 90% standard.10 200 µl of suspension was pipet- which the activities (zones of inhibition) ethanol and stored at 4ºC for future analy- ted onto the surface of Mueller-Hinton agar were measured. sis. in Petri dishes and spread with a sterile Extraction from biomass (cell pellet) spreader over the whole surface of the agar; Chemical characterization was also performed to obtain the antibiotic thereafter extract-containing discs were Analytical high-performance liquid compound left within the cells. Pellet left placed onto the agar surface containing the chromatography was used for initial analy- after filtration of the actinomycetes growth test microorganisms. Placement of disc sisonly of differences between the extracts with media was dried wrapped in the clean cloth were performed clockwise from a starting antibiotic activity and those without activi- and filter paper. After drying, the biomass point, beginning with the extracts that were ty. To separate the content of the extracts, was transferred to a clean vial, and mixed grown in neutral, then saline 1, saline 2, and reverse phase C18 column with increasing with 90% ethanol in 1g:3ml ratio, and left alkaline media, accordingly. We also per- acetonitrile gradient was used. 20 µl of sam- overnight at 4ºC. Next day, the biomass was formed the wells assay method as usean alter- ple was used per run and detected at 210 nm filtered out and liquid extract was evaporat- native method to compare the inhibitory wavelength. ed in the rotary evaporator at 97 mmHg pressure at 40ºC. The solvent was evaporated up to the volume equal to 2/100 times the volume of the filtrate (culture liquid), and stored at 4ºC. Each extract was labeled and given the numbers 1 and 2, which defines the origin of the extract, either from culture liquid (designated as 1) or biomass (designated as 2), respectively. For instance, extract #9-1 represents the extract from strain #9 extracted from culture liquid, and extract #9-2 is the extract obtained from the biomass of strain #9. The flowchart summa- rizing this method to obtain “crude extracts” used in the assays described below for antimicrobial activity andNon-commercial HPLC is shown in Figure 2.

In vitro antimicrobial assay Three levels of screening of the extract antibacterial activities were performed. The first level of screening was done after sepa- ration of culture liquid and biomass. Approximately 1-2 ml of culture liquid was taken for analysis before addition of n- butanol, and sample from biomass was Figure 2. Flowchart for the extraction of antibiotic method from actinomycetes species. taken after the addition of ethanol and Strains were cultured in broth media, then grown culture was filtered through the before evaporation. The second level of Büchner funnel with a cloth inside. So that the biomass can stay on the cloth. Afterwards, screening was performed using the final organic phase of a culture liquid was extracted and separated by addition of n-butanol in product (which were the extracts obtained a separatory funnel. Biomass was dried and suspended in 90% ethanol overnight. Finally, after evaporation and reconstitution). The solvents (n-butanol and ethanol) were evaporated in the rotary evaporator and resus- pended in 90% ethanol to a final volume of ~2-3-ml. Extracts were then given numbers procedure for both screenings is the same -1 or -2, which defines the origin of the extract, culture liquid or biomass, respectively. except that the samples tested are different. For the first and second levels of screen-

[Microbiology Research 2020; 11:8356] [page 13] Article

Molecular analysis machine and blast search analysis was per- Disc diffusion and wells assay For molecular characterization to iden- formed to identify the strains of actino- Wells assay showed significant tify the 5 actinomycetes strains, PCR was mycetes. inhibitory activity of extracts against the performed using the primers TCACGGA- conditionally pathogenic bacteria E. coli GAGTTTGATCCTG: and S. aureus. Results of secondary levels (forward rRNA actinomycetes) and GCG- of screening are shown in Table 1. Extracts GCTGCTGGCACGTAGTT (reverse rRN Results from strain #334 grown in saline media A actinomycetes) as previously report- Extracts obtained from culture media showed inhibition of both test microorgan- 11 ed, on Bio-Rad T100 thermal cycler and the biomass showed inhibitory antibac- isms, whereas extracts from the strain machine. The PCR conditions used were; terial activity. Most of the extracts were grown in neutral and alkaline media did not initial denaturation at 96°C, 5 min; denatu- either only active when grown in saline exhibit antimicrobial activity. Extracts from ration 96°C, 1 min; annealing 48 °C, 1min; media, or had better inhibitory activity culture broth of strains #536 and 550 grown extension 68°C, 1min, for 40 cycles; and against the selected test bacteria, when in all four media conditions showed activity final extension at 68°C, 5 min. The total grown with the increased pH growth condi- against S. aureus. Extract #78 was effective PCR product volume was 20 µl. The PCR tions. Preliminary chemical analysis against all five test microorganisms where- products were then sent for sequencing showed comparable chromatograms, which as extract #9 had minor inhibition of S. analysis to National Center for correlate with the activity of the extracts. aureus in alkaline medium. Results of the Biotechnology (NCB) in Nur-Sultan, All of the five strains were sequenced and third level of screening of these extracts Kazakhstan. Sequencing was performed appeared to be actinomycetes species, performed on the MDR pathogens A. bau- according to standard protocol using specifically belonging to the Streptomyces mannii and P. aeruginosa are summarized 3730XL Applied Biosystems sequencing and Nocardiopsis genera. in Table 2. Antibiogram for test bacterial

Table 1. Secondary screening using wells assay. Tests were done in duplicates. Average ZOI in mm is shown in table. Strain # Microorganism Neutral medium (mm) Saline 1 medium (mm) Saline 2 medium (mm) Alkaline medium (mm) 9-2 S. aureus 0 0 only 0 14bs E. coli 0 0 0 0 78-1 S. aureus 9 bc 10 bc 12 bs 0 E. coli 13 bc 10 bc 12 bc 11 bc 334-2 S. aureus 0 10 bc use 13 bc 0 E. coli 0 24 bc 12 bc 0 536-1 S. aureus 13 bc 14 bc 18 bc 15 bc E. coli 0 0 0 0 536-2 S. aureus 9 bc 12 bc 8 bc 0 E. coli 0 0 0 0 550-1 S. aureus 12 bs 16 bs 12 bs 20 bs E. coli 0 11 bc 13 bc 12 bc ZOI – zone of inhibition. bc – bactericidal effect (completely clear zones without any growth of test-microorganism). bs – bacteriostatic effect (slightly opaque zone or small colonies of test-microorganism grown within the zone of inhibition).

Table 2. Inhibition zones from activity test using wells method against pathogens. Tests were done in duplicates. Average ZOI in mm is shown in table. Strain # Microorganism Neutral medium (mm) Saline 1 medium (mm) Saline 2 medium (mm) Alkaline medium (mm) 9-2 A. baumannii 5.5 bc 0 0 0 P. aeruginosa (S) Non-commercial 0 0 10 bc 0 P. aeruginosa (R 0 0 0 0 78-1 A. baumannii 9.5 bc 8.5 bc 0 0 P. aeruginosa (S) 10 bc 10 bc 12 bc 11.5 bc P. aeruginosa(R) 13.5 bc 11 bc 10 bc 10.5 78-2 A. baumannii 0 0 0 0 P. aeruginosa (S) 9 bc 9.5 bc 9 bc 10 bc P. aeruginosa (R) 11 bc 10 bc 13 bc 12 bc 536-2 A. baumannii 0 0 0 0 P. aeruginosa (S) 0 10 bc 11 bc 10 bc P. aeruginosa(R) 0 0 0 0 550-1 A. baumannii 9.5 bc 10 bc 10 bc 0 P. aeruginosa (S) 11 bc 11.5 bc 11 bc 10.5 bc P. aeruginosa (R) 0 0 0 0 550-2 A. baumannii 4 bc 4 bc 4 bc 4 bc P. aeruginosa (S) 11.5 bc 4 bc 9.5 bc 10.5 bc P. aeruginosa(R) 0 0 0 0 ZOI – zone of inhibition. bc – bactericidal effect (completely clear zones without any growth of test-microorganism). bs – bacteriostatic effect (slightly opaque zone or small colonies of test-microorganism grown within the zone of inhibition)

[page 14] [Microbiology Research 2020; 11:8356] Article pathogens tested against standard antibiotics is shown in Table 3. For P. aeruginosa we tested our extracts against the drug sensitive as well as drug resistant strains. Extract #78-1 inhibited MDR resistant pathogens A. baumannii and P. aeruginosa, exhibiting bactericidal effect. Extracts # 78- 1, 78-2, 536-1, 550-1, and 550-2 all had activity against drug-sensitive P. aeruginosa. Extract #78-2 had promising effect against drug-resistant P. aeruginosa. Extracts from strain #550 had a weak yet promising activity against MDR resistant A. baumannii. Extracts #334-1 and 334-2 unfortunately, did not show any inhibition of MDR pathogens. All of the extracts showed clear zones, which suggests that they have completely killed the pathogen around the discs/wells, having bactericidal effect.

High-Performance Liquid Chromatography (HPLC) HPLC analysis showed the peaks which can be correlated with the activity of the extract. Comparing the extracts derived only from the same strain of actinomycetes but grown in different media, we were able to Figure 3. HPLC chromatogram for extract #334-2. A. Saline 2 media (50g salt); B. Alkaline media. There are notable peaks in Saline 2 media which also had activity against identify differential peaks that might be conditionally pathogenic bacteria S. aureus and E. coli, as well as resistant P. aeruginosa. associated with the antibacterial inhibitory Whereas the extract grown in alkalineuse media did not have activity in any test and, consis- activities of the extracts. For example, the tently, the chromatogram does not have same peaks. extract #334-2, which had activity in saline

Table 3. Antibiogram for Acinetobacter baumannii and Pseudomonas aeruginosa test microorganisms. Name of the microorganisms Acinetobacter Pseudomonas aeruginosa MRGN Pseudomonas Antibiotics baumanii MRGN Sensitivity of microorganisms aeruginosa Ampicillin sulbactam R Cephtazidime R R S Imipenem R R S Meropenem R R S Gentamycin R R S Ticarcillin Non-commercial R S Ciprofloxacin R R S Levofloxacin R R S Piperacillin R R S Piperacillin/ tazobactam R S Ticarcillin-clavulanate R Cefepime R R S Cefotaxime R Ceftriaxone R Aztreonam R S Amikacin S R S Tetracycline R Doxycycline R Trimetoprime/ sufometaxol R MRGN – multi-resistant Gram-Negative resistance to 4 types of antibiotics: ureapenicillins, 3-4 generation cefasporins, cabrophenes, fluoroquinolones. S- sensitive. R- resistant. A. baumannii and P. aeruginosa labeled as MRGN are resistant to 4 classes of antibiotics. One strain of P. aeruginosa is sensitive to all classes of antibiotics. This strains were taken for comparison with resistant test microorganisms, and see the potential of our crude extract.

[Microbiology Research 2020; 11:8356] [page 15] Article

1 medium (13 mm) and did not have the activity in alkaline medium (0 mm) had dis- tinguishable chromatograms with different peaks (boxed) (Figure 3). Extract #9-2 inhibited S. aureus in alkaline medium, whereas in other media there was no effect. Correspondingly in Figure 4 we can see that chromatogram of the extract in alkaline environment has a significant peak, whereas in extract from saline 2 medium there is a plateau at the same time point (boxed). Chromatograms of the extract #536-1 both have different contents in them (Figure 5); as shown in Table 1, secondary screening of this extract showed antibacterial activity in both saline and alkaline media against S. aureus.

Sequencing data Sequencing data was obtained as an alignment of nucleotide sequence for each strain. Basic Local Alignment Search Tool (BLAST) was used to find similarity of obtained sequences to those present in the database. Three of five strains (strain #9, 78, and 334) had 98 to 100% similarity to only Streptomyces species. Two strains (#536 Figure 4. HPLC chromatogram for extract #9-2. A. Saline 2 medium (50g salt); B. and 550) had similarity to Nocardiopsis Alkaline medium. Chromatograms differ according to the media they come from. Saline species. Most of the strains associated with 2 medium has significant peaks compared to the alkaline medium. This correlates with the inhibitory activity against S. aureus, 0mm in saline medium, and 14mm in alkaline our actinomycetes strains have antibiotic medium. use activity that were previously reported (Table 4).

Discussion Culture conditions for growing actinomycetes influence the production of bacterial inhibitory compounds by extremophilic actinomycetes. This was illustrated in the HPLC chromatogram that showed correlation of specific peaks to the antibacterial inhibitory activity tests (disc diffusion and wells assay) that were performed in this study. Extremophilic actinomycetes can potentially be the bacterial species that produce antibacterial agents that have notNon-commercial been previously identified and reported. It has been long known that scientists should come up with a new strategy to find new antibiotics compound and not re-discover known ones. Therefore, it is reasonable to try finding new sources of antibiotics using alternative methods to the tradi- tional approaches. Until now actinomycetes species have been the most abundant natural source that was found to produce antibiotic. However recently, the discovery of novel antibiotics from these organisms has waned. Since it has been found that actinomycetes can also live in extreme environ- Figure 5. HPLC chromatogram for extract #536-1. A. Saline 1 media (25 g salt); B. ments such as at high saline, high alkaline Alkaline media. Both chromatograms show presence of different compounds in the and temperature conditions, our research extract. There is an activity in both media, however, the chromatographic profiles are dif- focus has been to screen for antibiotic pro- ferent, which can mean that the bacteria secrete different compounds in response to the composition of the media. ducing organisms from those extremophiles

[page 16] [Microbiology Research 2020; 11:8356] Article instead of just actinomycetes isolated from ered a potential source of novel antibiotics. activities. All of these studies show that neutral environments, using specialized cul- Different studies have found such com- extreme environments, being desert, marine ture conditions that mimic the original envi- pounds as abenquines, chaxalactins, and ecosystems, or mud, contain unique acti- ronment. It was found that in such extreme others from actinomycetes isolated from nobacteria that still can produce antimicro- conditions, Actinobacteria are able to main- deserts and arid habitats.12 As such, Ramya bial, possibly novel, compounds. However, tain favorable environment for themselves et al. (2015),13 investigated the property of the strategy of these studies only directed to and inhibit growth of other bacteria by pro- actinomycetes to produce selenium isolate the bacteria from extreme environ- ducing special compounds. Halophilic and nanoparticles extracellularly, which have ments, but not grow them in similar envi- alkaliphilic actinomycetes are being consid- anti-biofilm, anti-cancer, and anti-viral ronments. To that end, the approach used in

Table 4. Sequencing results to elucidate actinomycetes strain identity Strain # Strain BLAST search match Strain species Presence in genome Reported antibiotic activity 9 Streptomyces sp. strain Streptomyces sparsus 99% - Has antimicrobial activity against Bacillus subtilis (ACCC 11060), Staphylococcus aureus (AS 1.72), Micrococcus luteus (ACCC 11001), ‘Sarcinalutea’ (AS 1.241) and Xanthomonas oryzae (AS 1.843).14,16 Streptomyces halotolerans 99% - N/A Streptomyces daliensis 98% - N/A Streptomyces luteoverticillatus 98% - N/A Kitasatospora aureofaciens 98% - N/A Streptomyces rimosus strain 98% - Oxytetracycline and other tetracycline antibiotics.15 Streptomyces tacrolimicus 98% - N/A 78 Streptomyces sp. strain Streptomyces pratensis 100% only - Methoxyphenyl-oximes. 17 Streptomyces lavendulae 100% - Streptothricin - Streptothricin like substances.18 Streptomyces cyaneofuscatus 100% - Anthracycline family.19 Streptomyces griseoplanus 98% use - Anticapsin.20 Streptomyces fimicarius 99% - Srystalline antibiotics21 Streptomyces flavogriseus 98% - Produce actinomycin D and holomycin22 334 Streptomyces sp. strain Streptomyces sparsus 99% - Has antimicrobial activity against Bacillus subtilis (ACCC 11060), Staphylococcus aureus (AS 1.72), Micrococcus luteus (ACCC 11001), ‘Sarcinalutea’ (AS 1.241) and Xanthomonas oryzae (AS 1.843) 14,16 Streptomyces halotolerans strain 99% - N/A 536 Nocardiopsis sp. strain Nocardiopsis dassonvillei 98% - Extracellular enzymes such as amylases, chitinases, cellulases, -glucanases, inulinases, xylanases andβ proteases23 Nocardiopsis synnemataformans 97% - N/A Nocardiopsis alba 98% - Thiopeptide Antibiotic24 550 Nocardiopsis sp. strain Nocardiopsis alba 98% - Thiopeptide Antibiotic24 Nocardiopsis exhalans 99% - Produce arabinose, cellobiose, fructose, gluconate, glucose, mannose, maltose, Non-commercial rhamnose, ribose, sucrose, trehalose, xylose, mannitol, putrescine, acetate, cis-aconitate, 4-aminobutyrate, azelate, citrate, fumarate, glutarate, 3-hydroxybutyrate, pyruvate, suberate, L-alanine, phenylalanine, proline, serine, 4-hydroxybenzoate, and phenylacetate, arbutin, galactose, melibiose, salicin, adonitol, inositol, maltitol, sorbitol, propionate, adipate, itaconate, lactate, malate, mesaconate, oxoglutarate, -alanine, aspartate, histidine, leucine, ornithine,β tryptophan, or 3-hydroxybenzoate, phosphatase and -glucosidase25 Nocardiopsis terrae 98% - N/A α Nocardiopsis dassonvillei 98% - Extracellular enzymes such as amylases, chitinases, cellulases, -glucanases, inulinases, xylanases andβ proteases23 Actinomycetes strains # 9, 78, and 334 that we used are Streptomycetes species, and strain #536 and 550 are Nocardiopsis species. Strain #78 has 100% similarity in its nucleotide sequence contents with Streptomyces pratensis, Streptomyces lavendulae, and Streptomyces cyaneofuscatus. Other strains do not have 100% match in the sequence, which can suggest that this is a new actinomycete strain, not explored yet. The tables also include information about the known antibiotics that are associated with each microorganism that potentially can be our strain. Some of the strains, such as Streptomyces halotolerans, Streptomyces daliensis , Streptomyces luteoverticillatus, Kitasatospora aureofaciens, Streptomyces tacrolimicus, Nocardiopsis synnemataformans, and Nocardiopsis terrae were not found to produce antibiotic compounds.

[Microbiology Research 2020; 11:8356] [page 17] Article our study was to use specific extreme cul- the sequence of which is similar to our organisms and possibly also other multi- ture media that mimic the organism’s natu- strains #9 and 334, have been reported to drug resistant microorganisms. The extracts ral environment for growing the have antibacterial activities against Bacillus that showed the best activity in this study extremophilic actinomycetes species. Those subtilis (ACCC 11060), Staphylococcus are extracts # 78, 536, and 550 will be strains such as the five chosen for this study aureus (AS 1.72), Micrococcus grown in scaled up experiments and further were screened through various screening luteus (ACCC 11001), Sarcinalutea (AS characterized to ensure reproducibility of levels. After these strains were shown to 1.241) and Xanthomonas oryzae (AS extract preparation that generate the active 14 consistently exhibit antibacterial properties 1.843). Streptomyces rimosus, which material for further study. Chemical charac- against our test organisms, these were fur- sequence was similar to our strain #9 is very terization for active compound identifica- ther characterized for active compound well-characterized species that is used to tion will be further enhanced by utilization 15 identification. produce industrial tetracycline antibiotics. of state of the art chemistry laboratories Extracts from strains #78, 536, and 550, However, there are also species that we available at our Nazarbayev University core which showed the greatest inhibitory activ- identified that have not been reported to facilities that would include use of prepara- ity were classified according to the previous produce antimicrobial agents. Those are for 8 tive HPLC, Mass spectrometry and LCMS research as a group I, meaning that actino- instance, species Streptomyces halotoler- runs and analyses. At this point in time, our mycetes species are able to grow in all three ance, Streptomyces daliensis and other (as research team has optimized growth culture conditions (neutral, saline, and alkaline). shown in Table 4). This indicates there is conditions, extract preparation, activity Based on the ability to show antagonism strong justification for continuation of our screening and strain identification protocols under different conditions, these three research to discover novel antibiotics from for future studies that will guide us in iden- extracts are classified into following sub- our producer organisms. We proceed with tifying potentially novel compounds from groups: extracts #78 and 550 are in sub- efforts to further characterize these strains our selected extremophilic strains with group IA and extract # 536 in subgroup IBc. and other promising extremophilic actino- activity against important MDR Both extracts #78 and # 550 show antago- mycetes in our collection with the ultimate nism in three habitats, whereas extract #536 goal to identify the active antibacterial com- show antagonism only in saline and alkaline pound from the species that were grown in habitats against tested ESKAPE pathogens. their specific antibacterial producing only Even though the other extracts did not show growth media. Conclusions very good inhibitory activity against hospi- There are several limitations in this Extracts derived from extremophilic tal strains of MDR pathogens, these did study whereby one of these is the low yield actinomycetes have shown significant show good antagonism against conditional- of extracts (3 mL of the extract out ofuse 300 inhibitory activity against conditionally ly pathogenic S. aureus and E. coli. mL of the cultural liquid and hence only 1% pathogenic bacteria. Some of the extracts Classification of those extracts was as fol- of the total growth media) and hence avail- exhibited a broad spectrum of activity lows; extract #334 belongs to subgroup ICb ability of extracts for testing in our small- inhibiting growth of both gram - positive and extract #9 in subgroup IBa. Mostly all scale research. In the future, we will scale and gram-negative test bacterial pathogens the extracts had antagonistic effect in two up production to enable higher amount of chosen for our study. On the other hand, habitats, whereby the saline environment extract production for testing and com- there were extracts that only inhibited shows the most inhibitions among all the pound identification. As shown in Table 2, growth of either gram - positive or gram- extracts prepared from the strains included the ZOI when testing our extracts against negative pathogens. Moreover, there was a in this study. the MDR pathogens P. aeruginosa and A. difference in activity of the extracts Extract #9-2 had only activity in alka- baumannii are not substantial however we obtained from strains grown in different line environment (Table 1), and correspond- are optimistic the results are significant growth media. HPLC results have shown ingly HPLC chromatogram showed inter- given that we used crude extracts from our correlation with the activity of the extracts esting contents in the same medium, but not producer organisms. We hypothesize that according to the activity test results. All the in others (Figure 4). It is possible that the the active compound within our chromatograms collectively show a general peak that showed up in the chromatogram is extremophiles actinomycetes producer profile of the extracts and their composition the compound that actually inhibits the organism will exhibit potent antibacterial with respect to the growth conditions. growth of pathogenic bacteria, and it is only activity once these are identified and isolat- Practical analysis shows that growth media produced under specific alkaline environ-Non-commercialed into its pure form. In this study, our com- ment. The same trend can be seen in extract pound characterization was limited to com- affects the production of antibiotic com- #334-2 (Figure 3). However, this strain parative peak identification of extracts from pounds, as either salt or alkalinity can trig- favors saline environment for production of the same strain grown in different growth ger the secretion of the antibiotic by actino- antibiotic compound, which can be seen media using analytical HPLC and hence no mycetes. Identification of a chemical com- both in the chromatogram (two clear peaks) information was made available about the position of the most potential antibiotic and the antimicrobial activity (Table 1). nature and identity of the active compound extract would answer most of the questions, Interestingly, extract #536-1 inhibited responsible for the antibacterial activities. such as the nature of the compound, the growth of conditionally pathogenic S. However, the analytical HPLC chro- mechanism of action, and presence or aureus in all four media. But the chromatograms generated from this study pro- absence of the resistance in pathogenic bac- matograms of this extract were substantially vided us with an overall profile of the teria. Dereplication process is a target different compared to each other (Figure 5). extracts, which have direct correlation with screening method to achieve good quality This can suggest that different contents are the activity-screening results. The data will and quick results in screening and identifi- released from bacteria depending on the guide us in planning for our next steps to cation of natural antibiotic producer com- medium they live in. achieve our ultimate goal of further charac- pounds, and will be utilized in our future Sequencing results display that most of terization and identification of the active studies. the matched species were previously report- antibacterial compounds. Overall, screening ed to produce antibiotics as described of the extracts shows promising results in below. For example, Streptomyces sparcus, inhibiting the growth of our test MDR

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