Nutritional value of vegetables and high prices of meat and meat originated food compel common people to consume plant originated food particularly salad vegetables. Microbial population of vegetable surfaces contains a large number of pathogenic bacteria including members of Enterobactereace like Escherichia coli (E. coli). A survey was conducted in three major markets of Rawalpindi, Pakistan. Tomato, lettuce, cabbage and cucumber samples were collected from three shops of each market. Each vegetable was analysed as unwashed and washed for total coliforms, faecal coliforms and E. coli by FAO (Food Quality Manual). About two hundred and fifty E. coli isolates were preserved, serotyped for presence of O157 serotype. Total coliforms, faecal coliforms and E. coli count exceeded the permissible limits in most samples. The highest Total coliforms were associated with cabbage (3.78 log10 cfu/g). Cucumber was the least contaminated by Total coliforms (2.15 log10 cfu/g). E. coli was detected in tomato, lettuce, cucumber and cabbage. Washed samples showed reduced bacterial population. Seventy six isolates of E. coli were biochemically characterized and serotyped for O157 antigen. A majority of strains could not be identified by serotyping. These findings conclude with high potentially pathogenic microbial load on salad vegetables and urge for preventive action on priority basis.
Foodborne illness has recently gained much attention worldwide due to its deleterious effects on human health and consequentially on national economy. Worldwide increased consumption of fresh vegetables in the form of raw and minimally processed salads has resulted in increase in foodborne outbreaks which sometimes may be fatal [
Foodborne outbreaks are witnessed by people of developed countries like the United States, Japan and Germany [3,4,5]. Coliform bacteria, particularly E. coli, are an index of water and food sanitation. Some of the strains are pathogenic like E. coli O157:H7 and are able to produce serious illness in humans including diarrhoea, vomiting, severe abdominal pain, hemorrhagic colitis (HC), and the acute hemolytic uremic syndrome (HUS) [6,7].
Foodborne illnesses seldom come into notice in developing countries like Pakistan. The proposed study is thought to assess the risk related to consumption of contaminated salad vegetables by detecting total coliforms, faecal coliforms and E. coli associated with salad vegetables sold at retail shops of Rawalpindi. Data generation for health policy makers and to have a view of difference between contamination level of unwashed and washed vegetables were also the research objectives.
Three markets of Rawalpindi were randomly selected i.e. Khannapul vegetable market, Chungi 22 vegetable market and Rajabazar vegetable market. Four salad vegetables (1 kg each) i.e. tomato, lettuce, cucumber and cabbage were sampled from three random markets of the city.
Each sample was coded and analysed for total coliforms, faecal coliforms and E. coli as (a) unwashed (b) washed. Sample was prepared and serially diluted in Butterfield phosphate buffer according to method described in FAO Manual (1992) [
Diluted Butterfield phosphate buffer and all media required were autoclaved at 121˚C and 15 pounds/sq. inch pressure for 15 - 20 minutes in Harvey Sterile Max autoclave (USA).
One ml blended sample was put in Pyrex screw cap test tubes containing 9 ml sterile Lauryl sulphate Tryptose (Oxoid) broth and inverted Durhum tubes and incubated at 35˚C for 24 - 48 hrs in Binder incubator. Production of gas in inverted Durhum tubes was the presumptive test for coliforms presence in the sample.
Positive cultures in LST tubes were transferred to sterile BGLB (Oxoid) tubes and incubated at 35˚C for 24 - 48 hrs in incubator. Gas production in inverted Durham tubes confirmed the presence of coliforms in sample. Their number was assessed by counting confirmed positive test tubes of consecutive three dilutions using MPN technique.
Positive LST culture was transferred to sterile 10 ml EC medium and incubated for 48± hours at 45.5˚C in GFL 1086 waterbath (Germany). MPN of faecal coliforms was calculated from positive EC tubes for consecutive three dilutions.
E.coli isolates were confirmed by performing tests for indole production, methyl red reactive compound tests, Voges Proskauer reactive compound tests, and citrate utilization. IMViC pattern of ++-- and -+-- was interpreted as Biotype I and II E. coli strains, respectfully. Some strains showing atypical characteristics were Gram stained.
All isolated strains of E. coli were tested for catalase production as described by Collins and Lyne (1980) [
In order to serotype the Enterohemorrhagic strain of E.coli, among the isolated strains, O157 Oxoid (DR120M) Dryspot Latex detection kit was used.
All isolates from fresh produce were coded as VB (Vegetable Bacteria) and given a number. All E. coli isolates from salad vegetables were preserved for short term on Nutrient agar (Sigma) slants and long term by freezedrying in 10% skim milk (Difco).
All experiments were performed in triplicate. The means, standard deviation and standard error of all experiments was determined. Graphs preparation and standard error calculation was performed in Excel 2010. Bifactoral Analysis of Variance (ANOVA) was applied to know the significance of washed and unwashed vegetables of different markets. Pairwise T test was applied (Statistix 8.1) to assess the difference between each vegetable in unwashed and washed conditions. Total Coliforms, Faecal Coliforms, E. coli values were also converted to log 10 before graph preparation.
A total of 36 samples were collected from their respective shops and markets and coded after transporting to the laboratory.