INHIBITORY ACTIVITY OF LACTIC ACID BACTERIA ISOLATED FROM TILAPIA GUT AGAINST STREPTOCOCCUS AGALACTIAE

Tilapia culture offers cheap high-quality protein for the human population demand. However, with intensification, the cultured tilapia become threatened by Streptococcus infection, and antibiotic resistance emergence in aquaculture. Therefore, probiotics were investigated to be used as an alternative for antibiotics to treat Streptococcus infections. In the current study, probiotic lactic acid bacteria count ranged from 5.67 to 5.78 log 10 CFU/g of tilapia gut microflora. The conventional biochemical tests were carried out for the lactic acid bacteria isolates identification. Only seven native isolates which exhibited inhibitory properties against Streptococcus agalactiae with an inhibition zone 26-50 mm in diameter were selected for characterization. All probiotic isolates were negative for hemolysis and produced both amylase and lipase enzymes. The selected strains tolerated the fish's gastrointestinal acidic and bile conditions. The promising features of the isolated strains indicate that probiotic bacteria of aquatic origin can be considered a safe alternative for pathogen control.


INTRODUCTION
Tilapia culture is the second most important finfish culture industry in the world (Cai et al., 2019).Nowadays, tilapia culture is expanding all over the world, cultured in more than 170 countries worldwide (FAO, 2020).Tilapia has been engaged in the development of many rural locations, poverty improvement, malnutrition alleviation, and help in the improvement of the human health population.Altogether, tilapia directly plays a critical role in the World Sustainable Development Goals achievement (El-Sayed and Fitzsimmons, 2023).The global tilapia production was estimated at almost 7 million tons in 2020 and is expected to reach 7.3 million tons by 2030, providing a low-cost, highly nutritious protein source (FAO, 2020).Approximately, 70 species of tilapia have been identified in the world, however, Nile tilapia is the most cultured tilapia fish (Abdel-Ghany et al., 2019).Due to its amazing features including its improved growth rate, and tolerance to extreme environmental circumstances (El-Sayed, 2006), fish farmers tend to culture this fish in intensified systems, to benefit from high marketability and consumer preference (Abdallah and Ismail, 2016).
Despite such benefits, bacterial infection of tilapia culture is the main hurdle against the sustainability of this industry and is considered a major public health significance for consumers (Mohamed andSaleh, 2010, Elkamel et al., 2011).Streptococcus species is considered one of the main pathogenic agents causing disease in cultured tilapia called "Streptococcosis" (Taukhid et al., 2021).Streptococci are Gram-positive cocci, catalase-negative non motile organisms.Several species have been identified to cause Streptococcosis in tilapia, but Streptococcus agalactiae is the biggest threat to tilapia culture (Zhang et al., 2022).Streptococcus agalactiae disease of tilapia is highly infectious, and can be horizontally spread through food and infected fish, causing high mortality and wide prevalence (Eissa et al., 2021).So, it was considered to be one of the most dangerous pathogens threatening tilapia culture in the world (Shoemaker et al., 2017).
Antibiotics are the first prime choice in combating this disease.The repeated use of these drugs leads to antibiotic-resistant development (Fang et al., 2018).Aquatic Streptococcus agalactiae bacteria can acquire antibiotic resistance.
Recently, two Streptococcus agalactiae isolates in tilapia aquaculture were identified (Kelany et al., 2024), these pathogens were detected in ready-for-marketing tilapia fish, exhibited multiple antibiotic-resistance for most of the evaluated antibiotics and one of them was resistant to amoxicillin which considered drug of choice, with a consequence of human risk.So, there was a critical need to search for non-antibiotic antimicrobial agents to control these resistant pathogens.
Probiotics have been recommended as a safe new alternative method in aquaculture, and several beneficial probiotic microorganisms have been approved to be used as probiotics in aquaculture (Ringø, 2020, Ringø et al., 2022).Probiotics are non-pathogenic microbial cells or microbial cell products that, when administered, the farmed species` survival and growth rates are enhanced (Jahangiri and Esteban 2018).The currently used terrestrial probiotics in the market have limited application effects in aquaculture (Wanka et al., 2018).Because host-derived probiotics are more promising (Lara-Flores and Olvera-Novoa, 2013), Therefore, indigenous probiotics are more advantageous (Husain et al., 2022).Lactic acid bacteria are the most common probiotic bacteria used in aquaculture (Dawood et al., 2019).The natural presence of lactic acid bacteria in the fish gut makes them a suitable probiotic candidate for aquaculture (Muthukumar andKandeepan, 2015, Reda et al., 2018).The selection of probiotic bacteria with extracellular enzyme production ability was considered the main principle for the candidate probiotics because of improving nutrient utilization to support the host growth.The effective probiotic candidates should survive the high bile salt concentration and acidic pH of the fish's gastrointestinal tract conditions (de Melo Pereira et al., 2018).So, the present study aimed to identify autochthonous lactic acid bacteria from Nile tilapia gut and used as potential probiotics with antimicrobial activity against Streptococcus agalactiae.

Tilapia Fish collection and preparation
Forty-eight healthy Nile tilapia were collected from cultured tilapia through 4 visits to three tilapia aquacultures in Assiut and Minia Governorates (Table 1).The visited farms were subjected to a questionnaire on some management data and the owners agreed to share the information orally.Each fish was cleaned from dirt with sterile distilled water, their body scales` were removed, the skin was disinfected by alcohol (70%), the fish body was aseptically opened with sterile scissors, and one gram of intestine was homogenized with homogenizer and suspended in 9 mL of sterile 0.85% Na Cl then diluted in tenfold serial dilution (Saikot, 2016).

Enumeration of lactic acid bacteria
The probiotic bacteria were counted according to Reda et al. (2018).Briefly, one hundred microliters of 1/10 2 , 1/10 3 , and 1/10 4 suspensions were poured into and dispersed over de Man, Rogosa and Sharpe (MRS) agar medium plate in pH 5.5, each dilution was represented by two plates.The inoculated volume was evenly spread using a sterile bent glass rod then a layer of soft agar (40 o C) was added above the inoculated sample (agar overlay method) and cultured anaerobically at 37 • C for 48 hours.All the steps were done under sterile conditions.

Probiotic lactic acid bacteria isolation
Single round convex and creamy colonies on MRS agar were considered to be lactic acid bacteria (Kaktcham et al., 2017), and were picked up to trypticase agar slants for further identification.The selected isolates were confirmed as illustrated by Bergey's manual® of Systematic Bacteriology (Vos et al., 2011).

Phenotypic identification
Provisional or tentative identification of lactic acid bacteria was achieved by Gram staining, cell morphology under the microscope, catalase reaction, and gas production from glucose.

I. Gram Staining
Gram staining was done (Collins et al., 2004).The purple cell color indicates a positive Gram reaction.

II. Catalase test
For the catalase reaction, each isolate inoculum was transferred and mixed with the previously added two drops of hydrogen peroxide (3% H2O2) on a clean glass slide.The formation of bubbles indicates a positive reaction, proving that the organism produced the catalase enzyme that converts hydrogen peroxide into water and oxygen (Collins et al., 2004).

III. Glucose Fermentation test
The ability of isolates to ferment glucose and produce co2 gas and organic acids end product was determined.Inoculate the glucose purple broth with isolated organisms, then incubate inoculated tubes in addition to control non-inoculated tube aerobically at 37ºC, for one day anaerobically.Observe for appearance of a yellow color in the medium (Razin and Cirillo, 2012).
Only pure Gram-positive catalase-negative, glucose fermentative isolates were considered potential probiotic lactic acid bacteria.All bacterial isolates were stored at − 20 °C using MRS broth supplemented with 25% glycerol.

Testing of Antimicrobial Activity
The antagonistic activities of the probiotic lactic acid bacteria strains against multiple antibiotic-resistant two Streptococcus agalactiae isolates were assessed using the agar spot method as Shokryazdan et al. (2014).

Hemolytic activity
Hemolytic action was determined according to Semedo et al., (2003), through streaking the isolates on blood agar medium with 5% (v/v) sheep blood.After incubation for 48 hours at 37•C, the presence or absence of zones of clearing around the colonies was observed.

II. Amylase Assay
The isolated bacteria were inoculated on starch-agar plates, which were incubated at 37°C for 24 hours.Extracellular amylase activities were evaluated by clear zones that appeared around the colonies (Poletto et al., 2018).

Acid and bile tolerance
Isolate pH and bile tolerance were performed according to Taj et al. (2022).An in-vitro acidic condition of the fish gastrointestinal tract was created with MRS broth pH 2. The absorbance measured at 0, 2, 3 hours.Cultures grown in MRS broth pH 07 served as control.The tolerance was calculated using the following formula:

RESULTS
The total number of probiotic lactic acid bacteria of tilapia gut was counted using serial dilution in MRS agar medium.The log10 value of the mean count of CFU/gm of lactic acid bacteria from the tilapia gut was presented in Table 2.The mean count of CFU/gm was 5.67 ± 4.3 in farm A, 4.83 ± 4.59 in farm B, 5.43 ± 4.71 and 5.78 ± 4.3 in farm C. Statistically there were significant differences in the mean count of lactic acid bacteria between farm visits.
Typical lactic acid bacteria colonies in MRS agar were creamy white, small convex colonies.Out of eighty isolates, only seven isolates met the lactic acid bacteria phenotypic properties.These colonies were Gram-positive cocci-bacilli, catalasenegative, glucose fermentative, and nonmotile.
Antimicrobial activity is one of the potential probiotic properties.In this study, the isolated lactic acid bacteria strains showed different degrees of inhibitory activity against the tested strains (Table 3).All the evaluated seven lactic acid bacteria strains exerted antagonistic activity against the two Streptococcus agalactiae strains with a zone of inhibition ranging from 26-50 mm in diameter.Furthermore, all isolates showed no zone around the colonies on the blood agar medium, so they were considered safe.
All the tested seven lactic acid bacteria strains have the power to secrete lipase and amylase enzymes.
As presented in Table 4, the tested strains showed variable tolerance to the acidic pH.Six strains adapted to the acidic pH, while after 3 hours only five strains survived.The survival rate in pH 2 ranged from 11-39% and 12-35% after 2 and 3 hours, respectively.As shown in Table 4 four strains were able to tolerate bile for 2 hours, and after 4 hours only 2 strains could adopt the bile stress.Moreover, the tested strains tolerated 0.3% bile with a survival rate of 3-45% after 2 hours and 4 -37% after 4 hours.

DISCUSSION
Despite the progress in industrial aquaculture, there are some problems such as increasing incidence and prevalence of diseases that might lead to excessive use of antibiotics which lead to the occurrence of antibiotic resistance in aquatics (Larsson and Flach, 2022).Therefore, the search for new alternatives is now recommended.Due to the host-specific application effect of probiotics, the commonly used probiotics in the market are not recommended for aquatic use (Wanka et al., 2018).Therefore, native probiotics identified from the Nile tilapia intestine are greatly recommended.

Probiotic lactic acid bacteria Count
The total lactic acid bacteria in the tilapia intestine using MRS agar was counted.The alimentary tract of fish is highly populated by microbes which play a vital role in the fish's immune function (Denev et al., 2009).

Probiotic lactobacillus bacteria isolation
We identified seven lactic acid bacteria isolates for potential probiotic characterization.
Seven lactic acid bacteria isolates were selected based on the characteristics of Dowarah et al. (2018) andFečkaninová et al. (2019).Lactic acid strains in our study is nearly similar to that selected by Maji et al.
(2016) from the intestine of five freshwater fish, and much lower than Govindaraj et al., (2021), who selected 33 lactic acid bacteria isolates from 120 colonies on the basis of acid production during fermentation.

Antimicrobial activity
All the lactic acid bacteria strains (100%) inhibited the growth of the two Streptococcus agalactiae strains with zones of inhibition ranging from 20-50 mm.
Antibacterial activity against pathogens is one of the significant important characteristics for probiotic strain selection (Chauhan and Singh, 2019).Probiotic antagonistic activity of lactic acid bacteria mainly attributed to the antimicrobial compounds they secret including bacteriocins, hydrogen peroxide, organic acids, short-chain fatty acids which are responsible for their antimicrobial activity (Thirunavukkarasu et al., 2022).Compared to our result, 935 of the lactic acid bacteria isolated from fish and fish products exhibited antimicrobial activity against Streptococcus agalactiae using qualitative stab-on-agar test as assayed by Muñoz-Atienza et al. (2013), with an inhibition zones of >10 mm.Also, 54% of lactic acid bacteria strains identified by Siangpro et al. (2023) from the gastrointestinal tract of fifteen Climbing perch, five Nile tilapia, three Asian sea bass, two Striped snakehead, one Soldier river barb, and one Common carp exhibited inhibitory effects against Streptococcus agalactiae with inhibition zone ranging from7-9 mm.

Hemolytic activity
The Hemolytic activity is one of the virulence factors of the bacteria, facilitates the infection by microbial invasion to their host (Sperandio et al., 2010).Therefore, hemolysis analysis is very important as a safety prerequisite (FAO/WHO, 2002).The seven selected lactic acid bacteria isolates were negative for hemolytic activity (100%).Our strain‫﮲‬s non hemolytic activity is similar to that obtained by Govindaraj et al. (2021) from freshwater fish intestine none of the lactic acid bacteria isolates exhibited hemolytic activity in blood agar (100%), but higher than that identified by Coulibaly et al. (2022) 7% of lactic acid bacteria isolates were non hemolytic.

Enzymatic activity
All lactic acid strains have the power to produce extracellular lipases and amylase enzymes.Enzyme production is a valuable character in the probiotic bacteria as this bacteria is considered as safe and their enzymes have valuable effects for the host, as it help in food digestion with improvement in feed intake and weight (Peivasteh-Roudsari et al., 2020).These extracellular enzymes produced by the beneficial intestinal microflora were imperative for several natural processes such as feed digestion as well as for metabolism (Kesarcodi-Watson et al., 2008).Lipases in the digestive tract act on the carboxyl ester bonds present in triacylglycerols to liberate fatty acids and glycerol (Gupta et al., 2004), While, amylase is important for starch degradation (Huddy and Coyne, 2015).Compared to our results, four lactobacillus isolates from tilapia gut showed amylase activity were obtained by Reda et al., (2018).

Acid and bile tolerance
Tolerance to acid and bile is a principal character in candidate strains to be selected as probiotics.The tested strains tolerated the acidic and bile stress with variable degrees.Compered to our results, seven lactic acid bacteria isolates evaluated by Govindaraj et al., (2021) tolerated pH 2 with survival rate 45.1-84.4%.While, Muthukumar and Kandeepan, (2015) recorded that only 5 lactic acid bacteria isolates from freshwater fish gut tolerated 0.3% bile for 2 hours.

CONCLUSION
The concept of disease control using probiotics has received a widespread attention for their safety and no resistance develops.Seven lactic acid bacteria isolated from Nile tilapia gut could form a promising competent for controlling Streptococcus agalactiae.The present study confirms that Nile tilapia gut can be considered a source of probiotics for future use.Additionally, full identification and in vivo studies are required to determine its applications in the aquaculture environment.The obtained probiotic lactic acid bacteria can be used in the aquaculture as a feed additive in the aquaculture feed.Molecular identification of the identified isolates is required in further research to determine the type of bacteria to the species level.

Table 1 :
The three tilapia aquacultures investigated in the current study.

Table 3 :
Antimicrobial activity of the isolated Probiotic lactic acid bacteria against Streptococcus agalactiae (2 isolates)