A Comparative Assessment of Bacteria Associated with Fried and Dry-Smoked Fish in Some Locations in Makurdi, Nigeria.

A Comparative Assessment of Bacteria Associated with Fried and Dry-Smoked Fish in Some Locations in Makurdi, Nigeria.

*Tyokusa, A. G., Hwande, N. S.

Microbiology Department, Joseph Sarwuan Tarka University (Formerly Federal University of Agriculture), Makurdi, Nigeria.

*Corresponding Author

DOI: https://doi.org/10.51584/IJRIAS.2024.905043

Received: 18 April 2024; Revised: 30 April 2024; Accepted: 04 May 2024; Published: 21 June 2024

ABSTRACT

Thirty-two samples of fried and smoked fish were collected from four different locations (Watada, High-level, North bank and Wurukum) in Makurdi. Serial dilutions were prepared and appropriate dilutions inoculated onto Nutrient and Salmonella/Shigella agar. The bacterial load of the samples was determined and the colony forming unit per gramme (cfu/g) of fried fish samples ranged from 2.0×10⁸ to 3.8×10⁸ cfu/g while that of dried fish samples ranged from 4.3×10⁸ to 7.8×10⁸ cfu/g indicating that the dried fish samples were more contaminated. A total of 9 bacterila species were isolated from the 32 samples which includes, E. coli, Staphylococcusaureus, Bacillus spp, Shigella spp, Salmonella spp, Klebsiella spp, Micrococcus spp, Listeriamonocytogenes, and Pseudomonas spp. The percentage (%) occurrence of the bacteria isolates indicates that E. coli, Staphylococcusaureus, and Bacillus spp, showed 100%, Micrococcus spp 43.75%, Klebseilla spp 31.25%, Shigella spp and Salmonella spp 21.88% each, Listeria monocytogenes 18.75%, and Psedomonas spp 12.5% percentage frequency respectively. Results from this study showed that, samples collected from Wadata were the most contaminated, followed by those from North Bank, then those from Wurukum and those from High Level had the least contamination. The antimicrobial susceptibility test on the bacterial isolates showed that, 3 of the isolates (Staphylococcus aureus, E. coli and Pseudomonas spp) were multi-drugs resistance. Staphylococcus aureus isolates were resistance to Penicillins and Glycopeptides as it showed 60.7%, 62.5%, and 25.5% resistance to Oxacillin, Penicillin G, and Vancomycin respectively.  E. coli and Pseudomonas spp isolates were both resistant to Tetracyclines and Penicillins. The resistance shown by E. coli isolates were 32.2% and 36.9% to Tetracycline and Ampicillin respectively while Pseudomonas spp isolates showed 54.5% and 79.3% to Tetracycline and Ampicillin respectively.

Key words: Fried fish, dried fish, bacterial load, antimicrobial, and susceptibility.

INTRODUCTION

Fish are aquatic animals that typically have paired fins, gills, a lengthy, scale-covered body, and are typically cold-blooded, some of which have no scales. Fish can be divided into three primary classes, groupings, or types: cartilaginous fish (Chondrichthyes), jawless fish (Agnatha), and bony fish (Osteichthyes).With over 33,000 different species, fish are the most varied group of vertebrates (Bales, 2023).Ichthyology is the branch of zoology devoted to the study of fish (Bond, 1999).

The flesh of fish is usually infected with a wide range of microbes present in the water body. While the precise source of the majority of microbes found in fish products remains unknown, some workers have linked microbial infections and/or contamination of fish and fish products to a variety of factors, including unfavourable fish system conditions, pollution, seasonal variations, and fish handling and processing, which frequently results in fish spoilage and nutritional deterioration (Mossel and Ingram,1995; Abolagbaet al., 2011).

Fish can be sold in various forms such as frozen fish, dried fish, smoked fish, fried fish, canned fish, pre-cooked fish, salt-cured fish, marinades fish, fermented fish products, and processed fish products such as fish balls ((NurSyahirah and Rozzamri, 2022).

In most Nigerian markets, fish are commonly sold in fried and dry-smoke form. Frying is considered one of the popular processes used in the food industry to provide unique sensory appetite and change the physical properties of the food products (NurSyahirah and Rozzamri, 2022).Frying in oil is one of the conventional procedures for reducing water activity(_aw), it is effective in fish preservation in the sense that the nature and gravity of fish spoilage depends proportionally on water activity (_aw), an important constituent of biological systems (Eyo, 2001).Smoking is one of the oldest methods of preserving fish. According to Arazu and Ogbeibu (2009), the preservation and processing of fish by smoking and drying are methods used by artisans’ fishermen to avoid spoilage of their catches, and is still the most popular way of preserving fish in many communities in Nigeria today.

Fish spoilage still happens even though preservation techniques like frying, smoking, drying, and other methods help to prevent it. Microbial contamination of fish and contaminated fish products pose a serious threat to the world’s growing demand for fish and fish products. Contamination and spoilage of fish due to bacteria is found to cause health hazards to man. Consequently, this work was aimed at examining the bacterial associated with both dried and fried fish sold in Makurdi, the Benue state, Nigeria as well as testing the antimicrobial susceptibility pattern of the bacteria isolates using some commercially available antibiotics.

MATERIALS AND METHODS

Sample Collection, Preparation and Inoculation

A total of 32 samples, 16 each of fried and smoked fish were collected from four different locations in Makurdi (Wurukum, North Bank, High Level and Wadata).  The samples were collected in sterile polythene bags, labelled appropriately and taken to the laboratory for bacteria examination. Serial dilutions were made by dissolving 1g of the sample into 9ml of sterile distilled water, from 10^-1 up 10^-9. The dilution factor of 10^-5 to 10^-9 were used to inoculate the nutrient agar and Salmonella/Shigella agar already prepared using spread plate method. All the plates were incubated at 37⁰C for 24 hours. After 24 hours, the colonies on the plates were counted using colony counter (Infitek Inc, China) and the results recorded as colony forming unit per grammme (cfu/g). The selected isolates were sub-cultured to obtain a pure culture.

The isolates were identified using morphological characteristics such as Gram staining/microscopy and biochemical characteristics (Franco-Duarte et al., 2019; ASM, 2020).

Antimicrobial Susceptibility Test on the Bacteria Isolates

The disk diffusion susceptibility method was used, and the direct colony suspension method was used for preparing the inoculum from colonies grown for 24 hours. The test was performed by applying a bacterial inoculum of approximately 1–2×10⁸cfu/ml to the surface of a Mueller-Hinton agar plate. Up to 6 for Gram-positive and 7 for Gram-negative commercially prepared, fixed concentration, paper antibiotic disks were placed on the inoculated agar surface. The antibiotics used for Gram-positive bacteria were; Cefazolin (30µg), Erythromycin (15µg), Gentamicin (10µg), Oxacillin (1µg), Penicillin G (10µg), and Vancomycin (30µg). That of Gram-negative bacteria were; Amikacin (30µg), Ampicillin (10µg), Cefoperazone (75µg), Cefazolin (30µg), Gentamicin (10µg), Tetracycline (30µg), and Tobramycin (10µg). Plates were incubated for 16–24 hours at 35°C prior to determination of results (Hudzicki, 2009; Reller et al., 2009; Bayot and Bragg, 2022).

RESULTS

The Results of the Total Bacteria Count from all the Samples

Table 1: The colony forming unit per grammme (cfu/g) of the bacteria from the fried fish.

Key: A= Wadata market, B= Wurukum market, C= North-Bank market, D=High-Level

Table 2: The colony forming unit per grammme (cfu/g) of the bacteria from the smoked-dried fish.

Key: A= Wadata market, B= Wurukum market, C= North-Bank market, D=High-Level

Results of Bacteria Isolated and Identified from all the Samples

Table3: Different bacteria species isolated from fried fish samples

Key: STA = Staphylococcus aureus, ECO = E. coli, BAC = Bacillus spp, SHI = Shigella spp, SAL = Salmonella spp, KLE = Klebsiella spp, MIC = Micrococcus spp, PSE = Pseudomonas spp., A = Wadata. B = North-Bank, C = Wurukum, and D = High-Level, + = present, – = absent

Table 4: Different bacteria species isolated from dried fish samples

Key: STA = Staphylococcus aureus, ECO = E. coli, BAC = Bacillus spp, SHI = Shigella spp, SAL = Salmonella spp, KLE = Klebsiella spp, MIC = Micrococcus spp, PSE = Pseudomonas spp., LIS = Listeria monocytogenes, A = Wadata. B = North-Bank, C = Wurukum, and D = High-Level, + = present, – = absent

Key: ECO = E. coli, STA = Staphylococcus aureus, BAC = Bacillus spp, MIC = Micrococcus sp, KLE = Klebsiella spp, SHI = Shigella spp, SAL = Salmonella spp, LIS = Listeria monocytogenes, and PSE = Pseudomonas spp.

Figure 1: Percentage (%) occurrence of the bacteria isolates from the samples

Results of Antimicrobial Susceptibility Test on the Bacterial Isolates

Table 5: Antimicrobial susceptibility pattern of Gram-positive bacterial isolates from the fish samples

Key:STA = Staphylococcus aureus, BAC = Bacillus spp, MIC = Micrococcus sp, LIS = Listeria monocytogenes, S = susceptible, and R = resistance

Table 6: Antimicrobial susceptibility pattern of Gram-negative bacterial isolates from the fish samples

Key: ECO = E. coli, SHI = Shigella spp, SAL = Salmonella spp,KLE = Klebsiella spp, PSE = Pseudomonas spp., S = susceptible, and R = resistance

DISCUSSION

The results from this investigation demonstrated that samples taken from each location contained bacteria (Table 1 and 2).Overall, smoked-dried fish samples had a greater bacteria load than fried fish. The fried fish samples showed a range of 2.0×10⁸ to 3.8×10⁸ colony forming units per gram (cfu/g), whereas the smoked-dried fish samples showed a range of 4.3×10⁸ to 7.8×10⁸ cfu/g (Table 1 and 2). According to Gutema and Hailemichael (2021), the high bacteria load in fish and fish products is probably due to poor handling. Many findings have been documented on the bacteria load of both dried and fried fish. According to Ebeniro and Nwosu (2020), the bacteria load of dried fish of samples collected from 3 local markets in Ngor-Okpara in Ebonyi state, Nigeria, ranged from 5×10⁴ to 1.8×10⁵ cfu/m. Various fried fish samples were observed to have cfu/g per plate ranging from 86×10⁵ to 165×10⁵ (Abolagba and Uwagbai, 2011).

A related finding was reported by Gutema and Hailemichael (2021), who found that samples of dried fish had a total viable bacterial count ranging from 4.20×10⁵ to 3.08×10⁷.A range of 1.08×10 to 2.43×10³ cfu/g was similarly found by Ohalete et al. (2019) for 24 fried fish samples that were taken from 8 different locations in Owerri, Imo state, Nigeria. Similar findings were made by Ahmad et al (2024), who examined 40 fried fish samples that were collected from various sites in Katsina, Nigeria, and found that the cfu/g ranged from 1.5×10⁶ to 7.2×10⁶.

Table 3 and 4 lists the nine species of bacteria that were isolated from the 32 samples. The bacteria that were isolated included E. coli, Staphylococcusaureus, Bacillus spp, Shigella spp, Salmonella spp, Klebsiella spp, Micrococcus spp, Listeriamonocytogenes, and Pseudomonas spp. All 32 samples had E. coli, Staphylococcusaureus, and Bacillus spp., however only 7 samples contained Salmonella spp. and Shigella spp. Ten samples had Klebsiella spp, 14 samples included Micrococcus spp, 4 samples contained Psudomonas spp, and 4 samples contained Listeria monocytogenes.

The isolation of bacterial species from smoke-dried and fried fish agreed with some earlier findings. Gutema and Hailemichael (2021), reported the presence of coliforms and other bacteria such as S. aureus and Salmonella spp in dried fish products. Similarly, Anihouvi et al (2019), also isolated Enterobacteriaceae, E. coli, Bacillus cereus, and Clostridium perfringens from smoked and smoke-dried fish. From dried fish, Hassan et al (2021), also isolated Salmonella spp., Bacillus spp., E. coli, and some unknown bacteria.

There have also been reports of the isolation of many bacteria from fried fish. For instance, Staphylococcus aureus, E. coli, Micrococcus spp., and Salmonella spp. were identified among the bacteria isolated from fried fish (Amarachi, 2020). From dried fish, Begun and Rhaman (2021), isolated seven distinct types of bacteria, including E. coli, Vibrio spp., Staphylococcusspp., Pseudomonas spp.,Salmonella spp, Shigella spp., and Klebsiella spp. Similarly, Ohalete et al (2019), reported 10 genera of bacteria Pseudomonasspp, E. coli, Aeromonasspp, Salmonella sp, Proteus spp, Vibrio spp, Bacillus spp Micrococcus spp, Streptococcus spp, and Staphylococcusspp from fried fish.

Each of the isolated bacteria is a human pathogen with a history of being linked to food-borne illnesses. For instance, it is known that six different types of E. coli can infect humans when they are found in food: diffusely adherent E. coli (DAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and enterohemorrhagic E. coli (EHEC) (Begum, 2024). Food poisoning is frequently caused by Staphylococcus aureus, which can produce seven different types of toxins (Krause, 2017). Bacillus spp like B. cereus causes two distinct food poisoning syndromes: a rapid-onset emetic syndrome characterized by nausea and vomiting, and a slower-onset diarrhoeal syndrome (Turnbull, 1996). According to Nunez (2014), the skin of warm-blooded animals, including humans, is a main reservoir for Micrococcus strains, which frequently contaminate foods of animal origin and can cause opportunistic infections. Klebsiella pneumoniae is not only a major hospital-acquired pathogen but also an important food-borne pathogen that can cause septicemia, liver abscesses, and diarrhoea in humans (Zhang et al., 2018).Shigellosis is a form of bacterial diarrhoea caused by the Shigella species. It is common in developing countries and is transmitted via the ingestion of contaminated food (Aslam and Okafor, 2022). According to Lee (2023), Salmonella is a genus of bacteria that commonly cause foodborne illness. An infection by the bacteria is called salmonellosis and could be got by consuming contaminated food products. Foodborne listeriosis is one of the most serious and severe foodborne diseases. It is caused by the bacteria Listeria monocytogenes(WHO, 2018). Pseudomonas aeruginosa is a leading human opportunistic pathogen and a prevalent causative agent of food infection (Gao et al., 2023).

The percentage (%) occurrence of the bacterial isolates indicates that E. coli, Staphylococcusaureus, and Bacillus spp, showed (100%), Micrococcus spp(43.75%), Klebsiella spp(31.25%), Shigella spp and Salmonella spp (21.88%) each, Listeria monocytogenes(18.75%), and Pseudomonas spp (12.5%) (Fig. 1).This is comparable to other researchers’ past findings. The percentage of isolated bacteria from fried fish, for instance, was documented by Amarachi (2020), as follows: 100% were Staphylococcus aureus, 85% were E. coli, 20% were Micrococcus spp, and 70% were Salmonella spp. Similarly, in their study, Begum et al (2021), identified seven different types of bacteria and the percentage occurrence from dried fish, including E. coli (21.43%), Vibrio spp (18.45%), Staphylococcus sp (17.86%), Pseudomonas spp (17.86%), Salmonella spp (12.5%), Shigella spp (8.93%), and Klebsiella spp (2.97%).

The 100% occurrence of E. coli, Staphylococcusaureus, and Bacillus spp in all the samples is a source of concern among the perceived consumers.Finding E. coli, Staphylococcus aureus, and Bacillus spp in all samples does indeed suggest a high level of contamination. E. coli is commonly found in faecal matter and indicates possible contamination by human or animal waste (Carson, et al., 2001). Staphylococcus aureus can cause various infections, and its presence suggests poor hygiene or improper food handling (Bencardino et al., 2021). Bacillus spp is a broad category of bacteria, some of which can be harmful if present in high quantities (Dilandro and Zundel, 2023). While finding these bacteria in all samples raises concerns, further analysis is necessary to determine the severity of the contamination and its potential risks to human health.

The antimicrobial susceptibility test of the bacterial isolates was also determined using Gram-positive and Gram-negative commercial antibiotics disks (Table 5 and 6).From Table 5, Staphylococcus aureusisolates were 100% susceptible to Cefazolin,Gentamicin, and Erythromycin. The investigation of Munawar et al. (2021) is comparable to this one in that they found that Staphylococcus aureus was susceptible to the following drugs: Ceftriaxone, Ciprofloxacin, Clindamycin, Augmentin, Erythromycin, and Cotrimoxazole.The Staphylococcus aureusisolates wereresistant to Oxacillin(60.7%), Penicillin G(62.5%), and Vancomycin(25.5%). Because the Staphylococcus aureus isolates were resistant to more than one class of antibiotics, including Glycopeptides (Vancomycin) and Penicillins (Oxacillin and Penicillin G), they are categorized as multidrug resistant(Bharadwaj et al., 2022). According to Nwankwo and Nasiru (2011), Staphylococcus aureus develops resistance very quickly and successfully to different antimicrobials over a period of time.

Micrococcus spp isolates were 100% susceptible to all the antibiotics used. The antimicrobial susceptibility pattern of Micrococcus spp isolates here is similar to that reported by Munawar et al (2021), with Gentamicin, Erythromycin, Ceftriaxone, Ciprofloxacin, Cotrimoxazole, and Augmentin. Bacillus sppand Listeria monocytogenes isolates were also 100% susceptible to the antibiotics used.

According to Table 6, the E. coli isolates were completely susceptible to Amikacin, Cefoperazone, Cefazolin, Gentamicin, and Tobramycin, while they were resistant to Ampicillin (36.9%) and Tetracycline (32.2%). The isolates can also be described as multi-drugs resistance hence their resistance to Tetracyclines and Penicillins.E. coli has a great capacity to accumulate resistance genes, mostly through horizontal gene transfer (Poirel et al., 2018).This outcome is comparable to that of Aabed et al. (2021), who found that isolates of E. coli had a percentage resistance of (70.8%) to both ampicillin and amoxicillin. It is however, at variance with Hassan et al (2023), that reported resistance to Amikacin (60.9%), Gentamicin (30%), Cefepime (51.8%), Cefotaxime (100%), Ceprofloxacin (77.8%), Ofloxacin (91%), Meropenem (11.4%), and Colistin (0.4%) respectively from E. coli isolates. The isolates of Pseudomonas spp. from Table 6 also showed 100% sensitivity to Amikacin, Cefoperazone, Cefazolin, Gentamicin, and Tobramycin, but resistance to Ampicillin (79.3%) and Tetracycline (54.5%). According to Lister et al (2009), Pseudomonas spp can develop resistance to antimicrobials either through the acquisition of resistance genes on mobile genetic elements or through mutational processes that alter the expression of chromosomally encoded mechanisms. Shigella spp, Salmonella spp, and Klebsiella spp isolates bothexhibited100% sensitivity to all the antibiotics used.

CONCLUSION

The findings of this study showed the presence of pathogens from all the samples. However, smoke-dried fish samples had higher bacterial load compared to the fried fish samples. The overall bacterial load exceeded the Food and Agriculture Organization’s (FAO) suggested threshold of 10⁵ cfu/g. Most bacterial isolates are likely to originate from the fish handlers as they are of feacal origin and members of Enterobacteriaceae. Therefore, in order to prevent such incidents, it is important to assign agencies in charge of public and personal hygiene to assist in educating vendors about safe handling practices for fish and fish products.The antimicrobial susceptibility test showed 3 of the bacterial isolates (Staphylococcus aureus, E. coli and Pseudomonas spp) to be multi-drugs resistance.Therefore, it’s crucial to implement strict infection control measures to prevent the spread of these resistant strains within healthcare facilities and the community. This can include practices such as proper hand hygiene, isolation protocols for infected patients, and prudent antibiotic use to minimize further development of resistance.

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