Producing Bacteria

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 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* 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 species (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 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 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. J. Microbiol. Biotechnol. Screening of Antibacterial Compound-Producing Bacteria 1443 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

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