1 Introduction

Fisheries and aquaculture products worldwide are important sources of high-quality aquatic animal proteins and good sources of income, foreign exchange, and employment. About 950 million people worldwide rely on fisheries and aquaculture directly or indirectly for their livelihoods. Globally, the consumption of fish and fishery products as a protein source has increased considerably over the years, constituting about 20% of total protein (FAO, 2020). The aquaculture industry grows at a fast rate when compared to all other animal food-producing sectors worldwide, with the world average annual growth rate of 8.8% /yr since 1970, compared with only 1.2% for capture fisheries and 2.8% for land-farmed animal production systems (FAO, 2020). However, as aquaculture production increases, aquaculture waste also increases, and this aquaculture wastewater harbours diverse pollutants, including pathogenic organisms, which are detrimental to public health when released into the environment, and this makes fish farmers treat aquaculture effluent with pesticides and antibiotics.

The use of antibiotics can result in drug-resistant strains of diseases, causing bacteria that can harm aquatic animal populations and consumers of aquaculture fish. There are also increasing concerns about foodborne hazards, such as chemicals and microbial contaminants, that might be present in fish. These concerns can also result in decreasing demand for farmed fish (Smallwood and Blaylock, 1991). In an approach to satisfy the growing demand for fish, food safety is a major factor to be considered since these animals can be routes for the transmission of various pathogens such as Salmonella spp., Vibrio spp., Aeromonas spp., Campylobacter spp., Shigella spp., Yersinia spp., Clostridium spp., Bacillus cereus, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and Plesiomonas shigelloides which have been responsible for numerous cases and outbreaks of food-borne diseases in humans worldwide (Cortes-Sanchez et al., 2021).

Plesiomonas shigelloides is an oxidase-positive, facultatively anaerobic, Gram-negative, motile, rod-shaped bacterium commonly found in aquatic environments (Jagger, 2010; Janda et al., 2016). Aquaculture-raised fish for commercial purposes appear to be strongly associated with the presence of P. shigelloides (Janda et al., 2016). Plesiomonas was a common pathogen in the gills, muscles and intestines of fish as well as in rearing waters and pond sediment (Pakingking et al., 2015). Many works of literature reported that Plesiomonas shigelloides caused diarrhoea/gastroenteritis in humans via water or fish contaminated with this pathogen (Gonzalez - Rey et al., 2000). Plesiomonads are mesophiles with growth temperatures ranging between 8 ℃~45 ℃ with (a) pH ranging between 4.5 and 9.0 (Janda et al., 2016). Several factors that encourage the growth of Plesiomonas shigelloides include(s) overcrowding, oxygen levels, temperature, and climatic conditions, as well as food sources (Jun et al., 2011). However, little or no information is documented on Plesiomonas shigelloides as a causal agent of enteritis in pond water and African catfish (C. gariepinus). Hence, this study aimed at evaluating the microbial loads, isolation and antimicrobial susceptibility of Plesiomonas shigelloides from experimental pond water and C. gariepinus.

2 Materials and Methods

2.1 Study area

This study was conducted at the Department of Fisheries and Aquaculture Technology’s Teaching and Research Farm, Olusegun Agagu University of Science and Technology, Okitipupa. Okitipupa is located in the Ondo State of Nigeria, and it is reported to have a geographical coordinate of 6º 27’ 25” N,4º 46’ 00” E.

2.2 Media preparation and sterilization of materials

All media (Inositol brilliant green agar, nutrient agar, Mueller Hinton agar, nutrient broth, blood agar and alkaline peptone water) were prepared according to the manufacturer's instruction; the media were weighed out accurately and dissolved in an appropriate volume of water. The prepared mixture was homogenized and sterilized by autoclaving at 121 ℃ for 15 min. All these media were allowed to cool after sterilization to about 45 ℃ before pouring into Petri dishes. Alkaline peptone water was used as an enrichment medium.

2.3 Sample collection and design

Experimental ponds (6) were randomly selected and used for this study. The experimental ponds were replicated twice and the pond water (aquaculture water) was collected from each experimental pond at 0, 2, 4, 6 and 8 weeks while fish tissues (gill, liver and intestine) were collected from each experimental pond at 0, 4 and 8 weeks. Three fish were sampled from each experimental pond every 4 weeks. The experimental design was completely randomized block design.

2.4 Water quality analysis

A water sample (50 mL) was taken at 25 cm below the water surface by using a Van Dom water sampler (Denmark) from each experimental pond. The water quality parameters such as pH, temperature and total dissolved solids were taken at 0, 2, 4, 6 and 8 weeks as described by Olaifa and Bello (2011).

2.5 Isolation of bacteria/ bacterial counts

One gram (1 g) of gills, intestine and liver sample of C. gariepinus were separately macerated and put into a sterile capped test tube containing 9 mL of sterilized alkaline peptone water and 1 ml of experimental pond water was dispensed into 9 mL of sterilized peptone water (Bello et al., 2012; Bello, 2014). The pond water was also enriched in alkaline peptone water for the isolation of the presumptive Plesiomonas shigelloides. Serial dilution was carried out and 0.1 mL each from 10^-4 and 10-⁵ dilution factor was dispensed into Petri dishes that were appropriately labelled and the molten sterilized medium was poured aseptically into a Petri dish. The plates were swirled gently for even distribution of inoculums and allowed to set/gel and then incubated at 37 ℃ for 24 h. The organism grew into visible different colonies after 24 h. Total viable counts were determined and the results were expressed in log₁₀ CFU/mL for pond water and log₁₀ CFU/g for fish tissues. Also, 3~5 colonies of presumptive P. shigelloides were picked, purified and stocked on nutrient agar slant for further study.

2.6 Identification of isolates

Identification of the isolates was based on the procedures described by Mohammed et al. (2026). After observing cultural growth indices, the positive culture was subjected to Gram staining to study staining properties and cellular morphology under a 100X objective of a light microscope. Mixed colonies and Gram-negative bacteria were subcultured on both broth and nutrient agar (Oxoid, UK) and further incubated aerobically for 24 h. Pure culture of single colony type from (both broth and) nutrient agar were transferred onto nutrient slant for a biochemical test including catalase, oxidase, urease test, motility test, indole reaction test and fermentative/oxidative tests, hemolysis on blood agar and Gram staining techniques as described by Quinn et al. (2002) and Medical Research Council (MRC, 2017).

2.7 Gram staining technique

Young growing cultures of 18~24 h of the test isolates were used to prepare smears on clean grease-free microscopic slides. This was done by first cleaning the glass slides with cotton wool soaked with ethanol. A distinct colony of the isolates was picked with a sterile wire loop and emulsified with distilled water to form a smear and fixed. The smear was then stained with aqueous crystal violet for 1 min and was rinsed off gently with water, 95% Lugol iodine was added. The smear was decolourized with acetone until there was no violet colour on the slide. This was then rinsed off gently with water again, counter-stained with safranin for about 30 seconds, and then rinsed with water. The slide was carefully dried and examined under an oil immersion microscope with a 100x objective (MRC, 2017).

2.8 Antibiotic susceptibility

The antibiotic susceptibility profile of presumptive P. shigelloides was determined by using the disc diffusion technique as described by Kirby-Bauer with some modified disc diffusion techniques using 12 antibiotic discs (Biotec Lab. the United Kingdom) corresponding to the drugs containing most used in the treatment of human and animal infections caused by bacteria. The antibiotic sensitivity results for presumptive P. shigelloides were interpreted using the recommended guidelines by the Clinical Laboratory Standard Institute (CLSI, 2020). The antibiotics include; Cotrimoxazole (COT) 25 μg, Cefuroxime (CRX) 30 μg, Tetracycline (TET) 10 μg, Gentamicin (GEN) 10 μg, Ceftazidime (CPZ) 30 μg, Chloramphenicol (CHL) 10 μg, Ceftriaxone (CTR) 30 μg, Ciprofloxacin (CPR) 5 μg, Cefotaxime (CTX) 30 μg, Vancomycin (VAN) 30 μg, Amikacin (AMK) 30 μg and Meropenem (MEM) 10 μg. An 18~24 h old culture of all the isolates was prepared, after which the standardized broth culture of the inoculum was used to inoculate solidified pre-sterilized Mueller Hinton Agar plates. The antibiotics disc containing a specific concentration of antibiotics was placed on the Mueller Hinton Agar using sterile forceps and incubated at 32 oC for 24 h. The diameter of zones of inhibition was measured in millimetres and interpreted using CLSI (2020) standard and classified as sensitive, intermediate sensitive and resistant (MRC, 2017).

2.9 Statistical analysis

Bacteriological characteristics and physiochemical analysis resulting from the experiment were subjected to one-way analysis of variance (ANOVA) using SPSS (Statistical Package for Social Sciences 2006 version 15.0). Duncan's multiple range test was used to compare differences among individual means.

3 Results

3.1 Water quality parameters of the experimental pond water

The physicochemical properties of the experimental pond water showed a pH range of 7.0~8.5, Temperature 21.3 ℃~33.0 ℃, and Total suspended solids 25.0~41.0 (mg/L), and there were significant differences (p < 0.05) among the treatments in pH, temperature and total dissolved solids (Table 1).

3.2 Bacteria counts of experimental pond water and fish tissues (gill, liver and intestine)

A total of 30 water samples from the experimental fish ponds were analyzed for total bacteria counts at 0, 2, 4, 6 and 8th week and 54 fish tissues (gills, intestine, liver) from C. gariepinus juveniles in all the experimental ponds were analyzed for total bacteria counts at 0, 4 and 8th week. Test for the presence of presumptive P. shigelloides revealed that the bacterium was present in C. gariepinus tissues and experimental pond water. The Plesiomonas count on C. gariepinus tissues (gills, liver and intestine) ranges between 6.4 to 7.0 log₁₀ CFU/g while the Plesiomonas count on experimental pond water ranges between 5.6 to 7.0 log₁₀ CFU/mL. There was no significant difference (p > 0.05) in the total bacteria observed in the gill, liver and intestine and experimental pond water among the experimental groups except for experimental pond water at the 6th week who recorded significant differences (p < 0.05) among the groups (Table 2).

3.3 Isolation of Plesiomonas shigelloides

A total of 250 isolates were obtained from both the aquaculture effluent (pond water) and fish tissues (gill, liver and intestine). Morphological identification was analyzed based on the shape, texture and colour of bacteria colonies on inositol brilliant bile green agar. The microscopic cell morphology analysis of the presumptive P. shigelloides however shows only 40 isolates obtained from the selective agar were round-ended, straight rod shapes which are motile (15 isolates from experimental pond water and 25 from fish tissues (Table 3 and Table 4).

3.4 Morphological and biochemical characteristics of isolates of experimental pond water and fish tissues

The 40 isolates further tested positive for oxidase, catalase, mannitol and citrate biochemical tests, they also tested negative for urease, methyl red and glucose biochemical tests (Table 5 and Table 6).

3.5 Antibiotic sensitivity test

The antibiotic sensitivity test for presumptive Plesiomonas shigelloides was interpreted using the recommended guidelines by the Clinical Laboratory Standard Institute (CLSI, 2020) and is shown in table 5. Presumptive Plesiomonas shigelloides that were observed showed 100% resistance to cefotaxime, and cefuroxime, which belongs to the antibiotic class of cephems, followed by meropenem 87.5%, which belong to the antibiotic class of Carbapenems, Ceftazidime 77.5%, which belong to the antibiotic class of cephems, Vancomycin 70% which belong to the antibiotic class of glycopeptides tetracycline 40% which belong to the antibiotic class of tetracycline, ceftriaxone 37.5% which belong to the antibiotic class of cephems, chloramphenicol 20% which belong to the antibiotic class of phenicols, ciprofloxacin 20% which belong to a class of fluoroquinolones, cotrimoxazole 17.5% which belong to a class of sulfonamides, gentamicin and amikacin 0% which belong to a class of aminoglycosides (Table 7).

3.6 Multiple antibiotic resistance phenotypes of Plesiomonas shigelloides

All presumptive P. shigelloides obtained from this study exhibited resistance to at least one antibiotic. Meanwhile, most of the isolates (85%) showed resistance to three (3) or more classes of antibiotics. Resistant to four (4) classes of antibiotics had the highest frequency of occurrence. Out of the seventeen isolates resisting the effect of four (4) classes of antibiotics, resistance to Tetracycline, Cephalosporins, Carbapenem and Glycopeptides (76.5%) was seen to be the highest compared to other phenotypes (Table 8).