Use Of Antibiotics In Aquaculture Research Articles

Identification of a Novel β-Defensin Gene in Gilthead Seabream (Sparus aurata)

The excessive use of antibiotics in aquaculture favors the natural selection of multidrug-resistant bacteria, and antimicrobial peptides (AMPs) could be a promising alternative to this problem. The most studied AMPs in teleost fish are piscidins, hepcidins, and β-defensins. In this work, we have found a new gene (defb2) encoding a type 2 β-defensin in the genome of gilthead seabream, a species chosen for its economic interest in aquaculture. Its open reading frame (192 bp) encodes a protein (71 amino acids) that undergoes proteolytic cleavage to obtain the functional mature peptide (42 amino acids). The genetic structure in three exons and two introns and the six characteristic cysteines are conserved as the main signature of this protein family. In the evolutionary analysis, synteny shows a preservation of chromosomal localization and the phylogenetic tree constructed exposes the differences between both types of β-defensin as well as the similarities between seabream and European seabass. In relation to its basal expression, β-defensin 2 is mostly expressed in the intestine, thymus, skin, and gonads of the gilthead seabream (Sparus aurata). In head kidney leucoytes (HKLs), the expression was very low and did not change significantly when stimulated with various immunocompetent agents. However, the expression was significantly down-regulated in the liver, head–kidney, and blood 4 h post-injection with the fish pathogen Vibrio harveyi. When infected with nodavirus, the expression was downregulated in brain at 7 days post-infection. These results denote a possible complementarity between the expression patterns of β-defensins and hepcidins. Further studies are needed to analyze gene duplications and expression patterns of β-defensins and describe their mechanism of action in seabream and other teleost fish.

A proteomics and transcriptome analysis provides insight into the molecular mechanisms of tea tree oil against Aeromonas hydrophila

Aeromonas hydrophila is a widespread fish pathogen. However, due to the extensive use of antibiotics in aquaculture, this bacterium has developed drug resistance. In recent years, tea tree oil, extracted from Melaleuca alternifolia, has gained widespread application in antibacterial research. Nevertheless, the underlying mechanism of action of tea tree oil on A. hydrophila remains unclear. In this study, the inhibitory effect of tea tree oil on A. hydrophila was analyzed, and its mechanism of action was elucidated through a combination of transcriptome and proteome analyses. In the current work, tea tree oil (0.039 mg/mL) significantly inhibited the growth of A. hydrophila and changed the permeability of the cell membrane. A total of 109 differentially expressed genes (DEGs) were identified by the combined analysis of the transcriptome and the proteome, of which 46 were upregulated and 63 were downregulated. These co-regulated DEGs were primarily enriched in metabolic pathways such as bacterial chemotaxis, pyrimidine metabolism, and the two-component system. Simultaneously, it was found that the target proteins of tea tree oil, such as secB, yajC, rpsP, rplL, and rpsT were significantly downregulated. These findings suggested that the potential of tea tree oil as an alternative to antibiotics in inhibiting the growth of A. hydrophila. It provides new insights into the molecular mechanisms underlying the antimicrobial activity of tea tree oil against A. hydrophila.

The CRISPR/Cas system as an antimicrobial resistance strategy in aquatic ecosystems.

With the growing population, demand for food has dramatically increased, and fisheries, including aquaculture, are expected to play an essential role in sustaining demand with adequate quantities of protein and essential vitamin supplements, employment generation, and GDP growth. Unfortunately, the incidence of emerging/re-emerging AMR pathogens annually occurs because of anthropogenic activities and the frequent use of antibiotics in aquaculture. These AMR pathogens include the WHO's top 6 prioritized ESKAPE pathogens (nosocomial pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), extended-spectrum beta lactases (ESBLs) and carbapenemase-producing E. coli, which pose major challenges to the biomagnification of both nonnative and native antibiotic-resistant bacteria in capture and cultured fishes. Although implementing the rational use of antibiotics represents a promising mitigation measure, this approach is practically impossible due to the lack of awareness among farmers about the interplay between antimicrobial use and the emergence of antimicrobial resistance (AMR). Nevertheless, to eradicate these 'superbugs,' CRISPR/Cas (clustered regularly interspersed short palindromic repeats/CRISPR associate protein) has turned out to be a novel approach owing to its ability to perform precise site-directed targeting/knockdown/reversal of specific antimicrobial resistance genes in vitro and to distinguish AMR-resistant bacteria from a plethora of commensal aquatic bacteria. Along with highlighting the importance of virulent multidrug resistance genes in bacteria, this article aims to provide a holistic picture of CRISPR/Cas9-mediated genome editing for combating antimicrobial-resistant bacteria isolated from various aquaculture and marine systems, as well as insights into different types of CRISPR/Cas systems, delivery methods, and challenges associated with developing CRISPR/Cas9 antimicrobial agents.

Antimicrobial Activity, Genetic Relatedness, and Safety Assessment of Potential Probiotic Lactic Acid Bacteria Isolated from a Rearing Tank of Rotifers (Brachionus plicatilis) Used as Live Feed in Fish Larviculture.

Aquaculture is a rapidly expanding agri-food industry that faces substantial economic losses due to infectious disease outbreaks, such as bacterial infections. These outbreaks cause disruptions and high mortalities at various stages of the rearing process, especially in the larval stages. Probiotic bacteria are emerging as promising and sustainable alternative or complementary strategies to vaccination and the use of antibiotics in aquaculture. In this study, potential probiotic candidates for larviculture were isolated from a rotifer-rearing tank used as the first live feed for turbot larvae. Two Lacticaseibacillus paracasei and two Lactiplantibacillus plantarum isolates were selected for further characterization due to their wide and strong antimicrobial activity against several ichthyopathogens, both Gram-positive and Gram-negative. An extensive in vitro safety assessment of these four isolates revealed the absence of harmful traits, such as acquired antimicrobial resistance and other virulence factors (i.e., hemolytic and gelatinase activities, bile salt deconjugation, and mucin degradation, as well as PCR detection of biogenic amine production). Moreover, Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) analyses unveiled their genetic relatedness, revealing two divergent clusters within each species. To our knowledge, this work reports for the first time the isolation and characterization of Lactic Acid Bacteria (LAB) with potential use as probiotics in aquaculture from rotifer-rearing tanks, which have the potential to optimize turbot larviculture and to introduce novel microbial management approaches for a sustainable aquaculture.

Intestinal Histopathological Aberrations in Oreochromis niloticus Juveniles upon Dietary Florfenicol Administration.

The aquaculture use of antibiotics can cause detrimental effects on fish organs and gut microbial dysbiosis. The impact of florfenicol (FFC) on fish intestinal histology, an approved antibiotic, remains unclear. This study aimed to investigate the effects of FFC on Oreochromis niloticus juveniles by administering FFC at 10mg and 30mg/kg biomass/day for 30 consecutive days to mimic long-term use. A dose-dependent reduction in feed intake, survival and biomass, with an upsurge in mortalities was observed. Even the therapeutic dose instigated mortalities on day 30 of FFC dosing (FD). Histopathological analysis revealed mild to moderate alterations, including loss of absorptive regions, epithelial degeneration, necrotized areas, intercellular enterocytic space and swollen laminar propria. Post-dosing, the observation of the detachment of lamina propria from the epithelium indicated imminent irritability. Goblet cells reduced drastically on day 30 FD, accompanied by an increase in intraepithelial lymphocytes. However, cessation of dosing for 13 days resulted in the reclamation of goblet cells and absorptive regions, indicating that the intestinal tissues underwent considerable repair after lifting antibiotic pressure. These findings suggested that O. niloticus can tolerate dietary FFC but emphasize the need for responsible use of antibiotics in aquaculture.

Чувствительность метода иммуномикрочиповой технологии при скрининговых исследованиях рыбы

There is currently growing concern about the presence of antibiotic residues in fish, which is an important food source for millions of people around the world. In light of this, screening methods for determining and quantifying antibiotic residues in fish are increasingly being investigated. Screening studies conducted in laboratories and research centers are of paramount importance in ensuring food safety by determining the presence and quantity of residual antibiotics. These studies help to form a complete picture of the state of the situation and identify potential problems associated with the use of antibiotics in aquaculture or fish processing.

Fungi and Actinobacteria: Alternative Probiotics for Sustainable Aquaculture

Aquatic animals are continually being exposed to stressors under farming conditions, increasing risks to gut health that result in dysbiosis. Since restrictions are imposed on the frequent use of antibiotics in aquaculture, there is emerging demand for economically viable, environmentally safe, and sustainable alternatives for the intensive production of aquaculture species. The application of beneficial microorganisms as probiotics has been suggested and widely practiced in recent times. Bacteria and fungi are ubiquitous microorganisms that can grow in various environments where organic substrates are available. Being rich in nutrients, a fish’s aquatic environment and gastrointestinal tract confer a favorable culture milieu for the microorganisms. However, the colonization and probiotic potential of fungi and mycelial bacteria resembling fungi (actinobacteria), either in the culture environment or within the gastrointestinal tracts of fish, have received less emphasis. Apart from bacilli and lactic acid bacteria, as the most conventionally used probiotics in aquaculture, numerous studies have focused on other promising alternatives. Diverse species of yeasts and molds belonging to the kingdom ‘Fungi’ have been characterized for their prospective roles in nutrition, immunomodulation, and disease prevention in fish. Bioactive compounds such as manno-oligosaccharides and β-glucans are recognized as fungal postbiotics that improve innate immunity and disease resistance in fish. Actinobacteria are known to possess different hydrolytic enzymes and novel secondary metabolites representing their probiotic attributes. The application of these groups in water quality amelioration has also been explored. Thus, this paper presents an overview of the present status of knowledge pertaining to the effects of yeasts (Candida, Cryptococcus, Debaryomyces, Geotrichum, Leucosporidium, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Sporidiobolus, Sporobolomyces, Trichosporon and Yarrowialipolytica), molds (Aspergillus spp.) and actinobacteria (Streptomyces) as probiotics in finfish aquaculture, as well as their occurrence within the gastrointestinal tracts of finfish. Furthermore, probiotic mechanisms, selection criteria, and future perspectives on using fungi and actinobacteria as promising probiotics are discussed.

Detection of multidrug resistant Vibrio parahaemolyticus and anti-Vibrio Streptomyces sp. MUM 178J

The growing global population has increased the demand for seafood, making the aquaculture industry a vital food source. However, climate change has negatively impacted the industry as natural inhabitants of aquatic environments such as Vibrio parahaemolyticus are thriving in the warming waters. These foodborne pathogens can cause disease in marine animals, resulting in lowered production rates and substantial economic losses. V. parahaemolyticus is transmitted from these marine animals in aquaculture to humans via raw or undercooked seafood, resulting in gastroenteritis outbreaks. In addition, the extensive use of antibiotics in aquaculture to treat infections within marine animals has led to the development of multidrug-resistant (MDR) V. parahaemolyticus, which threatens the effectiveness of antimicrobial treatments. Thus, it is vital to explore the alternatives of antibiotics in aquaculture to manage the spread of antibiotic resistance. This study aimed to investigate the prevalence of MDR V. parahaemolyticus to provide insight into the current antibiotic resistance patterns of this pathogen and to determine potential anti-Vibrio properties of Streptomyces MUM 178J which was previously isolated from the soils of a mangrove forest in Sarawak, Malaysia. Colony morphology and toxR-assay indicated that all samples tested positive for V. parahaemolyticus. Further identification with genomic analyses confirmed that 64% (64/100) of the isolates were V. parahaemolyticus. The multiple antibiotic resistance index (MAR) of the isolates ranged between 0.07 and 0.57, with 81% (52/64) of the isolates resistant to more than one antibiotic. The isolates were most resistant to ampicillin (98.4%), followed by ceftazidime (53.1%). In contrast, the isolates were highly susceptible to nalidixic acid (98.4%), tetracycline (93.8%), and sulfamethoxazole/trimethoprim (90.6%). They also demonstrated susceptibilities of 89.1% to levofloxacin, oxytetracycline, and gentamicin. Streptomyces sp. MUM 178J exhibited antagonistic effects on select V. parahaemolyticus strain, RP0132, with a minimum inhibitory concentration of 12.5 mg/mL and a minimum bactericidal concentration of 50 mg/mL.

Novel chitinolytic Bacillus spp. increase feed efficiency, feed digestibility, and survivability to Vibrio anguillarum in European seabass fed with diets containing Hermetia illucens larvae meal

Insect meals (IM) are being pointed out as a promising sustainable protein source. However, chitin in IM may negatively interfere with fish performance and nutrient availability. Given that carnivorous fish have a low or inexistent ability to digest chitin, novel strategies are needed to overcome chitin-imposed bottlenecks. Carbohydrolytic sporeforming probiotics offer a competitive industrial approach, being able to resist the harsh feed manufacturing process, transport, storage, and animal's gastrointestinal tract, while being able to increase the digestibility of otherwise indigestible components. Aiming to increase IM chitin digestibility, two chitinolytic sporeforming fish isolates (Fish isolates 645 (FI645) and 658 (FI658)), closely related to Bacillus licheniformis, were isolated from European sea bass (ESB) gastrointestinal tract, and incorporated (individually or as a mixture) in ESB diets with high defatted Hermetia illucens larvae meal (HM) levels (30% inclusion). FI were evaluated regarding their effects on ESB growth performance and digestibility. The spores were able to maintain their viability throughout the 180 days of diet storage at room temperature. Dietary inclusion of FI645 led to higher chitin digestibility. Concomitantly, the digestibility of dry matter, protein, and energy increased, overall leading to higher feed efficiency and protein efficiency ratio. Dietary inclusion of FI645 led to increased plasma N-acetylglucosamine (GlcNAc) levels. This is the first evidence for diet-mediated modulation of plasma GlcNAc concentrations. Dietary inclusion of FI645 also increased the expressions of the genes coding for N-acetylglucosamine kinase (nagk) and GlcNAc phosphomutase (pgm3), key enzymes from the GlcNAc salvage pathway and the hexosamine biosynthetic pathway (HBP). On the other hand, no differences were found for the expression of N-acetyl-D-glucosamine-6-phosphate deacetylase (amdhd2), a precursor enzyme for GlcNAc redirection to glycolysis. These results hint that free GlcNAc resulting from increased chitin digestibility might be preferentially redirected to protein O-GlcNAcylation processes through HBP in fish cells. Additionally, when challenged with Vibrio anguillarum, and after being feed for one month with the same diets, D645 also increased ESB survival from 52.5% to 77.5%, when compared to the control. Together, these results establish the potential of using FI645 as a probiotic for enhancing chitin digestion and acting as a prophylactic agent for ESB protection against V. anguillarum infection. This strategy opens novel possibilities for using commodities, including high chitin levels in aquafeeds, and reducing the use of antibiotics in aquaculture.

Metabonomics analysis of microalga Scenedesmus obliquus under ciprofloxacin stress

The wide use of antibiotics in aquaculture has triggered global ecological security issue. Microalgal bioremediation is a promising strategy for antibiotics elimination due to carbon recovery, detoxification and various ecological advantages. However, a lack of understanding with respect to the corresponding regulation mechanism towards antibiotic stress may limit its practical applicability. The microalga Scenedesmus obliquus was shown to be capable of effectively eliminating ciprofloxacin (CIP), which is a common antibiotic used in aquaculture. However, the corresponding transcriptional alterations require further investigation and verification at the metabolomic level. Thus, this study uncovered the metabolomic profiles and detailed toxic and defense mechanisms towards CIP in S. obliquus using untargeted metabolomics. The enhanced oligosaccharide/polyol/lipid transport, up-regulation of carbohydrate and arachidonic acid metabolic pathways and increased energy production via EMP metabolism were observed as defense mechanisms of microalgal cells to xenobiotic CIP. The toxic metabolic responses included: (1) down-regulation of parts of mineral and organic transporters; (2) electrons competition between antibiotic and NAD during intracellular CIP degradation; and (3) suppressed expression of the hem gene in chlorophyll biosynthesis. This study describes the metabolic profile of microalgae during CIP elimination and reveals the key pathways from the perspective of metabolism, thereby providing information on the precise regulation of antibiotic bioremediation via microalgae.

Evaluation of Probiotic Efficacy of Bacillus subtilis RODK28110C3 Against Pathogenic Aeromonas hydrophila and Edwardsiella tarda Using In Vitro Studies and In Vivo Gnotobiotic Zebrafish Gut Model System.

The indiscriminate use of antibiotics in aquaculture has led to the emergence of resistance; hence, eco-friendly, host-specific alternatives to mitigate bacterial infections have become imminent. In this study, bacteria that could possibly serve as probiotics were isolated and evaluated for their efficacy with in vitro experiments and in vivo zebrafish gut model. One isolate from each of the 23 rohu fish (Labeo rohita) was shortlisted after preliminary screening of several isolates and tested for their ability to inhibit two important warm water bacterial fish pathogens, Aeromonas hydrophila, and Edwardsiella tarda. An isolate (RODK28110C3) that showed broad-spectrum inhibitory activity against a battery of different isolates of the two fish pathogens included in this study and maintained in our repository was selected for further characterization. The culture was identified phenotypically as Bacillus subtilis and confirmed by 16S rDNA sequencing. The isolate was able to hydrolyze fish feed constituents that include starch, protein, and cellulose. Further in vitro tests ensured that the potential isolate with probiotic attributes could tolerate different gut conditions, which included a range of pH, salinity, and varying concentrations of bile salt. Exposure of 4days post fertilization zebrafish embryos to the RFP-tagged isolate confirmed the colonization of B. subtilis in the gut of the zebrafish embryo, which is an important attribute of a probiotic. The isolate was able to inhibit both A. hydrophila and E. tarda in gnotobiotic zebrafish embryo in triplicate. The study demonstrates the probiotic characteristics of the B. subtilis isolated from L. rohita and its ability to inhibit A. hydrophila and E. tarda using in vitro conditions and in the zebrafish gut and could serve as an effective alternative to antibiotics in aquaculture.

Vibrio parahaemolyticus: Exploring its Incidence in Malaysia and the Potential of Streptomyces sp. as an Anti-Vibrio Agent

As the world recovers from the COVID-19 pandemic, concern remains for future potential outbreaks because of the persisting effects of climate change, including the proliferation of infectious diseases. The frequent isolation of Vibrio parahaemolyticus in the surrounding environment is of concern as it can cause infections in marine animals and transmitted to humans. V. parahaemolyticus is the leading cause of foodborne gastroenteritis worldwide. Malaysia is one of the top seafood consumers and this places us at a higher risk of exposure to V. parahaemolyticus infections. Over the years, this foodborne pathogen has been isolated from various sources in Malaysia, mainly from seafood such as shellfish, shrimps, and fish. To make matters worse, there has been an emergence of antibiotic-resistant V. parahaemolyticus worldwide, which is attributed to the uncontrolled use of antibiotics in aquaculture to prevent and treat vibriosis. Therefore, it is vital to utilize alternatives such as probiotics to control V. parahaemolyticus to prevent further propagation of antibiotic-resistant strains of the bacteria. A potential candidate for probiotics is Streptomyces sp., a class of filamentous, Gram-positive bacteria that produce a variety of bioactive compounds during their life cycle, which can be useful in drug discovery. The bioactive compounds produced by Streptomyces sp. have been proven to have microbiota-modulating and stimulatory effects on the host, enhancing immunity and providing protective effects against V. parahaemolyticus infections. With the application of Streptomyces sp. as probiotics in aquaculture, the efficacy of the available antibiotics can be preserved, and the further spread of antibiotic resistance in the environment can be reduced.

Evaluation of the in vivo and in vitro interleukin-12 p40 and p35 subunit response in yellowtail (Seriola quinqueradiata) to heat-killed Lactobacillus plantarum strain L-137 (HK L-137) supplementation, and immersion challenge with Lactococcus garvieae

Dietary supplementation of immunostimulants might be effective to reduce the economic losses due to infectious diseases and the use of antibiotics in aquaculture. To investigate the immune response of interleukin-12 (IL-12) in yellowtail Seriola quinqueradiata to heat-killed Lactobacillus plantarum strain L-137 (HK L-137), we performed a leukocyte culture, feeding trial with diets containing L-137 and an immersion challenge with Lactococcus garvieae. IL-12 (IL-12p70) is a heterodimeric cytokine composed of IL-12p35 and IL-12p40 subunits. In the yellowtail-leukocyte culture, HK L-137 treatment stimulated the mRNA expression of one IL12p35 subunit (p35a) and all IL12p40 subunits (p40a, p40b, and p40c) in a dose-dependent manner. Furthermore, mRNA expression of type-I helper (Th-1) cytokine (tumor necrosis factor α, TNF-α, and interferon γ, IFN-γ) was also stimulated by HK L-137. After 6 weeks of feeding yellowtails with diets containing 0, 20, and 100 ppm of HK L-137, the mRNA expression of p35a and p40b in the spleen leukocytes increased with the dietary concentration of HK L-137, and that of p40b, p40c, and ifng in the head kidney leukocytes were the highest in the 20 ppm HK L-137 group. Survival rates in the 20 ppm HK L-137 group after immersion challenge with L. garvieae were significantly higher than the control (0 ppm of HK L-137). The 100 ppm HK L-137 group did not significantly suppress mortality. HK L-137 showed immunostimulant activity by increasing the expression of il-12, tnfa, and ifng mRNA in both in vitro and in vivo tests in yellowtail. Our results suggest that dietary supplementation with 20 ppm HK L-137 is the most efficient dose for improving immunity in yellowtail. Furthermore, a high dose of HK L-137 and/or long-term feeding of a diet containing HK L-137 might suppress the immune response, which probably decreases the survival rate of fish. To maintain a high immune response in yellowtail, the optimal dietary concentration of HK L-137 and/or feeding regime should be investigated further.

Effects of Florfenicol on Intestinal Histology, Apoptosis and Gut Microbiota of Chinese Mitten Crab (Eriocheir sinensis).

Excessive use of antibiotics in aquaculture causes residues in aquatic animal products and harms human health. However, knowledge of florfenicol (FF) toxicology on gut health and microbiota and their resulting relationships in economic freshwater crustaceans is scarce. Here, we first investigated the influence of FF on the intestinal health of Chinese mitten crabs, and then explored the role of bacterial community in FF-induced intestinal antioxidation system and intestinal homeostasis dysbiosis. A total of 120 male crabs (48.5 ± 4.5 g) were experimentally treated in four different concentrations of FF (0, 0.5, 5 and 50 μg/L) for 14 days. Responses of antioxidant defenses and changes of gut microbiota were assessed in the intestine. Results revealed that FF exposure induced significant histological morphology variation. FF exposure also enhanced immune and apoptosis characteristics in the intestine after 7 days. Moreover, antioxidant enzyme catalase activities showed a similar pattern. The intestinal microbiota community was analyzed based on full-length 16S rRNA sequencing. Only the high concentration group showed a marked decrease in microbial diversity and change in its composition after 14 days of exposure. Relative abundance of beneficial genera increased on day 14. These findings illustrate that exposure to FF could cause intestinal dysfunction and gut microbiota dysbiosis in Chinese mitten crabs, which provides new insights into the relationship between gut health and gut microbiota in invertebrates following exposure to persistent antibiotics pollutants.

Occurrence, distribution, and ecological risks of antibiotics in Honghu Lake and surrounding aquaculture ponds, China.

Antibiotics are largely applied in aquaculture to increase production and control diseases, while how the antibiotics used in pond farming influence the distribution of antibiotics in receiving water seasonally is still not well understood. In this study, the variations of 15 frequently used antibiotics in Honghu Lake and surrounding ponds were investigated seasonally to figure out the impact of pond farming on antibiotics distributions in Honghu Lake. Results showed that the antibiotic concentrations in fish ponds ranged from 11.76 to 389.8ng/L, while in crab and crayfish ponds were lower than 30.49ng/L. The predominant antibiotic in fish ponds was florfenicol, followed by sulfonamides and quinolones, with generally low concentrations. Sulfonamides and florfenicol were the main antibiotics in Honghu Lake, affected by the surrounding aquaculture water partially. The antibiotics residue in aquaculture ponds showed obvious seasonal characteristics, with the lowest in spring. From summer, the concentrations of antibiotics in aquaculture ponds gradually increased and reached a peak in autumn, and the seasonal variation of antibiotics in the receiving lake was also related to the antibiotics in the aquaculture ponds. Risk assessment analysis showed that antibiotics such as enrofloxacin and florfenicol in fish ponds posed a medium and low risk to algae, and Honghu Lake acted as a natural reservoir of antibiotics and poses increased risks to algae. In general, our study demonstrated that aquaculture represented by pond farming brought significant risks of antibiotic pollution to natural water bodies. Therefore, reasonable control of the fish antibiotics usage in autumn and winter, as well as the rational use of antibiotics in aquaculture and the use of antibiotics before pond cleaning, is required to reduce the migration of antibiotics from aquaculture surface water to the receiving lake.

In Vitro Evaluation of Postbiotics Produced from Bacterial Isolates Obtained from Rainbow Trout and Nile Tilapia against the Pathogens Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida

The use of antibiotics in aquaculture leads to the proliferation of multidrug-resistant bacteria, and an urgent need for developing new alternatives to prevent and control disease has, thus, arisen. In this scenario, postbiotics represent a promising tool to achieve this purpose; thus, in this study, isolation and selection of bacteria to further produce and evaluate their postbiotics antibacterial activity against fish pathogens was executed. In this respect, bacterial isolates from rainbow trout and Nile tilapia were obtained and tested in vitro against Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida. From 369 obtained isolates, 69 were selected after initial evaluation. Afterwards, additional screening was carried out by spot-on-lawn assay to finally select twelve isolates; four were identified as Pediococcus acidilactici, seven as Weissella cibaria, and one as Weissella paramesenteroides by matrix assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS). Selected bacteria were used to obtain postbiotic products to test their antagonistic activity through coculture challenge and broth microdilution assays. The influence of incubation time prior to postbiotic production on antagonistic behavior was also recorded. Two isolates identified as W. cibaria were able to significantly reduce (p < 0.05) A. salmonicida subsp. salmonicida's growth in the coculture challenge up to 4.49 ± 0.05 Log CFU/mL, and even though the reduction in Y. ruckeri was not as effective, some inhibition on the pathogen's growth was reported; at the same time, most of the postbiotic products obtained showed more antibacterial activity when obtained from broth cultures incubated for 72 h. Based on the results obtained, the preliminary identification of the isolates that expressed the highest inhibitory activity was confirmed by partial sequencing as W. cibaria. Through our study, it can be concluded that postbiotics produced by these strains are useful to inhibit the growth of the pathogens and could, thereby, be applicable in further research to develop suitable tools as feed additives for disease control and prevention in aquaculture.

High occurrence of antibiotic resistance genes in intensive aquaculture of hybrid snakehead fish

The increasing abundance of antibiotic resistance genes (ARGs), which are regarded as new pollutants, has raised public health concern. The use of antibiotics in aquaculture has promoted the evolution and spread of ARGs. The occurrence and abundance of ARGs in aquaculture has attracted extensive attention. However, the distribution and transmission of ARGs in aquaculture require further study. This study analyzed water and sediment from intensive culture of hybrid snakehead fish farm in Zhongshan, South China. Twenty-two types of ARGs were detected in all environmental samples. The relative abundance of sulfonamide resistance genes (sul1 and sul2) was the highest, ranging from 3.37×10-2 to 8.53×100 copies/16S rRNA gene. High occurrence of quinolones, phenicols, tetracycline resistance genes, and class 1 integrase gene (intI1) was also observed in pond water samples. This implies that pond water is one of the main reservoirs and origins of ARGs in the aquatic environment. Proteobacteria was the most abundant phylum in all the environmental samples, and its relative abundance ranged from 24.05% to 41.84%. Network and canonical correspondence analyses showed that a high abundance of ARGs (int1, sul1, sul2, qacEΔ1, aac6, and oqxA) was positively correlated with Proteobacteria, Cyanobacteria, and Bacteroidetes, and the abundance of Proteobacteria, Actinobacteria, and Patescibacteria was positively correlated with environmental factors (sulfide, nitrite nitrogen, pH, free chlorine, and ammonia nitrogen). These findings demonstrate the prevalence and persistence of ARGs in intensive fish farming in southern China. This suggests that ARG levels and microbiological community composition in aquaculture should be conventionally determined to assess potential risks to public health.

Antimicrobial Activity of Eucalyptus (Eucalyptus camaldulensis) Essential Oil Against Fish Pathogen Bacterium, Aeromonas caviae

The intensive use of antibiotics in aquaculture has resulted in increased resistance among fish pathogens, and this situation has led researchers to investigate the antibacterial properties of natural resources. The present study focused on an essential oil isolated from the leaves of Eucalyptus camaldulensis as a potential antibacterial that could be used against Aeromonas caviae. Eighteen compounds were identified in the essential oil, representing 86.68 % of the total oil. The components were found to be p-cymene (20.09%), β-phellandrene (18.61%), α-phellandrene (7.50%), α-terpineol (6.02%), terpinen-4-ol (5.50%), Crypton (5.36%), spathulenol (4.26%), linalool (3.56%), 1,8-cineole (2.77%), farnesol (2.31%), Cumin aldehyde (2.13%), limonen (2.12%), α-thujene (1.94%), fellendral (1.13%), γ-terpinene (1.10%), sabinene (0.97%), α-pinene (0.68%) and α-terpinen (0.63%). The antibacterial efficiency of essential oils against Aeromonas caviae was determined using Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values, ranging from 200 µg/ml to 400 µg/ml respectively. Our findings revealed the potential of essential oils isolated from Eucalyptus (Eucalyptus camaldulensis) as a natural antibacterial agent that could efficiently contribute to the control of Aeromonas caviae infection in fish.

The bacterial flora of Oreochromis niloticus and Clarias gariepinus from earthen ponds in Sagana and Masinga, Kenya

Abstract. Karimi RD, Ngeranwa JJN, Njagi ENM, Kariuki S. 2022. The bacterial flora of Oreochromis niloticus and Clarias gariepinus from earthen ponds in Sagana and Masinga, Kenya. Intl J Bonorowo Wetlands 12: 63-73. Food-borne diseases traced to fish consumption have been reported globally, including in Kenya. The aspect of food quality as far as fish consumption is concerned is underestimated in Kenya though aquaculture has been promoted. The bacterial flora of Tilapia (Oreochromis niloticus Linnaeus, 1758) and Catfish (Clarias gariepinus Burchell, 1822) from Masinga Dam and earthen ponds at Sagana fish farm was determined in this study to determine the anti-microbial response of the pathogenic bacteria. Tilapia fish and Catfish samples were collected from Masinga Dam and Sagana farm in the dry and rainy seasons. The fish were skinned, and gut content was taken for laboratory tests. The water and water sediment samples from these two study sites were also collected. Those samples were processed and cultured in MacConkey agar, and the selective media were subcultured in the colonies and then subjected to morphological examination from cultures. Then, the biochemical tests were carried out using commercially available API kits. The study showed the presence of bacterial species belonging to Enterobacter spp. (n=34), Pseudomonas spp. (n=6), Aeromonas spp. (n=5), Vibrio spp. (n=3) and Acinetobacter spp. (n=2) isolates during the dry season, while bacterial species belonging to Enterobacter spp. (n=31), Pseudomonas spp. (n=6), Aeromonas spp. (n=4) isolates during the dry season. The anti-microbial susceptibility analysis showed that the highest resistance rates were found against Ampicillin (Amp) (61.5% of isolates), Amoxicillin (AmL) (65.9% of isolates), Tetracycline (Te) (31.8% of isolates), and Chloramphenicol (C) (27.5% of isolates) while the lowest was Nalidixic acid (Na), Cefuroxime (Cxm) and Streptomycin (S) at (4.4% of isolates) each. All isolates were sensitive to Gentamycin (Gen), Ciprofloxacin (Cip), and Cefotaxime (CTX). The presence of the above organisms, some potentially pathogenic to humans, indicates that improperly handled, undercooked, or consumed raw fish may cause disease in susceptible individuals. At the same time, some isolates’ anti-microbial resistance indicates that the use of antibiotics in aquaculture to promote growth should be studied further with a view to policy formulation.

THE USE OF ENVIRONMENTALLY-SAFE POLUMS IN AQUACULTURE

Introduction. In the conditions of anthropogenic pollution of water, environment-safe drugs and implementing of immunomodulatory drugs are becoming increasingly common. Schemes of their use in fisheries are being developed. The article presents and analyzes the potential use of biologically active additives (BAA), namely probiotics, prebiotics and yeasts, both domestically and internationally produced, in terms of their effect on the fish body.&#x0D; Materials and methods of research. Search for literature data on the use of biologically active substances in fish farming, namely probiotics, prebiotics and yeast.&#x0D; Research results. Considering the negative impact of prophylactic and therapeutic use of antibiotics in aquaculture, the use of dietary immunostimulants has been proposed as an alternative to antimicrobial drugs. In this sense, functional dietary supplements, including pre-, probiotics and yeasts, are receiving increasing attention as an environmental strategy to improve fish health.&#x0D; Probiotics are the objects of comprehensive scientific research and an important product on the world market. The use of probiotics as biocontrol agents in aquaculture is increasing. The benefits of such additives include increased nutritional value, inhibition of pathogens and enhanced immune response by increasing white blood cells and phagocytosis. They improve the quality of the growing environment, protect fish from biological hazards, and modulate physiological processes that ultimately contribute to the health and welfare of fish in aquaculture. Probiotics also enhance growth performance and feed utilization in aquatic animals by increasing the activity of digestive enzymes.&#x0D; The beneficial effects of prebiotics are due to by-products resulting from the fermentation of intestinal commensal bacteria. Among the many health benefits attributed to prebiotics is the modulation of the immune system. They directly enhance the innate immune response, including activation of phagocytosis, neutrophils, alternative complement system, and increased lysozyme activity.&#x0D; Another environmentally friendly product that has been proposed as a dietary supplement is yeasts. Research on yeast products in fish diets has focused on their role in nutritional and functional supplements that contribute to the immune responses and gut health of fish.&#x0D; Conclusions. Various studies of pro- and prebiotics in fish have shown the following results: effects on growth, gut microbiota, resistance to pathogenic bacteria and parameters of innate immunity such as alternative complement activity (ACH50), lysozyme activity, natural hemagglutination activity, respiratory burst, superoxide dismutase activity and phagocytic activity.All the above studies demonstrate that the addition of nutritional supplements to feed, such as immunostimulants, is an alternative method for the prevention and control of various diseases in aquaculture.