GENOTYPING AND HEMOLYTIC CHARACTERIZATION OF PATHOGENIC BACTERIA FROM SOME RAW AND COOKED FOODS

This study aimed to identify pathogenic bacteria in contaminated food from cafeterias and restaurants at the University of Kerbala. Thirty-nine bacterial samples were collected from various foods, such as salads, falafel, and meat products (burger, kebab, and shawarma), before cooking. Bacteria were serially diluted, isolated on selective media, and identified based on biochemical characteristics, and 16S rDNA sequencing. Hemolysin production, seen in most bacteria from raw food samples, was determined using blood agar. Genomic DNA was extracted from all bacterial samples, and their 16S rDNA were analyzed through PCR, gene sequencing, and phylogenetic tree construction. Twenty-seven genetic variants representing both gram-positive and gram-negative bacteria were identified. Most of the bacterial isolates produced α or β hemolysin and are likely important causes of food poisoning. These results highlight the need for strict quality control in the cafeterias and restaurants at the university, improving the public’s awareness of food safety issues, and possible routine medical examination of those who handle food at these locations.


INTRODUCTION
Food is an excellent medium for growth and reproduction of microbes, making the bacteriological quality of both uncooked and cooked food important to consumers (18).Specifically, the extensive handling of crude sustenance, such as vegetables, increases the likelihood of contamination of these products by pathogens (34).In 2016, one study found an uncommon strain of E. coli O157:H7 in mixed salad that caused mild to bloody diarrhea and acute abdominal pain.The latest outbreak consisted of 161 cases, including 16 hospitalizations and two deaths (26).====Foodborne illnesses or diseases are classified as intoxication ("food poisoning") or infectious depending on the specific cause of the illness.The causative agent must be present in sufficient numbers to cause symptoms, such as food contaminated with bacteria and/or bacterial toxins (35,15,02).For example, Staphylococcus spp.must reach 10 5 CFU per gram of food to yield enough toxin to cause emergence of symptoms (02).One important virulence factor produced by many pathogenic foodborne bacteria are hemolysins, a group of pore-forming toxins that destroy red cells and are often produced by Staphylococcus aureus.Hemolysins are classified as alpha, beta, gamma, delta, or epsilon based on their mechanism of action and effect on red blood cells (5,6,9,24,25,27) and are produced by both gram-positive and gram-negative bacteria.Microbiological investigations of food traditionally rely on identifying pathogenic organisms by culturing on selective media (e.g.Mannitol Salt Agar, Salmonella-Shigella Agar) and biochemical testing, but these approaches are often unwieldy and time-intensive (1,2,15,19).More recently, molecular methods for identifying pathogenic bacteria in foods have been adopted due to their rapidity and accuracy.In this approach, the polymerase chain reaction (PCR) amplifies 16S rDNA from samples of interest, which can then be sequenced for diagnostic purposes (4,13).The bacterial 16S rDNA gene has been widely used in phylogenetic studies because of its universality, conserved nature, and sufficient length for reliable sequence analysis (approximately 1500 bp) (23).In this study, we isolated bacteria from potentially contaminated foods, inferred their phylogeny based on 16S rDNA sequences, and determined their ability to produce hemolysins.

MATERIALS AND METHODS Sample collection and culture-based identification:
Twenty-eight bacterial isolates were collected from raw and processed foods, including salads, falafel, and meat products (burger, kebab, and shawarma) before cooking in cafeterias and restaurants at the University of Kerbala between May and November 2016.Samples were stored and transported to the laboratory at 4°C.Bacteria were collected from the sample by scraping 11 g of food and adding this to 99 ml pepton water (0.1%), then mixing with a blender for 2 min before serially diluting the sample.From the final dilution of 10^-3 , 1 ml was added to duplicate petri dishes, then selective agar media at 45°C was added (e.g., MacConkey, Mannitol Salt, and Salmonella-Shigella agars) and the plates slowly moved for mixing.After the plates solidified, they were incubated at 37°C for 48 hours.After the incubation period, colonies were counted.Bacteria were isolated and tenatively identified based on their culture characteristics as previously described (8,14,16 and 19).

Determination of hemolysin production:
Hemolytic activity of the bacterial samples was measured by culturing on blood agar prepared according to the manufacturer's instructions (Himedia).Briefly, the agar base was sterilized by autoclaving at 121°C for 15 min at 15 psi and cooled to 50°C before cattle blood was added to a final concentration of 5%.Bacteria were isolated by quadrant streaking, and plates were incubated at 37°C for 24 h.The presence and type of hemolysin(s) produced were determined as described previously (3).

DNA extraction
Genomic DNA was extracted from the bacterial samples using a total DNA G-spin iNtron kit (Korea) according to the manufacturer's instructions (11).DNA concentration and purity were determined by spectrophotometry at A 260/280 .Amplification of 16S Rdna 16S rDNA genes were PCR amplified using the universal primer set 8F: 5'-AGAGTTTGATCCTGGCTCAG-3' and U1492R: 5'-GTTACCTTGTTACGACTT-3'.Reactions also consisted of Maxime PCR premix (iNtron, Korea) 5 U/µl Taq polymerase, 2.5 mM dNTPs, 1X buffer, and 1X loading dye that was added to 2 µl DNA template and 10 pmol of each primer to a final volume of 16 µl.PCR was carried out with an iCycler (Bio-Rad Laboratories, Hercules, CA, USA) at an initial denaturation of 94°C for 5 min, 35 cycles of 94°C for 45 sec, 62°C for 1 min, and 72°C for 1 min, and a final extension of 72°C for 5 min.Amplicons were then electrophoresed and visualized on a 2% agarose gel (11).

16S rDNA sequencing and alignment
Amplicon sequencing was performed by Macrogen (Canada) using the forward primer for each reaction.Sequences reaching 1500bp were scrutinized with FinchTV v.1.4.0 (Geospiza, Waltham, UK).Each 1500-bp amplicon was evaluated using a quality value, trimmed to 521 bp, and compared with sequences in the National Center for Biotechnology Information (NCBI) database with the BLASTN tool (www.ncbi.nlm.nih.gov)(36).A phylogenetic tree was constructed with MEGA v.6 (32) using parameters described by Saitou and Nei (31).

RESULTS AND DISCUSSIONS
16S rDNA was successfully amplified from 28 bacterial samples as shown in Fig. 1, similar to what has been found in other studies (23,33).Figure 2 shows the putative neighbor-joining relationships between the bacteria isolated in our study based on their 16S rDNA homology with sequences in the NCBI database (31).One of the isolates yielded a poor-quality 16S rDNA sequence and was excluded from further analysis.The optimal tree had a branch length sum of 0.68765434, and evolutionary distances were calculated using the maximum likelihood method (33) and represent the number of base substitutions per site.Our phylogenetic analysis clustered the 27 isolates into monophyletic groups descending from ancestors such as F22 (Providencia alcalifaciens MG063180), F2 (Pseudomonas putida MG063164), and F27 (Pseudomonas entomophila MG063183) (7).Polyphyletic groups in Clade 1 (gram-negative bacteria) consist of various species that lack a common ancestor, including F6 (Raoultella terrigena ).16S rDNA-based identification of pathogenic foodborne bacteria enables accurate diagnosis that can be accomplished by even small laboratories.An additional benefit of this genotyping method allows the classification of such bacteria into phylogenetic groups to study their relationships with each other.Moreover, the impact of environmental conditions on bacterial evolution and (sub)speciation can be inferred by identifying polymorphisms in the organisms' 16S rDNA genes (11,28).

Figure 1 .
Figure 1.16S rDNA amplicons of 28 bacterial samples collected from various food sources are shown.(M, marker) The value of the 16S rDNA gene in taxonomic studies relies on its universality in both gramnegative and gram-positive bacteria (12,13).Moreover, sequencing the entire 16S rDNA gene can distinguish bacteria at the strain level(21,29,30).

Table 1 . Homology of gram-positive bacterial 16S rDNA sequences from our study with sequences from the NCBI database. Table 2. Homology of gram-negative bacterial 16S rDNA sequences from our study with sequences from the NCBI database.
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