J. Microbiol. Biotechnol. (2017), 27(8), 1441–1448 https://doi.org/10.4014/jmb.1703.03012 Research Article Review jmb

A Rapid and Efficient Screening Method for Antibacterial Compound- Producing 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*

1Korea Institute of Ocean Science & Technology, Jeju Special Self-Governing Province 63349, Republic of Korea 2Department of Marine Biology, Korea University of Science and Technology, Jeju Special Self-Governing Province 63349, Republic of Korea 3College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea

Received: March 8, 2017 Revised: May 25, 2017 Antibacterial compounds are widely used in the treatment of human and animal diseases. The Accepted: June 13, 2017 overuse of antibiotics has led to a rapid rise in the prevalence of drug-resistant bacteria, First published online making the development of new antibacterial compounds essential. This study focused on June 16, 2017 developing a fast and easy method for identifying marine bacteria that produce antibiotic

*Corresponding author compounds. Eight randomly selected marine target bacterial (Agrococcus terreus, Phone: +82-64-798-6102; Bacillus algicola, Mesoflavibacter zeaxanthinifaciens, Pseudoalteromonas flavipulchra, P. peptidolytica, Fax: +82-64-798-6039; E-mail: [email protected] 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 S upplementary data for this diffusion assay. P. peptidolytica, P. piscicida, and P. rubra showed antagonistic activity against paper are available on-line only at http://jmb.or.kr. 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 pISSN 1017-7825, eISSN 1738-8872 screening for antibacterial compound-producing bacteria. Copyright© 2017 by The Korean Society for Microbiology Keywords: Colony picking, colony scraping, broth culture, screening, antibacterial compounds and Biotechnology

Introduction estimated that microorganisms produce more than 22,000 bioactive compounds in nature, the identification and In recent years, research on drug discovery and characterization of new and useful metabolites can be development has increasingly focused on naturally occurring difficult [6]. products [1]. Nature undoubtedly harbors a huge arsenal Identification of bacteria that potentially produce bioactive of organisms capable of producing compounds that will compounds, extraction of these compounds, and subsequent prove beneficial to humans [2]. In particular, marine testing against various pathogenic bacteria are the necessary microorganisms are proving to be an important source of first steps in the process of screening for new antibacterials. novel microbial compounds that display antibacterial, Screening systems used for the identification of new antiviral, and antitumor activities, as well as anticoagulant antibacterial compounds should be simple, rapid, reproducible, and cardiodynamic properties [3]. Marine microbes from and inexpensive [7]. The earliest screening methods to be extreme ecological niches can be good sources of novel adopted were microbial inhibition assays, which are still pharmaceutical materials [4], since their adaptive strategies widely used for the detection of antibiotic residues [8]. and metabolism have been optimized for survival in highly Bioautographic, diffusion, and dilution methods are also diverse and challenging environments [5]. While it is commonly used; the diffusion method is often used as a

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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 some of them have shown bactericidal effects in previous rRNA sequencing analysis and stored at −80°C in 40% glycerol [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 18] and H. smyrnensis and V. alginolyticus are gram negative with fresh marine broth, followed by spreading onto marine agar 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.

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H. smyrnensis 1.64 × 108 CFU/ml, and V. alginolyticus 2.14 × 108 Standards Institute [25], with several modifications. Lawn CFU/ml) was spread onto marine agar Petri dishes. Sterile cultures of target bacteria were prepared by spreading 60 µl of wooden toothpicks were used to introduce pure single colonies of fresh broth culture of B. cereus (0.71 × 108 CFU/ml), B. subtilis target bacteria onto the marine agar dishes with different test (1.7×108 CFU/ml), and H. smyrnensis (0.4 × 108 CFU/ml) onto bacteria (Fig. 1). The plates were incubated at 30°C for 16 h, after marine agar plates. Sterile paper disks (8 mm diameter; Toyo which zones of inhibition were observed and measured. 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 Colony Scraping Method of 10 mg/ml ampicillin and sterile marine broth were used as Target bacteria that exhibited antagonistic activities were positive and negative controls, respectively. The plates were cultured on marine agar plates. Approximately 0.1 g (wet weight) incubated at 30°C for 16 h and the diameter of inhibition zones of each bacterial colony was collected using a sterile scraper and around the disks was measured. The test was performed in transferred to a labeled sterile 1.5 ml tube. Distilled water (500 µl) duplicates (n =2). 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 Results and Discussion were filtered through a 0.45 µm disposable membrane filter unit (Toyo Roshi Kaisha Ltd., Japan). Colony Picking Method Colony picking was used to identify test bacteria exhibiting Broth Culture Method antibacterial activity. The growth of pure colonies of test Pure colonies of each target bacterium were inoculated into bacteria on lawn cultures of target bacteria was observed 4 ml of marine broth and incubated at 30°C, 170 rpm for 16 h. after incubation. The presence and size of clear zones These cultures were transferred to 100 ml of fresh marine broth and incubated under the same conditions to obtain 100 ml of broth indicated the degree of antibacterial activity in the test culture of each target bacterium. The cultured broth was separated bacteria. Four target bacteria belonging to the genus to supernatant and cell pellet by centrifugation at 7,500 ×g for Pseudoalteromonas inhibited the growth of the two Bacillus 10min. The supernatant was concentrated 10 times after freeze sp. and H. smyrnensis in preliminary screening (Table 2). drying, and was filtered through a 0.45 µm disposable membrane These bacteria did not exhibit any activity against filter unit. The collected cell pellet (approximately 0.1 g) was mixed V. alginolyticus. The other four bacterial species (A. terreus, with 500 µl of distilled water and sonicated at 40% amplitude for M. zeaxanthinifaciens, B. algicola, and Z. atlantica) did not 1 min. The contents were transferred to a new 1.5 ml tube and exhibit any antibacterial activity against the test bacteria. centrifuged at 7,500 ×g for 2 min. The supernatants were separated P. flavipulchra and P. piscicida exhibited comparatively and filtered through a 0.45 µm disposable membrane filter unit. large clear zones on lawns of B. cereus and B. subtilis Both samples were used for the antagonistic activity test by disk (Fig. 2). Clear zones are the result of test bacteria inhibiting diffusion assay. the growth of target bacteria. Test bacteria that produced Disk Diffusion Assay clear zones could therefore be initially identified as The antagonistic activity of each sample was tested by the disk producing antibacterial compounds. diffusion method, as described by the Clinical and Laboratory In the colony picking test, test bacterial colonies come

Table 2. Antagonistic activity of picked colonies of target bacteria against test bacteria. Test bacteria Target bacteria B. cereus B. subtilis H. smyrnensis V. alginolyticus A. terreus -- - - B. algicola -- - - M. zeaxanthinifaciens -- - - P. flavipulchra ++ + - P. peptidolytica ++ + - P. piscicida ++ + - P. rubra ++ + - Z. atlantica -- - - (+) Clear zone observed; (-) no clear zone observed.

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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 than the cross-streak method as a primary screening. Colony between bacteria enhance the activation of cryptic genes picking is thus suitable as a rapid method for screening responsible for producing various types of metabolites antibacterial compound-producing bacteria. It is also more [26]. Antagonism depends on multiple antibacterial factors cost-effective than other methods, as it does not require in test bacteria; for example, exopolysaccharide and biofilm paper disks [7], filter papers, or additional incubation. formation, compound concentration, co-culture effect, and the polarity of the compound produced (water solubility) Colony Scraping Method [27-29], and most of these factors influence the results of The colony scraping method was introduced as another the colony picking test. Instead of using a broth culture and quick approach to check the antibacterial activity of test testing supernatants and cell pellets, as in the commonly bacteria. Pure colonies of test bacteria were scraped from used broth culture screening method, colony picking offers agar plates and tested for antibacterial ability. The efficacy a quick approach, encompassing the factors mentioned above, of this method was compared with that of the broth culture for primary screening to identify antibacterial compound- method. In P. peptidolytica, antibacterial activity against producing bacteria. Moreover, this method provides space H. smyrnensis was only detected by the colony scraping for more than 10 target bacteria within one agar plate. method. Colony scraping also revealed a higher degree of Therefore, it differs from the existing cross-streak method, antibacterial activity in P. flavipulchra and P. piscicida than which should be drawn perpendicular lines on agar plate did the broth culture method (Table 3). Both methods with target bacteria against the test bacteria [30]. The test revealed the same degree of antagonistic activity in P. rubra can be performed more rapidly and concurrently on a against B. cereus, whereas the antibacterial activity of number of target bacteria compared with the cross-streak P. piscicida was only revealed by the colony scraping method. It also facilitates direct contact of test bacteria with method (Fig. 3A). The antibacterial activity of P. rubra target bacteria and indicates obvious clear zones. Therefore, against B. subtilis was also only detected by the colony the colony picking method is more organized and precise 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±0.711.0±0.0 10.5±0.717.5±0.7 14.0±0.0 P. piscicida 11.5±0.7- - - 16.0±1.4 11.5±0.7 P. rubra 11.5±0.711.5±0.710.5±0.7 - 15.5±0.7 16.0±1.4

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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 Fig. 3. Comparison of colony scraping and broth culture colonies and broth culturing were subjected to sonication methods for detecting the antagonistic activity of test bacteria against target bacteria. 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 Although P. peptidolytica showed some antibacterial may have already secreted extracellular compounds. activity in the preliminary screening, it did not produce an Therefore, whereas the colony picking test relies on inhibition zone against B. subtilis in either the colony extracellular bactericidal compounds, the colony scraping scraping or the broth culture method (Fig. 3). All test method depends on both extracellular and intracellular bacteria exhibited inhibition zones against H. smyrnensis in bactericidal compounds. Cell extractions of broth culture both methods, except P. peptidolytica in broth culture result in inhibition zones owing to intracellular bactericidal (Fig. 3C). The antagonistic activity of all test bacteria on all compounds. target bacteria was higher in the colony scraping method than in the broth culture method, with the exception of Antibacterial Activity of Tested Bacteria P. rubra against H. smyrnensis (Fig. 4). P. piscicida, P. rubra, Among the eight marine bacteria used in the colony and P. peptidolytica exhibited antagonism against B. cereus, picking test, four were identified as producing antibacterial B. subtilis, and H. smyrnensis, respectively, only in the compounds. All of these belonged to the genus colony scraping method (Fig. 3). Pseudoalteromonas, in the class Gammaproteobacteria. Genus The 10 times concentrated broth of P. peptidolytica and Pseudoalteromonas produces a large number of antibacterial P. flavipulchra exhibited minor antagonistic activity against compounds [36]. It has also been reported that several marine

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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 sonicating the scraped pure colonies of test bacteria. Therefore, proteins [37]. There are two main groups of Pseudoalteromonas; it indicates the collective effects of both extracellular and one pigmented and the other not [38]. All bacteria showing intracellular bactericidal compounds of target bacteria. It antibacterial activity in the present study were pigmented. provides a more concentrated cell extraction than does Pigments and extracellular compounds produced by broth culture, which leads to more obvious zones of Pseudoalteromonas species exhibit bioactivity against various inhibitions in disk diffusion assay. The commonly used pathogens [39]. P. flavipulchra, which produces a golden- broth culture method exhibited insignificant zones of yellow pigment, possesses excellent antibacterial activities inhibitions (extracellular secretions) with its 10 times against bacteria in marine aquaculture [40]. In the present concentrated cell-free supernatants. However, the sonicated study, although P. peptidolytica exhibited antagonistic activity cell extractions (intracellular cmpounds) exhibited significant against only H. smyrnensis in the colony scraping method, zones of inhibition. Dilution of the bactericidal compounds all target bacterial species were antagonized by P. flavipulchra. in broth is the major reason for minor zones of inhibition in Although many earlier publications have described the the broth culture method. antagonistic activity of Pseudoalteromonas on genus Bacillus The results of this study indicate that colony picking and [4, 41, 42], this study is the first report of antagonism by colony scraping from bacterial cultures on solid agar are P. peptidolyica, P. flavipulchra, and P. piscicida against the more effective methods of primary screening for antibacterial genus Halomonas. This study also revealed the first direct compound-producing marine bacteria. antibacterial activity of P. peptidolytica against B. subtilis. In conclusion, this study was conducted to find an efficient Acknowledgments method to screen antibacterial compound-producing marine bacteria. The colony picking method provides a fast, efficient, This research was supported by a research grant (PE0129C, and inexpensive antibacterial compound-producing bacteria PM59732) from the Korea Institute of Ocean Science & screening process. Furthermore, the colony picking test Technology (KIOST) and Marine Biotechnology Program enables direct contact of test and target bacteria and gives Funded by the Ministry of Ocean and Fisheries. We thank precise clues about the bactericidal extracellular secretions Dr. Won-Bo Shim (Texas A&M University, USA) for the of test bacteria. The colony scraping method was done with helpful discussion and careful reading of this manuscript.

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