In vitro and in vivo exploration of palmitic acid from Synechococcus elongatus as an antibiofilm agent on the survival of Artemia franciscana against virulent vibrios

https://doi.org/10.1016/j.jip.2017.09.001Get rights and content

Highlights

  • Palmitic acid (PA) effectively promotes the loosening of biofilm architecture of vibrios.

  • Anti-biofilm potential of PA can be related to Quorum Sensing interruption.

  • PA enhanced the survival rate of vibrios challenged Artemia franciscana larvae.

  • PA could be exploited as anti-biofilm agent against seafood borne pathogens in aquaculture.

Abstract

Biofilm formation of Vibrio spp. has been demonstrated as a potentially important mechanism contributing antibiotic treatment failure in aquaculture. In the present study, the effect of palmitic acid (PA) identified from Synechococcus elongatus was assessed for the inhibition of quorum sensing (QS) regulated biofilm formation in aquatic bacterial pathogens. The biofilm inhibitory concentration (BIC) of PA against Vibrio spp. was found to be 100 µg ml−1. In this concentration, PA exhibited a significant inhibition in biofilm biomass of Vibrio harveyi MTCC 3438, V. parahaemolyticus ATCC 17802, V. vulnificus MTCC 1145 and V. alginolyticus ATCC 17749 without hindering their planktonic growth. Also, PA displayed gradual decrease in bioluminescence production of V. harveyi. The results of extracellular polymeric substances quantification, microbial adhesion to hydrocarbons and Fourier transform infrared spectroscopic (FT-IR) analyses suggested that PA positively interferes with the initial adhesion stages of biofilm formation. In addition, confocal and scanning electron microscopic analysis substantiates the antibiofilm efficacy of the PA. The transcriptomic analysis revealed the down-regulation of QS mediated response regulator genes expression in V. harveyi. Concomitantly, PA reduced the intestinal colonization of vibrios in brine shrimp larvae and thereby attenuates the biofilm assemblage and its associated virulence. In vivo studies using brine shrimp larvae manifested the reduction in adherence and virulence, which prompts further investigation about the potential of PA for the treatment of vibriosis.

Introduction

Vibriosis is one of the most prevalent diseases affecting a number of aquatic organisms including fishes, molluscs and crustaceans worldwide (Dubert et al., 2016). It is considered as a major hitch in aquaculture, which leads to severe economical and quality loss (Austin and Zhang, 2006). Vibrio species such as Vibrio harveyi, V. parahaemolyticus, V. vulnificus and V. alginolyticus are the major cause of vibriosis and are ubiquitous in marine environment globally (Longyant et al., 2008, Igbinosa and Okoh, 2008). V. harveyi, a bioluminescent bacterium is the most important etiological agent for the mass mortalities in Penaeus monodon and Litopenaeus vannamei shrimp culture systems. Equally, V. parahaemolyticus is a halophilic marine bacterium strewn in temperate water and often isolated from a range of seafoods including codfish, clam, octopus, shrimp, crab, lobster and oysters (Liston, 1990). It causes specifically early mortality syndrome in Penaeid shrimp (de Schryver et al., 2014) and acute gastroenteritis in humans via consumption of contaminated uncooked seafood (Matsumoto et al., 2000, Kaysner and DePaola, 2001). Similarly, V. vulnificus and V. alginolyticus are also well renowned to cause gastrointestinal infections in humans through the ingestion of uncooked seafood and could also cause wound infections in marine organisms (Liu et al., 2006). Consumption of raw or undercooked seafood, particularly shellfish, contaminated with vibriosis may lead to development of acute gastroenteritis characterized by diarrhoea, headache, vomiting, nausea, abdominal cramps and low fever in human (Kaysner and DePaola, 2001). Vibriosis is ranked as the foremost infectious cause of human mortality by Centre for Disease Control and Prevention.

In order to combat the consequences of Vibrio infections, the farmers rely on the usage of antibiotics (Swain et al., 2009). Nevertheless, due to the unsystematic and over dosage of antibiotics, vibrios are becoming resistant to several broad as well as wide spectrum antibiotics. Moreover, excessive antibiotic usage leads to the accumulation of toxic materials in the edible tissue of farmed fish and shellfish, causing long term adverse health effect to humans and other animals (Verschuere et al., 2000, Cabello, 2006). Vibrio spp. is known to possess a broad resistance to FDA approved common antibiotics, including chloramphenicol, tetracycline, cotrimoxazole, ampicillin, erythromycin and streptomycin (Soto-Rodriguez et al., 2006). Therefore, the ineffectiveness and failure of existing chemotherapeutic agents against vibriosis causing bacterial pathogens makes it vital to search for a safe and cost-effective alternative approach to combat multi-drug resistant Vibrio spp (Pande et al., 2013).

Several approaches are being followed to protect the aquaculture organisms from pathogenic bacteria as alternative measures to antibiotics. One such approach reported is the targeting of quorum sensing (QS) mechanism to control the pathogenic bacterial infections among aquatic organisms. So far, three different types of QS systems (LuxM/N, LuxS/PQ and CqsA/S) have been reported in Vibrio spp. QS is regulated by three cognate signaling molecules such as acylhomoserine lactones (AHLs), cholera-autoinducer-1 (CAI-1) and a mixture of molecules collectively called autoinducer-2 (AI-2) (Brackman et al., 2011). Further, there are three core enzymes involved in the synthesis of these molecules and synchronize the genes responsible for various important phenotypic characters such as bioluminescence, protease, hemolysin, chitinase, siderophore productions and most importantly biofilm formation (de Kievit and Iglewski, 2000, Whitehead et al., 2001, de Windt et al., 2003, Natrah et al., 2011). Bacterial biofilm is defined as bacterial cells encapsulated by the hydrated self-secreted extracellular polymeric matrix by which the bacterial cells aggregate and interact with each other forming stable, multi-cell clusters. Moreover, in Vibrio spp. QS mediated biofilm formation plays a vital role in causing multiple diseases to marine and brackish water animals (Randall et al., 2004, Yildiz and Visick, 2009). It has also been reported that bacterial cells present inside the biofilm often were 1000 fold resistant to antibiotic action, biocides and heavy metals than their free living planktonic counterparts (Brooun et al., 2000). Hence, it is worth to note that biofilm plays major path for more than 80% of bacterial infections. Several reports have already designated the biofilm as an important cause of persistence, survival and antibiotic resistance of Vibrio spp. in aquaculture systems. As a consequence, the biofilm shed of marine organisms could contaminate the whole farm and enhance the prevalence of vibriosis in the environment (You et al., 2007, Chen et al., 2010).

In recent years, several reports showed that marine cyanobacteria are capable of producing bioactive compounds with immense pharmaceutical applications. Although cyanobacteria are considered to be a proliferative source of bioactive compounds, the exploration of quorum sensing inhibitory (QSI) compounds from this natural resource is still in its infancy. Very few reports have renowned the discovery of QSI compounds from marine cyanobacteria that protect against infectious bacterial pathogens (Clark et al., 2008, Dobretsov et al., 2010). Recently, the QSI potential of S. elongatus extract was reported against Vibrio spp. and the major active molecule present in it was characterized as palmitic acid (PA) through HPLC and GC–MS analyses (Santhakumari et al., 2016). PA has been reported to possess various pharmacological properties such as anti-cancer, anti-inflammatory, analgesic, cytotoxic and apoptotic property to human cancer cells (Soni et al., 2008, Keawsa-ard et al., 2012). With this contextual, the current study aims to divulge the antibiofilm efficacy of PA from marine cyanobacterium S. elongatus against biofilm formation by vibrios and explicate its mode of action. The in vitro studies were further validated with in vivo approach using brine shrimp larvae (Artemia franciscana), a facile and widely used live marine animal model organism for host-pathogen interactions and toxicity studies in aquaculture.

Section snippets

Bacterial strains and culture conditions

Four strains of vibrios such as V. harveyi MTCC 3438, V. parahaemolyticus ATCC 17802, V. vulnificus MTCC 1145 and V. alginolyticus ATCC 17749 were grown in Zobell Marine Broth (ZMB) (pH 7.5 ± 0.2) at 30 °C. For experimental use, all these bacterial strains were subcultured in ZMB [(0.4 OD at 600 nm (OD600)].

Effect of PA on biofilm development

The stock solution (50 mg ml−1) of Palmitic acid (PA; Catalogue no. P0500-10G, Sigma-Aldrich, Switzerland) was prepared in absolute ethanol and stored at 4 °C till further use. The effect of PA

Determination of BIC

In quantitative analysis, the BIC of PA was examined against the test Vibrio strains. The obtained results clearly revealed a highest level of antibiofilm effect up to 64%, 63%, 78% and 71% in V. harveyi, V. parahaemolyticus, V. vulnificus and V. alginolyticus, respectively at 100 μg ml−1 of PA. As all the tested concentrations gave nearly comparable results, the lesser concentration of 100 μg ml−1 was fixed as the BIC (Fig. 1).

Interference with bioluminescence production of V. harveyi

Bioluminescence production is one of the important phenotypic factors

Discussion

In aquaculture, bacterial diseases remain as one of the major constraints due to lack of effective and benign treatment strategies. In order to establish a successful infection, vibrios needs to ascertain the major pathogenic factors such as biofilm, motility, cell adhesion, secretion of toxins and hydrolyzing enzymes (Milton, 2006). Therefore, targeting such factor production would constitute an interesting alternative control method to bacterial infections. One such type of modern microbial

Conclusion

Regulation of virulence and biofilm formation by QS is a multifaceted phenomenon which plays an important role in the colonization and pathogenesis of aquatic bacterial pathogens. The identification of natural molecules with the knack to inhibit biofilms affords an effective alternate approach to combating the virulence and resistance of these infectious pathogens. In this study, PA unveiled as strong antibiofilm compound against pathogenesis of Vibrio species. Moreover, inhibition of QS

Ethical standards

Authors declare that, in the current study there is no concern of experimental animals and/or human volunteer participants for research purpose.

Acknowledgements

The authors acknowledge the computational and bioinformatics facility provided by the Alagappa University, Bioinformatics Infrastructure Facility [funded by the Department of Biotechnology, Government of India Grant No. BT/BI/25/015/2012 (BIF)]. The technical assistance in CLSM analysis by Mr. S. Gowrishankar, Assistant Professor, Department of Biotechnology, Alagappa University is thankfully acknowledged. Financial assistance rendered to S. Santhakumari in the form of Senior Research

Competing interest

The authors declare that there is no competing interest.

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