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Discovery of Actinomycetes from 
Extreme Environments with 
Potential to Produce Novel 
Antibiotics  
 

Lyudmila Trenozhnikova1, Azliyati 
Azizan2 

 
 

1Institute of Microbiology and Virology, 
Almaty, Kazakhstan;  
2Nazarbayev University School of 
Medicine, Astana, Kazakhstan 
 

 
Vol. 7, No. 1 (2018)   |   ISSN 2166-7403 (online)  
DOI 10.5195/cajgh.2018.337 |   http://cajgh.pitt.edu 

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Central Asian Journal of Global Health 
Volume 7, No. 1 (2018) |  ISSN 2166-7403 (online) | DOI 10.5195/cajgh.2018.337|http://cajgh.pitt.edu 

 
 

Abstract 

Introduction: Antimicrobial-resistant pathogens pose serious challenges to healthcare institutions and health of the public. Thus, 
there is an urgent need for the discovery of new and effective antimicrobial agents.  Microorganisms that exist in extreme 
environments such as those with high salinity or alkalinity, are known as extremophiles, and include various species of 
actinomycetes. The goal of this study is to discover novel antibiotics from extremophiles found in Kazakhstan that are effective 
against drug resistant pathogens. 
Methods: Soil from extreme environments of Kazakhstan was collected, and pure cultures of actinomycetes were isolated and 
cultured in modified Bennett’s broth with either high concentrations of salt or high pH to mimic extreme environments. Extracts 
obtained from selected actinomycetes strains were used to test for antimicrobial activity against Staphylococcus aureus, Escherichia 
coli and Aspergillus niger. 
Results: A total of 5936 strains of extremophile actinomycetes were isolated; from these, 2019 strains were further isolated into 
pure cultures. Of these, 415 actinomycetes strains that demonstrated antagonistic antibacterial activities were selected.  These 
actinomycetes were further classified into groups and subgroups based on their responses to different culture conditions. 
Antimicrobial antagonism activity for some of the actinomycetes strains was dependent on culture conditions and development of  
aerial mycelia under extreme conditions.  
Conclusion: We identified several interesting candidate extracts with putative antimicrobial activities against several strains of 
drug resistant pathogens. Our research of the actinomycetes’ ability to produce antibiotics in the near-natural conditions provides 
a great opportunity to assess their biodiversity and distribution in the Central Asian region and to develop new methodological 
approaches to the screening of new antimicrobial agents. 

Keywords: Extremophiles, Actinomycetes, Antibiotics, Natural Products, Pathogens, Antibiotic Resistance 

 
Discovery of Actinomycetes from 
Extreme Environments with Potential 
to Produce Novel Antibiotics  
 

Lyudmila Trenozhnikova1, Azliyati 
Azizan2 

 
 

1Institute of Microbiology and Virology, 
Almaty, Kazakhstan;  
2Nazarbayev University School of Medicine, 
Astana, Kazakhstan 
 
 

Research 

Introduction 

Although antibiotics have saved millions of 
lives over the past 70 years, their indiscriminate use has 
led to the emergence of antibiotic-resistant organisms. 
This concerns many medical experts who caution that we 
may soon return to the pre-antibiotic era1,2. 

Actinomycetes, which occur in both terrestrial 
and aquatic habitats, are among the most common groups 
of gram-positive microorganisms in nature. 
Actinomycetes decompose organic matter and display 
antagonism against other bacteria and fungi, with which 
they compete for nutrients. Actinomycetes have 
incredible abilities to survive under extreme conditions 
in their natural environment and have long been the focus 

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of scholarly attention and have been harnessed as 
valuable sources of natural compounds, such as 
antibiotics, enzymes, and vitamins. More than 90 percent 
of chemotherapeutic antibiotics have been isolated from 
actinomycetes3-5. 

The large-scale screening of antagonistic 
actinomycetes under neutral conditions (with a pH of 7.0 
and no addition of salt [NaCl, Na2CO3] to the growth 
media) has led to the discovery of virtually all modern 
medical antibiotics1,2. During screening, the strains of 
actinomycetes that did not show activity in neutral 
environments were likely disregarded. Microorganisms 
that produce antimicrobials under extreme growth 
conditions may have gone undetected while being 
screened under neutral growth conditions, preventing the 
discovery of potentially valuable novel compounds from 
these strains. 

Advances in PCR technology have revealed that 
actinomycetes do not always exhibit antagonistic 
properties when cultivated in vitro despite the presence 
of genes that are involved in antibiotic synthesis in the 
genome of the strain. This suggested that dormant genes 
may be present but not expressed. Certain natural 
conditions may exist under which these genes are 
expressed, and identification of these conditions is 
important when screening for the new natural antibiotic 
substances. Modern screening programs should take into 
account the relevant characteristics of the actinomycetes 
and should also include alternative conditions for 
antibiotic isolation. To that end, we undertook a study 
utilizing an alternative method of screening to enhance 
the chances of discovering unique bacterial strains from 
the environment that are potentially producing novel 
antibiotics for therapeutic use.   

This study focuses on actinomycetes strains 
isolated from the extreme habitats of Kazakhstan. We 
investigated the characteristics of actinomycetes in the 
three most commonly occurring natural 
habitats/conditions (which are neutral, saline, and 
alkaline). We believe that this approach, which is 

different than the conventional screening methodology, 
enhances the chance of identifying actinomycetes strains 
from extreme environments that harbor the potential to 
produce novel antimicrobial agents. Kazakhstan 
possesses unique natural resources that can be sources of 
promising and novel biologically-active substances with 
antibiotic properties. Soil and water resources of the 
Republic of Kazakhstan provide a great variety of natural 
habitats for extremophilic microorganisms (such as 
solonchaks, solonetzes, and saline takyrs). These habitats 
differ both in morphological features and salinization 
(chloride, sulfate, soda, and mixed). The water resources 
of Kazakhstan present a wide variety of habitats for 
extremophiles; this includes the Caspian Sea, the Aral 
Sea, Balkhash Lake, and numerous saline lakes in which 
water salinity can reach up to 335 g/l. The area of saline 
soils in Kazakhstan (including solonetzes, alkaline soils 
and combinations with other soils) is 111.55 million 
hectares (0.4307 million square miles), or 41% of the 
entire territory of the country (1.052 million square 
miles)6. Most of these areas are marked by natural 
mineralization due to the presence of marine sediments. 
Overall, research on extremophilic microorganisms in 
Kazakhstan has been limited. The goal of this publication 
is to describe the process of screening for antagonistic 
activity against drug-resistant pathogens and characterize 
actinomycetes strains which are the potential producers 
of novel antibiotics. 

 

Materials and methods 

Sampling sites and collection of soils  

Natural substrate samples (soils, muds, and 
rhizosphere) were collected from the extreme habitats of 
Northern (Kostanay region, Auliekol and Mendykara 
district) and Southern (Almaty region, Balkhash district) 
Kazakhstan during several field trips in summer 2010-
2011 (Fig. 1A). The soil samples collected were of the 
solonchaks, solonetz and takyr types. Solonchaks are 
strongly saline soils, which are usually light colored and 

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are typically developed in poorly drained arid or semiarid 
areas vegetated mostly by halophytes7. Solonetz are soils 
with a high content of exchangeable sodium and/or 
magnesium ions8. Takyr is usually formed in a shallow, 
depressed area with a heavy clay soil which is submerged 
by water after seasonal rains; after the water evaporates, 
a dried crust with fissures forms on the surface9. The soil 

samples were collected at the depth of 10 cm (~4 inches). 
The samples were packed in sterile plastic containers, 
transported to the laboratory, and refrigerated at 4°C 
(39°F) until ready for analysis. Natural substrate samples 
(soils, muds, and rhizosphere) were collected from the 
extreme habitats of Northern and Southern Kazakhstan 
during several field trips in the summer of 2010-2011.

 

Figure 1A. Map of field trips collecting soil samples 

We chose the most common factors in nature, 
which are sodium chloride and pH, and grew the same 
actinomycetes in different growth media to mimic the 
natural environment of extremophiles. The conditions 
these organisms were originally isolated from are neutral, 
saline, and alkaline environments.  Strains of 
actinomycetes from extreme environments of 
Kazakhstan were cultured in neutral and alternative 
(saline and alkaline) conditions and their antimicrobial 
and morphogenetic properties were studied.  Saline and 
alkaline conditions were created by using inorganic salts 
(NaCl, Na2CO3) and рН modulation. 

Isolation and maintenance of Actinomycetes  

Soil samples were plated following the standard 
microbiological dilution plating method. Actinomycetes 

samples were isolated on two variants of modified 
Bennett’s agar:  glucose (0.2%), peptone (0.2%), yeast 
extract (0.1%), and agar (2.0%) with 5% NaCl, рН 7,2 
(variant #1) or with 0.5% Na2CO3, рН 9.0 (variant #2). 
The medium was adjusted with NaOH to pH 9.0 after 
sterilization.  

The plates prepared with variants of modified 
Bennett’s agar were incubated at 28°C and examined for 
growth after 1-2 weeks of incubation. The colonies with 
different cultural-morphological characteristics were 
inoculated from the variants of modified Bennett’s agar 
(variant #1 and variant #2) into slants with the same 
variants of the medium. The purity of isolated strains was 
confirmed by standard microbiological methodologies 
whereby strains were isolated in pure cultures from 

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separately growing colonies obtained from plates seeded 
with soil samples. Each colony was further tested by 
screening on specific agar growth media as described. 

Purified isolates were maintained on the variants 
of modified Bennett’s agar slants at 4°C for further 
antagonism tests and morphogenesis investigation. The 
percent of the total number of strains isolated in the 
relevant region was determined. 
 
In vitro antimicrobial assay 

Actinomycetes isolates were tested for their 
antagonistic activity against the selected microorganisms 
by the disc diffusion agar method10.  

The first stage of screening: The isolates were 
cultured on the variants of modified Bennett’s agar (#1 
or # 2) for 10 days at 28°C. Agar discs (7 mm) were cut 
by a cork borer and transferred to the surface of agar 
plates, previously inoculated with bacterial test 
organisms (hospital strain MRSA # 3316). The petri 
dishes were kept in a refrigerator for 3 hours before 
incubation to permit the diffusion of antimicrobial 
substances. The diameters of the inhibition zones were 
measured after incubation for 24 hours at 37°C. 

The second stage of screening: The isolates with 
antagonistic properties, selected based on the results of 
the first screening stage, were cultured on three variants 
of modified Bennett’s agar (#1-3) for 10 days at 28°C. 
Variant # 3 of modified Bennett’s agar corresponded to a 
neutral habitat:  glucose (0.2%), peptone (0.2%), yeast 
extract (0.1%), and agar (2.0%) at рН 7.2. Agar discs (7 
mm) were cut off by a cork borer and transferred to the 
surface of agar plates, previously inoculated with the test 
organism (MRSA # 3316, Escherichia coli pMG223, and 
Aspergillus niger). The petri dishes were kept in a 
refrigerator for 3 hours before incubation to permit the 
diffusion of antimicrobial substances. The diameters of 
inhibition zones were measured after incubation for 24 
hours at 37°C for bacteria and for 72 hours at 28°C for 
fungi. Each test was repeated three times and the activity 

was expressed as the mean diameter of the inhibition 
zones (mm). 

Study of morphogenesis  

The degree of aerial mycelium development in 
isolates of actinomycetes was studied visually on three 
variants of modified Bennett’s agar (#1-3). 

Bacterial and fungal pathogens  

The following bacterial strains were used in this 
study as testers for the antimicrobial activity of the 
isolated actinomycetes strains:  hospital strain MRSA # 
3316, Escherichia coli (pMG223) and Aspergillus niger 
(wildtype isolate).  

 

Results 

In Northern Kazakhstan (Kustanai region), 
samples were collected from the soils of the steppe and 
forest zones, sor solonchaks (non-perennial salt lakes in 
the area of Aman-Karagai forest), solods, solonets 
(steppe and meadow), rhizospheres of plants from 
halophytic meadows, and muds from the salt lakes (Fig. 
1B). In Southern Kazakhstan (Almaty region), samples 
were collected from the soils of the arid zone, typical 
meadows and sor solonchaks, salinized takyrs, takyr-like 
salinized soils, and rhizospheres of the arid zone plants 
(Fig 1C). We collected 36 samples of natural substrates 
from extreme ecosystems in Northern Kazakhstan and 50 
samples from extreme ecosystems in Southern 
Kazakhstan. 

We identified a link between the ability of 
actinomycetes to grow under conditions reflective of 
three habitats, the ability to antagonize in each of these 
conditions, and the ability to form aerial mycelia. In the 
course of our research, we focused on three main types 
of ecological niches: neutral habitats (рН 7.0), saline 
habitats (рН 7.0), and alkaline habitats (рН 9.0).  

A total number of 5936 actinomycetes strains 
were isolated on the two variants of modified Bennett’s

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Figure 1B. The unusual ecosystem of Northern 
Kazakhstan. In Northern Kazakhstan (Kustanai region), 
samples were collected from the soils of the steppe and forest 
zones, sor solonchaks (non-perennial salts lakes in the area of 
Aman-Karagai forest), solods, solonets soils (steppe and 
meadow), rhizospheres of plants from halophytic meadows, and 
muds from the salt lakes.  

 

Figure 1C. The unusual ecosystem of Southern 
Kazakhstan. In Southern Kazakhstan (Almaty region), 
samples were collected from the soils of the arid zone, typical 
meadows and sor solonchaks, salinized takyrs, takyr-like 
salinized soils, and rhizospheres of the arid zone plants. 
 

 

agar; from these, 2019 strains of extremophile 
actinomycetes grew in media #1 and #2 and differed in 
cultural-morphological characteristics. These strains 
were further isolated in pure cultures (756 strains from 
Northern Kazakhstan and 1263 strains from Southern 
Kazakhstan). The actinomycetes from the extreme 
environments of Kazakhstan were analyzed based on 
their ability to show antagonism against MRSA in saline 
or alkaline conditions. A total 415 strains with 
antagonistic properties were selected: 127 strains from 
Northern Kazakhstan and 288 strains from Southern 
Kazakhstan. These strains showed antagonistic 
properties against bacterial pathogens when grown under 
extreme conditions in media # 1 and #2. 100 percent of 
these 415 strains showed antagonism against 
clinical MRSA # 3316, 21.6 percent against E. coli 
(pMG223), and 28.4 percent against A. niger.   

The correlation of the changes in growth, 
morphogenesis, and antagonism of 415 strains of 

extremophile actinomycetes was determined under three 
conditions, modeling the most common natural habitats: 
neutral, saline, and alkaline. The actinomycetes were 
further classified into groups, subgroups, and 
variants (Table 1) based on their ability to antagonize 
(exhibiting antimicrobial activities of test organisms 
MRSA, E. coli and A. niger) in all three habitats 
(Subgroup IA), only two habitats (Subgroup IB), or only 
one habitat (Subgroup IC).   

Subgroups IIa showed growth in neutral and 
saline conditions, IIb showed growth in neutral and 
alkaline conditions, and IIс showed growth in saline and 
alkaline conditions. 

In each group, we established the following 
subgroups. Three subgroups were identified in Group I: 
IА, IB, and IC (Fig. 2). The subgroups of I are as follows; 
IА subgroup – actinomycetes that show antagonism in 
three conditions (neutral, saline and alkaline): IB 
subgroup – actinomycetes that show antagonism in two 

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conditions:  IBa – antagonism in neutral and saline 
conditions: IBb – antagonism in neutral and alkaline 
conditions: IBс – antagonism in saline and alkaline 
conditions: IC subgroup – actinomycetes that show 
antagonism in one medium: ICa - antagonism in neutral 
conditions: ICb – antagonism in saline conditions, and 
ICс – antagonism in alkaline conditions. 

Two subgroups were identified in Group II: IIA 
and IIB. The subgroups of Group II are as follows; IIA 
subgroup – actinomycetes that show antagonism in two 
media: IIAа – antagonism in neutral and saline 
conditions: IIAb – antagonism in neutral and alkaline 
conditions, and IIAс – antagonism in saline and alkaline 
conditions: IIB subgroup – actinomycetes that show 
antagonism in one medium: IIBa – antagonism in neutral 
conditions, IIBв – antagonism in saline conditions, IIBс 
– antagonism in alkaline conditions.  

In summary, the groups of actinomycetes differ 
from one another in their ability to grow in different 
habitats, which are neutral, saline and alkaline. We define 
subgroups by the ability to produce antibiotics, or they 
may lose this ability to produce antibiotics in these 
specific habitats (neutral, saline or alkaline). The 
magnitude of the inhibition zone was determined by us, 
but for this study, only the presence or total absence of 
antibiotic production was important for the classification 
of actinomycetes that we identified in this study. Since it 
was important to determine the conditions under which 
antibiotic production is possible for each of the producer 
organisms, the strains were classified into these specific 
groups, subgroups and variants. 

Table 2 summarizes the data on the quantitative 
content of actinomycetes-antagonists of different group I 
subgroups in conditions mirroring those of Southern and 
Northern Kazakhstan. The data on the actinomycetes of 
Subgroup II is not discussed here, as the group did not 
have a sufficient size to be represented accurately.  

We observed differences in the occurrence of 
Group I antagonists in the natural substrates of the 

Southern and Northern Kazakhstan. The antagonism 
against gram-positive bacteria in both researched regions 
is characterized by the predominance of Subgroup IA 
(55.4 – 72.3%); Subgroup IB was less common (21.0 – 
34.0%), while Subgroup IC had the smallest amount of 
antibacterial activity against gram-positive bacteria (6.7 
- 10.6%). The antagonism against gram-negative bacteria 
and mycelial fungi also varied by region. In Southern 
Kazakhstan, the Subgroup IA prevailed (41.2 – 41.4%), 
while the Subgroups IB and IC occurred less commonly. 
In Northern Kazakhstan, the antagonism against gram-
negative bacteria was characterized by the predominance 
of the Subgroups IA (41.7%) and IC (38.9%). The 
antagonism against mycelial fungi in the researched 
substrates of Northern Kazakhstan is characterized by the 
predominance of the Subgroup IB (40.7%), while in 
Southern Kazakhstan, Subgroup IA predominates 
(41.4%). 

Our studies of the dependence of antagonism on 
the degree of development of the aerial mycelium of 
actinomycetes under extreme conditions show that 
actinomycetes can be represented by the two main 
variants, which we designated as F and Q (Table 3) 
whereby “F” represents the “Fighters” and “Q” 
represents the “Quitters” which we describe further 
below. 

The variant Q is represented by the 
actinomycetes whose antagonism is associated with good 
growth and abundant development of the aerial 
mycelium. Reduction of sporulation leads to a decrease 
in the production of antibiotics, and absence of aerial 
mycelium to the loss of this ability. 

The variant F is represented by the 
actinomycetes that exhibit antagonistic properties only in 
the conditions when the formation of aerial mycelium is 
inhibited, and its absence is associated with the 
maximum formation of antibiotics. 

The actinomycetes from the extreme 
environments of Kazakhstan were analyzed based on

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Table 1. Classification of actinomycetes based on their ability to grow under different conditions 

Group of actinomycetes Growth in neutral habitat Growth in saline habitat Growth in alkaline habitat 

I (all habitats) + + + 
 II (two habitats) 

IIa + + - 

IIb + - + 

IIc - + + 

+ means growth; - means no growth 

Table 2. The quantitative content of Group I antagonists in the extreme ecosystems of Kazakhstan 

Subgroups of 
actinomycetes 

Quantitative content of antagonists, %1 

Antibacterial activity 

(gram-positive bacteria, S. 

aureus) 

Antibacterial activity 

(gram-negative bacteria, 

Е. coli) 

Antifungal activity (A. 

niger) 

2Southern Kazakhstan            N = a                   N = b                   N = c 

Subgroup IA 55.4 41.2 41.4 

Subgroup IВ 34 37.3 29.3 

Subgroup IС 10.6 21.5 29.3 

3Northern Kazakhstan             N’ = x                  N’ = y                  N’ = z 

Subgroup IA 72.3 41.7 29.6 

Subgroup IВ 21 19.4 40.7 

Subgroup IС 6.7 38.9 25.9 

 
1Note – Total activity for the three subgroups IA, IB, and IC add up to 100%, for each kind of antibacterial or antifungal 
activity tested. 
2Note-Total number (N) of actinomycetes strains from Southern Kazakhstan tested was 1,263. 
3Note-Total number (N) of actinomycetes strains from Northern Kazakhstan tested was 756. 
 

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Table 3. The quantitative content of variants of actinomycetes in the extreme ecosystems of Kazakhstan 

Type of soil Variants of actinomycetes 
Variant F1 Variant Q2 

Solonchaks 67.2 32.8 
Takyr-like salinized soils 56.7 43.3 
Salinized takyrs 51.8 48.2 
Solonets soils 50.4 49.6 
Solods 75.0 25.0 
Total 60.2 39.8 

1F (“Fighters”) – antagonism in the absence of aerial growth (i.e., when they are fighting to survive);  
2Q (“Quitters”) – antagonism associated with good aerial growth (i.e., quit producing antibiotics when growth is poor) 
 

Figure 2. The classification of actinomycetes from extreme ecosystems into groups and subgroups 

Group IA -  antagonism in three habitats (neutral, saline and alkaline) 

 

 
 
 
 

 
 
 
 
 

Subgroup IBа - antagonism in neutral and saline habitats 

 

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Subgroup IBb - antagonism in neutral and alkaline habitats 

 
 
 
 
 
 
 
 
 
 
 

Subgroup IBс - antagonism in saline and alkaline habitats 
 
 
 
 

 
 
 
 
 
 
 
 

Subgroup ICb - antagonism in saline habitat 

 

 

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Subgroup ICc - antagonism in alkaline habitat 

their ability to show antagonism against MRSA in saline 
or alkaline conditions. The isolates with antagonistic 
properties, selected based on the results of the first 
screening stage, were cultured on three variants of 
modified Bennett’s agar (#1-3) for 10 days at 28°C. 
Variant #1 mimics a saline habitat, while variant # 2 
mimics an alkaline habitat and variant # 3 mimic neutral 
habitats. Agar discs (7 mm) were cut by a cork borer and 
transferred to the surface of agar plates, previously 
inoculated with the test organism (MRSA # 3316, 
Escherichia coli pMG223, and Aspergillus niger). The 
diameter of inhibition zones was measured after 
incubation for 24 hours at 37°C. Each test was repeated 
three times and the activity was expressed as the mean of 
diameter of the inhibition zones (mm). The 
actinomycetes were classified into these groups; I 
(growth in all three habitats) and II (growth in two 
habitats). The actinomycetes group I, showing the ability 
to grow in all researched habitats, had a greater 
variability of antagonistic properties. These 
actinomycetes groups were further classified into 
subgroups based on their ability to antagonize in the 
saline, alkaline and neutral habitats. Group IA - 
antagonism in three habitats (neutral, saline and 
alkaline). Subgroup IBа - antagonism in neutral and 
saline habitats. Subgroup IBb - antagonism in neutral and 
alkaline habitats. Subgroup IBс - antagonism in saline 
and alkaline habitats. Subgroup ICb - antagonism in 

saline habitat. Subgroup ICc - antagonism in alkaline 
habitat. 

 

Discussion  

We have identified 415 strains of actinomycetes 
that demonstrate varying degrees of antifungal and 
antibacterial activities in saline and alkaline habitats. 
Some of these strains may be producing previously 
unknown antibiotics. 

Traditionally, researchers have studied the 
diversity of actinomycetes using neutral growth media. 
Similarly, isolation, research of their properties, and 
production of natural, biologically active substances have 
been associated with strains grown in neutral media and 
conditions. These neutral conditions served as the basis 
of the discovery of contemporary natural antibiotics. 
However, the prevalence of drug resistance to known 
antibiotic substances necessitates the expansion of the 
boundaries of screening and changes in screening 
methods in order to identify new and promising 
antimicrobials11,12. Thus, unusual natural substrates and 
extreme ecosystems have currently become the most 
popular targets of research as they are the most likely to 
yield new microorganisms with unique properties. 
Marine environments, as well as soils with high levels of 
salinity and alkalinity, are being actively studied as 
sources of new secondary metabolites13 With their unique 

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metabolic pathway formed in special environments, 
microorganisms from extreme environments produce 
many special bioactive substances, such as enzymes and 
antibiotics14-17. The molecules formed by extremophilic 
microorganisms have unique properties that offer ample 
opportunities for a variety of applications18-22. 

There are several limitations in this study. When 
investigating antagonism under changing conditions, we 
did not conduct studies in an acidic environment. In 
addition, in nature there may be other growth conditions 
that are not represented by the three media that we used 
to grow actinomycetes in the laboratory, therefore we 
may have missed the detection of other potential 
antibiotics produced by actinomycetes. The screening 
does not take into account the possibility of re-discovery 
of known antibiotics from certain strains, therefore we 
plan to develop approaches to rule out this possibility in 
future studies. The strains studied were not identified at 
the level of genus and species; however, in future studies 
we plan to perform these studies using traditional 
microbiology methods as well as molecular (PCR) 
methodologies. 

It is important to determine how actinomycetes 
interact with their changing environment and with other 
microorganisms in relation to their ability to consume 
nutrients and produce antibiotics. The main 
characteristics of interest of actinomycetes from extreme 
environments of Kazakhstan are their growth, ability to 
form aerial mycelia, and their ability to antagonize 
bacteria and fungi. 

We have determined that the most widespread 
antibiotic-producing organisms were the actinomycetes 
belonging to group I; they grew in each of the selected 
habitats – neutral, saline, and alkaline. This important 
characteristic accounts for the wide distribution of the 
actinomycetes in soils and water. These actinomycetes 
also constituted the majority of those isolated in our 
research. Actinomycetes of group II were very rare. 
When tested for growth in three different conditions 
(neutral, saline or alkaline), all strains grew in at the least 

two or three conditions. We did not find any isolate that 
grew only in one condition. Their absence may be a result 
of the isolation method used. However, their absence also 
indicates that despite their isolation from extreme 
habitats, actinomycetes are highly adaptable to different 
environmental conditions, including the neutral media. 
Thus, screening from any extreme natural substrates, 
including the less explored marine sources, will enable 
the isolation of actinomycetes that belong to the 
subgroups IA, IBa, IBb, which grow and secrete 
antimicrobials in neutral conditions and, perhaps also, 
secrete already known antibiotic compounds. Our 
proposed classification allows researchers to concentrate 
their efforts on studying antibiotic-producing 
actinomycetes that belong to the specific groups IBс, 
ICb, ICс, IIAc, IIBb, and IIBс, which do not show 
antagonism under neutral conditions but do show 
antagonism under saline and/or alkaline conditions; these 
actinomycetes have the greatest potential for yielding 
new and unexplored antibiotic compounds.  

Actinomycetes are an unusual group of gram-
positive bacteria that have differentiated mycelia, both 
substrate and aerial. Information on the relationship of 
morphogenesis and antibiotic formation in actinomycetes 
is often contradictory. Some studies have correlated 
initial stages of antibiotic production with inhibition of 
aerial mycelium formation, while others indicate that the 
formation of antibiotics is linked to its abundant 
formation23-28. We found two variants of actinomycetes 
whose antagonism is inversely related to their 
morphogenesis. In the actinomycetes of the variant Q 
"quitters" (which constituted 39.8% of total 
actinomycetes isolated), antagonism was correlated to 
good growth and formation of aerial mycelium. The 
reduction of sporulation resulted in a decrease of 
antibiotic production, and its absence resulted in the loss 
of this ability. Actinomycetes of the variant F "fighters" 
(60.2% of total actinomycetes) exhibited antagonistic 
properties only when growth was poor and the production 
of aerial mycelium was inhibited, and its absence was 
associated with the highest level of antagonism. 

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Accordingly, in each previously selected subgroup based 
on the type of antagonism exhibition, the actinomycetes 
may be represented by these variants.  Actinomycetes 
of the variant Q are more attached to certain habitats that 
ensure their comfortable existence, while the 
actinomycetes of variant F are capable of freer movement 
in a variety of habitats and colonization of new 
environments. We can conclude that in any natural 
environment members of both groups can be present 
simultaneously and can engage in certain relationships 
that ensure the survival of the actinomycetes community 
as a whole.  

The research of the actinomycetes’ ability to 
produce antibiotics in near-natural conditions provides a 
great opportunity to assess their biodiversity and 
distribution in the various regions of the world and to 
develop new methodological approaches to the screening 
of new antibiotics. Those groups of actinomycetes that 
produce antibiotics only under extreme conditions, even 
if these are neutrophile strains of the variant F, may be 
the most promising for modern screening. Once their 
growth is inhibited, the neutrophiles of the variant F may 
produce previously unknown antibiotics in saline and 
alkaline habitats. We consider the following subgroups to 
hold the greatest potential for screening: IBс, ICb, ICс, 
IIAc, IIBb, and IIBс. In future studies, we hope to further 
characterize the nature of these unknown antibiotics and 
to test them against known fungal and bacterial 
pathogens, particularly those strains that may be resistant 
to antibiotics currently available in the market. 
Additionally, one of our long-term goals of this work is 
to identify known strains of actinomycetes that exhibit 
interesting characteristics to the species level (which 
would be relevant to this study particularly for strains that 
produce potentially novel antibiotics), and/or to 
potentially identify novel actinomycetes strains. 

An organism’s environment may vary greatly, 
and a neutral habitat may gradually become saline, 
alkaline or acidic. Accordingly, actinomycetes ability to 
survive is largely determined by their adaptability. The 

study of the behavior of actinomycetes, which use 
antibiotics for their survival in changing habitats may 
allow for the development of new approaches to the 
identification of pharmaceutically valuable drugs. This 
study has a significant global health implication. The 
search for new antibiotic-producing strains will require 
collaborations of many countries and will significantly 
enhance global health if new antibiotic agents become 
available. The actinomycetes classification we propose 
here is neither complete nor indisputable, and it can be 
changed and supplemented with new data and hypotheses 
that make it possible to significantly expand it.  

 

Acknowledgements  

We thank Dr. Nancy Touchette and Dr. Ranjan 
Gupta from NIAID/NIH for their review and helpful 
suggestions on this manuscript, following up from the 
NIAID Scientific Writing Workshop held in Tbilisi, 
Georgia in May 2017.   

This research was partly supported by an NIH-
ISTC grant no. K-2239. 

 

References 

1. Singer RS, Finch R, Wegener HC, Bywater R, 
Walters J, Lipsitch M. Antibiotic resistance--the 
interplay between antibiotic use in animals and 
human beings. The Lancet Infectious diseases. 
2003;3(1):47-51. 

2. Talbot GH, Bradley J, Edwards JE, Jr., Gilbert D, 
Scheld M, Bartlett JG. Bad bugs need drugs: an 
update on the development pipeline from the 
Antimicrobial Availability Task Force of the 
Infectious Diseases Society of America. Clinical 
infectious diseases : an official publication of the 
Infectious Diseases Society of America. 
2006;42(5):657-668. 

3. Newman DJ, Cragg GM. Natural products as sources 
of new drugs over the last 25 years. Journal of 
natural products. 2007;70(3):461-477. 

4. Tobias Kieser MJB, Mark J. Buttner, Keith F. 
Chater, David A. Hopwood. Practical Streptomyces 

http://www.library.pitt.edu/
http://www.pitt.edu/
http://www.library.pitt.edu/articles/digpubtype/index.html
http://www.upress.pitt.edu/upressIndex.aspx


 
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of its D-Scribe Digital Publishing Program and is cosponsored by the University of Pittsburgh Press. 
 

Central Asian Journal of Global Health 
Volume 7, No. 1 (2018) |  ISSN 2166-7403 (online) | DOI 10.5195/cajgh.2018.337|http://cajgh.pitt.edu 

 
 

Genetics.  
https://www.jic.ac.uk/science/molmicro/Strepmanu
al/Manual.htm. Accessed 11 Jul, 2018. 

5. Demain AL. Pharmaceutically active secondary 
metabolites of microorganisms. Applied 
microbiology and biotechnology. 1999;52(4):455-
463. 

6. VM B. Formation of saline soils and 
halogeochemical provinces of Kazakhstan. Alma-
Ata, Science (Nauka). 1982. 

7. Merriam-Webster. Solonchak definition.  
https://www.merriam-
webster.com/dictionary/solonchak. Accessed Nov 
10, 2018. 

8. Paul Driessen WAU, International Institute for 
Aerospace Survey and Earth Sciences (ITC), Jozef 
Deckers, Catholic University of Leuven Otto 
Spaargaren, International Soil Reference and 
Information Centre Freddy Nachtergaele, FAO. 
Mineral Soils conditioned by a (semi-)Arid Climate.  
http://www.fao.org/docrep/003/Y1899E/y1899e09.
htm. Accessed Nov 10, 2018. 

9. Wikipedia. Takyr definition.  
https://en.wikipedia.org/wiki/Takir_(soil). Accessed 
Nov 10, 2018. 

10. Egorov NJMM. Fundamentals of theory of 
antibiotics. 2004. 

11. Basilio A, Gonzalez I, Vicente MF, et al. Patterns of 
antimicrobial activities from soil actinomycetes 
isolated under different conditions of pH and 
salinity. Journal of applied microbiology. 
2003;95(4):814-823. 

12. Natural Products. Humana Press; 2005. 
13. Paul EA. Soil microbiology, ecology and 

biochemistry. Academic press; 2014. 
14. Ningthoujam DS, Kshetri P, Sanasam S, 

Nimaichand S. Screening, identification of best 
producers and optimization of extracellular 
proteases from moderately halophilic 
alkalithermotolerant indigenous actinomycetes. 
World Appl Sci J. 2009;7(7):907-916. 

15. Suthindhiran K, Kannabiran K. Cytotoxic and 
antimicrobial potential of actinomycete species 
Saccharopolyspora salina VITSDK4 isolated from 
the Bay of Bengal Coast of India. Am J Infect Dis. 
2009;5(2):90-98. 

16. Vasavada S, Thumar J, Singh SJCS. Secretion of a 
potent antibiotic by salt-tolerant and alkaliphilic 

actinomycete Streptomyces sannanensis strain RJT-
1. 2006:1393-1397. 

17. Dietera A, Hamm A, Fiedler HP, et al. Pyrocoll, an 
antibiotic, antiparasitic and antitumor compound 
produced by a novel alkaliphilic Streptomyces 
strain. J Antibiot (Tokyo). 2003;56(7):639-646. 

18. Kokare C, Mahadik K, Kadam S, Chopade BJCs. 
Isolation, characterization and antimicrobial activity 
of marine halophilic Actinopolyspora species AH1 
from the west coast of India. 2004:593-597. 

19. Manam RR, Teisan S, White DJ, et al. Lajollamycin, 
a nitro-tetraene spiro-beta-lactone-gamma-lactam 
antibiotic from the marine actinomycete 
Streptomyces nodosus. Journal of natural products. 
2005;68(2):240-243. 

20. Fiedler HP, Bruntner C, Bull AT, et al. Marine 
actinomycetes as a source of novel secondary 
metabolites. Antonie van Leeuwenhoek. 
2005;87(1):37-42. 

21. Jensen PR, Williams PG, Oh D-C, Zeigler L, Fenical 
W. Species-specific secondary metabolite 
production in marine actinomycetes of the genus 
Salinispora. Applied and environmental 
microbiology. 2007;73(4):1146-1152. 

22. Phoebe CH, Jr., Combie J, Albert FG, et al. 
Extremophilic orgainisms as and unexplored source 
fo antifungal compounds. The Journal of antibiotics. 
2001;54(1):56-65. 

23. Demain AL. Microbial secondary metabolism: a 
new theoretical frontier for academia, a new 
opportunity for industry. Ciba Foundation 
symposium. 1992;171:3-16; discussion 16-23. 

24. WC Champness KC. Regulation and integration of 
antibiotic production and morphological 
differentiation in Streptomyces. Regulation of 
bacterial development. 1994. 

25. Demain A. Why do microorganisms produce 
antimicrobials? Paper presented at: SYMPOSIA-
SOCIETY FOR GENERAL 
MICROBIOLOGY1995. 

26. Demain AL, Fang A. Emerging concepts of 
secondary metabolism in actinomycetes. 
Actinomycetologica. 1995;9(2):98-117. 

27. Bibb MJ. Regulation of secondary metabolism in 
streptomycetes. Current opinion in microbiology. 
2005;8(2):208-215. 

http://www.library.pitt.edu/
http://www.pitt.edu/
http://www.library.pitt.edu/articles/digpubtype/index.html
http://www.upress.pitt.edu/upressIndex.aspx
https://www.jic.ac.uk/science/molmicro/Strepmanual/Manual.htm
https://www.jic.ac.uk/science/molmicro/Strepmanual/Manual.htm
https://www.merriam-webster.com/dictionary/solonchak
https://www.merriam-webster.com/dictionary/solonchak
http://www.fao.org/docrep/003/Y1899E/y1899e09.htm
http://www.fao.org/docrep/003/Y1899E/y1899e09.htm
https://en.wikipedia.org/wiki/Takir_(soil


 
 

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Volume 7, No. 1 (2018) |  ISSN 2166-7403 (online) | DOI 10.5195/cajgh.2018.337|http://cajgh.pitt.edu 

 
 

28. Size AFJA. Structure of the population of 
Streptomyces hygroscopicus and characteristics of 
its variants. 1993;4(Part 3):65-71. 

 

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	Abstract
	Keywords: Extremophiles, Actinomycetes, Antibiotics, Natural Products, Pathogens, Antibiotic Resistance
	Research

