Susceptibility of fish-associated Flavobacterium spp. isolates to cinnamaldehyde, vanillin and Kigelia africana fruit extracts

Phytochemicals are being explored as therapeutic alternatives in aquaculture since they have destressing, growth-promoting, appetite-increasing, immune-stimulating, and antimicrobial properties. The susceptibility of 28 Flavobacterium johnsoniae-like isolates and nine selected Flavobacterium spp. isolates to three phytochemicals, viz.: cinnamaldehyde (10 250 μg/ml), vanillin (5 500 μg/ml) and four crude Kigelia africana extracts (4 – 10 mg/ml ethyl acetate, dichloromethane, methanol and hexane), were assessed using disk diffusion assays and compared to standard antimicrobial agents, ampicillin and tetracycline using activity indices. Cinnamaldehyde (250 μg/ml) was more effective than 250 μg/ml vanillin, which was ineffective even at higher concentrations. K. africana extract (4 mg/ml) antibacterial efficacy decreased in the following order: Ethyl acetate, methanol, dichloromethane and hexane. The 10 mg/ml methanolic K. africana extract was most effective, with 100% of isolates displaying susceptibility, irrespective of the isolation source. Methanolic extract (10 mg/ml) activity indices ≥ 1 were obtained for 67.9 and 71.4% of isolates, respectively, relative to AMP10 and TE30. Cinnamaldehyde and the K. africana methanol extract are promising candidates to be tested for their efficacy in the treatment of Flavobacterium-associated fish infections. These phytochemicals might be environmentally-friendly, cost-effective alternatives to antimicrobial agent use in aquaculture, with a lesser potential of resistance development.


INTRODUCTION
Aquaculture is one of the fastest growing industries with ~80 million tons of farmed fish and shellfish being produced annually.Over-crowding and increased stress levels in aquaculture systems predispose fish to develop flavobacterial infections, leading to significant economic losses (Flemming et al., 2007;Rattanchaikunsopon and Phumkhachorn, 2009;Schrader, 2008).A number of Flavobacterium spp.(Gram-negative, oxidase-positive, yellow-pigmented, and non-fermenting rods with gliding motility) are pathogenic or regarded as opportunistic pathogens in a wide variety of organisms, including plants and fish.Flavobacterium columnare, F. psychrophilum, F. branchiophilum, F. aquatile and F. johnsoniae have been associated with fish disease and have also been detected in surrounding water in the presence of disease outbreaks.Flavobacterium johnsoniae-like isolates appear to be significant in the Southern African context as aquaculture pathogens (Flemming et al., 2007;Basson et al., 2008), being isolated predominantly from diseased cultured trout.Prevention of flavobacteria epizootics is hampered by the ubiquitous presence of Flavobacterium spp. in soils and waters and the identification of multidrug resistant strains (DeClerq et al., 2013), which makes control of flavobacterial disease outbreaks a significant challenge.F. johnsoniae isolates contain chromosomally located β-lactamase genes (Naas et al., 2003) and chloramphenicol-inducible resistance-nodulation-division family multidrug efflux pump system, FmeABC1 (Clark et al., 2009).
Thus the appropriate choice of effective antimicrobial agents for treatment of Flavobacterium spp. is challenging due to their decreased susceptibility to many antimicrobial agents (Clark et al., 2009).Although these infections may be resolved using synthetic antimicrobial agents, the dangers of multi-drug resistance, environmental pollution, residues in fish and the need for organic aquaculture has stimulated the search for bioactive materials from plants that have potent antimicrobial/anti-virulence activity (Chakraborty and Hancz, 2011).
Phytochemicals are plant-derived compounds, which based on their chemical structure, can principally be categorized into alkaloids, flavonoids, pigments, phenolics, terpenoids, steroids and essential oils.Phytochemicals have multiple effects, with the antibacterial active components of plants being able to lyse the cell wall, block protein synthesis and DNA synthesis (Chakraborty and Hancz, 2011), while the anti-virulence components inhibit enzyme secretions and interfere with the signaling mechanisms of the quorum sensing pathway (Chenia, 2013;Packiavathy et al., 2012).Cinnamaldehyde or 3phenyl-2-propenal occurs naturally in the bark and leaves of cinnamon trees of the genus Cinnamomum.This potent aromatic compound, with GRAS status, demonstrates a broad spectrum of antimicrobial activity (Nuryastuti et al., 2009).Cinnamaldehyde acts by inhibiting the proton motive force, respiratory chain, electron transfer and substrate oxidation, resulting in uncoupling of oxidative phosphorylation, inhibition of active transport, loss of pool metabolites, and disruption of synthesis of DNA, RNA, proteins, lipids, and polysaccharides.
The resulting extensive leakage from bacterial cells or the exit of critical molecules and ions leads to cell death (Nuryastuti et al., 2009).
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a major component of natural vanilla, (a widely used flavoring material with GRAS status), obtained from the bean of the tropical orchid Vanilla planifolia (Kappachery et al., 2010).The mode of action of the phenylpropene phenolic aldehyde, vanillin, is not well understood, but it appears to be bacteriostatic.The inhibitory action of vanillin on E. coli, Lactobacillus plantarum and Listeria innocua cells was due to its ability to negatively affect cell membrane integrity, which resulted in a loss of the ion gradient, pH homeostasis and an inhibition of respiration (Fitzgerald et al., 2004).Kigelia africana (Lam.)Benth., commonly known as the sausage tree, of the Bignoniaceae family is found in South, Central and West Africa.This plant is used against dysentery, venereal diseases and as a topical application on wounds and abscesses in many African countries.The antimicrobial properties of K. africana leaves, fruits and stem-bark against Gramnegative and Gram-positive bacteria (Grace et al., 2002;Eldeen and Van Staden, 2007;Jeyachandran and Mahesh, 2007;Shai et al., 2008;Saini et al., 2009) and the anti-quorum sensing potential of K. africana fruit extracts (Chenia, 2013) have been investigated, giving credence to the use of this plant in traditional medicine.Plant-based antimicrobials provide a vast untapped source of natural antimicrobial agents with potential therapeutic use in fish culture practice because they may be used for the effective treatment of infectious fish diseases; enhancing fish health and food safety and quality while conserving the aquatic environment (Chakraborty and Hancz, 2011).Thus, screening and proper evaluation of phytochemicals in medicinal plants that may be both sustainable and environmentally-acceptable, could offer possible alternatives to chemotherapeutic agents, reducing the side-effects of applying antimicrobial agents and the cost of therapy.Therefore, this study investigated the antimicrobial activity and efficacy of the phytochemicals cinnamaldehyde, vanillin and four crude K. africana fruit extracts on selected Flavobacterium spp.type strains, as well as 28 F. johnsoniae-like isolates from diverse South African aquaculture systems.
Activity indices of each extract were calculated by comparing zones of inhibition obtained with each of the extracts with those obtained with the standard antimicrobial agents, ampicillin and tetracycline.The following equation was used: Relative activity index (RAI) = Inhibition dia-meter (mm) with test extract/Inhibition diameter (mm) with standard antimicrobial agent (Jeyachandran and Mahesh, 2007).

Statistical analysis
The variations between experiments were estimated by standard deviations, and statistical significance of changes was estimated by one way repeated measures analysis of variance, with p ≤ 0.05% being regarded as statistically significant (SigmaStat V3.5, Systat Software, Inc, CA, USA).
Resistance was also observed to 5 and 25 μg/ml of vanillin (Tables 2 and 3), with no zones of inhibition being observed for both selected Flavobacterium spp.isolates as well as F. johnsoniae-like isolates.Although zone diameters ranged from 0 to 13 mm for Flavobacterium spp.isolates with 250 μg/ml of vanillin, only the F johnsoniae ATCC 17061 T isolate displayed intermediate resistance.
Based on zones of inhibition obtained with phytochemicals and standard antimicrobial agents, the RAIs were determined (Table 5).An extract was considered effective against an isolate if the RAI was ≥ 1 (Table 6).Ampicillin was regarded as a poor standard for comparison since 46.43% (13/28) of the study isolates exhibited resistance (Table 3).
The percentage of selected Flavobacterium spp.and F. johnsoniae-like isolates demonstrating RAIs ≥ 1 is indicated in Table 6.

Common treatments of
Flavobacteriumassociated infections include bath treatment with chemicals such as copper sulfate and potassium permanganate in response to epizootics or the inclusion of antimicrobial agents such as oxytetracycline and related compounds in fish feed (prophylactic treatment).However, their efficacy is impacted by water quality variables, they are highly phytotoxic, they have a broad-spectrum toxicity, consequently also killing beneficial organisms, and most importantly, the bioaccumulation of these compounds in the environment and fish tissue is problematic (Schrader, 2008).Although a number of antimicrobial agents are used as feed additives, prophylactically or therapeutically, the increasing incidence of drug-resistant pathogens, zoonoses, accumulation of antimicrobial agents in both fish and the environment and horizontal gene transfer of antimicrobial resistance determinants suggest the need for alternative, sustainable, cost-economic and environmentally-friendly therapeutic options (Chakraborty and Hancz, 2011).Phytochemicals are thus being investigated as alternative therapeutic options for the treatment of Flavobacterium-associated infections in aquaculture (Rattanchaikunsopon andPhumkhachorn, 2009, 2010;Seong Wei et al., 2009).Castro et al. (2008) observed that F. columnare was the microorganism most susceptible to majority of the 46 tested methanolic Brazilian plant extracts, in comparison to Aeromonas hydrophila and Streptococcus agalactiae isolates.
The majority of isolates in the present study were susceptible to tetracycline while displaying resistance to ampicillin, which was expected because of the presence of chromosomal β-lactamases (Naas et al., 2003).F. johnsoniae-like and selected Flavobacterium spp.isolates appeared to be resistant to 10 to 125 µg/ml cinnamaldehyde, although an increase in susceptibility was observed with 250 µg/ml cinnamaldehyde.This is in agreement with Chang et al. (2001) who observed that Cinnamomum osmophloeum essential oil extract B with 76% cinnamaldehyde had excellent antibacterial activity against diverse Gram-negative and Gram-positive bacteria at 250 to 1000 µg/ml concentrations.Ooi et al. (2006) also observed that cinnamaldehyde inhibited both Gramnegative and Gram-positive bacteria at concentrations from 75 to 600 µg/ml.Cinnamaldehyde appeared to work in a concentration-dependent manner, so concentrations >250 µg/ml could potentially demonstrate increased inhibitory activity against F. johnsoniae-like isolates.An added advantage is that cinnamaldehyde is a legally registered flavouring and foodstuff with international food safety organisations (Zhou et al., 2007), making its potential application in aquaculture more acceptable.
At 5 and 25 µg/ml, vanillin did not inhibit study isolates.
Exposure to 250 µg/ml of vanillin was not significantly inhibitory in the present study with 92.86% of isolates displaying resistance.Kappachery et al. (2010) and Ponnusamy et al. ( 2009) also observed that vanillin did not have an antimicrobial effect against A. hydrophila at concentrations ranging from 63 to 250 µg/ml but rather inhibited quorum sensing and biofilm development.Fitzgerald et al. (2004) observed that vanillin had a time of exposure, concentration and species-specific dependency to its antimicrobial activity.In this light, exposure to 400 to 500 µg/ml vanillin reduced resistance of isolates in the present study to 85.71%.Ripe or unripe K. africana fruits are often dried and powdered and applied directly or in topical preparations to treat a variety of skin, digestive and genito-urinary tract infections (Grace et al., 2002).A furanone derivative, eleven iridoids, 3b, 19a-dihydroxyurs-12-ene-28oic acid, caffeic acid, chlorgeric acid, and 6-p-coumaroyl-sucrose, together with a diverse group of phenylpropanoid and phenylethanoid derivatives and a flavonoid glycoside, have been isolated from the fruit (Saini et al., 2009).At 4 mg/ml, extract antibacterial efficacy decreased in the following order: ethyl acetate > methanol > dichloromethane > hexane.According to Grace et al. (2002), the antibacterial activity of K. africana fruits against Gram-positive and Gram-negative bacteria was due to a mixture of three fatty acids in an ethyl acetate fruit extract.In the present study, 4 mg/ml of the ethyl acetate extract (EX 1) moderately inhibited study isolates while the dichloromethane extract (EX 2) proved less effective against study isolates with resistance being displayed by 35.7% of isolates, while the hexane extract (EX 4) was the least effective.While K. africana methanol extract (EX 3) demonstrated limited inhibitory activity at 4 mg/ml, exposure to 10 mg/ml was most effective with 100% of isolates displaying susceptibility.This is in keeping with data from Agyare et al. ( 2013) who obtained MICs of 5.5 and 7.5 µg/ml following challenge of E. coli and Pseudomonas aeruginosa, respectively, with methanolic K. africana leaf and stem bark extracts.
A notable efficacy of the K. africana methanolic extract on Gram-negative bacteria has been previously reported (Eldeen and Van Staden, 2007;Jeyachandran and Mahesh, 2007;Shai et al., 2008).This might be a result of methanol being the best solvent for extraction since most phytochemical components are either polar or non-polar and methanol is thus able to solubilise the antibacterial components of medicinal plants (Jeyachandran and Mahesh, 2007).The antibacterial and antifungal activity of methanolic K. africana root and fruit extracts might be due to naphthoquinones, kigelinone, iso-pinnatal, dehydro-αlapachone, and lapachol, and the phenylpropanoids, pcoumaric acid and ferulic acid (Saini et al., 2009).The antimicrobial activity of the crude K. africana extracts is most likely the result of the synergistic action of the multiple bioactive compounds found within them.
The susceptibility of bacteria to any given antimicrobial agent may be dependent on the bacterial species or strain, extraction process or mode of action (Rattanachaikunsopon and Phumkhachorn, 2010).Strainand species-specific differences in susceptibility were observed for cinnamaldehyde, vanillin and K. africana extracts in this study.Chryseobacterium and Aeromonas spp.isolates also display differences in their responses to K. africana extracts as well as cinnamaldehyde and vanillin (unpublished data).
It appears that 10 mg/ml of the methanolic extract (EX 3) is an alternative to ampicillin and tetracycline as an antimicrobial agent against F. johnsoniae-like isolates, while 4 mg/ml of the ethyl acetate EX 1 was more effective than ampicillin.The present study, which is the first examining the efficacy of cinnamaldehyde, vanillin and K. africana extracts against Flavobacterium spp.isolates indicates that cinnamaldehyde and the K. africana ethyl acetate and methanol extracts are promising candidates to be tested for their efficacy in the treatment of Flavobacterium-associated fish infections.Dada et al. (2010) compared the effect of incorporation of 50 to 200 g K. africana fruit meal to one kg fish feed (five isonitrogenous diets) and observed positive effects on fecundity, hatching rate and percentage survival of catfish, Clarias gariepinus, following the use of 100 g dried K. africana fruit meal/kg feed.Thus, the extract has an important role as fertility enhancer, minimizing the use of synthetic fertility-enhancing drugs, in addition to its antimicrobial effect.The use on catfish indicates the nontoxic nature of the K. africana fruit (upto 200 g fruit meal/kg feed) against fish and highlights its potential beneficial application in an aquaculture setting.Further investigations will have to be carried out to ascertain the effects of antimicrobial agent synergy with K. africana phytochemical compounds and practical phytotherapy of Chenia and Singh 2063 infected fish with these phytochemicals.

Table 1 .
Zones of inhibition (mm) obtained with cinnamldehyde, vanillin and four crude Kigelia africana extracts as well as standard antimicrobial agents, ampicillin and tetracycline, against fish-associated F. johnsoniae-like isolates. *

Table 2 .
Susceptibility analysis of nine selected Flavobacterium spp.isolates to phytochemical extracts and standard antimicrobial agents.

Table 3 .
Susceptibility analysis of 28 F. johnsoniae-like isolates to phytochemical extracts and standard antimicrobial agents.