Molecular Identification, Enzymatic Activity and Antibiotic Resistance Profiles of Bacteria Isolated from Merlangius merlangus

Year 2025, Volume: 8 Issue: 5, 1634 - 1644, 15.09.2025

Huseyin Sezgin Caglar , Semra Saygın , Hayrettin Saygin

https://doi.org/10.34248/bsengineering.1749516

Abstract

The present study intended to examine the cultivable bacterial microbiota of Merlangius merlangus, an ecologically valuable fish species from the Black Sea. Serial dilution and pre-enrichment steps were undertaken before isolation. Ten different isolates were isolated from muscle, gill, and intestinal tissues of fresh specimens. Molecular identification by 16S rRNA gene sequence showed all isolates to be members of Enterobacteriaceae, comprised of the genera Morganella, Providencia, Proteus, Klebsiella, and Escherichia. As one of the isolates (S09) was found to exhibit the highest similarity with a validly published species within the genus Morganella, with a relatively low value of 99.11%, it could be a new taxon. Extracellular enzyme tests showed variable presence of lipase and urease activities among isolates but no detectable amylase, caseinase, lecithinase, or DNase activities. Antimicrobial susceptibility testing was consistent with high rates of multidrug resistance, with eight isolates being resistant to three or more antibiotic groups. Resistance was greatest against amoxicillin/clavulanic acid, tetracycline, and sulfamethoxazole/trimethoprim, with all isolates showing susceptibility to imipenem. The observations point to the occurrence of metabolically active, environmentally adapted, and resilient bacterial forms in M. merlangus, with food safety implications and concern for antimicrobial resistance dissemination in aquatic ecosystems. Further, the uniqueness of the phylogenetic status of isolate S09 suggests an additional polyphasic taxonomic study. Future research involving metagenomes will be required to characterize host-associated bacterioplankton communities comprehensively, including their ecological functions.

Keywords

Fish microbiota , Merlangius merlangus , Enterobacteriaceae , Extracellular enzymatic activity , Antibiotic resistance , 16S rRNA gene phylogeny

Ethical Statement

In accordance with national and institutional guidelines, ethical approval was not required for this study, as the fish specimens (Merlangius merlangus) were obtained post-mortem from commercial fishers during routine fishing activities. No experimental procedures were performed on live animals. The collection and use of these samples did not involve any intervention, manipulation, or distress to living organisms, thereby exempting the study from formal ethical review.

Supporting Institution

Türkiye Bilimsel ve Teknolojik Araştırma Kurumu (TÜBİTAK)

Project Number

1919B012309020

Thanks

This research was supported by The Scientific and Technological Research Council of Türkiye (TÜBİTAK), [grant number 1919B012309020].

References

• Albejo AL, Hamza TA. 2017. Isolation and characterization of thermostable amylase producing bacteria from hot spring at Arba Minch Nech Sar National Park, Southern Ethiopia. Int J Novel Res Interdiscip Stud 4: 9-16.
• Algammal AM, Mabrok M, Ezzat M, Alfifi KJ, Esawy AM, Elmasry N, El-Tarabili RM. 2022. Prevalence, antimicrobial resistance (AMR) pattern, virulence determinant and AMR genes of emerging multi-drug resistant Edwardsiella tarda in Nile tilapia and African catfish. Aquaculture 548: 737643. https://doi.org/10.1016/j.aquaculture.2021.737643
• Alikunhi NM, Batang ZB, AlJahdali HA, Aziz MA, Al-Suwailem AM. 2017. Culture-dependent bacteria in commercial fishes: Qualitative assessment and molecular identification using 16S rRNA gene sequencing. Saudi J Biol Sci 24: 1105-1116.
• Asciutto E, Maioli F, Manfredi C, Anibaldi A, Cimini J, Isailović I, Marčeta B, Casini M. 2024. Spatio-temporal patterns of whiting (Merlangius merlangus) in the Adriatic Sea under environmental forcing. PLoS One 19: e0289999. https://doi.org/10.1371/journal.pone.0289999
• Banerjee G, Ray AK. 2017. Bacterial symbiosis in the fish gut and its role in health and metabolism. Symbiosis 72: 1-11. https://doi.org/10.1007/s13199-016-0441-8
• Birolli WG, Lima RN, Porto AL. 2019. Applications of marine-derived microorganisms and their enzymes in biocatalysis and biotransformation, the underexplored potentials. Front Microbiol 10: 1453. https://doi.org/10.3389/fmicb.2019.01453
• Brauge T, Bourdonnais E, Trigueros S, Cresson P, Debuiche S, Granier SA, Midelet G. 2024. Antimicrobial resistance and geographical distribution of Staphylococcus sp. isolated from whiting (Merlangius merlangus) and seawater in the English Channel and the North Sea. Environ Pollut 345: 123434. https://doi.org/10.1016/j.envpol.2024.123434
• Bunpa S, Sermwittayawong N, Vuddhakul V. 2016. Extracellular enzymes produced by Vibrio alginolyticus isolated from environments and diseased aquatic animals. Procedia Chem 18: 12-17. https://doi.org/10.1016/j.proche.2016.01.002
• Cabello FC. 2006. Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environ Microbiol 8: 1137-1144. https://doi.org/10.1111/j.1462-2920.2006.01054.x
• Calì F, Stranci F, La Mesa M, Mazzoldi C, Arneri E, Santojanni A. 2023. Whiting (Merlangius merlangus) grows slower and smaller in the Adriatic Sea: New insights from a comparison of two populations with a time interval of 30 years. Fishes 8: 341. https://doi.org/10.3390/fishes8070341
• Cantas L, Sørby JRT, Aleström P, Sørum H. 2012. Culturable gut microbiota diversity in zebrafish. Zebrafish 9: 26-37. https://doi.org/10.1089/zeb.2011.0712
• Cheng TH, Ismail N, Kamaruding N, Saidin J, Danish-Daniel MJB. 2020. Industrial enzymes-producing marine bacteria from marine resources. Biotechnol Rep 27: e00482.
• Chiarello M, Villeger S, Bouvier C, Bettarel Y, Bouvier T. 2015. High diversity of skin-associated bacterial communities of marine fishes is promoted by their high variability among body parts, individuals and species. FEMS Microbiol Ecol 91: fiv061. https://doi.org/10.1093/femsec/fiv061
• Clinical and Laboratory Standards Institute. 2023. Performance standards for antimicrobial susceptibility testing. CLSI supplement M100, Clinical and Laboratory Standards Institute, Wayne, PA, USA, pp: 428.
• Cui MJ, Teng A, Chu J, Cao B. 2022. A quantitative, high-throughput urease activity assay for comparison and rapid screening of ureolytic bacteria. Environ Res 208: 112738. https://doi.org/10.1016/j.envres.2022.112738
• de Bruijn I, Liu Y, Wiegertjes GF, Raaijmakers JM. 2018. Exploring fish microbial communities to mitigate emerging diseases in aquaculture. FEMS Microbiol Ecol 94: fix161. https://doi.org/10.1093/femsec/fix161
• Dewi RR, Hassan L, Daud HM, Matori MF, Zakaria Z, Ahmad NI, Aziz SA, Jajere SM. 2022. On-farm practices associated with multi-drug-resistant Escherichia coli and Vibrio parahaemolyticus derived from cultured fish. Microorganisms 10: 1520.
• Dhayalan A, Velramar B, Govindasamy B, Ramalingam KR, Dilipkumar A, Pachiappan P. 2022. Isolation of a bacterial strain from the gut of the fish, Systomus sarana, identification of the isolated strain, optimized production of its protease, the enzyme purification, and partial structural characterization. J Genet Eng Biotechnol 20: 24. https://doi.org/10.1186/s43141-022-00299-3
• Drzewiecka D. 2016. Significance and roles of Proteus spp. bacteria in natural environments. Microb Ecol 72: 741-758. https://doi.org/10.1007/s00248-015-0720-6
• Egerton S, Culloty S, Whooley J, Stanton C, Ross RP. 2018. The gut microbiota of marine fish. Front Microbiol 9: 873. https://doi.org/10.3389/fmicb.2018.00873
• FAO. 2020. The State of Mediterranean and Black Sea Fisheries 2020. General Fisheries Commission for the Mediterranean, Rome, Italy. pp: 129. https://doi.org/10.4060/cb2429en
• Farooq S, Ganai SA, Ganai BA, Mohan S, Uqab B, Nazir R. 2022. Molecular characterization of lipase from a psychrotrophic bacterium Pseudomonas sp. CRBC14. Curr Genet 68: 243-251 https://doi.org/10.1007/s00294-021-01224-w
• Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x
• Froese R, Pauly D. 2024. FishBase. URL: https://www.fishbase.se (accessed April 01, 2025).
• Gauthier DT. 2015. Bacterial zoonoses of fishes: A review and appraisal of evidence for linkages between fish and human infections. Vet J 203: 27-35.
• Ghaly AE, Dave D, Budge S, Brooks MS. 2010. Fish spoilage mechanisms and preservation techniques: Review. Am J Appl Sci 7: 859-877.
• Hatosy SM, Martiny AC. 2015. The ocean as a global reservoir of antibiotic resistance genes. Appl Environ Microbiol 81: 7593-7599. https://doi.org/10.1128/AEM.00736-15
• Hoppe HG, Arnosti C, Herndl GJ. 2002. Ecological significance of bacterial enzymes in the marine environment. In: Burns RG, Dick RP, editors. Enzymes in the Environment: Activity, Ecology, and Applications. Marcel Dekker, New York, USA. pp: 73-107.
• Hossain A, Habibullah-Al-Mamun M, Nagano I, Masunaga S, Kitazawa D, Matsuda H. 2022. Antibiotics, antibiotic-resistant bacteria, and resistance genes in aquaculture: risks, current concern, and future thinking. Environ Sci Pollut Res Int 29: 11054-11075.
• Huang Q, Sham RC, Deng Y, Mao Y, Wang C, Zhang T, Leung KM. 2020. Diversity of gut microbiomes in marine fishes is shaped by host‐related factors. Mol Ecol 29: 5019-5034. https://doi.org/10.1111/mec.15699
• Hudzicki J. 2009. Kirby-Bauer disk diffusion susceptibility test protocol. Am Soc Microbiol 15: 1-23
• ICES. 2024. Whiting (Merlangius merlangus) in divisions 7.b–c and 7.e–k (southern Celtic Seas and western English Channel). ICES Advice 2024, whg.27.7b-ce-k. https://doi.org/10.17895/ices.advice.25019729
• Ikeda-Ohtsubo W, Brugman S, Warden CH, Rebel JM, Folkerts G, Pieterse CM. 2018. How can we define “optimal microbiota?”: A comparative review of structure and functions of microbiota of animals, fish, and plants in agriculture. Front Nutr 5: 90. https://doi.org/10.3389/fnut.2018.00090
• Jackman J. 2012. The microbe: the basics of structure, morphology, and physiology as they relate to microbial characterization and attribution. In: Cliff J, Kreuzer H, Ehrhardt C, Wunschel D, editors. Chemical and Physical Signatures for Microbial Forensics, pp: 13-34.
• Janda JM, Abbott SL. 2021. The changing face of the family Enterobacteriaceae (Order: “Enterobacterales”): New members, taxonomic issues, geographic expansion, and new diseases and disease syndromes. Clin Microbiol Rev 34: e00021-19. https://doi.org/10.1128/cmr.00174-20
• Justé A, Thomma BPHJ, Lievens B. 2008. Recent advances in molecular techniques to study microbial communities in food-associated matrices and processes. Food Microbiol 25: 745-761. https://doi.org/10.1016/j.fm.2008.04.009
• Köker L, Aydın F, Gaygusuz Ö, Akçaalan R, Çamur D, İlter H, Ayoğlu FN, Altın A, Topbaş M, Albay M. 2021. Heavy metal concentrations in Trachurus mediterraneus and Merlangius merlangus captured from Marmara Sea, Turkey and associated health risks. Environ Manag 67: 522-531. https://doi.org/10.1007/s00267-020-01352-y
• Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35: 1547-1549. https://doi.org/10.1093/molbev/msy096
• Kyule DN, Maingi JM, Njeru EM, Nyamache AK. 2022. Molecular characterization and diversity of bacteria isolated from fish and fish products retailed in Kenyan markets. Int J Food Sci 2022: 2379323.
• Muus BJ, Nielsen JG. 1999. Sea Fish. Scandinavian Fishing Year Book, Hedehusene, Denmark. pp: 340.
• Ng’eno E, Cobos ME, Kiplangat S, Mugoh R, Ouma A, Bigogo G, Ouma S, Peterson AT. 2024. Long-term antibiotic exposure landscapes and resistant Escherichia coli colonization in a densely populated setting. PLoS One 19: e0302521. https://doi.org/10.1371/journal.pone.0302521
• Nissar S, Bakhtiyar Y, Arafat MY, Andrabi SA, Bhat AA, Yousuf T. 2023. A review of the ecosystem services provided by the marine forage fish. Hydrobiologia 850: 2871-2902. https://doi.org/10.1007/s10750-022-05033-1
• Ntushelo K. 2013. Identifying bacteria and studying bacterial diversity using the 16S ribosomal RNA gene-based sequencing techniques: A review. Afr J Microbiol Res 7: 5533-5540. https://doi.org/10.5897/AJMR2013.5966
• O'Neill J. 2014. Antimicrobial resistance: Tackling a crisis for the health and wealth of nations. Rev Antimicrob Resist 20: 1-16
• Paarup T, Nieto JC, Peláez C, Reguera JI. 1999. Microbiological and physico-chemical characterisation of deep spoilage in Spanish dry-cured hams and characterisation of isolated Enterobacteriaceae with regard to salt and temperature tolerance. Eur Food Res Technol 209: 366-371. https://doi.org/10.1007/s002170050511
• Pękala-Safińska A. 2018. Contemporary threats of bacterial infections in freshwater fish. J Vet Res 62: 261-267. https://doi.org/10.2478/jvetres-2018-0037
• Pepi M, Focardi S. 2021. Antibiotic-resistant bacteria in aquaculture and climate change: A challenge for health in the Mediterranean area. Int J Environ Res Public Health 18: 5723. https://doi.org/10.3390/ijerph18115723
• Pitout JD, Laupland KB. 2008. Extended-spectrum β-lactamase-producing Enterobacteriaceae: An emerging public-health concern. Lancet Infect Dis 8: 159-166. https://doi.org/10.1016/S1473-3099(08)70041-0
• Pratte ZA, Besson M, Hollman RD, Stewart FJ. 2018. The gills of reef fish support a distinct microbiome influenced by host-specific factors. Appl Environ Microbiol 84: e00063-18. https://doi.org/10.1128/AEM.00063-18
• Preena PG, Swaminathan TR, Kumar VJR, Singh ISB. 2020. Antimicrobial resistance in aquaculture: A crisis for concern. Biologia 75: 1497-1517. https://doi.org/10.2478/s11756-020-00456-4
• Rabbani MAG, Howlader MZH, Kabir Y. 2017. Detection of multidrug resistant (MDR) bacteria in untreated waste water disposals of hospitals in Dhaka City, Bangladesh. J Glob Antimicrob Resist 10: 120-125. https://doi.org/10.1016/j.jgar.2017.04.009
• Rameshwaram NR, Singh P, Ghosh S, Mukhopadhyay S. 2018. Lipid metabolism and intracellular bacterial virulence: Key to next-generation therapeutics. Future Microbiol 13: 1301-1328. https://doi.org/10.2217/fmb-2018-0013
• Ramkumar R, Ravi M, Jayaseelan C, Rahuman AA, Anandhi M, Rajthilak C, Perumal P. 2014. Description of Providencia vermicola isolated from diseased Indian major carp, Labeo rohita (Hamilton, 1822). Aquaculture 420: 193-197. https://doi.org/10.1016/j.aquaculture.2013.11.013
• Ray AK, Ghosh K, Ringø E. 2012. Enzyme‐producing bacteria isolated from fish gut: A review. Aquac Nutr 18: 465-492. https://doi.org/10.1111/j.1365-2095.2012.00943.x
• Romero J, Ringø E, Merrifield DL. 2014. The gut microbiota of fish. In: Merrifield D, Ringø E, editors. Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics. Wiley-Blackwell, Oxford,USA. pp: 75-100.
• Rutherford JC. 2014. The emerging role of urease as a general microbial virulence factor. PLoS Pathog 10: e1004062. https://doi.org/10.1371/journal.ppat.1004062
• Saitou N, Nei M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406-425.
• Santos RA, Oliva-Teles A, Pousão-Ferreira P, Jerusik R, Saavedra MJ, Enes P, Serra CR. 2021. Isolation and characterization of fish-gut Bacillus spp. as source of natural antimicrobial compounds to fight aquaculture bacterial diseases. Mar Biotechnol 23: 276-293.
• Sheng L, Wang L. 2021. The microbial safety of fish and fish products: Recent advances in understanding its significance, contamination sources, and control strategies. Compr Rev Food Sci Food Saf 20: 738-786. https://doi.org/10.1111/1541-4337.12671
• Song J, Rensing C, Holm PE, Virta M, Brandt KK. 2017. Comparison of metals and tetracycline as selective agents for development of tetracycline resistant bacterial communities in agricultural soil. Environ Sci Technol 51: 3040-3047. https://doi.org/10.1021/acs.est.6b05342
• Stead D. 1986. Microbial lipases: Their characteristics, role in food spoilage and industrial uses. J Dairy Res 53: 481-505.
• Stefani FO, Bell TH, Marchand C, De La Providencia IE, El Yassimi A, St-Arnaud M, Hijri M. 2015. Culture-dependent and -independent methods capture different microbial community fractions in hydrocarbon-contaminated soils. PLoS One 10: e0128272. https://doi.org/10.1371/journal.pone.0128272
• Stratev D, Daskalov H, Vashin I. 2015. Characterisation and determination of antimicrobial resistance of β-haemolytic Aeromonas spp. isolated from common carp (Cyprinus carpio L.). Rev Med Vet (Toulouse) 166: 54-61
• Trincone A. 2017. Enzymatic processes in marine biotechnology. Mar Drugs 15: 93. https://doi.org/10.3390/md15040093
• Uchiyama H. 2000. Distribution of Vibrio species isolated from aquatic environments with TCBS agar. Environ Health Prev Med 4: 199-204. https://doi.org/10.1007/BF02931258
• Wanja DW, Mbuthia PG, Waruiru RM, Mwadime JM, Bebora LC, Nyaga PN, Ngowi HA. 2019. Bacterial pathogens isolated from farmed fish and source pond water in Kirinyaga County, Kenya. Int J Fish Aquat Stud 7: 295-301
• World Health Organization. 2019. No time to wait: Securing the future from drug-resistant infections. Report to the Secretary-General of the United Nations. World Health Organization, Geneva, pp: 25.
• Yang JH, Sheng WH, Hsueh PR. 2020. Antimicrobial susceptibility and distribution of extended-spectrum β-lactamases, AmpC β-lactamases and carbapenemases among Proteus, Providencia and Morganella isolated from global hospitalised patients with intra-abdominal and urinary tract infections: Results of the Study for Monitoring Antimicrobial Resistance Trends (SMART), 2008–2011. J Glob Antimicrob Resist 22: 398-407. https://doi.org/10.1016/j.jgar.2020.04.011
• Yang Y. 2018. Detection, activity measurement and phylogeny of ureolytic bacteria isolated from elasmobranch tissue. Masters Thesis, University of Southern Mississippi,USA. pp: 89.
• Yildiz T, Uzer U, Yemişken E, Karakulak FS, Kahraman AE, Çanak Ö. 2021. Conserve immatures and rebound the potential: stock status and reproduction of whiting (Merlangius merlangus [Linnaeus, 1758]) in the Western Black Sea. Mar Biol Res 17: 815-827. https://doi.org/10.1080/17451000.2021.2019787
• Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J. 2017. Introducing EzBioCloud: A taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol 67: 1613-1617. https://doi.org/10.1099/ijsem.0.001755
• Yukgehnaish K, Kumar P, Sivachandran P, Marimuthu K, Arshad A, Paray BA, Arockiaraj J. 2020. Gut microbiota metagenomics in aquaculture: Factors influencing gut microbiome and its physiological role in fish. Rev Aquac 12: 1903-1927. https://doi.org/10.1111/raq.12416
• Zhang C, Kim SK. 2010. Research and application of marine microbial enzymes: Status and prospects. Mar Drugs 8: 1920-1934. https://doi.org/10.3390/md8061920