Elsevier

Fish & Shellfish Immunology

Volume 110, March 2021, Pages 23-34
Fish & Shellfish Immunology

Full length article
Octominin: An antibacterial and anti-biofilm peptide for controlling the multidrug resistance and pathogenic Streptococcus parauberis

https://doi.org/10.1016/j.fsi.2020.12.017Get rights and content

Highlights

  • Octominin displayed strong antibacterial activity against Streptococcus parauberis.

  • Octominin involved inhibition and eradication of biofilm in S. parauberis.

  • S. parauberis exposed larval zebrafish are protected by Octominin treatment.

Abstract

Streptococcus parauberis is a pathogenic gram-positive bacterium that causes streptococcosis infection in fish. Since S. parauberis is becoming resistant to multiple antibiotics, the development of alternatives, such as antimicrobial peptides, has gained great attention. Octominin, derived from the defense protein of Octopus minor, showed a significant antimicrobial activity against multidrug resistance S. parauberis, with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 50 and 100 μg/mL, respectively. Furthermore, time-kill kinetics, agar diffusion, and bacterial viability assays confirmed the concentration-dependent antibacterial activity of Octominin against S. parauberis. Field emission scanning electron microscopy analysis showed morphological and ultra-structural changes in S. parauberis upon Octominin treatment. Moreover, Octominin treatment demonstrated changes in membrane permeability, induced reactive oxygen species (ROS), and its binding ability to genomic DNA, suggesting its strong bactericidal activity with multiple modes of action. We confirmed the inhibition of biofilm formation and the eradication of existing biofilms in a concentration-dependent manner. Additionally, Octominin on S. parauberis at transcriptional level exhibited downregulation of membrane formation (pgsA and cds1), DNA repairing (recF), biofilm formation (pgaB and epsF) genes, while upregulation of ROS detoxification (sodA) and DNA protecting (ahpF) related genes. An in vivo study confirmed a significantly (P < 0.05) higher relative percentage survival in Octominin-treated larval zebrafish exposed to S. parauberis (93.3%) compared to the control group (20.0%). Collectively, our results confirm that Octominin could be a potential antibacterial and anti-biofilm agent against S. parauberis.

Introduction

Antimicrobial peptides (AMPs) are defense molecules produced by a variety of microbes, plants, and animals, which act against pathogenic viruses, bacteria, fungi, and parasites. In addition, most AMPs have multiple functions, such as anticancer, anti-inflammatory, and immunomodulatory actions [1]. AMPs can act against pathogenic microbes alone, or synergistically with other antimicrobials, showing different modes of actions, including morphological changes, membrane permeability alteration, reactive oxygen species (ROS) generation, antibiofilm activity, DNA damage, and inhibition of protein synthesis [2,3]. Even though pathogenic microbes develop resistance toward antibiotics rapidly, resistance development for AMPs is limited due to their multiple modes of actions and inhibitory activity against multiple classes of pathogens [4]. AMPs are either natural or synthetic; they differ from each other with respect to amino acid sequence, molecular weight, secondary and tertiary structure, yet almost all AMPs share common characteristics, including net charge, higher hydrophobicity, and amphipathic nature [5]. Because of these specific characteristics, AMPs exhibit a relatively low toxicity toward the host, and subsequently produce specific and efficient antimicrobial actions [6]. Although current findings showed that AMPs exhibit a broad spectrum of antimicrobial activity, most AMPs have only been tested against gram-negative bacteria, and limited data are available for those that control gram-positive infections [7]. Therefore, there is an urgent need to discover or synthesize AMPs that control infections caused by gram-positive bacteria and derive their mechanisms of action.

Streptococcosis is a common bacterial infective disease in fish presenting with hemorrhagic septicemia, meningitis, abdominal distention, etc. S. parauberis, which was introduced by Williams and Collins (1990), is one of the major causative bacteria for streptococcosis, and is classified as S. uberis genotype II. S. parauberis is a gram-positive, non-motile, alpha-hemolytic, cocci bacterium that mainly infects rainbow trout (Oncorhynchus mykiss) and olive flounder (Paralichthys olivaceus) [8]. In a number of previous studies, S. parauberis was identified as an antibiotic resistant bacterium. The S. parauberis strain, which was found in an olive flounder from a fish farm in Republic of Korea, had high resistance to multiple antibiotics, including amikacin, ciprofloxacin, and kanamycin [9]. Moreover, S. parauberis is capable of forming biofilms, which can be considered as one of its major defense mechanisms against commercially available antibiotics, and promotes the development of antibiotic resistance [10]. Therefore, a promising novel drug candidate is essential for the control of S. parauberis infections.

Since invertebrates lack an adaptive immunity, AMPs play a significant role in their innate immunity to protect against pathogenic microorganisms. Several studies have been conducted on invertebrates including, Cornu aspersum [11], Helix lucorum [12], and Rapana venosa [13], for their self-synthesized AMPs. In contrast, marine invertebrates, such as tunicates (e.g., Halocynthia aurantium), crustaceans (e.g., Homarus americanus), Chelicerates (e.g., Tachypleus tridentatu), and mollusks (e.g., Mytilus edulis) have become a novel source for a number of AMPs [14]. Very recently, Nikapitiya et al. [15] synthesized an AMP named Octominin, which is based on the defense protein of Octopus minor; it showed an antifungal activity against Candida albicans. Moreover, a recent study showed that Octominin had an inhibitory effect on lipopolysaccharide (LPS)-induced chemokine and pro-inflammatory cytokine secretion [16]. Since Octominin contained the basic characteristics of AMPs, in this study, we tested its antibacterial activity against S. parauberis. Initially, we tested the antibiotic susceptibility of S. parauberis, antimicrobial activity of Octominin, and Octominin's possible modes of action, including morphological and structural alterations, membrane permeability changes, ROS generation, DNA binding capacity, anti-biofilm activity, and transcriptional changes of selected genes in S. parauberis. Finally, efficiency of Octominin in controlling an S. parauberis infection was investigated by in vivo, using zebrafish larvae.

Section snippets

Determination of the antibiotic resistance profile of S. parauberis

S. parauberis (lab strain) was cultured using tryptic soy agar (TSA)/broth (TSB) (Becton, Dickinson and Company, USA) containing 2% sodium chloride, at 28 °C. To investigate antibiotic resistance, we selected 19 different antibiotics belonging to 13 antibiotic classes, and performed the disc diffusion assay, according to the method described by Nikapitiya et al. [17]. Briefly, S. parauberis was spread by swabbing on TSA plates, and antibiotic discs (6 mm) (BD BBL™ Sensi-Disc™, Becton, Dickinson

Antibiotic resistance profile of S. parauberis

To detect the possible drug resistance of S. parauberis, antibiotic susceptibility tests were conducted using 19 antibiotics. It was shown that S. parauberis has developed resistance for 11 (57.8%) selected antibiotics including streptomycin, gentamycin, amikacin, tetracycline, vancomycin, erythromycin, tobramycin, penicillin, ampicillin, clindamycin, and sulfamethoxazole/trimethoprim (Table 2). Among them, four antibiotics (streptomycin, erythromycin, penicillin, and ampicillin) had the

Discussion

AMPs are successful alternatives to antibiotics, and provide a promising solution for controlling MDR pathogens, by disturbing their multiple physiologic and metabolic processes. However, resistance development for some AMPs has been reported recently [7]. Gram-positive bacteria are coated with an acidic polymer of teichoic acid associated with a peptidoglycan layer, which forms a net negative charge on the outer surface. In addition, it acts as a major defense against environmental threats [23

CRediT authorship contribution statement

E.H.T. Thulshan Jayathilaka: Methodology, Investigation, Formal analysis, Writing - original draft, Writing - review & editing. T.D. Liyanage: Investigation, Formal analysis, Writing - review & editing. D.C. Rajapaksha: Methodology, Investigation, Formal analysis, Writing - review & editing. S.H.S. Dananjaya: Investigation, Formal analysis. Chamilani Nikapitiya: Methodology, Supervision, Writing - review & editing. Ilson Whang: Conceptualization, Funding acquisition, Project administration,

Declaration of competing interest

The authors declared that no conflicts of interest.

Acknowledgments

This work was supported by the Research Program of the National Marine Biodiversity Institute of Korea (MABIK2021M00600), funded by the Ministry of Oceans and Fisheries, and the National Research Foundation of Korea (NRF) grants, funded by the Korean government (MSIT) (2019R1A2C1087028 and 2018R1A2B6007841).

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