jmb

J. Microbiol. Biotechnol. (2017), 27(8), 1441–1448

https://doi.org/10.4014/jmb.1703.03012 Research Article

Review

A Rapid and Efficient Screening Method for Antibacterial Compound-

Producing Bacteria S
Sachithra Amarin Hettiarachchi1,2, Su-Jin Lee1, Youngdeuk Lee1, Young-Kyung Kwon1, Mahanama De Zoysa3,
Song Moon1, Eunyoung Jo1, Taeho Kim1, Do-Hyung Kang1,2, Soo-Jin Heo1,2, and Chulhong Oh1,2*
1
Korea Institute of Ocean Science & Technology, Jeju Special Self-Governing Province 63349, Republic of Korea
2
Department of Marine Biology, Korea University of Science and Technology, Jeju Special Self-Governing Province 63349, Republic of Korea
3
College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea

Received: March 8, 2017

Revised: May 25, 2017
Accepted: June 13, 2017
First published online
June 16, 2017
*Corresponding author
Phone: +82-64-798-6102;
Fax: +82-64-798-6039;
E-mail: och0101@kiost.ac.kr
S upplementary data for this
paper are available on-line only at
http://jmb.or.kr.
pISSN 1017-7825, eISSN 1738-8872
Copyright © 2017 by
The Korean Society for Microbiology
and Biotechnology
Antibacterial compounds are widely used in the treatment of human and animal diseases. The
overuse of antibiotics has led to a rapid rise in the prevalence of drug-resistant bacteria,
making the development of new antibacterial compounds essential. This study focused on
developing a fast and easy method for identifying marine bacteria that produce antibiotic
compounds. Eight randomly selected marine target bacterial species (Agrococcus terreus,
Bacillus algicola, Mesoflavibacter zeaxanthinifaciens, Pseudoalteromonas flavipulchra, P. peptidolytica,
P. piscicida, P. rubra, and Zunongwangia atlantica) were tested for production of antibacterial
compounds against four strains of test bacteria (B. cereus, B. subtilis, Halomonas smyrnensis, and
Vibrio alginolyticus). Colony picking was used as the primary screening method. Clear zones
were observed around colonies of P. flavipulchra, P. peptidolytica, P. piscicida, and P. rubra tested
against B. cereus, B. subtilis, and H. smyrnensis. The efficiency of colony scraping and broth
culture methods for antimicrobial compound extraction was also compared using a disk
diffusion assay. P. peptidolytica, P. piscicida, and P. rubra showed antagonistic activity against
H. smyrnensis, B. cereus, and B. subtilis, respectively, only in the colony scraping method. Our
results show that colony picking and colony scraping are effective, quick, and easy methods of
screening for antibacterial compound-producing bacteria.
Keywords: Colony picking, colony scraping, broth culture, screening, antibacterial compounds

Introduction estimated that microorganisms produce more than 22,000

In recent years, research on drug discovery and
development has increasingly focused on naturally occurring
products [1]. Nature undoubtedly harbors a huge arsenal
of organisms capable of producing compounds that will
prove beneficial to humans [2]. In particular, marine
microorganisms are proving to be an important source of
novel microbial compounds that display antibacterial,
antiviral, and antitumor activities, as well as anticoagulant
and cardiodynamic properties [3]. Marine microbes from
extreme ecological niches can be good sources of novel
pharmaceutical materials [4], since their adaptive strategies
and metabolism have been optimized for survival in highly
diverse and challenging environments [5]. While it is
bioactive compounds in nature, the identification and
characterization of new and useful metabolites can be
difficult [6].
Identification of bacteria that potentially produce bioactive
compounds, extraction of these compounds, and subsequent
testing against various pathogenic bacteria are the necessary
first steps in the process of screening for new antibacterials.
Screening systems used for the identification of new
antibacterial compounds should be simple, rapid, reproducible,
and inexpensive [7]. The earliest screening methods to be
adopted were microbial inhibition assays, which are still
widely used for the detection of antibiotic residues [8].
Bioautographic, diffusion, and dilution methods are also
commonly used; the diffusion method is often used as a

August 2017 ⎪ Vol. 27 ⎪ No. 8

1442 Hettiarachchi et al.

qualitative method of screening for antibacterial compounds. produce antibacterial compounds.

However, chemical methods are generally too target-
specific and expensive in the primary screening step [9], Materials and Methods
where the main focus is on determining the presence or
absence of antimicrobial activity in bacteria. All strains of marine bacteria (Table 1) used in this study were
All target marine bacteria were randomly selected for the from stocks maintained at Jeju International Ocean Science Center
experiment. They belong to five genera (Agrococcus, Bacillus, for Research & Education (Korea Institute of Ocean Science &
Mesoflavibacter, Pseudoalteromonas, and Zunongwangia) and Technology, Korea). The strains were previously identified by 16S
rRNA sequencing analysis and stored at −80°C in 40% glycerol
some of them have shown bactericidal effects in previous
[24] and marine broth (1:1 (v/v)). Strains used for this study were
studies. Bacterial species in genus Agrococcus and Bacillus were
cultured on marine agar (Becton Dickinson Company, USA) and
gram positive [10, 11] and in Mesoflavibacter, Pseudoalteromonas,
in marine broth (Becton Dickinson Company) at 30°C. Eight strains
and Zunongwangia were gram negative [12-14]. Among of test bacteria and four of target bacteria were used in this study.
them, genus Pseudoalteromonas spp. have been subjected to
more research regarding their biofilm-inhibiting compounds, Colony Picking Method
including bactericides [15]. In particular, the gram negativity Pure colonies of target and test bacteria were prepared by
of marine bacteria has led them to thrive in harsh streaking a loopful of stock onto agar plates. Test bacteria were
environments, resulting in ranges of structurally diversified inoculated in 4 ml of marine broth and incubated at 30°C, 170 rpm
secondary metabolites [16]. Bacillus cereus, Bacillus subtilis, for 16 h in a shaking incubator (Vision Scientific Co. Ltd., Korea).
Halomonas smyrnensis, and Vibrio alginolyticus were used as The concentration of each culture in colony forming units (CFU)
test bacteria. B. cereus and B. subtilis are gram positive [17, per milliliter was measured by serial dilution of broth cultures
with fresh marine broth, followed by spreading onto marine agar
18] and H. smyrnensis and V. alginolyticus are gram negative
Petri dishes. A 60 µl aliquot of broth culture of each test bacterium
[19, 20]. B. cereus causes foodborne illnesses in humans [21]
(B. cereus 2.34 × 108 CFU/ml, B. subtilis 1.42 × 108 CFU/ml,
and B. subtilis is important as a probiotic bacterium in the
rumen of some animals [22]. Some species in genus
Halomonas and V. alginolyticus are pathogenic [20, 23].
The rapid spread of multidrug resistance in bacteria is
leading to reemerging and newly emerging infectious
diseases, creating an urgent need for new classes of antibiotics
[4]. In this study, we tested two preliminary screening
methods for the rapid identification of marine bacteria that

Table 1. Bacterial strains used in this study.

Bacteria Accession No.
Target bacterial species
Agrococcus terreus KY366352
Bacillus algicola KY366351
Mesoflavibacter zeaxanthinifaciens JF800672
Pseudoalteromonas flavipulchra KY366349
Pseudoalteromonas peptidolytica KY366348
Pseudoalteromonas piscicida KY366347
Pseudoalteromonas rubra KY366346
Zunongwangia atlantica KY366350
Test bacterial species
Bacillus cereus KY366356
Bacillus subtilis KY366353
Fig. 1. Steps of the colony picking method.
Halomonas smyrnensis KY366355 ① Spreading test bacteria on agar plate; ② Picking pure single colony
Vibrio alginolyticus KY366354 from each target bacterium and placing on test bacteria loan culture.

J. Microbiol. Biotechnol.

H. smyrnensis 1.64 × 108 CFU/ml, and V. alginolyticus 2.14 × 108
CFU/ml) was spread onto marine agar Petri dishes. Sterile
wooden toothpicks were used to introduce pure single colonies of
target bacteria onto the marine agar dishes with different test
bacteria (Fig. 1). The plates were incubated at 30°C for 16 h, after
which zones of inhibition were observed and measured.
Colony Scraping Method
Target bacteria that exhibited antagonistic activities were
cultured on marine agar plates. Approximately 0.1 g (wet weight)
of each bacterial colony was collected using a sterile scraper and
transferred to a labeled sterile 1.5 ml tube. Distilled water (500 µl)
was added to each tube and sonicated at 40% amplitude for 1 min.
All tubes were centrifuged at 7,500 ×g for 2 min, and supernatants
were filtered through a 0.45 µm disposable membrane filter unit
(Toyo Roshi Kaisha Ltd., Japan).
Broth Culture Method
Pure colonies of each target bacterium were inoculated into
4 ml of marine broth and incubated at 30°C, 170 rpm for 16 h.
These cultures were transferred to 100 ml of fresh marine broth
and incubated under the same conditions to obtain 100 ml of broth
culture of each target bacterium. The cultured broth was separated
to supernatant and cell pellet by centrifugation at 7,500 ×g for
10 min. The supernatant was concentrated 10 times after freeze
drying, and was filtered through a 0.45 µm disposable membrane
filter unit. The collected cell pellet (approximately 0.1 g) was mixed
with 500 µl of distilled water and sonicated at 40% amplitude for
1 min. The contents were transferred to a new 1.5 ml tube and
centrifuged at 7,500 ×g for 2 min. The supernatants were separated
and filtered through a 0.45 µm disposable membrane filter unit.
Both samples were used for the antagonistic activity test by disk
diffusion assay.
Disk Diffusion Assay
The antagonistic activity of each sample was tested by the disk
diffusion method, as described by the Clinical and Laboratory
Table 2. Antagonistic activity of picked colonies of target bacteria against test bacteria.
Test bacteria
Target bacteria B. cereus B. subtilis
A. terreus - -
B. algicola - -
M. zeaxanthinifaciens - -
P. flavipulchra + +
P. peptidolytica + +
P. piscicida + +
P. rubra + +
Z. atlantica - -
(+) Clear zone observed; (-) no clear zone observed.
Screening of Antibacterial Compound-Producing Bacteria 1443
Standards Institute [25], with several modifications. Lawn
cultures of target bacteria were prepared by spreading 60 µl of
fresh broth culture of B. cereus (0.71 × 108 CFU/ml), B. subtilis
(1.7 × 108 CFU/ml), and H. smyrnensis (0.4 × 108 CFU/ml) onto
marine agar plates. Sterile paper disks (8 mm diameter; Toyo
Roshi Kaisha Ltd.) were placed on the plates, and 30 µl of test
bacterial culture was loaded onto the disks. Paper disks with 60 µl
of 10 mg/ml ampicillin and sterile marine broth were used as
positive and negative controls, respectively. The plates were
incubated at 30°C for 16 h and the diameter of inhibition zones
around the disks was measured. The test was performed in
duplicates (n = 2).
Results and Discussion
Colony Picking Method
Colony picking was used to identify test bacteria exhibiting
antibacterial activity. The growth of pure colonies of test
bacteria on lawn cultures of target bacteria was observed
after incubation. The presence and size of clear zones
indicated the degree of antibacterial activity in the test
bacteria. Four target bacteria belonging to the genus
Pseudoalteromonas inhibited the growth of the two Bacillus
sp. and H. smyrnensis in preliminary screening (Table 2).
These bacteria did not exhibit any activity against
V. alginolyticus. The other four bacterial species (A. terreus,
M. zeaxanthinifaciens, B. algicola, and Z. atlantica) did not
exhibit any antibacterial activity against the test bacteria.
P. flavipulchra and P. piscicida exhibited comparatively
large clear zones on lawns of B. cereus and B. subtilis
(Fig. 2). Clear zones are the result of test bacteria inhibiting
the growth of target bacteria. Test bacteria that produced
clear zones could therefore be initially identified as
producing antibacterial compounds.
In the colony picking test, test bacterial colonies come
H. smyrnensis V. alginolyticus
- -
- -
- -
+ -
+ -
+ -
+ -
- -
August 2017 ⎪ Vol. 27 ⎪ No. 8
1444 Hettiarachchi et al.

Fig. 2. Antagonistic activity of test bacterial colonies against target bacteria (1, A. terreus; 2, P. rubra; 3, P. flavipulchra; 4,
P. peptidolytica; 5, P. piscicida; 6, M. zeaxanthinifaciens; 7, B. algicola; 8, Z. atlantica).

into direct contact with target bacteria. Interspecies interactions
between bacteria enhance the activation of cryptic genes
responsible for producing various types of metabolites
[26]. Antagonism depends on multiple antibacterial factors
in test bacteria; for example, exopolysaccharide and biofilm
formation, compound concentration, co-culture effect, and
the polarity of the compound produced (water solubility)
[27-29], and most of these factors influence the results of
the colony picking test. Instead of using a broth culture and
testing supernatants and cell pellets, as in the commonly
used broth culture screening method, colony picking offers
a quick approach, encompassing the factors mentioned above,
for primary screening to identify antibacterial compound-
producing bacteria. Moreover, this method provides space
for more than 10 target bacteria within one agar plate.
Therefore, it differs from the existing cross-streak method,
which should be drawn perpendicular lines on agar plate
with target bacteria against the test bacteria [30]. The test
can be performed more rapidly and concurrently on a
number of target bacteria compared with the cross-streak
method. It also facilitates direct contact of test bacteria with
target bacteria and indicates obvious clear zones. Therefore,
the colony picking method is more organized and precise
than the cross-streak method as a primary screening. Colony
picking is thus suitable as a rapid method for screening
antibacterial compound-producing bacteria. It is also more
cost-effective than other methods, as it does not require
paper disks [7], filter papers, or additional incubation.
Colony Scraping Method
The colony scraping method was introduced as another
quick approach to check the antibacterial activity of test
bacteria. Pure colonies of test bacteria were scraped from
agar plates and tested for antibacterial ability. The efficacy
of this method was compared with that of the broth culture
method. In P. peptidolytica, antibacterial activity against
H. smyrnensis was only detected by the colony scraping
method. Colony scraping also revealed a higher degree of
antibacterial activity in P. flavipulchra and P. piscicida than
did the broth culture method (Table 3). Both methods
revealed the same degree of antagonistic activity in P. rubra
against B. cereus, whereas the antibacterial activity of
P. piscicida was only revealed by the colony scraping
method (Fig. 3A). The antibacterial activity of P. rubra
against B. subtilis was also only detected by the colony
scraping method (Fig. 3B).

Table 3. Antagonistic activity of sonicated cell-free supernatants of scraped colonies and broth cultures of test bacteria on target
bacteria (mean ± SD).
Diameter of inhibition zones of each target bacterium (mm)
Test bacteria B. cereus B. subtilis H. smyrnensis
Colony scraping Broth culture Colony scraping Broth culture Colony scraping Broth culture
P. peptidolytica - - - - 10.0±0.0 -
P. flavipulchra 14.0±1.4 11.5±0.7 11.0±0.0 10.5±0.7 17.5±0.7 14.0±0.0
P. piscicida 11.5±0.7 - - - 16.0±1.4 11.5±0.7
P. rubra 11.5±0.7 11.5±0.7 10.5±0.7 - 15.5±0.7 16.0±1.4

J. Microbiol. Biotechnol.

Fig. 3. Comparison of colony scraping and broth culture
methods for detecting the antagonistic activity of test bacteria
against target bacteria.
Although P. peptidolytica showed some antibacterial
activity in the preliminary screening, it did not produce an
inhibition zone against B. subtilis in either the colony
scraping or the broth culture method (Fig. 3). All test
bacteria exhibited inhibition zones against H. smyrnensis in
both methods, except P. peptidolytica in broth culture
(Fig. 3C). The antagonistic activity of all test bacteria on all
target bacteria was higher in the colony scraping method
than in the broth culture method, with the exception of
P. rubra against H. smyrnensis (Fig. 4). P. piscicida, P. rubra,
and P. peptidolytica exhibited antagonism against B. cereus,
B. subtilis, and H. smyrnensis, respectively, only in the
colony scraping method (Fig. 3).
The 10 times concentrated broth of P. peptidolytica and
P. flavipulchra exhibited minor antagonistic activity against
Screening of Antibacterial Compound-Producing Bacteria 1445
B. cereus and only P. peptidolytica against B. subtilis (see
Supplementary data). The antagonistic activity of the test
bacteria was more obvious in colony scraping than in the
broth culture method (cell-free supernatants of broth
cultured cell extraction). A possible explanation for this
discrepancy may be the inactivation or reduction of
antibacterial mechanisms due to dilution of the organisms
in a large volume of broth culture [31].
During our experiment, we also observed that P. rubra
produced a dark red pigment on solid agar and a light pink
one in broth culture, indicating a difference in secondary
metabolite production between colony and broth cultures.
Furthermore, Dheilly et al. [32] described how different
exopolysaccharides and proteins secreted by Pseudoalteromonas
sp. colonies affect antibacterial activity. Surface-attached
marine bacteria exhibit higher levels of antibacterial
activity than do bacteria suspended in broth culture [33].
Our colony scraping method thus may be seen to facilitate
the activity of antibacterial factors such as pigments,
exopolysaccharides, and inner cellular compounds. It also
provides reliable results and a cost-effective method of
testing a large number of samples in a short time.
Extracellular vs. Intracellular Effects
Antagonistic activity of the colony picking method and
cell-free supernatant of broth culture depends entirely on
the extracellular bactericidal secretions. In the colony
picking test, the secretion process is further controlled by
direct association of test bacteria (cell-to-cell interactions)
[34]. However, the cells obtained from scraping the pure
colonies and broth culturing were subjected to sonication
for the purpose of exposing the intracellular water soluble
compounds to the supernatants [35]. In the colony scraping
method, when the colonies are scraped, those pure colonies
may have already secreted extracellular compounds.
Therefore, whereas the colony picking test relies on
extracellular bactericidal compounds, the colony scraping
method depends on both extracellular and intracellular
bactericidal compounds. Cell extractions of broth culture
result in inhibition zones owing to intracellular bactericidal
compounds.
Antibacterial Activity of Tested Bacteria
Among the eight marine bacteria used in the colony
picking test, four were identified as producing antibacterial
compounds. All of these belonged to the genus
Pseudoalteromonas, in the class Gammaproteobacteria. Genus
Pseudoalteromonas produces a large number of antibacterial
compounds [36]. It has also been reported that several marine
August 2017 ⎪ Vol. 27 ⎪ No. 8
1446 Hettiarachchi et al.
Fig. 4. Visual comparison of inhibition zones made by colony scraping (CS) and broth culture (BC) methods against target bacteria
(1, P. rubra; 2, P. piscicida; 3, P. peptidolytica; 4, P. flavipulchra; 5, marine broth as a negative control; 6, 10 mg ml−1 ampicillin as a
positive control).
Gammaproteobacteria strains can synthesize antibacterial
proteins [37]. There are two main groups of Pseudoalteromonas;
one pigmented and the other not [38]. All bacteria showing
antibacterial activity in the present study were pigmented.
Pigments and extracellular compounds produced by
Pseudoalteromonas species exhibit bioactivity against various
pathogens [39]. P. flavipulchra, which produces a golden-
yellow pigment, possesses excellent antibacterial activities
against bacteria in marine aquaculture [40]. In the present
study, although P. peptidolytica exhibited antagonistic activity
against only H. smyrnensis in the colony scraping method,
all target bacterial species were antagonized by P. flavipulchra.
Although many earlier publications have described the
antagonistic activity of Pseudoalteromonas on genus Bacillus
[4, 41, 42], this study is the first report of antagonism by
P. peptidolyica, P. flavipulchra, and P. piscicida against the
genus Halomonas. This study also revealed the first direct
antibacterial activity of P. peptidolytica against B. subtilis.
In conclusion, this study was conducted to find an efficient
method to screen antibacterial compound-producing marine
bacteria. The colony picking method provides a fast, efficient,
and inexpensive antibacterial compound-producing bacteria
screening process. Furthermore, the colony picking test
enables direct contact of test and target bacteria and gives
precise clues about the bactericidal extracellular secretions
of test bacteria. The colony scraping method was done with
J. Microbiol. Biotechnol.
sonicating the scraped pure colonies of test bacteria. Therefore,
it indicates the collective effects of both extracellular and
intracellular bactericidal compounds of target bacteria. It
provides a more concentrated cell extraction than does
broth culture, which leads to more obvious zones of
inhibitions in disk diffusion assay. The commonly used
broth culture method exhibited insignificant zones of
inhibitions (extracellular secretions) with its 10 times
concentrated cell-free supernatants. However, the sonicated
cell extractions (intracellular cmpounds) exhibited significant
zones of inhibition. Dilution of the bactericidal compounds
in broth is the major reason for minor zones of inhibition in
the broth culture method.
The results of this study indicate that colony picking and
colony scraping from bacterial cultures on solid agar are
more effective methods of primary screening for antibacterial
compound-producing marine bacteria.
Acknowledgments
This research was supported by a research grant (PE0129C,
PM59732) from the Korea Institute of Ocean Science &
Technology (KIOST) and Marine Biotechnology Program
Funded by the Ministry of Ocean and Fisheries. We thank
Dr. Won-Bo Shim (Texas A&M University, USA) for the
helpful discussion and careful reading of this manuscript.

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