Faculty of Science
CHARACTERIZATION OF THE ANTIMICROBIAL SECONDARY
METABOLITES
PRODUCED
BY
THE
CAVE
BACTERIA
STREPTOMYCES SP. STRAIN ICC1
2021 | APRIL LEIGH-ANN READ

B.Sc. Honours thesis – Biology

CHARACTERIZATION OF THE ANTIMICROBIAL SECONDARY METABOLITES
PRODUCED BY THE CAVE BACTERIA STREPTOMYCES SP. STRAIN ICC1
by
APRIL LEIGH-ANN READ

A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF SCIENCE (HONS.)
in the
DEPARTMENT OF BIOLOGICAL SCIENCES
(GENERAL BIOLOGY)

This thesis has been accepted as conforming to the required standards by:
Dr. Heidi Huttunen-Hennelly (Ph.D.), Thesis Supervisor, Dept. Physical Sciences
Dr. Naowarat Cheeptham (Ph.D.), Co-Supervisor, Dept. Biological Sciences
Dr. Donkor Kingsley (Ph.D.), Co-Supervisor, Dept. Physical Sciences
Dr. Dipesh Prema, Co-Supervisor (Ph.D.), Dept. Physical Sciences
Dr. Eric Bottos, Co-supervisor (Ph.D.), Dept. Biological Sciences
Dr. Jonathan Van Hamme (Ph.D.), External Examiner, Dept. Biological Sciences
Dated this 27th day of April, 2021, in Kamloops, British Columbia, Canada
© April Leigh-Ann Read, 2021

ABSTRACT
Streptomyces is one of the most abundant microbial genera in cave environments. The
characteristic most notable of Streptomyces is its ability to produce bioactive secondary
metabolites

–

such

as

antifungals,

antivirals,

antitumorales,

anti-hypertensives,

immunosuppressants, and antibiotics. Through the present research, secondary metabolites
produced by the cave-dwelling Streptomyces sp. strain ICC1 were examined; a strain which is
prevalent in the isolated environment of the Iron Curtain Cave in Chilliwack, British Columbia.
Secondary metabolites secreted by Streptomyces sp. ICC1 have demonstrated antimicrobial
properties, effective against both multi-drug resistant strains and common laboratory strains of
Escherichia coli and Staphylococcus aureus. Liquid organic solvent extractions as well as
preparative and analytical reverse-phase high performance liquid chromatography suggest that
the bioactive secondary metabolites are polar with hydrophobic substituents. Further instrumental
analysis is required to determine the antimicrobial active secondary metabolites structure and
mode of action.

Thesis Supervisor: Naowarat Cheeptham
ii

ACKNOWLEDGEMENTS
I would like to extend my sincere gratitude to my supervisor Dr. Heidi Huttunen-Hennelly for her
help even during her absence. To Dr. Naowarat Cheeptham for her continued support, patience,
understanding, as well as laboratory space and supplies. I would also like to extend thanks toward
professors and lab technicians who provided me guidance throughout my research. To Dr. Eric
Bottos for his knowledge on microbiology and problem-solving abilities. To Trent Hammer for
his assistance in instrumental set up and analysis, particularly with HPLC techniques. To Dr.
Dipesh Prema and Dr. Kingsley Donkor for their instruction with NMR analysis and valuable
chemistry expertise. Further, to Jon Van Hamme for his role as an external examiner.

iii

TABLE OF CONTENTS
Abstract……………………………………………………………………………………………ii
Acknowledgements…………………………………………………………………………….... iii
Table of Contents…………………………………………………………………………………iv
List of Figures……………………………………………………………………………………...v
List of Tables………………………………………………………………………………………v
Introduction………………………………………………………………………………………. 1
The Iron Curtain Cave……………………………………………………………………. 1
Genus Streptomyces………………………………………………………………………..3
Secondary Metabolite Production…………………………………………………………8
Materials and Methods…………………………………………………………………………...12
Recovery from Dormant Colonies………………………………………………………..12
Preparations for Escherichia coli #15-124……………………………………………….12
Bioassay Screening for Antimicrobial Activity…………………………………………..13
Active Compound(s) Separation and Preliminary Structural Analysis…………………..14
Results……………………………………………………………………………………………16
Bioassay for Antimicrobial Activity……………………………………………………...16
Compound Separation Via Reverse-Phase HPLC……………………………………….17
Structural Elucidation of Bioactive HPLC Collections by 1-D 1H-NMR……………….20
Discussion………………………………………………………………………………………..23
Conclusions and Future Work……………………………………………………………………26
Literature Cited…………………………………………………………………………………...29
Appendix…………………………………………………………………………………………33

iv

LIST OF FIGURES
Figure 1. Map of the Iron Curtain Cave in Chilliwack, BC, where Streptomyces sp. ICC1, which
is the strain of interest, was retrieved from Point 1.……………………………………………….3
Figure 2. The developmental life cycle of Streptomyces from spore to aerial hypha .……………4
Figure 3. Scanning electron micrograph of hyphae and spores of Streptomyces coelicolor………5
Figure 4. Scanning electron micrograph of Streptomyces sp. ICC1……………………………….7
Figure 5. Macroscopic morphology of Streptomyces sp. ICC1 on nutrient agar………………….7
Figure 6. Filtered secondary metabolites produced by Streptomyces sp. ICC1………………….10
Figure 7. Kirby-Bauer disk diffusion test against multi-drug resistant Escherichia coli #15-124
showing bioactivity after diethyl ether extractions……………………………………………….17
Figure 8. Analytical reverse-phase HPLC chromatograms of bioactive secondary metabolites in
methanol measured at 272 nm……………………………………………………………………18
Figure 9. Preparative reverse-phase HPLC of bioactive secondary metabolites in methanol……19
Figure 10. Kirby-Bauer disk diffusion test against multi-drug resistant Escherichia coli #15-124
showing bioactivity on Peak 2 and 4 of preparative HPLC collections………………………….20
Figure 11. 1-D1 H NMR spectra of Peak 2 dissolved in deuterium oxide…………..……………22

LIST OF TABLES
Table 1. Analytical and Preparative HPLC parameters for compound separation………………14
Table 2. NMR parameters for structural elucidation……………………………………………..14
Table A1. The diameters of zones of inhibition from Kirby-Bauer disk diffusion assays………34

v

INTRODUCTION

Drug resistance microbes have presented an issue to human healthcare since the first observed
resistance from a strain of Staphylococcus against penicillin in 1942 (Lobanovska and Pilla
2017). Since then, the discovery of new antibiotics has been necessary to combat the spread of
bacteria that are resistant to available pharmaceuticals. Understanding the evolution and origins
of antibiotic resistance genes is vital to predicting, preventing and managing this global health
problem. Studies over the past decade have revealed that antibiotic resistance is common in
contemporary and ancient environmental bacteria, and that the associated genes are similar or
identical to those circulating in pathogens (Perry et al. 2014). Most notable was a study
performed by Bhullar and colleagues (2016) where microbial samples from a region of the
Lechuguilla Cave in New Mexico, which was thought to have been isolated from surface input
for over 4 million years, displayed resistance to commercially available antibiotics. While
resistance is a natural phenomenon, what has changed since the beginning of the antibiotic era is
a marked increase in selection for mobile resistance elements and consequently, increase of drug
resistant pathogenic and non-pathogenic bacteria (Pawlowski et al. 2016). According to the
World Health Organization at least 700,000 individuals die each year due to drug-resistant
diseases (Sarkar et al. 2017). Therefore, an overwhelming demand for novel treatment presents
itself; the likelihood of multi-resistant microbes surpassing the development of antibiotics
escalates in a coevolutionary race between humans and bacteria.

The Iron Curtain Cave
The Iron Curtain Cave is located near Chilliwack, British Columbia. It was discovered by caver
Rob Wall in 1993 and named for its iron-rich sediment. The cave is decorated with large curtains
1

made of calcium carbonate and limestone structures. While the sediment contains iron that gives
it a unique reddish coloration, it also has a clay consistency that indicates higher levels of
moisture (Ghosh et al. 2017). Studies of unique environments have demonstrated that metals play
a pivotal role in determining subsurface living communities (Parnell et al. 2015). Iron is one of
the most abundant metals in the Earth’s crust and is present in various biomolecules (Parnell et
al. 2015). Iron is essential for the function of many fundamental, conserved biochemical
processes, such as the electron transport and Krebs cycle (Bonis and Gralnick 2015). Many cave
bacterial species use iron in a variety of ways, for instance the genus Thiobacillus can obtain
energy by oxidizing sulfur and ferrous iron compounds (De Mandal et al. 2017). The oxidation
state of iron also influences soil structure, dissolved carbon stability, and enzyme activity,
affecting microbial communities and soil fertility (Bonis and Gralnick 2015).

There are six pools of water present from the entrance and throughout the Iron Curtain cave,
which creates a humid environment. The temperature ranges between 4–12 °C depending on the
time of year (Ghosh et al. 2017). The cave has been exposed to a limited number of people as it is
gated and locked, and any access to it needs prior permission from the cave custodian, Rob Wall.
Within the cave there is a specific path to limit human impact as well as to preserve the features
and habitat of the cave. The microbe of importance in this study, Streptomyces sp. ICC1, was
originally isolate 22.47 m from the entrance of the cave, depicted as point 1 in Figure 1 illustrated
by Ghosh et al. (2017). The unique conditions of the Iron Curtain Cave – including high
humidity, relatively low but stable temperatures, and low nutrients, create a highly selective
environment. Energy-starved conditions of caves often lead to the production of various
secondary metabolites that enhance their producers’ likelihood of survival (Gosse et al. 2019).

2

Streptomyces sp. ICC1 is able to thrive in such desolate conditions due to their unique physiology
and specialized metabolic pathways.

Figure 1. Map of the Iron Curtain Cave in Chilliwack, BC, where Streptomyces sp. ICC1,
which is the strain of interest, was retrieved from Point 1 (Ghost et al. 2017).

Genus Streptomyces
Streptomyces is

a

genus

of

the

family Streptomycetaceae belonging

to

the

order

Actinomycetales and the class Schizomycetes. Streptomyces are Gram-positive bacteria that have
a DNA GC content of 69–78 mol% (Korn-Wendisch & Kutzner, 1992). This genus grows
predominately in soil but is also found in various other locations including marine environments
and caves (Flärdh and Buttner 2009). The morphology of most soil dwelling Streptomyces
resembles filamentous fungi due to the production of an extensive branching substrate and aerial
mycelia. The substrate hyphae are approximately 0.5 to 1.0 µm in diameter and often lack crosswalls during the vegetative phase (Anderson and Wellingtonn 2001). Growth occurs at the hyphal
3

apices and is accompanied by branching, thus producing a complex tightly woven matrix of
hyphae during the vegetative growth phase. As the colony ages, aerial mycelia are produced,
which develop into chains of spores by the formation of cross walls in the multinucleate aerial
filaments. This is followed by separation of individual cells directly into spores. The
morphological features of aerial mycelium are regarded as more significant for characterization
than vegetative mycelium. The aerial mycelium include the mode of branching, the configuration
of the spore chains, and the surface of the spores.

Figure 2. The developmental life cycle of Streptomyces from spore to aerial hypha (Flärdh
and Buttner 2009).

4

Figure 3. Scanning electron micrograph of hyphae and spores of Streptomyces coelicolor
(Tschowri 2015).
The spores in soil germinate to produce mycelium under favorable conditions. Upon germination,
the cells show complex vegetative hyphal growth, which aids the microbes’ ability to colonize
the soil. Under conditions of high concentration of nutrient and energy sources, the hyphae are
highly branched and excrete a number of extracellular enzymes in order to obtain the required
nutrients for growth (Avignone-Rossa et al. 2013). In contrast, conditions of nutrient scarcity
restrict cell growth and aerial mycelium is formed, eventually differentiating into spores and
therefore restarting the cycle. This morphological change is accompanied by metabolic changes,
which result in the induction of secondary metabolism. The formation of spores, a semi-dormant
stage in the life cycle of the bacterium, serves the double purpose of resisting unfavorable
environments and spreading the organism. Cave dwelling actinomycetes, such as Streptomyces,
have been studied less compared to soil strains.

5

The strain of interest in the present study is Streptomyces sp. ICC1, which was collected from the
Iron Curtain Cave in Chilliwack, British Columbia. Preliminary 16S rRNA gene sequencing of
ICC1 confirmed that the microbe belongs to the genus Streptomyces, which is supported by
morphology and growth characteristics of the bacteria (Gosse et al. 2018). The scanning electron
micrograph captured by Ghosh et al. (2017) shown below as Figure 2 demonstrates that
Streptomyces ICC1 shares the general trends displayed across the genus, with its dense rodeshaped filaments and colonial appearance. Whole genome sequencing of Streptomyces sp. ICC1
by Illumina MiSeq (Illumina, United States) with de novo genome assembly using PATRIC
generated a draft genome of 9,034,309 nucleotide base pairs containing a guanine and cytosine
content of 72%. BLAST was used to compare the genomic profile of Streptomyces sp. ICC1 with
other genomes on the database. The closest genome match was Streptomyces lavendulae strain
CCM 3239 with 54% sequence alignment to Streptomyces sp. ICC1 and an average nucleotide
identity of 87.9% between the organisms. (Gosse et al. 2018). In addition, RAST annotation
predicted 8235 coding regions for Streptomyces sp. ICC1, which indicates very similar predicted
protein content and function between the strain of interest and S. lavendulae (Gosse et al. 2018).
While Streptomyces lavendulae was found to have the closest genomic match to Streptomyces sp.
ICC1, due to a low percent similarity of 87.9% it remains undetermined if they are the same
species. Current research regarding different strains Streptomyces lavendulae is being performed
by Dr. Christopher Boddy at the University of Ottawa in the Department of Chemistry and
Biomolecular sciences. Dr. Boddy was involved in the whole genomic sequencing and
metabolomic study of Streptomyces sp. ICC1 as well as another bacterium originally isolated
from the Iron Curtain Cave, Streptomyces sp. ICC4.

6

Figure 4. Scanning electron micrograph of Streptomyces sp. ICC1 showing a dense mass of
interwoven filaments with irregular rugose surface collected from the Iron Curtain Cave
(Ghosh et al. 2017).

Figure 5. Macroscopic morphology of Streptomyces sp. ICC1 on nutrient agar after fourteen
days of growth in a 15°C incubator.

7

Secondary Metabolite Production
A large variety of compounds are produced by microorganisms through means of specialized
biosynthetic pathways. Secondary metabolites are low-molecular-mass organic compounds that,
unlike primary metabolites, are not directly involved in the growth, development, or reproduction
of the producing organisms. The small diffusible molecules disperse from the microorganism and
may provide its producer an advantage by adapting to the extracellular conditions to some degree
(Cihak et al. 2017). They may also aid in defense, competition, signaling, or interspecies
interactions, depending on the environmental cues, thus increasing the likelihood of survival in
an inhospitable environment. In natural conditions, secondary metabolites are primarily present at
low concentrations (Netzker et al. 2015). Secondary metabolites may also play a role in
transferring information between cells. Secondary metabolites create signaling networks that aid
in intra‐ and inter‐species communication, as well as in regulation of many aspects of
metabolism, virulence, physiology, competence, motility, symbiosis, and other functions (Dufour
and Roa 2011). Chemical communication occurs between bacteria-bacteria or bacteria-fungi
since they form close communities in nature. Successful co-existence can be mediated through
these signaling molecules in low concentration where the fungal mycelia and bacterial filaments
of both microorganisms are connected together (Abdalla et al. 2017). For instance, αButyrolactone is present in most Streptomyces and was the first Streptomyces sporulation factor
discovered. α-butyrolactones are analogous to homoserine lactones and have been deduced to
play an important role as signals as well as markers for the setting-in of morphological and
physiological differentiation (Safari et al. 2014). It was first characterized as an activator of
streptomycin production and spore formation in Streptomyces griseus (Miguélez et al. 2010).

8

An interesting property of Streptomyces is its ability to produce bioactive secondary metabolites,
such as antifungals, antivirals, antitumorals, anti-hypertensives, immunosuppressants, and
antibiotics (Procópio et al. 2012). Streptomyces are regarded as the most prolific source of
bioactive compounds in particular commercially available antibiotics since they produce
approximately two-thirds of commercially available antibiotics (Barka et al. 2016). The
production of most antibiotics is species specific, and these secondary metabolites are important
for Streptomyces species in order to compete with other microorganisms that come in contact,
even within the same genus. The bioactivity of the small molecules is mostly achieved by
affecting transcription in receiving cells. The genes for the biosynthesis of Streptomyces
secondary metabolites are mostly clustered and their expression is highly regulated (Tanaka et al.
2013). The biosynthetic genes of most secondary metabolites have previously been shown to be
expressed during germination (Strakova et al. 2013). Secondary metabolite production
predominately occurs at the aerial hyphae developmental stage and are isolated during stationary
phase of growth in liquid cultures (Seipke et al. 2012). The setoff of those physiological states,
and therefore of the associated synthesis of secondary metabolites, is linked to the depletion of
nutrients. A decrease in growth rate may be the signal for triggering secondary metabolism.

The secondary metabolites produced by Streptomyces often have dark pigmentation due to the
synthesis and excretion of melanins or melanoid pigments. Melanin compounds are irregular,
dark brown polymers that are hydrophobic and negatively charged. Various microorganisms
produce melanins through fermentative oxidation or oxidative polymerization of phenolic or
indolic compounds. These compounds play important roles in microorganisms against thermal,
chemical, and biochemical stresses. Specifically, melanins have radioprotective and antioxidant
properties that can effectively protect the living organisms from ultraviolet radiation, heavy
9

metals, and oxidizing agents (Nagger and Ewasy 2017). In addition, the melanin synthesized by
microbes help maintain their water and ion storage through increasing the structural rigidity of
their cell wall (Allam and Zaher 2012).

Figure 6. Brown pigmentation characteristic of Streptomyces sp. ICC1 secondary
metabolites grown in nutrient broth for nine days at 15°C.

In the present study secondary metabolites produced by Streptomyces sp. strain ICC1 have been
examined. Secondary metabolites secreted by Streptomyces sp. ICC1 have shown antimicrobial
properties, effective against both multi-drug resistant strains and common laboratory strains
of Escherichia coli and Staphylococcus aureus. The bioactive secondary metabolites produced
by Streptomyces sp. ICC1 strain were separated out from culture via diethyl ether liquid-phase
organic solvent and water extractions. The metabolite containing solution was purified and
separated

via

analytical

and

preparative

reversed-phase

high

performance

liquid

chromatography (HPLC) techniques. Each peak from the HPLC was tested for antimicrobial
activity through disk diffusion and well assay methods. One-dimensional proton nuclear
magnetic resonance (NMR) spectroscopy was used in attempt to elucidate the structure of each
active fraction that was identified by HPLC. Organic extractions with diethyl ether suggest the
10

bioactive compounds are polar but also contain hydrophobic substituents due to how the activity
stays within the less polar organic layer relative to water. In addition, the reverse-phase HPLC
results suggest that the secondary metabolites are polar due to short retention times with the
elution of a polar mobile phase. Together, the findings of the present study indicate that the
secondary metabolites secreted by Streptomyces sp. ICC1 are polar and with some hydrophobic
substituents. Future work to further the understanding of the bioactive compounds involve
additional instrumentation techniques, such as carbon-13 NMR spectroscopy and mass
spectrometry, as well as comparisons to secondary metabolites produced by other members of
the Streptomyces genus.

11

MATERIALS AND METHODS
Storage and Growth of Streptomyces sp. ICC1
Dormant cells of Streptomyces sp. ICC1 strain were stored in glycerol stock at -80°C. For
recovery, a sterile loop was used to scrape material from the top of the frozen stock and streaked,
in triplicate, onto nutrient agar plates. The plates were incubated at 15°C for growth and
secondary metabolite production. Visible colonies were present after seven days of incubation
and were irregular shaped, dry texture, rugose surface, and opaque cream in color. For liquid
cultures, 150 mL of nutrient broth was inoculated with a single colony of Streptomyces sp. ICC1,
followed by incubation in a shaker incubator at 150 rpm and 15°C for 5 to 40 days. Plates were
sealed with parafilmed and kept at 4°C until fresh stocks were made.

Growth of Escherichia coli #15-124
Multi-drug resistant Escherichia coli #15-124 was prepared with antibiotic mixed into the
nutrient agar in order to prevent mutations that decreased resistance. A stock solution of
chloramphenicol was produced by weighing 0.8078g into 5 mL of ethanol for a final
concentration of 0.05 M. From this stock solution, 100 µL was added to 100 mL of molten
nutrient agar for a final concentration of 50 µM. The nutrient agar was poured into four petri
dishes and allowed to solidify before thawed freezer stock of multi-drug resistant Escherichia
coli #15-124 was four-way streaked. The plates were incubated at 37°C for 24 h prior to storing
in a 4°C fridge.

Bioassay Screening for Antimicrobial Activity
Target organisms were cultured overnight at 37°C to 0.60-0.90 OD600 to suggest active
logarithmic division was occurring. The target organisms used include Escherichia coli #15-124,
12

Escherichia coli #59, laboratory Staphylococcus aureus, and methicillin-resistant Staphylococcus
aureus ATCC-43300.

A 10ml aliquot of the Streptomyces sp. ICC1 broth culture was retrieved from 15°C incubator
after 5-40 days at 150 rpm. Brown pigmentation was apparent after 5 days of incubation with
bioactivity occurring at this time or anywhere up to 40 days after. The sample was centrifuged at
10,000 rpm for 20 minutes. The supernatant was collected and filtered through a sterile
hydrophilic low-protein-binding polyether sulfone membrane syringe filter with 0.22 μm pore
size. The filtered supernatant contained the Streptomyces sp. ICC1 secondary metabolites. Liquid
extractions were performed with filtrate aliquots of 1:1 broth to diethyl ether by mixing them in a
separatory funnel for one minute with frequent pauses for ventilation.

Kirby-Bauer disk diffusion assays were used to determine whether a sample had antimicrobial
activity by the presence or absence of a zone of inhibition around a solution infused disk.
Bioassay screening was performed in 15 mm x 150 mm polystyrene Petri dishes. Eighty mL of
autoclaved molten nutrient agar was inoculated with a ratio of 1% volume to volume of the
exponentially dividing target organism, OD600 between 0.600-0.900. Autoclaved 6 mm paper
disks (Toyo, Advantech) were saturated with filtered broth, diethyl ether, post-extraction aqueous
filtrate or organic diethyl ether layer and dried completely prior to addition to the bioassay plate.
For the Kirby-Bauer disk diffusion method, disks containing different controls and experimental
conditions were evenly placed onto nutrient agar that had been inoculated with a target organism.
Antimicrobial activity was determined by whether the target organism was able to grow around
the disk. The Kirby-Bauer disk diffusion method was supplemented with Well-assays to
determine antimicrobial activity. For the Well-assay technique, Pasteur pipettes were used to
13

make wells in the molten agar. The depth of the wells were approximately midway between the
bottom of the top and bottom of the agar, 100 µl of filtered secondary metabolite broth as well as
controls was pipetted into the wells.

Diethyl ether was used as a control to ensure diethyl ether did not inhibit the growth of the
bacteria lawn. In addition, a disk containing10 µg of ampicillin was used to confirm that the
target bacteria were resistant to common pharmaceuticals such as ampicillin.

All bioassay

screening was performed within a biosafety cabinet to reduce contamination. Five minutes of
ultraviolet radiation was used to sterilize glassware, picks, loops, and plastic equipment prior to
use. The bioassay plates were incubated at 37°C for 24 h to ensure optimal growth of the target
bacterial lawn. The diameter or absence of zones of inhibition were recorded for each
experimental condition.

Active Compound(s) Separation and Preliminary Structural Analysis
The organic layer of the diethyl ether extraction was known to possess antimicrobial activity
against the previously mentioned target organisms based on the bioassays performed. Liquid
extractions with diethyl ether were performed on aliquots of the Streptomyces sp. ICC1 broth
culture collected between 5 and 40 days of incubation at 15°C at 150 rpm. One portion of broth
culture was extracted with equal portions of diethyl ether three times. The organic layers from
each extraction performed on the same day, which were one third of the total extraction volume,
were combined and evaporated. Extracts were dissolved from the dry flasks in 10 mL of HPLCgrade methanol and 2 mL of water prior to injection into the Agilent 1220 Infinity II high
performance liquid chromatography (HPLC) system. Separation was performed according to the
analytical reversed-phase HPLC conditions summarized in Table 1. Following method
14

development, individual fractions were collected via the preparative HPLC conditions listed in
Table 1. The resulting four separated components were divided among Eppendorf tubes and the
eluent evaporated on a Vacufuge® 5301 Centrifugal Vacuum Concentrator at 45°C. One sample
from each separation was resuspended in deionized 18MOhm water and analyzed for
antimicrobial activity via Kirby Bauer disk diffusion assays. Remaining samples were
resuspended in deuterium oxide for structural elucidation with one-dimensional proton nuclear
magnetic resonance (NMR) spectroscopy via a 500 MHz Bruker Avance AMIII 500
Spectrometer.

Table 1. Analytical and preparative HPLC parameters for compound separation.
Instrument
Column
Dimensions
Detector
Flow Rate
Injection Volume

Analytical HPLC Parameters
Agilent 1220 Infinity HPLC
Kinetex® 2.6 μm EVO C18
100Ȧ
100 x 3.0 mm
272 nm
0.4 mL/min
5.0 μL

Preparative HPLC Parameters
Waters HPLC 486
Phenomenex® Jupiter 10µ C18
300Ȧ
250 x 21.20 mm
272 nm
5 mL/min
1.0 mL

Eluent

A: 5 mM NH4OAc in 18 MOhm H2O
B: Acetonitrile

Program Gradient

1-21 min 90% A 10% B
21-35 min 100% B
35-47 min 90% A 10% B

Table 2. NMR parameters for structural elucidation.
Instrument
Operating Frequency
Solvent
Number of Scans

Bruker Avance AMIII 500 Spectrometer
500 MHz
D2O
1024

15

RESULTS
Bioassay for Antimicrobial Activity
Well assays and Kirby-Bauer disk diffusion assays were performed on nutrient agar with all
target organisms listed. Zones of inhibition occurred 5 days post-inoculation and decreased in
diameter when samples were incubated for longer durations. Zones of inhibition ranged from 8 to
6.5 mm in diameter for the organic layer after diethyl ether extraction and filtered broth culture.
After antimicrobial activity was confirmed, liquid organic solvent extractions were performed to
elucidate the relative polarity of the bioactive antimicrobial secondary metabolites. Extractions
were performed with diethyl ether, which was previously shown by Alysha Milward (2019) to be
the most successful organic solvent compared to ethyl acetate, hexane, and 1-octanol. Solvent
success was determined by the antimicrobial activity being maintained in either the aqueous or
organic layer post extraction. Solvents were deemed unsuccessful if bioactivity against the target
organism was either inconsistently observed or absence. As depicted in Figure 7, activity was
present in the organic layer of the extraction as well as filtered broth when all conditions
including diethyl ether control, ampicillin control, and aqueous layer of the extraction were tested
using a Kirby-Bauer disk diffusion assay. Also, it should be noted that bioactivity was
inconsistent throughout the present research, for example the same culture would be sporadically
successful and unsuccessful against the same target bacteria at different points of incubation.
Furthermore, some liquid cultures produced from the same plate of Streptomyces sp. ICC1 would
have detectable bioactivity while others did not.

16

Figure 7. Kirby-Bauer disk diffusion test for Streptomyces sp. ICC1 secondary metabolites
against multi-drug resistant Escherichia coli #15-124 showing bioactivity in the organic
layer after diethyl ether extractions.
Compound Separation via Reverse-Phase HPLC
In order to begin separating compounds in the diethyl ether extract from Streptomyces sp. ICC1,
reverse-phase HPLC was used to produce fractions of the sample based on differing polarities.
The program gradient for both the analytical and preparative reverse-phase HPLC was as follows:
during the first 21 minutes the initial 90:10 ammonium acetate buffer to acetonitrile ratio moved
to 100% acetonitrile, which remained at 100% between 21 to 35 minutes, after 35 minutes the
program gradient transitioned slowly back to 90:10 buffer to acetonitrile ratio that occurred at 47
minutes. Absorbance was measured at 272nm for reverse-phase HPLC techniques since this
wavelength was previously found by Alysha Milward (2019) to provide a chromatogram with a
stronger baseline and sharper peaks. Analytical revers-phase HPLC was used for method
development to estimate when the sample eluted and provide information regarding how long the
preparative reverse-phase HPLC should be run for. Blank runs with water were first run to
17

determine baseline activity. Figure 8 shows the chromatogram from the bioactive secondary
metabolites resuspended in methanol. The majority of the sample eluted quickly from the column
while the rest eluted at 38.434 minutes when the program gradient returned to a 90:10 buffer to
acetonitrile ratio.

Figure 8. Analytical reverse-phase HPLC chromatograms of bioactive secondary
metabolites in methanol measured at 272 nm.

The preparative reversed-phase HPLC chromatogram, shown below in Figure 9, had different
absorbance readings and separation from that of the analytical chromatogram as a result of the
different column volumes. The Phenomenex® Jupiter 10u C18 column has a non-polar stationary
phase, therefore polar substituents are the first to elute due to their high affinity for the polar
mobile phase. Five runs were performed on the same culture sample to ensure the results were
repeatable. Fractions covered a broad range of materials Fraction 1 was a broad short peak, which
had a retention time between 8 to 10 minutes in each of the five trials run. Fraction 2 was the
tallest peak of the four observed and had a consistent height between samples. The trail of peaks
following Fraction 2 were denoted Fraction 3 since the number and height of each peak within
this cluster varied between runs. Fraction 4 was a relatively small peak that occurred
approximately at 42 minutes after injection. Due to the high-volume capacity of the preparative

18

Phenomenex® Jupiter 10u C18 column, it can be reasoned that all of the fractions collected were
likely eluted in 90% 5 mM ammonium acetate buffer and 10% acetonitrile.

Figure 9. Preparative reverse-phase HPLC of bioactive secondary metabolites in methanol.
The four fractions were evaporated to remove all the remaining HPLC solvent, which left a
brown pigmented solid residue in the collection tubes. Following eluent evaporation, the
bioactivity of each of the four factions collected, which were dissolved in methanol, were tested
via Kirby-Bauer disk diffusion assays. As shown below in Figure 10, the bioactive secondary
metabolites were separated into Fraction 2 and Fraction 4 using the preparative reversed-phase

19

HPLC conditions used. The clear zones of inhibition against the multi-drug resistant strain
Escherichia coli #15-124 are indicative of antimicrobial agents having been absorbed into the
applied paper disk. Fraction 2 was further analyzed for structural elucidation via one-dimensional
1

H NMR analysis. Whereas Fraction 4 did not produce enough sample to be effectively used for

1-D 1H NMR analysis.

Figure 10. Kirby-Bauer disk diffusion test against multi-drug resistant Escherichia coli #15124 showing bioactivity on Peak 2 and 4 of preparative HPLC collections.
Structural Elucidation of Bioactive HPLC Collections by 1-D 1H-NMR
The spectrum obtained from analyzing Fraction 2 in D2O at 1024 scans by the 500 MHz Bruker
NMR spectrometer can be seen below in Figure 11. Chloroform was initially used in attempt to
dissolve solid Fraction 2 after evaporation; however, this solvent was ineffective so D2O was
used to dissolve the Fraction for the remaining trials. The large singlet at 4.75ppm was the
solvent residual peak of D2O. The signal peak at 2.0 ppm was likely acetonitrile residue, which
was used in preparative HPLC. The peaks present at 0.8 and 1.2 ppm are likely grease carried
over from greasing the joints of the evaporation apparatus. The following interpretations of the
20

various splitting observed in Figure 11 should be confirmed by running 1-D1H NMR
spectroscopy with a larger quantity of sample. The singlet at approximately 3.20 ppm may
correspond to a tert-butyl methyl ether group due to its position in the spectrum. Additionally, the
complicated splitting observed in the peaks appearing following the OCH3 from 3.5-4.0 ppm may
be a result of protons being nearby electron-withdrawing groups, such as alkenes, or
electronegative oxygen or nitrogen atoms. The quadruplet at approximately 4.2 ppm is possibly
an ethyl proton adjacent to a methyl group due to its’ relatively deshielded position. Also
apparent on the spectra are a quadruplet and triplet at 1.35 ppm and 4.30 ppm, respectively. No
peaks were observed downfield of the D2O singlet at 4.75 ppm. The sample did not appear to
decompose after being left in a 4 ⁰C refrigerator for 48 hours in D2O since the sample run for 128
scans the first day showed the same distinctive peaks after being run for 1024 scans two days
later. A larger number of scans was used to produce sharper peaks that were more easily
distinguished from the baseline.

21

A

B

Figure 11. A) 1-D1 H NMR spectra of Peak 2 dissolved in deuterium oxide, NS=1024. B)
zoomed in portion of A from 0 to 5 ppm.
22

DISCUSSION
Through antimicrobial assays, the secondary metabolites produce by Streptomyces sp. ICC1 have
been notably effective against Gram-negative and Gram-positive target bacterium, namely
laboratory and multi-drug resistant strains of Escherichia coli and Staphylococcus aureus. The
antimicrobial activity demonstrated by Streptomyces sp. ICC1 is comparable to other members of
the Streptomyces genus, which are among the most numerous and most versatile soil
microorganisms. The genus Streptomyces is characterized by their large metabolite production
rate, biotransformation processes, capability of degrading lignocellulose and chitin, and their
fundamental role in biological cycles of organic matter (Procopio et al. 2012). The genome of S.
coelicolor, for example, encodes many secreted proteins, including 60 proteases, 13
chitinases/chitosanases, eight cellulases/endoglucanases, three amylases, and two pactato lyases
(Procopio et al. 2012). In addition, the results relate to other cave studies for instance Turkish
karstic caves were reported to harbor Actinobacteria, for which over half of the isolates, were
active against several microbial pathogens including Gram positive bacteria, Gram negative
bacteria, yeast, and filamentous fungi (Yücel and Yamac 2010). One of the Actinobacteria,
Streptomyces sp. 1492, had strong activity against clinical strains of MRSA, VRE,
and Acinetobacter baumannii. Furthermore, Streptomyces E9 isolated from Helmcken Falls cave
in British Columbia could inhibit the growth of Paenibacillus larvae, a causative agent of
American foulbrood disease in honeybees (Kay et al. 2013). The bioactive compounds examined
in this research should be tested against additional infectious microbes to determine their
limitations and effectiveness.

23

Following liquid organic extractions with the aqueous metabolite-containing broth culture,
bioactivity was present with in the organic, less polar layer of the diethyl ether-water extraction.
Diethyl ether has a bent molecular structure that produces a small dipole moment, this property of
diethyl ether allows the essentially polar liquid to be used to isolate both non-polar and polar
compounds in aqueous extractions. The secondary metabolites were maintained in the organic
layer following extractions, which suggests that the compounds are polar and may contain
hydrophobic functional groups.

Analytical and preparative reversed-phase high performance liquid chromatography analysis
revealed mainly polar Fractions within the diethyl ether extracts. The compounds within
Fractions 1, 2 and 3 likely possess more of a hydrophilic, polar nature due to how they eluted
when the program gradient contained ammonium acetate in the polar mobile phase. Ammonium
acetate buffer is more polar relative to acetonitrile, which means compounds that eluted during
this program gradient were polar since they were able to elute in the more polar mobile phase
compared to 100% acetonitrile. Fraction 4 eluted once the program gradient returned to a 90:10
ammonium acetate buffer to acetonitrile ratio. It can be inferred that Fraction 4 was less polar
since it adhered to the column until the mobile phase returned to a polarity it was able to dissolve
in. From additional Kirby-Bauer disk diffusion assays, it was determined that the antimicrobial
secondary metabolites from Streptomyces sp. ICC1 were separated into Peaks 2 and 4. It unclear
whether these peaks contain entirely different bioactive compounds or if there was carry-over
from the high-volume capacity of the preparative Phenomenex® Jupiter C18 column. The
chromatogram suggests there may be incomplete chromatographic separation due to the close
proximity of the Fractions observed. A longer program gradient at a higher aqueous

24

concentration could potentially establish whether these peaks possess different bioactive
molecules.

1-D1H NMR spectroscopy should be repeated with higher concentrations of the analyzed sample.
Further analysis may intensify present peaks or reveal additional peaks, such as the
highly deshielded signals found in past work by Alysha Milward (2019). The 1-D1H NMR
spectrum demonstrates insufficient sample may have been used due to the small size of sample
peaks compared to solvent residual peaks. The high level of splitting and peaks present could
suggest that the Streptomyces sp. ICC1 bioactive compounds are complex, however they are
unlikely proteinaceous in nature due to the absence of amine groups on the spectrum. The 1-D1H
NMR spectrum also provides supports that the bioactive compounds are polar with hydrophobic
substituents due to the hydrophobic functional groups, which contain hydrogen atoms, illustrated
in the spectrum. In future work sodium phosphate should be added to the deuterium oxide to
mimic physiological conditions.

Comparing the compounds produced by Streptomyces sp. ICC1 to those synthesized by
streptomyces from the literature does not offer much insight since they exhibit extreme chemical
diversity. The compounds range from simple amino acid derivatives to high molecular weight
proteins, and macro lactones from simple eight membered lactones to different condensed macro
lactones (Harir et al. 2018). For example, actinomycin was first isolated from Streptomyces
antibioticus and is produced by many Streptomyces strains. The actinomycins are a family of
bicyclic chromopeptide lactones sharing the chromophoric phenoxazinone dicarboxylic acid to
which are attached two pentapeptide lactones of nonribosomal origin (Singh et al.
2020). Actinomycin D acts as a transcription inhibitor, binding to DNA duplexes at
25

the transcription initiation complex and preventing RNA polymerase elongation (Koba and
Konopa 2005).

Throughout the present research bioactivity was lost in the secondary metabolite solution, further
analyses are required to understand the loss of activity. The inability to inhibit the target bacteria
may be due to Streptomyces sp. ICC1 mutation during growth in the 15 ⁰C incubator,
contaminants being present, or the bioactive compounds not being excreted in sufficient
concentrations. In addition, after centrifuging the Streptomyces sp. ICC1 nutrient broth solution,
the pellet could contain the bioactive compounds due to adherence to the bacteria cell walls. The
pellet was discarded after centrifugation and was not tested for antimicrobial activity.

CONCLUSIONS AND FUTURE WORK
The progression of antibiotic-resistant microorganisms has hindered the therapeutic efficiency of
various commercially available pharmaceuticals. Therefore, an overwhelming demand for novel
treatment presents itself as the likelihood of multi-resistant microbes surpassing the development
of antibiotics escalates in a coevolutionary race between humans and bacteria. Extreme
environments are starting to be considered as valuable sources of microbial capability,
particularly in the production of pharmaceutically significant metabolites (Rangseekaew and
Pathom-aree 2019). The findings of the present study aids in furthering knowledge regarding
bioactive compounds produced by cave dwelling microbes.

Laboratory growth conditions should be optimized for Streptomyces sp. ICC1. Previous research
performed by Alysha Milward (2019) found the cave-dwelling strain produced the largest amount
of bioactive secondary metabolites when grown in 15 mL of nutrient broth compared to Hickey
26

Tesner broth and Reasoner’s 2A broth. However, other media should be examined since media
used for the isolation of cave actinobacteria range from routine cultivation media such as
International Streptomyces Project medium 2 (yeast malt extract agar, ISP2) or tryptic soy agar
(TSA) to selective media including humic acid vitamin agar (HV), starch casein agar (SC), starch
casein nitrate agar (SCN), peptone-yeast extract/brain-heart infusion medium (PY-BHI), R2A
medium, actinomycete isolation agar (AI), and Gauze's medium No.1 (Rangseekaew and
Pathom-aree 2019). Moreover, isolation media that mimic the conditions of low concentration
nutrients in caves such as tap water agar, 1/100 ISP2 and oligotrophic medium (M5) were also
successfully used for the isolation of actinobacteria. (Lee et al., 2000b; Velikonja et al.,
2014; Covington et al., 2018; Passari et al., 2018). High concentration of nutrients in standard
cultivation media were reported to cause cell death in cave-associated bacteria due to osmotic
stress (Ghosh et al. 2017).

In addition, optimization for the seed should be determined. The seed refers to stock cultures
from which samples are taken and move into another media for selection of the stationary phase
of the growth cycle and production of secondary metabolites. Optimal growth conditions likely
differ to maintain the culture at logistic versus stationary growth phase. The optimal pH,
temperature and oxygen content should also be established for Streptomyces sp. ICC1. Pervious
work performed by Alysha Milward (2019) examined the bioactive present after incubation in 8
⁰C, 15 ⁰C, and 37 ⁰C. A temperature of 15⁰C was found to be most effective at promoting the
production of secondary metabolites as determined by larger diameters of zones of inhibition
against target bacteria compared to the other temperatures. However, the Iron Curtain Cave
ranges between 4–12 °C depending on the time of year, which suggests a larger variety of
temperatures should be tested to determine the optimal growth conditions for Streptomyces sp.
27

ICC1 (Ghosh et al. 2017).

Streptomycetes are regarded to prefer neutral to alkaline

environmental pH, the optimal growth pH range being 6.5–8.0 (Kutzner 1986; Locci 1989).
However, acidophilic and alkalophilic streptomycetes have also been found, such as those
causing potato scabies have represented phylogenetically diverse group, and included alkalophilic
strains (Kutzner 1986; Takeuchi et al. 1996). Among streptomycetes common habitats, soil and
hay have a low mean pH of 3.5–6.8 and pH of 5.5–6.5, respectively (Kutzner 1986;
Erviöet al. 1990). In buildings suffering water damage and mould growth, pH varies from acid
and

neutral

values

of

building

materials

to

highly

basic

pH

of

concrete

(Nevalainen et al. 1991; Andersson et al. 1998; Kontro et al. 2005).

The methodology practiced should be repeated to produce greater quantities of bioactive
compounds to validate and ensure the repeatable of the results found here. Future instrumental
analyses should be executed on the purified samples, primarily 13C NMR and mass spectrometry,
to elucidate the bioactive molecular structure of the compounds. Additionally, the minimum
inhibitory concentration of the produced bioactive molecules should be established and
investigated against several target strains, both Gram-positive and Gram-negative. Further
bioactive screening on the pellet of the centrifuged product could explain the lost bioactivity. The
findings should be compared to commercially available secondary metabolites produced by the
Streptomyces genus to assess the efficiency of a cave-dwelling strain to the more common soil
species. Further analyses should be performed on the secondary metabolites secreted by cave
dwelling Streptomyces sp. ICC1 to determine its potential as an antibiotic producing organism for
clinical use.

28

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32

APPENDIX
Table A1. The diameters of zones of inhibition =from Kirby-Bauer disk diffusion assays
using Streptomyces sp. ICC1 broth culture before and after diethyl ether extraction, with E.
coli #15-124 as the target organism.
Experimental Condition
Filtered broth
Filtered broth
Filtered broth
Filtered broth
Filtered broth
Filtered broth
Organic layer
Organic layer
Filtered broth
Filtered broth
Filtered broth
Organic layer
Organic layer
Peak 2
Peak 4

Incubation time (days)
7
7
10
10
10
15
15
15
21
21
23
23
23
40 +
40+

Diameter (mm)
7.0 mm
6.5 mm
6.9 mm
7.0 mm
6.6 mm
6.6 mm
6.8 mm
6.8 mm
6.6 mm
6.6 mm
6.7 mm
6.5 mm
6.6 mm
8.0 mm
7.8 mm

33