Detection of Escherichia Coli in Freshly Harvested Spinach Samples Collected from Five Different Markets in Zaria

Introduction

Escherichia coli are natural predominant facultative anaerobes common in the lower intestines of endothermic animals and it grows in most laboratory media at an incubation temperature of (370C) optimum [1]. They colonize the gastrointestinal tract hours after birth or days after [2]. Nevertheless, some strains have evolved the capability to cause both intestinal and extra intestinal illnesses [3,4]. Not all the strains are harmless, as some can cause debilitating and sometimes fatal diseases in humans as well as mammals and birds [5]. Faecal–oral transmission is the major route through which pathogenic strains of the bacterium cause disease. Cells of the bacteria can survive outside the body for a limited amount of time, which makes them potential indicator organisms to test environmental samples for faecal contamination [6,7]. In Zaria, Tijani et al. [8], Kabiru et al. [9] reported the prevalence of Escherichia coli 0157:H7 in spinach grown around abattoir waste dumpsite, manure farm and soil, irrigated with raw abattoir wastewater. This suggests that spinach are vegetables that can potentially harbor Escherichia coli when manured with contaminated faeces. The use of raw manure or slurry (liquid manure) on or near fruit and vegetable crops, particularly those to be eaten raw is a potential hazard for Escherichia coli especially 0157:H7 [8] and hence the need for work to be done on the prevalence of E.coli in spinach meant for human consumption in Zaria and its environs. Studies have shown that Escherichia coli especially 0157:H7 can survive in faeces for extensive periods of 70-100 days at the temperature in the region of 45 0C [10]. Thereby establishing that faeces serving as manure to most subsistence and commercial farmers growing spinach at backyard or irrigational farms at dry season may be a potential vehicle for transmission of the organism into the environment [11].

Materials and Methods

Study Area

The study was carried out in Samaru Campus of ABU, Zaria, Sabon Gari Local Government Area of Kaduna State. Located on Latitude (11°11´N, 07, 38´E, 686m above sea level) in the Northern Guinea Savannah with temperature ranging from 13.8° to 36.7 °C. Annual rainfall of 1092.8mm [12]. Farming system in Zaria can be divided into two types: Rainfall (from May to October) and irrigation farming in the dry season (from November to April). Dry season farming is the second most prevalent Agricultural activity in Zaria with vegetables being the common produce, but in some cases, fruits are sandwiched among cereal crops [13].

Sample Collection

A total of 100 samples of freshly harvested spinach leaves were procured randomly in sterile polythene bags from 5 different open markets in Zaria metropolis. Twenty (20) samples each were collected from Sabon Gari, Samaru, Hayin-Dogo, Palladan and PZ markets and transported on ice cold packs of the laboratory and analyzed immediately.

Isolation and Identification

Freshly plucked spinach samples were purchased from five different markets in Zaria Metropolis, the stem and roots were removed and discarded, while the leaves were washed thoroughly with distilled water and the washing was placed in universal bottles. Five mls of the spinach washing was inoculated into 5mls of double strength Mac Conkey broth and inoculated for 24 hours at 37 0C. A loop full of the positive colonies was subcultured on EMB Agar and incubated for 24h at 37 0C a greenish metallic sheen on the surrounding medium were observed. Pure culture of all colonies on EMB agar that exhibited typical dark purple red colour with metallic sheen which characterized E. coli were again subculture on Mac Conkey agar for identification and purification of lactose fermenting colonies. Biochemical tests such as indole, citrate, carbohydrate fermentation tests and MR-VP test were done on all lactose fermenting colonies to confirm E. coli. All the Lactose fermenting colonies were picked and stored in nutrient Agar slants (Oxoid) at 4 0C for further test. Biochemical test that were carried out to isolate E. coli, includes: Triple Sugar ion (TSI), Indole, Urease, Citrate, Motility, Methyl Red (MR), Vokes Proskaur (VP) and H4S production.

Biochemical tests

The conventional biochemical test was conducted according to [14]. Five different biochemical media were used, and all media were prepared according to manufacturer’s instructions.

Citrate utilization test

This test is based on the ability of an organism to use citrate as its source of carbon. A sterile wire loop was used to inoculate the isolate onto the Simon’s citrate agar medium, which was already prepared as slant in test tubes, and incubated at 370C for 24hours after which it was examined for color change. A bright blue color in the medium was observed at against green coloration.

Methyl red voges proskauer test (MRVP)

A loop full of the organism was inoculated into a MRVP media and incubated at 370C for 24 hours. The turbid positive broth was divided into two halves, 2-3 drops of methyl red was added to one broth and observed for formation of pink color coloration, while to the remaining half, 5drops of 40.0% potassium hydroxide (KOH) and 5drops of 5.0% of alcoholic alpha-45 Naphthol were added and shaken vigorously for homogenization. The cap of the tube is placed in a sloping position. The development of a red color starting from the liquid air interface within 15-30minutes was indicative of a positive test.

Urease test

Urease hydrolyzes urea to give alkaline ammonia (NH5) and carbon dioxide. Urease liquid media was poured in three to five milliliters portion in a sterile screw-top glass tube. The tubes were inoculated with a loop full of the positive isolate using a sterile wire loop, but the tubes were placed in a slanting position and incubated at 37 0C for 24 hours. A pink color indicating positive test while a yellow colouration was considered negative.

Indole test

A sterile wire loop was used to inoculate a colony of the positive organism into a prepared SIM (Sulphide, Indole and Motility) media. The tube was capped and incubated at 37 0C for 24 hours. After incubation, four drops of Kovac’s reagent was added to the medium using a sterile pasteur pipette to examine for the production of indole by the organism. Observation of red coloration on the surface layer within ten minutes gave a positive result. The suspected isolates when inoculated into the medium using a sterile straight wire followed by incubation by 370C for 24 hours. The upper layer of the inoculated medium and line of stabbing was observed for the presence of dark coloration which will be indicative of a positive H4S test.

Motility test

Motility test medium was poured into test tubes and allowed to solidify. The tubes were inoculated with a colony of the test organism using a sterile wire pin and incubated at 37 oC for twenty-four hours before tests were read.

Triple sugar iron agar

Carbohydrates fermentation test in triple sugar iron agar slants were performed from commercially prepared TSI mix and poured in to a test tube which is placed in such a manner to allow the butt of the agar to be four centimeters deep, the slants were inoculated with a colony of the test organism using a sterile wire loop. Observable color changes as red and pink were interpreted as alkaline and acids with or without gas formation.

Results

Tables 1 & 2 shows the frequency and distribution of suspected E. coli isolated from spinach gotten from five different sites in Zaria Metropolis, Kaduna State. Using EMB, while Table 1, further confirms the presence of the E. coli, using the conventional Biochemical Test method. Out of the 100 samples of spinach screened for E. coli 119 (47.6) % were positive with typical growth on EMB as shown in Table 1. The results showed that Hayin Dogo had the least number of E. coli positive isolates on EMB 14 (28.0%). In addition, Palladan had the highest positive isolates on EMB 28 (56%). Further screening of the isolate with conventional biochemical tests showed a decrease in total number and percentages of confirmed E. coli isolate on both EMB agar from 119 (47.6) and 16(6.4) respectively.

Table 1:Frequency (%) distribution of suspected E. coli isolates obtained from five different markets were spinach is sold in Zaria metropolis, Kaduna State.

Key: EMB: Eosin Methylene Blue

Table 2:Frequency (%) distribution of confirmed E. coli isolates after conventional biochemical tests from five markets sold in Zaria metropolis, Kaduna State.

Key: EMB: Eosin Methylene Blue

Discussion

The result from this research showed that Escherichia coli O157: H7 were cultured from spinach sourced from five different open markets in Zaria metropolis, Kaduna State. An occurrence of 119 (47.6%) E. coli O157: H7 and 16 (6.4%) E. coli O157: H7 respectively was observed which is higher than the occurrence reported by Reuben & Makut [15], Enabulele & Uraih [16] with a prevalence rate of 17.5% and 19.5% in Lafia and Benin Metropolises respectively. In other parts of the World, Mora et al. [17] and Benard [18] reported occurrence rates of 18.2% and 21.66% respectively. The high occurrence of E.coli as observed in this research could be attributed to high microbial load on vegetables especially those grown around faecal waste dumpsite during e dry season when there is no rainstorm-washing to reduce the microbial load on the vegetables as reported by [19]. Solomon et al. [20] and Kabiru et al. [9] also opined that manure application and irrigation with contaminated water as frequently done during the dry season in Zaria metropolis could have been another reason for this finding in which isolates were confirmed highly E. coli positive. This also agrees with earlier isolation records by Brown et al. [21] who reported that E. coli especially 0157:H7 is more common in warmer months of the year and sampling plans that could include these times of the year, will result in higher recovery rates. The isolation of the pathogen (E. coli O157: H7) in Zaria metropolis suggests that contamination of spinach sold to consumers exposes them to food-borne hazards. The socio-economic implication is that resources and time are wasted on medication. The effects of food-borne infections are also greatly felt by immune-compromised individuals such as people living with HIV/AIDS, pregnant women, children, diabetic patients [22].

Leafy vegetables are an essential component of a healthy diet; however, they have been associated with high-profile outbreaks causing severe illnesses [23,24]. In addition to E. coli capacity to cause infection to humans, the ingestion of contaminated foods, water and vegetables offered to livestock may contribute to the prevalence of infections in farm animals with a resultant contamination of the environment [25-29].

Conclusion

The findings of this research have indicated the occurrence of Escherichia coli O157:H7 in spinach sourced from five open markets in Zaria Metropolis. This portends public health consequences to the unsuspecting general populace who would usually ingest such vegetable. There is therefore the need to educate the public on personal and environmental hygiene and adherence to good food handling practices such as washing of hands and disinfecting of all vegetables thoroughly before consumption. It is also the authors’ opinion that further research work be conducted to characterize at molecular level, the E. coli from spinach in local markets in Zaria.

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