Review of the Role of Probiotic and Herbal Supplements as Antibacterial, Antioxidant, and Immunomodulatory Against Aeromonas hydrophila )

: Aeromonas hydrophila ( A. hydrophila ) are classified as zoonotic and opportunistic bacteria. Natural immunostimulants such as herbs are biodegradable and safe for the environment and host health. There has been no systematic review of using these natural ingredients to treat or prevent A. hydrophila infection. This review aims to demonstrate the role of herbs and probiotics as antibacterial, antioxidant, and immunostimulatory against A. hydrophila infection. This study deployed systematic and comprehensive literature from PubMed, Google Scholar, and Scopus from 2012 to 2022, with keywords: A. hydrophila , herbal, probiotics, antioxidant, immunomodulator, antibacterial. Data are obtained from journals, proceeding of research results, excluding article reviews. The review results indicate that the utilization of these two ingredients (herbs and probiotics) is potential to prevent A. hydrophila infection in animal models with variation action mechanisms and dose administration. However, an inappropriate combination of herbs and probiotics leads to the development of resistance and toxicity profile, thereby requiring further research to standardize such combination prior to application for humans or animals.

Vaccination and antibiotics have the potential to control A. hydrophila infection (Abdel-Tawwab et al., 2018). Antibiotics effectively kill bacteria, despite counterindicating harmful effects on biotics in waters and humans Abdelkhalek et al. (2020) due to reducing the host's immune system (Ahmadifar et al., 2019). In addition, consuming fishery products containing residues presents harm Devi et al. (2019), as it is undegradable, triggering pathogenic bacteria in the environment. The application of natural products is urgently required as an alternative to minimize the administration of chemical drugs to prevent bacterial resistance, improve the host immune response and reduce free radicals (Dawood et al., 2020). However, the effective administration of vaccines for prevention against certain bacteria must be in the form of a boosted dose (Hardi et al., 2016).
Probiotics and herbs serve to prevent or treat disease. Herbs act as prophylaxis by preventing the early emergence of clinical symptoms to prevent disease proliferation (Adeniyi et al., 2020). However, prior reviews that systematically collect data for the treatment or prevention of A. hydrophila infection by herbs and probiotics have been limited. Hence, this review aims to demonstrate the importance of probiotic and herb administration to prevent A. hydrophila infection along with providing their properties as health supplements.

Method
Article searches were generated from sources such as PubMed, Science Direct, Springer Link, and Google Scholar in 2012-2022. The keyword searches (in English) included Aeromonas hydrophila, herbs, probiotics, antioxidant, immunomodulatory, and antibacterial. The obtained articles were in the form of title and abstract only or full text. Article search criteria were based on PICO (Population, Intervention, Comparison, Outcome), in which Population: studies in animal model (fish and rodent); Intervention: therapy with herbs and probiotics (lactic acid bacteria (LAB) or yeast); Comparison: herbal and/or LAB/yeast vs. controls; Outcome: Antibacterial, antioxidant, and immunomodulatory effects. Inclusion criteria included herbs or probiotics to control in English. Exclusion criteria were as follows: bacteria or yeast strain not acting as probiotics. Antibacterial tests, such as MIC (minimum inhibition concentration) and MBC (minimum bactericidal inhibition), were excluded.

Herbs
Most herbal plants that act as health supplements contain essential oils and metabolic secunder. In this review, the leaves and roots of herbs were examined. The herbs were prepared in whole extract, nanoparticles and nanoencapsulation to accommodate intestinal absorption. In this review, the administration of herbs against A. hydrophila was more allocated to fish than to rodents. The application of the herbs was orally administered at a dose of 50 mg -0.75 g/kg in each feeding while bathing at 5-40 mg/L water for 2-12 weeks (see Table 1). Herbal nanocapsules require lower doses and shorter applications than those in herbal extract preparations. The administration of herbs can be single or in combination. In experimental fish, herbal plants were evident to trigger the growth of fish. The administration of herbs was mainly prophylactic, not therapeutic.

Antimicrobial activity and application
The results of a research review indicated that the antibacterial properties of herbal plants against A. hydrophila were based on bactericidal serum tests. The seaweed (Gracilariopsis persica) carbohydrate fraction contains antimicrobial activity against A. hydrophila due to the presence of sulfate molecules, such as carrageenan, fucans, and laminarin (Khosravi et al., 2018). However, A. hydrophila bacteria are resistant to multiple antibiotics (El-Adawy et al., 2018). Lessappropriate administration of antibiotics in cultured fish leads to rejection of overseas shipments due to the effects of antimicrobial residues found on human and aquatic organisms (Babu et al., 2016). A. hydrophila is classified as a gram-negative bacterium that is more resistant to essential oils than gram-positive bacteria because the protection of a thicker outer membrane (lipopolysaccharide) is hydrophobic (Bhargava et al., 2015). Antioxidant activity and application A. hydrophila bacteria contain a potential prooxidant compound in the liver through increased protein oxidation and inhibition of antioxidant enzymes, generating clinical symptoms (Baldissera et al., 2017). Plant bioactivity contains a therapeutic effect that improves health and physiological status (Alagawany et al., 2021). The review results of prior studies reported that antioxidant activity was navigated based on free radical product tests, MDA (malondialdehyde) and NO (nitric oxide) and antioxidant enzymes such as SOD, CAT and GPx. Curcumin boosts growth and modulates innate immunity as well as anti-inflammatory cytokines such as IL-10 but decreases the expression of proinflammatory genes in Ctenopharyngodon idella infected with A. hydrophila. The optimal dose of curcumin is 438.20 mg/kg diet (Ming et al., 2020).
Antioxidant enzymes inhibit the production of free radicals by removing their precursors (Dawood et al., 2020). Carotenoids serve as a part of antioxidant systems that work to protect against oxidative damage through two mechanisms, including (1) attenuating singlet oxygen and (2) scavenging free radicals Anbazahan et al. (2014), thereby preventing damage to cellular lipids, proteins, DNA, and polysaccharides. Hence, balancing free radicals and antioxidants is deemed pivotal to maintain physiological functions by adding exogen antioxidants (Tanvir et al., 2017).

Immunomodulator and application
The immunomodulator test was based on the review results of articles focusing on phagocytic activity, such as NBT, to determine the production of superoxide ions, lysozyme activity, respiratory burst, complement activity, leucocyte count and lymphocyte count as an innate immune response by preventing adhesion and colonization of bacteria hindering disease (Adel et al., 2016). Macrophages and neutrophils exhibit respiratory burst activity by producing free radicals such as superoxide anions, hydrogen peroxide, and NO in the phagocytosis process (Adel et al., 2016). Nitric oxide is classified as a highly reactive free radical generating pathological reactions such as damage to the immune system and physiological cells (Abdel-Tawwab et al., 2018).
In contrast, the adaptive immune response test was performed based on IgM and IgA measurements. Grass carp and Nile tilapia fed pellets containing 2 g kg -1 Lycium barbarum have the potential to improve total immunoglobulin, bactericidal activity and anti-protease activity when challenged with A. hydrophila bacteria (Mo et al., 2016). However, excessive administration leads to adverse effects on health (Ming et al., 2020). However, a dose of 2 g kg -1 resulted in significant weight loss of grass carp and Nile tilapia fish (Mo et al., 2016).

Probiotics
Probiotics consist of LAB (producing antimicrobial peptides such as enterocin, polysaccharides, and acids) and yeast containing glucans and mannanoligosaccharides, peptides, nucleotides, cell solubles and vitamins (Abu-Elala et al., 2013). In this study, LAB were prepared in the form of microencapsulated LAB at a dose of 10 8 -10 11 CFU/g of diet, while yeast was administered at a dose of 10 gr yeast/kg of feed. Additionally, microbial administration was available in a single form or combination with herbs (synbiotics) (see Table 2), with an average feeding time of 7-90 days. Antioxidant testing was performed based on antioxidant enzymes by neutralizing free radicals (hydroxyl radical scavenging and DPPH scavenging activity). The immune response to herbs was observed based on nonspecific and specific immunity. The prepared strains (probiotic agents) included Lactobacillus, L. plantarum, L. fermentum, L. rhamnosus, L. acidophilus, and L. delbrueckii (Behbahani et al., 2019). Only a few yeasts are used as probiotics in commercial formulations, such as Saccharomyces cerevisiae, S. boulardii, S. bayanus, S. florentius, S. pastorianus, S. sake and S. unisporus (Saber et al., 2019), due to their nonpathogenic nature, lack of plasmid-encoded antibiotic resistance genes, and resistance to bile and acidic pH (Abu-Elala et al., 2013).

Antimicrobial activity and application
The LAB applied in this study were classified as excellent probiotic candidates that were host-specific (Kuebutornye et al., 2020) and nonpathogenic, tolerating various extreme environments, growing, surviving and occupying the gastrointestinal tract of the host (Ramesh et al., 2015), and effortlessly adapted to compete with pathogenic bacteria for nutrients and space (Maeda et al., 2014). A sole administration of Lactococcus lactis (L. lactis) L19 or in combination with Enterococcus faecalis (E. faecalis) W24 isolated from the gut of Channa argus (C. argus) was evident to improve digestive enzyme activity, antioxidant enzyme activity and intestinal microbiota, thereby increasing the generation of probiotics in C. argus fish infected by A. hydrophila (Kong et al., 2021). Probiotic 3.5 × 10 9 CFU/g of feed (Bifidobacterium sp), 3.5 × 10 9 CFU/g of feed (L. acidophilus) and 3.5 × 10 9 CFU/g of feed (E. faecium) 0,3 g/kg of feed Reduced mortality, preserved gut microbiota modulation, weight gain + MOS (4 g/kg of feed) for 63 days L. plantarum L-137 and βglucan (BG) Dawood et al., (2020) β-glucan

Antioxidant activity and application
Probiotic strains could lower reactive radicals by stimulating intestinal saccharolytic metabolism and reduce stress-induced oxidation of doxorubicin . The mechanisms of action of probiotics as antioxidants are through direct neutralization of oxidants in the intestinal tract by antioxidant enzymes and stimulation of the immune system by decreasing oxidative stress-induced cytokines and inhibiting intestinal pathogens. Furthermore, probiotics could increase the absorption of micro-and macronutrients, including antioxidants (Kleniewska et al., 2016). A probiotic strain when heated could kill Pseudomonas aeruginosa VSG2 isolated from the intestines of Indian carp Labeo rohita, expressing antioxidant genes (SOD, GPx, and Cat) in C. carpio infected with A. hydrophila at a dose of 30 mg kg−1 for 8 weeks (Giri et al., 2020).

Immunomodulator and application
Live cell mixture and supernatant of Lactococcus lactis of Z-2 (5 × 10 8 cells mL -1 ) for 7 days decreased NO levels and pro-inflammatory cytokines but increased levels of anti-inflammatory cytokines (IL-10, TGF-β) in carp . Yeast hydrolysate refers to yeast cells such as S. cerevisiae supplemented with acid enzymes or other hydrolysis methods containing more polysaccharides and amino acids. However, due to the peptide's smaller size, it is easy to digest, acting as an immunostimulant, compared to yeast culture. Administration of 1.5% yeast hydrolysate for 56 days could increase the expression of IL-10, SOD, CAT, and IgM, decreasing MDA levels postchallenge with A. hydrophila in largemouth bass fish. Yeast hydrolysate has a positive impact on growth (Gong et al., 2019).
Among the available probiotics, LAB are acknowledged to improve the immune system and antibacterial activity (Dong et al., 2018). Some of the most critical human intestinal probiotics include Lactobacillus and Bifidobacterium as anti-inflammatory, immunomodulator and antibacterial agents (James & Wang, 2019). Lactobacillus produces antibacterial agents such as bacteriocin and galactosidase enzymes (Behera et al., 2020).
Synbiotics consist of an incorporated mixture of probiotic and prebiotic agents that provide a synergistic effect on the host to trigger metabolism and to improve microbial biota in the digestive tract (Devi et al., 2019) and in the immune system through interaction with the host's pattern recognition receptors. However, factors including the dose, time of administration, composition, and type of probiotics are required for supplementation considerations dealing with various body responses (Cavalcante et al., 2020). The combination of prebiotics and probiotics must be synergistic and appropriate based on the mechanism of action, dose, and duration, as an inappropriate combination leads to problems in animal physiology and microbial flora diversity. The combination of Bacillus subtilis + inulin (10 7 CFU/g + 10 g/kg) in the diet for four weeks caused inflammation, decreasing the gut microbiome in the gut of gilthead seabream (Sparus aurata L.) infected A. hydrophila.

Conclusion
This review serves as a scientific basis regarding the administration of herbs and probiotics to develop new drugs against A. hydrophila infection. Significant findings from the review of research results indicate that herbs and probiotics are evidently beneficial as antibacterial, antioxidant and immunomodulatory agents and drugs. Hence, this study suggests the preparation of nanoencapsulation as a drug rather than extracts due to longer availability when absorbed in the body. However, an inappropriate combination and dose leads to the development of resistance and the formation of free radicals, generating tissue damage. Thus, further research is encouraged to standardize the aforementioned compounds and to investigate the toxicity profiles prior to human and animal application.