Sampling area
The study was conducted in and around BAU campus located in Mymensingh city of Bangladesh. Fifty restaurants distributed in seven places were identified in and around the BAU campus. Among these places, four were selected on the basis of a higher number of restaurants as well as consumers’ preference. The sample collection places within the study area were Kamal-Ranjit market (from two restaurants), Jabbarer mor (from six restaurants), Fossiler mor (from two restaurants), and Shesh mor (from two restaurants). The sampling program was comprised with the following segments (i) microbiological sampling of RTE vegetables salads that was ready to serve at these restaurants, (ii) a questionnaire-based study regarding salad handling practices, and (iii) verbal questioning of consumers to evaluate the consumption pattern of salads.
Microbiological samples
A total of 60 RTE vegetables salads were collected from twelve (12) randomly selected restaurants on five different time points. In all cases, each sample was placed in a labeled sterile polyethylene bag and transported to the laboratory in the icebox. Then, 15 gm of the sample was ground aseptically and mixed with 135 ml phosphate-buffered saline and ten-fold serial dilution was made followed by spread onto nutrient agar, mannitol-salt agar, salmonella-shigella agar, and MacConkey agar to determine total number of bacteria, Staphylococcus spp., Salmonella spp., and E. coli per gm of samples, respectively. Identification of the bacterial species was further performed by cultural and morphological examinations [15].
Assessment of salad handling practices
General observations were gotten from different restaurant workers through observational questions to assess the salad handling practices during serving. During sampling of salad, following observations were made: (i) Types of salad served in that restaurant; (ii) The place where salads are stored during serving; (iii) How the salads are kept in the eateries, (iv) Types of utensil used to serve food; and (v) Practice of the food handlers during serving of salads. Along with these, the vendors were asked about whether they received any training regarding hygienic food preparation and handling.
Verbal assessment of salads consumption patterns
For assessing consumption patterns of consumers, a total of 200 questionnaires were handed out to 100 male and 100 female consumers. They were asked to answer the following questions: (i) How old they were; (ii) From which source they frequently consume the salads; and (iii) How frequently they consume salads from the sources. The questionnaires were collected instantly after they filled the questionnaire themselves.
Microbial risk assessment
Microbial risk assessment of food involves four steps consisting of hazard identification, hazard characterization, exposure assessment, and risk characterization. In order to assess microbial risk, this study was focused on the production chain of vegetable salads and at the post salad preparation condition in various restaurants to the point of consumption following the study design described by Amoah.
Hazard identification
The process of microbiological risk assessment starts with hazard identification. The purpose of hazard identification was to identify the micro-organism(s) present in RTE vegetables salad samples. Three organisms as Staphylococcus spp., Salmonella spp., and E. coli were selected as the model QRMA organisms as they are frequently found in salads samples.
Hazard characterization (dose-response assessment)
Hazard characterization defines the adverse health effect related to the consumption of microorganisms. For dose-response assessment, an exponential dose-response model [18] was used to predict the probability of Salmonella spp., Staphylococcus spp., and E. coli infection on the basis of laboratory output of selected microorganisms. Mathematically, the equation used for determining probability is illustrated.
Exposure assessment
This step deals with the amount of salads consumed and the frequency of consumption of salads for a specified period of time, to assess the consumer’s exposure to microorganisms with salads for a defined time period. On the basis of consumption frequency, three exposure scenarios were assessed as frequent (daily), average (weekly), and occasional (monthly) consumption of RTE vegetables salad by consumers, as described by Amoah. In all three scenarios, it assumed that pathogen levels were not reduced aside a potential growth from post salad production to the point of utilization because the items were served as raw materials and were put at room temperature for quite a while during sales. Subsequently, any expansion of pathogens numbers during sales will be transferred to the consumer.
Statistical analysis
All the data were incorporated in Statistical Package for the Social Sciences 25.0 (IBM Corporation, USA) for performing descriptive analysis. Experimental data were analyzed by one-way analysis of variance, appropriate and significant differences among different restaurants were made at 95% confidence level (p < 0.05) by Tukey’s multiple comparison test. For each exposure scenario, Monte Carlo simulation was run using Microsoft excel@risk software version 7.5.0 (Palisade Corporation) sampling 10,000 iterations to measure the annual risk of infection.