Antibiotic Sensitivity Pattern of Microorganisms Isolated from Smoked and Frozen Fishes Sold in Benin and Warri Metropolis

Antibiotic sensitivity pattern of microorganisms isolated from smoked and frozen fishes sold in Benin and Warri metropolis were investigated. Adopting microbiological standard techniques, the results of the bacterial counts and fungal counts ranged from 5.4 x 10 6 (Ekpan market) to 25.1 x 10 6 (Ekpan market) and 1.1 x 10 5 (Ekpan and New Benin market) to 2.9 x 10 5 (Effurun market). Six bacterial species namely; Staphylococcus spp., Escherichia coli , Bacillus spp., Pseudomonas sp., Corynebacterium sp., Micrococcus sp. and four fungal species namely; Aspergillus spp., Fusarium sp., Mucor sp. and Penicillium spp. were identified. Majority of the bacteria were isolated from fish samples from Benin City Markets (51.1%, n = 23) while (48.9%, n = 22) were obtained from Warri Markets. Bacillus spp. were the most predominant (35.6 %) and Micrococcus sp. was the least predominant among the bacterial isolates (2.2%). Aspergillus sp. was recorded the most predominant (37.1%) while Penicillium sp. (14.3%) was the least predominant among the isolated fungi. The isolated bacteria ( Escherichia coli, Bacillus sp., Staphylococcus sp., Pseudomonas sp., Corynebacterium sp. and Micrococcus sp.) were sensitive to ciprofloxacin and showed resistance to zinnacef and rocephin. These findings show that fish samples from different markets harbour varied levels of potentially pathogenic organisms which could pose health threat to consumers. Proper handling, processing and storage methods should be put in place to reduce potential health risk.


Introduction
Fishes are known to be highly nutritious and excellent sources of animal protein, which is consumed by a larger percentage of the world's population because of its availability and palatability (Foran et al., 2005;Sutharshiny and Sivashanthini, 2011). Marketing of fish in Nigeria is mostly carried out by local fish sellers (Okoro et al., 2010), hence it's harvesting, handling processes and distribution provides livelihood for millions of people. Fish flesh contains significantly low lipid and high water compared to that of beef or chicken (Nestel, 2000) and rich in vitamins and minerals for both young and old consumers (Edem, 2009;Koffi-Nevny et al., 2011).
Contamination of fish and other fish products by microbes have been a serious threat to human health. The improper handling and storage processes of fish pose serious effects thereby limiting the shelf life of the products (Sallam, 2007). The quality of fish products degrades due to complex processes in which physical, chemical and micro-biological forms are implicated (Sallam, 2007). Doyle (2007) had reported that storage and processing conditions affect microbial growth. Thus the need for proper handling, storage facilities and the knowledge of spoilage pattern at tropical fishes and their shelf life under ambient condition is very imperative (Okoro et al., 2010).
The smoking of fish from smoldering wood for its preservation dates back to civilization (Olokor, 2007). It's also noted that apart from giving the product a desirable taste and odour, smoking provides longer shelf life through its anti-bacterial and oxidative effect, lowering of pH, imparting, desirable coloration as well as accelerating the drying process and acting as antagonist to spoilage agents (Sengor et al., 2004;Eyo, 2001;Homer, 1997). Martin (1994) stated that organisms such as Staphylococcus aureus, Saccharomyces cerevisiae, Penicillium and Aspergillus were the commonest microorganisms associated with smoked fish. Freezing is a widely used method for the preservation of fish which involve the use of refrigeration, cold rooms and icing etc. Freezing generally slows down the deterioration of fish at low storage temperature (Sivertsvik et al., 2002). The aim of this study was to investigate the microbiological qualities and antibiotic sensitivity pattern of the isolated bacteria from smoked and frozen sold in markets in Benin-City and Warri.

Materials and methods
Collection of samples: Smoked and frozen fish samples were obtained from two different Markets in Warri (Ekpan and Effurun Markets) and Benin City (Uselu and New-Benin Markets). The fish samples were collected in labelled aseptic containers and transported to the laboratory for analysis.
Enumeration and isolation of microbial isolates: Ten grams of fish sample was weighed into 90 ml of sterilized peptone water for serial dilutions processes (10 -4 and 10 -5 ) were prepared and used for serial dilution. Appropriate dilutions were plated in duplicates on sterile nutrient agar and potato dextrose agar for total heterotrophic bacterial and fungal counts respectively. The PDA agar plates were made selective for fungal growth by the addition of 1 ml of 250 mg/ml chloramphenicol solution prior to pouring of the molten medium. The agar plates were incubated aerobically at 30 o C for 48 hours and 5 days at room temperature (28 o C ± 5 o C) for total heterotrophic bacterial and total fungal counts respectively. After incubation, counts obtained were recorded and expressed as the colony forming unit per gram (cfu/g) of the original sample .The various isolates were further identified and characterized using the methods of Barnett and Hunter, 1975;Cheesbrough, 2006.

Discussion
The presence of microorganisms in fishes is not out of place, when viewed as a variable substrate for the proliferation of these organisms. However, when considered from the public health stand point, growth and presence of microorganisms in commercially sold smoke and frozen fishes is unacceptable. The results of this study demonstrated the significant presence of saprophytic and pathogenic microbial flora in commercially sold smoked and frozen fishes from major Markets in Benin City and Warri.
The isolated microbial isolates included six bacterial and four fungal isolates. They were Staphylococcus spp., Bacillus spp., Pseudomonas sp., Micrococcus sp., Escherichia coli, Corynebacterium sp., Aspergillus spp., Fusarium spp., Penicillium spp., and Mucor sp. The presence and high number of these microbial genera could be explained by the fact that these organisms can withstand the adverse processing conditions. These bacterial and fungal isolates have earlier been implicated as major pathogens associated with fish spoilage (Gram and Huss, 2001). According to Adebayo-Tayo et al. (2011), contamination of fish may occur from food handlers and retailers who sell these items to the public for consumption. Bacillus sp. and Aspergillus sp. had the highest frequency of occurrence among the various bacterial and fungal isolates which may have been occasioned by poor handling or cross contamination (Gupta et al., 2006). Also, organisms like Bacillus and fungal genera such as Aspergillus and Penicillium spp. were reported to survive for a long time in a dormant state due to the spore forming state.
The presence of indicator organisms like Escherichia coli might be as a result of possible contamination during sales and unhygienic handling of fish products. The isolation of Pseudomonas sp. from the fish samples is of high importance because it plays a considerable role as a potential pathogenic bacteria for human, also as an indicator of food quality and as a good spoilage index (Jeyesekaran et al., 2006;Yagoub, 2009), and it was prevalent in the fish sampled from Warri market.  7mm KEY: PEF = Perfloxacin, CN = Gentamycin, APX = Ampiclox, Z = Zinnacef, AM = Amoxacilin, R = Rocephin, CPX = Ciprofloxacin, S = Streptomycin, SXT = Septrin, E = Erythromycin, Nil = no zone of inhibition Staphylococcus sp. produce a variety of extra cellular enzymes and toxins that have been found to be responsible for food poisoning and can rapidly develop resistance to many anti-microbial agents and pose a therapeutic problem. Aspergillus sp. and related mould generally grow faster and are more resistance to high temperature and low water activities and tends to dominate spoilage in warmer climate (Doyle, 2007). Ogunshe and Olabode (2009) reported antibiotic resistance among bacterial species isolated from the fish (Clarias gariepinus). According to Malik and Ahmad (1994), high levels of bacterial resistance to antibiotics are indications of abuse and misuse of antibiotics in the society. This involves situation where individuals purchase and consume antibiotics with out and medical advice. Bacterial groups co-habiting a common environment may express a similar antibiotics pattern if they share in a common pool of R-factor plasmids (Spanggard et al., 1993). All isolates were sensitive to ciprofloxacin and showed resistance to rocephin and zinnacef except Micrococcus sp.
This study therefore confirms the presence of some pathogenic bacterial and fungal isolates from the frozen and smoked fish which are of public health significance and indicative of health risk in contacting diseases associated with these organisms. Compliance with standard microbiological measures to prevent contamination by these organisms becomes very necessary and should be enforced.