Isolation and identification of bacterial pathogens in minimally processed vegetable salads in some Saudi restaurants using DNA sequencing

The genetic diversity and relationship amongst the bacterial populations were determined in fresh salad samples (Tabbouleh, Fattoush, Hummus, Mutabbel and Caesar) collected from various restaurants located in five different areas (west, north, south, east and center) in Riyadh (the capital city of Saudi Arabia). Isolated colonies found were identified via molecular methods. Total number of identified isolates in the vegetable salads Tabbouleh, Fattoush, Mutabbel, Hummus and Caesar were 24, 20, 18, 16 and 12, respectively. Pseudomonas sp., Bacillus cereus and Enterobacter aerogenes were recorded in the five types of vegetable salads. Escherichia coli, Klebsiella sp., Kluyvera cryocrescens and Kluyvera ascorbata were recorded in all types of vegetable salads except Caesar. Vegetable salads collected from all sites were contaminated with E. coli, Enterobacter sp. and Enterobacter aerogenes. Elevated level of contamination with E. coli was recorded in the samples collected from the north and east areas, while high level of contamination with Enterobacter aerogenes was recorded in the samples collected from central area. Estimation of bacterial genetic relationships was determined using DNA sequencing, phylogenetic tree and bioinformatic techniques. The 36 isolates were arranged in one main cluster including 34 bacterial species and two bacterial species Pseudomonas and Bacillus cereus were out groups with similarity index range of 0.0080.063 and 0.008-0.125, respectively, to the main group. This cluster was subdivided into two subgroups: subgroup A included Acinetobacter sp. and Stenotrophomonas sp.; subgroup B included 32 bacterial species. Subgroup B contains one major subgroup C comprising 31 bacterial species with similarity index range of 0.061-0.019 to Hafnia alvei. Subgroup C was subdivided into subgroup D including 15 bacterial species and subgroup E including 16 bacterial species. The inter-specific analysis of the sequences showed that most bacterial species exhibit a homogeneous sequence indicating a low level of polymorphism, which is congruent with the reduced variability found in these bacterial species from other salad sources. The identification of intra-specific variation suggests that DNA sequencing had a potential to aid in quick identification and classification of bacterial isolates in salads. The reproducibility generated by this technique may lead to the development of specific molecular markers for characterization of these microbes.