Isolation, identification and antibiogram of verotoxin producing Escherichia coli from raw salad vegetables at Jashore, Bangladesh

Verotoxin-producing E. coli are getting an emergence for human health and day by day, it is also becoming more alarming in the fact that, they are acquiring multidrug-resistant profiles. This study aimed at isolation and identification of verotoxigenic E. coli followed by the antibiogram of the species from salad vegetables at Jashore district in Bangladesh. A total of 119 raw vegetable salad samples were collected from different areas of the Jashore district. The collected samples were initially enriched in nutrient broth and then used for streak plating on MacConkey and followed by Eosin Methylene Blue (EMB) agar media. Escherichia coli species were isolated and identified following observation of the cultural, microscopic, and biochemical characteristics and further analyzed to determine the presence of verotoxin (VT) producing genes through polymerase chain reaction (PCR) using specific primers (VT1, VT2, and Eae). A panel of antibiotics were tested following the disc diffusion method for determining the antibiotic profile. Total 55 (46.22%) E. coli was isolated phenotypically. 6 (10.9%) isolated E. coli showed positive by amplifying verotoxin-producing gene vt1, but none of the samples were positive vt2 and eaeA gene. Antibiogram for 55 E. coli isolates showed the highest resistance to Erythromycin (83.64%), Streptomycin (78.18%), Ceftriaxone (70.91%). On the other hand, the most heightened susceptibility was observed for Nalidixic acid (76.36%). Moreover, six vt1 positive isolates showed a multidrug-resistant profile. Thus, the presence of toxigenic genes in the isolates suggests the proper maintenance and regular monitoring of raw salad items should be increase to ensure healthy life in this region.


INTRODUCTION
Escherichia coli is one of the most common bacteria on the planet. Theodor Escherich, a German bacteriologist, was the first to discover it in 1885. As a result, this bacteria was named after him. E. coli is a Gram-negative *Corresponding author. E-mail: nigar@just.edu.bd.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License facultative anaerobe that does not generate spores (Parvej et al., 2018). The cells are generally rod-shaped, with a diameter ranging from 0.2 to 1 μm and a length of around two μm. Flagella-containing strains are motile, and the flagella are frequently arranged in a peritrichous pattern. It is typically found in animal excrement, mammalian lower intestines, and on the outskirts of hot springs. They thrive at 37°C. These bacteria live mainly in the intestines of warm-blooded mammals. E. coli contains a broad spectrum of strains with actions ranging from harmful to helpful (Law, 2000).
Verotoxin-producing E. coli (VTEC) is an unusual E. coli strain. O26:H11 was the most prevalent VTEC isolated from sporadic instances of hemolytic uremic syndrome (HUS) (Foxman, 2010). Verotoxins, also known as Shiga toxin, initially found in Shigella dysentery. However, the genes for their manufacture are easily spread across E. coli strains by toxin-encoding bacteriophages (Herold et al., 2004). The antigenically unique toxins VT1 and VT2 are encoded by two individual special lysogenic lambdoid bacteriophages (933J and 933W) introduced into the chromosome of E. coli 933 (Nazmul et al., 2008). VT phages have also been found in strains belonging to serogroups O111, O119, O128, and O157 (Ranjbar et al., 2017). Although this virulence factor (VT) may be found in a variety of serotypes, only a few well-characterized bio-serotypes and clones, especially O157:H7 and O26:H11, are the most significant hosts for the verotoxin phage(s) and are distributed globally (Nazmul et al., 2007).
Infections produced by isolates of VTEC serotypes other than the O157 have received more attention in recent years. Non-O157 VTEC infections are becoming more common in several countries. Many non-O157:H7 isolates linked to outbreaks do not have eae or the pO157 plasmid (Gyles, 2007), suggesting more non-O157 VTEC strains are not yet non-pathogenic. As a result, enhancing our understanding of these organisms requires efficient detection, isolation, and characterization of non-O157 VTEC isolates. The universality of the Polymerase Chain Reaction (PCR) test for amplification of vt1 and vt2 gene sequences has been proven, with both toxin genes being identified and distinguished across a wide range of verotoxigenic E. coli. The PCR approach allows for the quick, reliable, and low-cost identification of these critical toxin genes in clinical samples and potentially contaminated foods (Hamed et al., 2017).
Recently all over the world, food safety issues are given much concern because of the increased number of foodborne illnesses (WHO, 2002;Peattie, 2006). According to World Health Organization (WHO), Foodborne illness is referred to as hazardous for human health when the pathogen-associated contaminated food enters the human body (WHO, 2002). VTEC isolates are commonly present in food. Predictions are made of the possible increase in problems associated with these emerging pathogens (Younus et al., 2020;Parvej et al., 2018). As time passed, the food habits of ordinary people have changed rapidly in Bangladesh. Nowadays it is more popular in this country to take a meal in a nearby hotel or restaurant rather than eating homemade food. Here in Bangladesh, Restaurant and hotels are not only just food places but also assessed as refreshment sites. In restaurants, hotels, roadside food carts, along with different fast or Bengali foods, salad are common for all types of dishes. Mainly seasonal vegetables are used for making this salad, such as tomato, carrot, cucumber, coriander leaf, radish, and green chili are very common among them (Younus et al., 2020).
Jashore is the third-largest city in the Khulna Division and one of the most populous cities in Bangladesh's southwestern area. It is the second most developed city in the Khulna Division and Bangladesh's most industrious and progressive city. According to the Jashore municipality's records, it has a population of around 0.298 million people. There are various roadside food stores or restaurants that offer a range of foods with a vegetable salad throughout the city. A vast variety of microorganisms, particularly verotoxin-producing E. coli, can be found in vegetables. This organism has been associated with outbreaks connected to fresh vegetable intakes, such as lettuce, spinach, carrots, sprouts, and alfalfa (Younus et al., 2020). According to outbreak research, E. coli may live and thrive in a variety of minimally processed vegetables (Kabir et al., 2014). It can also withstand freezing temperatures and extreme environmental conditions. Still now there is no study on the microbiological aspect of this vegetable salad in this region. It is necessary to evaluate the food safety issue of this served vegetable salad from a microbial aspect. So, this study was conducted for isolation, identification, and antibiogram of verotoxin-producing E. coli from raw salad vegetables at Jashore, Bangladesh.

Sample collection
In this study, raw vegetables were selected as they are often eaten raw without any heat treatment, sometimes without washing and peeling. Total 119 vegetable samples (25 carrots, 22 cucumbers, 27 tomatoes, 20 radishes, 15 coriander leaves, and 10 green chilies) were collected from different locations at Jeshore in Bangladesh. Sterile zip bags (wiped with 70% ethanol) were used to collect salad vegetables, and the samples were carried to the laboratory without any delay.

Isolation and identification
After processing the collected samples, the nutrient broth was used for preliminary enrichment of the bacteria where the incubation temperature was 37°C for overnight. Overnight incubated test tubes then steaked on MacConkey and Eosin Methylene Blue (EMB) (Both were HiMedia, India) agar media for isolation of the   (Punom et al., 2020).

Biochemical tests
Oxidase test and citrate utilization test were performed with culturepositive isolates according to the methods described in the Microbiology Laboratory Manual (Cheesbrough, 2006). Different sugar fermentation tests such as Dextrose, Mannitol, Lactose, Maltose, and Sucrose were done to confirm the organism (Punom et al., 2020).

Molecular detection
Genomic DNA was extracted by the conventional crude boiling method (Mishra et al., 2020). In short, 2-3 fresh cultured colonies were taken in a sterile 1.5 ml microcentrifuge tube and added 200 µl sterile Milli-Q water. Vortexed thoroughly and heated the microcentrifuge tube at 99°C for 10 min, followed by rapidly frozen at −20°C and centrifuged at 13,000 rpm for 5 min. Finally, 100 µl supernatant was collected and used as the DNA template in a PCR. The oligonucleotide primers used to amplify the genes by PCR are mentioned in Table 1, and the PCR reaction mixture was prepared as the author described in their study. The PCR products were visualized on a gel documentation system (Biometra, Jena, Germany) after electrophoresis with 1.5% agarose gel (SeaKem® LE Agarose from Lonza) as described by other researcher .

Antimicrobial susceptibility test
The antimicrobial susceptibility was carried out for all the phenotypically positive isolates using the standardized agar disc diffusion method recommended by the Clinical Laboratory and Standards Institute (CLSI, 2018). Commercially available disc and Mueller Hinton Agar (Both were HiMedia, India) were used for the antimicrobial assay. Total 10 antibiotics disc from a different group of antimicrobial agents (Amoxicillin, 10 µg; Azithromycin, 15 µg; Ceftriaxone, 30 µg; Chloramphenicol, 30 µg; Ciprofloxacin, 5 µg; Erythromycin, 15 µg; Gentamycin, 10 µg; Nalidixic acid, 30 µg; Streptomycin, 10 µg and Tetracycline, 30 µg) were used in this study. Interpretation of the test results was made according to the CLSI guidelines and recorded in an excel file.

RESULTS AND DISCUSSION
Out of 119 vegetable samples, 55 samples were positive for the presence of E. coli. These positive samples were phenotypically confirmed where cultural characteristics on different agar media, microscopic examination, and biochemical test results were the parameters. The present study showed that the overall prevalence of E. coli was 46.22% (n=55/119). The highest and lowest incidence of E. coli were 60% and 31.81% found in the radish and cucumber, respectively (Table 2). E. coli produced small, round, smooth, and greenish colonies onto EMB agar and pink-colored colonies on Mac-Conkey agar. They were Gram-negative and small rod-  shaped under microscopic view. Oxidase and Citrate tests were both negative for these isolates. All the suspected isolates fermented the sugars without producing any gas. The findings of microscopic exam and biochemical results were similar to the previously described in Punom et al. (2020), Younus et al., (2020 and Hasan et al. (2018). A total of 6 isolates (cucumber-6a, cucumber-15c, carrot-11a, radish-4a, coriander leaf-13d, and coriander leaf-14a) were confirmed verotoxin producing E. coli by amplification of the vt1 gene (Figure 1), but none of the samples were positive for vt2 and eaeA verotoxin producing genes. Antibiogram study of all E. coli isolates revealed that the highest was 83.64% (46), 78.18% (43), and 70.91% (39) isolates found resistant to Erythromycin, Streptomycin, and Ceftriaxone, respectively. 76.36% (42) and 69.09% (38) isolates showed susceptibility to Nalidixic acid and Amoxicillin antibiotics (Table 3 and Figure 2). Isolates of verotoxin-producing E. coli were found multidrug-resistant.
For maintaining a healthy life there is no alternative to take fresh fruit and vegetables. Because of their low fat and high vitamin, mineral contents, day by daily total consumption ratio is increasing (Rekhy and McConchie, 2014). Moreover, fresh fruit and vegetables are also prescribed to cardiovascular, cancer, diabetes-like diseased patients (Wang et al., 2014). But it must be ensured first that the consumable fruit and vegetables are free from all types of contamination. In this study, it had been found that Vegetable salad is working as a potential source of E. coli. It is predicted that foods are primarily contaminated during post-harvest handling and unhygienic processing environment. (Chowdhury et al., 2014;Alam et al., 2015). Moreover, the excess amount of use of antibiotics in farm areas is increasing the number of MDR organisms. It has been found that pesticide residue, which is used for agricultural purposes, may affect the body homeostasis of animals (Kobir et al., 2020).
During this investigation, one hundred nineteen raw vegetable samples were collected from a different market in Jashore, Bangladesh. Out of the 119 samples, E. coli was isolated from 55 samples, with a prevalence of 46% for this investigation. Isolation of E. coli from raw vegetables is also published in Dhaka city by several researchers (Islam et al., 2015;Kabir et al., 2014). But the prevalence ratio is relatively low. It might be the knowledge gap on the community level in this region, where farmers do not maintain the necessary hygienic measures during the production and harvesting of their products. The use of untreated wastewater and manure as fertilizers for producing fruits and vegetables is a major contributing factor to contamination (Rai and Tripathi, 2007). Also, restaurants, and hotel kitchens' hygienic condition is responsible for being so high prevalence in this area. Another source might be the middle man who is unconscious about the hygienic state, which plays a crucial role in contamination.
The vt gene was also detected in E. coli utilizing a PCR methodology and its molecular characterization in this investigation. In all, 55 E. coli isolates were tested for the presence of the vt gene using three sets of primers (VT1, VT2, and Eae). 6 (10.9%) E. coli isolates were discovered to possess the vt1 gene, but no isolates carried the vt2 and eaeA gene. According to these findings, the vt1 gene is the most frequent verotoxin found in vegetable strains. In bovine isolates, the vt1 gene is the most frequent toxin, while in porcine isolates, the vt2 gene is the most prevalent (Gyles, 2007). The presence of just the vt1 gene in this study might be related to the geographical dispersion of the isolates or members of a single clone that has spread globally.
The antibiotic susceptibility test yielded that the isolates exhibited resistance to Erythromycin 83.64% (46), Streptomycin 78.18% (43), Ceftriaxone 70.91% (39). Nalidixic acid 76.36% (42), Amoxicillin 69.09% (38), Tetracycline 63.64% (35) antibiotics showed the highest susceptibility to the isolates. Antibiotic pattern normally depends on the use of antibiotics on an area. For example, in the Chittagong district, one study says the E. coli isolates from salad samples are highly resistant to Gentamycin, Ampicillin, and Streptomycin, where our isolate showed only 38.18% (21) resistant to Gentamycin. Their isolate showed 100% sensitivity to Ciprofloxacin. But in our study, we found only 52.72% (29) susceptible to this antibiotic (Nipa et al., 2011). For any specific region, a class of antibiotics may be successful in any outbreak, whereas it may work as resistant in another region. To eradicate or successfully treat any outbreak, along with genetic information, location-specific information (data containing which antibiotic is mostly resistant or susceptible in any location) is compulsory . These developments of antibiotic resistance can be deadly to both humans and other animals. In the investigation, the highest resistance was found against Erythromycin and Streptomycin. Uncontrolled usage of antibiotics in the treatment of animals and their integration in animal feeds has been believed to account for the increase in antibiotic resistance (Kundu et  to the Penicillin group of antibiotics has been on the higher side and is increasing day by day in different parts of the world. Nalidixic acid, the most efficient antibiotic against the isolates, prevents protein chain elongation by inhibiting the peptidyl transferase activity of the bacterial ribosome.

Conclusion
It might be concluded from this study that the raw salad vegetables collected from different areas are contaminated with antibiotic-resistant E. coli. The highest resistance was found against Erythromycin 83.64% (46)