Revista Cienﬁca, FCV-LUZ / Vol. XXXV Recibido:17/10/2024 Aceptado:25/12/2024 Publicado: 28/02/2025 hps://doi.org/10.52973/rcfcv-e35537 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 7 Molecular characterizaon and idenﬁcaon of Shewanella putrefaciens and isolaon and morphological characterizaon of its lyc phage Caracterización molecular e idenﬁcación de Shewanella putrefaciens y aislamiento y caracterización morfológica de su fagolíco Oguzhan Kuzucu 1 , Mikail Özcan 2 * 1 Birecik District Agriculture Directorate of The Turkish Ministry of Agriculture and Forestry, Sanliurfa, Turkey 2 Kahramanmaras Sütcü Imam University, Agriculture Faculty, Department of Animal Science, Kahramanmaras, 46040 Turkey * Corresponding author: mikailozcan@ksu.edu.tr ABSTRACT The studies concerning bacteria isolated using tradional di- agnosc methods, which have been employed for many years for the detecon of pathogens in aquaculture, were restrict- ed to 15-20 bacterial species unl the 1990s. Conversely, the number of bacteria idenﬁed has reached 70 through the ul- isaon of 16S rDNA sequencing, which has been idenﬁed as the gold standard for idenﬁcaon in recent years. Shewanella putrefaciens is a pathogenic bacterium that has been isolated from both marine and freshwater ﬁsh. The number of reports concerning Shewanella putrefaciens has increased markedly with the applicaon of this method, and it has been iden- ﬁed as an opportunisc pathogen in aquaculture. In this study, Shewanella putrefaciens bacteria were isolated from rainbow trout (Oncorhynchus mykiss) in cages in Karkamış Dam Lake and idenﬁed through the use of 16S rDNA sequencing. Fur- thermore, the lyc phages of this bacterium were isolated from the same dam lake and visualized by ﬁeld emission elec- tron microscopy. It was determined that the phages obtained exhibited an icosahedral head structure, lacked a tail, and were approximately 50-60 nm in length. One of the primary factors inﬂuencing the eﬃcacy of phage therapy studies is the prev- alence of opportunisc pathogens. This study demonstrated that the lyc phages of the opportunisc pathogen Shewanella putrefaciens, which has been increasingly reported in recent years, may have the potenal to be ulized in phage therapy models. Key words: Aquaculture; Shewanella putrefaciens; bacteriophage RESUMEN Hasta la década de 1990, los estudios sobre bacterias aisla- das mediante los métodos de diagnósco tradicionales, que se han empleado durante muchos años para la detección de patógenos en la acuicultura, se limitaban a 15-20 especies bac- terianas. Por el contrario, el número de bacterias idenﬁcadas ha llegado a 70 mediante el uso de la secuenciación del 16S rDNA, que se ha idenﬁcado como el estándar de oro para la idenﬁcación en los úlmos años. Shewanella putrefaciens es una bacteria patogénica que fue aislada de peces marinos en agua dulce. El número de informes sobre Shewanella putrefa- ciens ha aumentado notablemente con la aplicación de este método, y se ha idenﬁcado como un patógeno oportunista en la acuicultura. En este estudio, la bacteria Shewanella putre- faciens se aisló de la trucha arcoíris (Oncorhynchus mykiss) en jaulas en el lago de la presa Karkamış y se idenﬁcó mediante el uso de la secuenciación del rDNA 16S. Además, los fagos lí- cos de esta bacteria se aislaron del mismo lago de la presa y se visualizaron mediante microscopía electrónica de emisión de campo. Se determinó que los fagos obtenidos exhibían una estructura de cabeza icosaédrica, carecían de cola y tenían una longitud de aproximadamente 50-60 nm. Uno de los factores principales que inﬂuyen en la eﬁcacia de los estudios de tera- pia con fagos es la prevalencia de patógenos oportunistas. Este estudio demostró que los fagos lícos del patógeno oportu- nista Shewanella putrefaciens, del que se ha informado cada vez más en los úlmos años, pueden tener el potencial de ser ulizados en modelos de terapia con fagos. Palabras clave: Acuicultura; Shewanella putrefaciens; bacteriófago

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION The genus of Shewanella bacteria encompasses more than 35 species, which are variously classiﬁed as Pseudomonas, Ach- romobacter, or Alteromonas [1]. Shewanella bacteria can be widely isolated in the environment, parcularly in freshwater and seawater, and play an important role in the degradaon of organic maer. Some species of this genus have been docu- mented to cause infecon in aquac environments. Moreover, some have been documented to cause soﬅ-ssue infecons in humans [2]. Shewanella putrefaciens is a gram-negave and mole-pos- ive bacterium with rod-shaped cells belonging to the Altero- monadaceae family. Despite the fact that this bacterium is par- cularly prevalent in the bacterial ﬂora of marine ﬁsh intesnes, studies examining its isolaon in freshwater ﬁsh are scarce. Con- versely, the role of Shewanella putrefaciens in ﬁsh pathology re- mains incompletely understood. To date, only a limited number of studies have been published on the subject of ﬁsh infecons caused by this microorganism. These studies were conducted on seahorse species, including Siganus rivulatus and Dicentrarchus labrax, as well as two freshwater ﬁsh, namely carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) [2]. In recent years, there has been a notable increase in the re- porng of these bacteria as an opportunisc pathogen in ﬁsh. Addionally, these bacteria have been idenﬁed as a novel e- ological agent of a disease known as “Shewanellosis,” which presents with symptoms such as numbness, skin discoloraon, and lesion formaon [2 , 3 , 4 , 5]. The ﬁrst documented case of a disease outbreak in freshwater ﬁsh related to Shewanellosis was reported in Poland in 2004 [4]. Subsequently, it has been idenﬁed as a pathogen in a number of freshwater ﬁsh species [2]. The objecve of this study was to isolate and characterize Shewanellosis, which has recently been idenﬁed as a pathogen in aquaculture, from sick rainbow trout samples sourced from aquaculture facilies operang in Karkamış Dam Lake. Addion- ally, the study aimed to visualize its lyc phages with potenal prophylacc and therapeuc applicaons through electron mi- croscopy following their isolaon from the same dam lake using the double layer agar method. MATERIALS AND METHODS Bacterial isolaon The study encompasses 20 farms situated in the vicinity of the Karkamış Dam Lake. In the course of the ﬁeld survey, the managers of each enterprise were interviewed to obtain the necessary anamnesis informaon. A total of 60 ﬁsh were col- lected, including three samples of ﬁsh that were observed to be stagnant in the cages, not taking feed, showing darkening of the skin colour and exophthalmus symptoms. A total of 420 ﬁsh were sampled seven mes throughout the year and trans- ported to the laboratory in ice-ﬁlled containers in a manner that ensured they did not come into contact with the ice. The ﬁsh were weighed in the laboratory. An autopsy was conducted on ﬁsh with an average weight of 250 grams, with a detailed ex- aminaon of the internal organs. For the purposes of bacterial isolaon, the external lesions, liver, spleen, kidneys and intes- nes of all samples were inoculated on Trypc Soy Agar (TSA), Brain-Heart Infusion Agar (BHIA) and Nutrient Broth (NB) media. The inoculated media were incubated at 20-24°C (Lovibond BOD Incubator, TC 135 S, UK) for 48 hours. To obtain pure cultures of the bacteria, the inoculaons were repeated three mes, and the colonies were stored at -22°C with a mixture of Trypc Soy Broth (TSB) and 20% glycerol for later use. Bacterial idenﬁcaon Bacterial samples extracted from the stock were isolated into pure culture aﬅer revival in appropriate media. Commer- cial DNA isolaon kit (Vivans GF-BA-100) was used for molec- ular idenﬁcaon of bacterial samples. For the idenﬁcaon of the isolated DNA samples, a universal bacterial primer called BAK2-F from the Internal Transcribed Spacer (ITS) gene regions: 5’AGTTTGATC(A/C)TGGCTCAG 3’BAK11-F: 5’GGACTAC(C/T/A) AGGGTATCTAAT 3’primer pairs were used. PCR mix protocol (10x PCR Buﬀer, MgCl 2 , Primer F -Bak2, Primer R -Bak 11, DNA (Tem- plete), dNTP, Taq polymerase, ddH 2 O) was applied for ampliﬁ- caon of DNA samples under appropriate PCR condions and ampliﬁcaon of DNA bands for DNA gene sequence analysis. Af- ter the preparaon of the mix protocol, for the ampliﬁcaon of bacterial DNA (Thermo Scienﬁc™, PCR Master Mix (2X), USA), denaturaon was performed at 95 o C for 5 min, followed by 30 s at 95 o C, 45 s at 48 o C, 1 min at 72 o C, repeated as 35 x and the PCR (Thermo Scienﬁc ARKTIK Thermal Cycler Type 5020 96 Well Lab, USA) study protocol was completed with 10 min at 72°C. Aﬅer that, Exsosap puriﬁcaon for DNA gene sequence analy- sis was performed by mixing 2.5 µL of PCR product and 1 µL of Exsosap and the PCR protocol was applied at 37 o C for 30 min and 80 o C for 15 min. Aﬅer Exso-SAP puriﬁcaon, the sequence PCR method was applied and nucleodes were ﬂuorescently labeled with BigDye 3.1. All samples were then prepared with Cycle Sequencing PCR reacon mix and Cycle Sequencing PCR Protocol was applied. Before sequencing, all samples were Sep- hadex puriﬁed and loaded into AB3130XL16 Genec Analyzer (Hitachi, Japan). For Sephadex puriﬁcaon; 1 g of Sephadex gel was dissolved in 14 mL of deionized ultrapure water and trans- ferred into 700 µL receiver column and centrifuged at 1800 G for 2.5 min to remove the liquid part of the sephadex mixture. Sequence PCR products were added to the resulng cola and centrifuged (McKesson, 1015031, USA) at 2127.5 G for 2.5 min and the lower part aﬅer centrifugaon was loaded into the se- quencer and run. For the evaluaon of sequence data, raw data were processed with Bioedit 7.2.5 soﬅware [6] and compared with reference databases using NCBI - Basic Local Alignment Search Tools (BLAST) program. All samples were blasted in du- plicate aﬅer consensus was reached. Phage isolaon and idenﬁcaon For phage isolaon, water samples were taken with the help of a Nansen bole from 3 diﬀerent depths (surface-middle-bot- tom) into 250 mL sterile capped laboratory boles from around the cage during the visits to the facilies where sick ﬁsh were sampled and transported to the laboratory under asepc con- dions [7]. The obtained samples were incubated at 15 o C for 3 h aﬅer mixing for 10 min with the help of a sterile swab. During incubaon, the boles were frequently inverted and the water samples were taken into 20 mL sterile falcon centrifuge tubes and centrifuged at 1850 G, 4 o C for 5 min. The supernatants were mixed with the same amount of liquid medium (NB) and incu- bated in Jeio-Tech brand shaking incubator (Jeio Tech, ISS-4075, Korea) at 64.75 G, 22 o C for 24 h. Aﬅer incubaon, the samples were transferred to 15 mL falcon tubes and aﬅer adding 0.5 mL chloroform to each sample, they were inverted and mixed for 10 min. The prepared tubes were centrifuged at 1850 G for 15 2 of 7

Molecular characterizaon of Shewanella putrefaciens / Kuzucu and Özcan UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico min at +4°C and the supernatants were ﬁltered with the help of a 0.20 µm pore diameter injector ﬁlter and the water sample obtained was considered as potenal phage lysate and stored in separate sterile tubes at +4°C [8]. The determinaon of the presence of the phages in samples isolated and enriched was carried out using the double-layer agar plate evaluaon method with minor diﬀerences. In the method carried out to determine the presence of phages in the samples collected, 2-stage casng was performed in the pe- tri dish and the lower agar was hard and the upper agar was a mixture of water sample and bacteria. Trypc Soy Agar (TSA) was used as the boom agar forming the boom of the petri dish and 15 mL was poured equally into a 90 mm sterile petri dish and allowed to dry. The top agar was prepared by adding 0.7% agar to Nutrient Agar (NA) and sterilized in autoclave and kept ready in a water bath at 30-40 o C. The previously stocked Shewanella putrefaciens was removed from the stock and each bacterium was used as a potenal host for phage isolaon. For each bacterial isolate, 200 µL of phage suspension was mixed in sterile vials with 200 µL of samples kept in liquid medium over- night. The mixture was incubated at room temperature for 5-10 min to allow the phages to adsorb the host cells and 5 mL of the liquid medium kept in the water bath was added to this mix- ture and the mixture was inverted several mes. The mixture was poured onto the prepared sub-agar and the mixture was allowed to form a thin plate for 15 min. All prepared petri dishes were incubated at 15 o C for 24-48 h and the presence of plaques was analyzed. Formed plaques were evaluated as an indicator of the presence of lyc phages [9]. The plates were taken with a sterile pasteur pipee and 1 mL SM Buﬀer and 50 µL chloroform were added and the tubes were vortexed gently. The mixture was incubated in a Thermal Cycler (Thermo Scienﬁc, ARKTIK Thermal Cycler Type 5020 96 Well Lab, USA) at 22 o C and 92.5 G for 2 h and the mixture formed at the end of incubaon was tested again using the double layer agar inoculaon method. The same process was repeated 3 mes and the isolated phages were puriﬁed [10]. The resulng puriﬁed phage suspension was ﬁltered with a 0.20 µm pore diameter injector ﬁlter; 10 µL of phage suspension was mixed with 10 mL of host bacterial cul- ture grown in liquid medium and incubated at 64.75 G +22°C overnight. After incubation, it was filtered again and 50 % glyc- erol and 10 % SM Buﬀer were added to the ﬁltrate and stocked as puriﬁed phage stock at - 22°C [11]. The examinaon of the phage by ﬁeld emission scanning elec- tron microscope The isolated and puriﬁed bacteriophage extracts were dilut- ed at 1:10 rao with SM buﬀer (2.0 g/L MgSO 4 , 5.8 g/L NaCl, 5 mL/L pre-sterilised 2% gelan, pH 7.5, 50 mL/L of 1 M Tris) and ﬁltered with a 0.20 µm pore diameter injector ﬁlter. The ﬁltrates obtained were taken to Kayseri Erciyes University Tech- nology Research and Applicaon Center (TAUM) and imaged by a ﬁeld emission scanning electron microscope (ZEISS, GEMINI 500, Germany). 20 µL of the samples were dropped onto car- bon-coated 300 mesh grids on a paraﬁlm surface and allowed to dry overnight. The grids were then washed twice with dislled water and observed and recorded with the ﬁeld emission scan- ning electron microscope. RESULTS AND DISCUSSION Bacterial isolaon and idenﬁcaon For bacterial isolaon, sick ﬁsh samples were grouped ac- cording to the facilies where they came from and their symp- toms were recorded by taking photographs. As with the pre- viously reported [12]. Symptoms of shewanellosis in rainbow trout, Aeromonas sobria and Shewanella putrefaciens were isolated from the samples with necroc lesions and exophthal- mus in the macroscopic examinaon (FIG. 1). Aﬅer the external examinaon of the ﬁsh grouped in the laboratory, they were cleaned with 70% ethyl alcohol in a sterile cabinet and dissect- ed. Addionally, as reported by Pekala et al. [4] in a study on the pathogenicity of Shewanella putrefaciens, intense haemorrhag- es in the peritoneum and internal organs and enlargement of the spleen were observed in ﬁsh (FIG. 2). FIGURE 1. Fish samples showing disease symptoms for bacterial isolaon FIGURE 2. Necropsy for bacterial isolaon The specimens were collected from the liver, spleen, kidneys and intesnes of the necropsied ﬁsh in a sterile cabinet with the help of a sterile core and cultured on previously prepared me- dia (Trypc Soy Agar, Brain-Heart Infusion Agar, Blood Agar and Nutrient Agar). Aﬅer incubaon in TSA and BHIA for 48 hours, smooth and rounded cream-colored bacterial colonies were ob- tained (FIG. 3). Bacteria detected in all samples. 3 of 7

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 3. Bacterial isolaon media In similar studies, Shewanella putrefaciens idenﬁcaon was performed with API 20E and biochemical idenﬁcaon of collected Shewanella putrefaciens strains as well as enzymac acvies tested in API Zym were determined [13]. In this study, the idenﬁcaon of the target bacteria was carried out using the 16s rDNA gene sequencing method, which is accepted as the golden method in bacterial idenﬁcaon. Aﬅer bacterial isola- on, passaged and puriﬁed colonies were prepared for bacterial idenﬁcaon according to the Vivans bacterial DNA isolaon kit protocol and PCR protocols were applied to the isolated DNAs. The products obtained were visualized on agarose gel for control before sequencing. The results obtained aﬅer bacterial gene sequencing were conﬁrmed to be 97% similar to the strain with accession number ‘CP028435.1’ in NCBl and DNA sequence chromatograms consis- tent with Shewanella putrefaciens as shown in FIG. 4. FIGURE 4. The chromatograms of Bacterial DNA Sequence The sequence results were compared with reference strains of Shewanella putrefaciens in the Naonal Center for Biotech- nology Informaon (NCBI) database to ascertain the degree of similarity (FIG. 5). The most closely related species was idenﬁed as Shewanella putrefaciens WS13 (sequence ID: CP028435.1). The highest degree of similarity was observed with Shewanel- la putrefaciens WS13, with a similarity percentage of 661/680 (97%). There were two gaps in the alignment, represenng 0% of the total number of gaps. The total score was calculated as 1.179 bits (638). The similarity values and alignment compari- son are provided in FIG. 6. FIGURE 5. A comparison of the sequence of one Shewanella putrefaciens with the sequ- ences of reference strains of Shewanella putrefaciens in the Naonal Center for Biote- chnology Informaon (NCBI) database was conducted to ascertain the level of similarity FIGURE 6. Alignment result for the taxon Shewanella putrefaciens (Query: Shewanella putrefaciens, Sbjct: Shewanella putrefaciens WS13 (Sequence ID: CP028435.1) 4 of 7

Molecular characterizaon of Shewanella putrefaciens / Kuzucu and Özcan UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico There are a few publicaons on Shewanella putrefaciens bacterium in Turkey related to soﬅ skin infecon in human ﬁeld; in aquaculture, it has been reported in eels (Anguilla anguilla) in Antalya Bay, carp (Cyprinidae) in Çoruh River, oysters in Marma- ra Sea and endemic ﬁsh species in Deriner Dam Lake [14 , 15]. The idenﬁcaon of this bacterium, which was reported for the ﬁrst me in Karkamış Dam Lake, was carried out by 16s rDNA gene sequencing method in the present study unlike the studies in the literature. The isolaon and examinaon of the lyc phages of She- wanella putrefaciens The supernatants obtained from the water samples collect- ed from around the net cages of the enterprises operang in the reservoir were isolated by enriching with liquid medium at a rao of 5X (NB). The isolaon obtained was recorded with the double layer agar method and the presence of plaque was con- ﬁrmed and recorded as shown in FIG. 7. In several studies on about lyc phage of Shewanella putrefaciens, it was isolated 5 from 6 Shewanella putrefaciens strains [16] and 1 from 3 She- wanella putrefaciens strains isolated from marine sources [17] by invesgang their prophages. FIGURE 7. The double layer agar plate image for plaques of Shewanella putrefaciens The plates visualized in FIG. 7. were obtained with a sterile pasteur pipee and the phages were single dropped by using the double layer agar inoculaon method (FIG. 8). Single fallen phage plates were collected with the help of a sterile glass pas- teur pipee and ﬁltered with 1mL SM buﬀer using a 0.20 µm pore diameter syringe ﬁlter and stocked into sterile eppendorf tubes at +40 °C. FIGURE 8. Diluted and single-dropped phage plaque images The isolated and puriﬁed phage ﬁltrates were diluted with SM Buﬀer and the grids prepared were observed by electron microscopy and phage parcles were measured and recorded as shown in FIG. 8. The imaged phages were found to have ico- sahedral head structure, tailless and 50-60 nm in size (FIG. 9). In another study in which Shewanella lyc phages were imaged by TEM, it was reported that the SFCi1 phage isolated in the mor- phological analysis of the images obtained belonged to Myoviri- dae family with an icosahedral capsid diameter of approximate- ly 70 nm [18]. FIGURE 9. Size measurement of phages of Shewanella putrefaciens by electron micros- copy 5 of 7

Revista Cienﬁca, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Shewanella putrefaciens has been reported as an opportu- nisc pathogen in aquaculture in recent years. Classical micro- biological diagnosc methods used in the diagnosis of bacteri- al diseases in aquaculture are reliable but may not be fast and praccal and may not give reliable results in the diagnosis of such bacteria. The 16S rDNA sequence analysis method, which was preferred for bacterial idenﬁcaon in this study, is consid- ered to be the gold standard for the diagnosis of bacterial infec- ons and for the idenﬁcaon of bacteria at genus and species level [19]. Bacteriophages, also the subject of this study, are a subgroup of prokaryoc viruses that speciﬁcally invade bacterial cells. Shortly aﬅer their discovery in 1917, they were tested as anbacterial agents against bacterial infecons in animals and humans, but remained in the background for many years due to the discovery and widespread use of anbiocs [20 , 21]. CONCLUSION The increasing incidence of infecon due to anbioc-resis- tant bacteria in recent years has led to a renewed interest in phages and phage therapy. Although diﬀerent phage isolaon and idenﬁcaon studies have been conducted on Shewanella putrefaciens, the number of studies evaluated in terms of aqua- culture is relavely limited. In this study, Shewanella putrefaciens and its lyc phages, which have been reported as pathogens in aquaculture in recent years, were isolated and the phages were visualized by electron microscopy. Pathogenic bacteria causing many primary and secondary infecons reported in aquaculture and, more phages should be isolated and more therapy models should be developed for prophylacc/therapeuc use. ACKNOWLEDGEMENTS Acknowledgements to Kahramanmaras Sütcü Imam Univer- sity (KSÜ), for the support and infrastructure. Ethical Approval All animal studies were approved by the Animal Ethics Com- miee of KSÜZİRHADYEK and Research Instute (Protocol num- ber: 2022/1). Availability of data and material The data that support the ﬁndings of this study are available from the corresponding author upon reasonable request. Conﬂict of interest The authors have no conﬂict of interest that could be per- ceived as prejudicing the imparality of the study reported. BIBLIOGRAPHIC REFERENCES [1] Buller NB. Bacteria and fungi from ﬁsh and other aquac animals: a praccal idenﬁcaon manual. 2nd ed. Pondi- rechi, India. CAB Internacional. 2014; 863 p. [2] Qin L, Zhu M, Xu J. First report of Shewanella sp. and Listonella sp. infecon in freshwater cultured loach, Mis- gurnus anguillicaudatus. Aquac. Res. [Internet]. 2014; 45(4):602-608. doi: hps://doi.org/f537n3 [3] Paździor E. 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