Molecular Characterization of E . coli Isolated from Raw Vegetable

Received: Revised: Accepted: 2013–11–1


INTRODUCTION
Use of animal manure for cultivation is a common practice and it is still considered to be better and safer than chemical fertilizers in India.Recently, association between fresh vegetables and outbreaks of food borne infections has led to a greater concern about contamination of vegetables with fecal pathogenic bacteria like E. coli, Vibrio, Salmonella, norovirus, Shigella, Listeria etc (Mahima et al., 2013) and safety of using animal manures as fertilizer in vegetable production (Pell, 1997;Tauxe et al., 1997).The animal faeces and manure contains number of bacteria that are zoonotic, such as Escherichia coli, Campylobacter spp.Salmonella spp.and Mycobacterium spp.(McGarvey et al., 2004;Dhama et al., 2013;Malik et al., 2013;Singh et al., 2013).Therefore, it is imperative to test the vegetables for indicator bacteria that are always present in bovine manure and have survival characteristics comparable to those of fecal enteric pathogens.The E. coli is one of the important enteric pathogens among the intestinal microflora of human and warm-blooded mammals.Hence, the present study was designed to study E. coli contamination of vegetables that are generally consumed raw or served as salad on dining table using conventional and molecular methods.

Collection and Preparation of Samples
The study was conducted on the vegetables that are generally consumed raw as salad.Total of 80 samples consisting 15 each of tomato, cucumber, radish and coriander leaves and 20 samples of green chilies were collected from the local retail shops.The samples were collected in sterile polybags and brought to laboratory.In laboratory, the samples were washed with sterile PBS under sterile conditions and the washings were allowed to stand for ten minutes.The supernatant was discarded; the sediment was collected in sterile glass vials and stored at 4 º C until processed for isolation of E. coli.

Isolation and Identification of E. coli
The enrichment was done by inoculating a loopful of the sediment into MacConkey lactose broth at 37 0 C for 18hr and then streaked on MacConkey Lactose Agar (MLA).Following 24 hr of incubation at 37 0 C, the lactose fermenting rose-pink colonies were picked up and streaked onto Eosin Methylene Blue (EMB) agar plates.The samples producing a greenish metallic sheen on EMB plates after 24 hr incubation at 37 º C, were transferred on to nutrient agar slants and further processed for biochemical identification as described by Cruickshank et al. (1975).The isolates were identified on the basis of their cultural, morphological and biochemical characters (Edwards and Ewing, 1972) and those preliminarily screened out as E. coli were serotyped at National Salmonella and Escherichia Center, Central Research Institute, Kasauli, H.P., India.

Preparation of DNA Template
The snap chill technique described by Ibenyassine et al. (2007) was used for preparation of DNA template for amplification of virulence genes.For this, 2ml nutrient broth culture of the field isolates was centrifuged at 4,000 rpm for 15 min.The supernatant was discarded; the pellet was suspended in 1ml of sterile triple glass distilled water (TGDW) and centrifuged further for 15min.The resultant pellet was suspended in 1 ml of TE buffer (pH 8.0) and centrifuged at 9,000 rpm for 6 min.The

Molecular Characterization of E. coli Isolated from Raw Vegetable
ISSN: 2307-8316 (Online); ISSN: 2309-3331 (Print) pellet so obtained was re-suspended in 500 µl of sterile TGDW, boiled for 10 min in a boiling water bath and then rapidly chilled at -20 º C. The supernatant was directly used as a template for further amplification of target sequence.

Primers
The published primers (Table 1) based on specific portion of mdh, elt, est, stx-1 and stx-2 genes of E. coli, were synthesized by Merk (India) and used in the present study.
The simplex PCR assays were carried out in 50µl of reaction mixture containing 25µl of 2X master mix (Merck), 5µl of DNA template, 10 picomoles of forward and reverse primers.
A negative control without template DNA was included in each experiment.PCR cycling was performed in a thermal cycler (Genie TC-3000) at initial denaturation for 3 min at 94 º C, followed by 35 cycles of 94 º C for 20 sec., annealing at 60 º C for 30 sec., extension at 72 º C for 30 sec and final extension at 72 º C for 5 minutes.The amplified product was electrophoresed on a 1.5% agarose gel in Tris-Borate-EDTA buffer at 80Volts.The gels were stained with ethidium bromide and the bands were visualized under UV gel doc system (UVtech, UK).1997;Buck andWalcott, 2003, Ibenyassine et al., 2007).There are reports that provide the evidence that E. coli could be transmitted to vegetables from manure (Wang and Doyle 1998;Natvig et al., 2002), contaminated irrigation water (Fattal et al., 1986;Rai and Tripathi, 2007) and direct contamination from animal and human faeces (Johannesen et al., 2002).Solomon et al. (2002) also reported the survival of E. coli O157:H7 on lettuce leaves for more than 20 days and concluded that the organism migrated to the internal location in plant tissue and was protected from action of sanitizing agent.
In the present study, E. coli isolates of serogroup O4, O5, O11, O17, O20, O60, O63, O69, O71, O116, O132, O138 and O147 were recovered from vegetable washings.Of these, O69 and O147 serogroups were also reported from cases of bovine diarrhea (Diwakar et al., 2008) and serogroups O17, O60, O69, O147 from poultry (Dwivedi et al., 2007;Yadav et al., 2009) from the same study area.Similarly, serogorup O5, O20, O60 and O138 have been reported from diarrheic pigs by Dutta et al. (2010).These findings clearly indicate the association of manure with the isolation of E. coli from vegetables.Besides this, serogroups O4, O20, O60 and O132 have been reported to be associated with human cases of urinary tract infections, pyelonephritis and renal failure (Johnson, 1991;Blanco et al., 1997;Johnson et al., 2005;Kausar et al., 2009).This indicates that the vegetables sold in domestic market pose serious public health risk as these are injured while peeling, slicing, shredding during salad preparation and usually consumed without heat treatment (Beuchat, 2002).Furthermore, the predominance of O69 serogroup in this study warrants a special attention to study their role in the development of diarrhea because serogroup O69 is an established shiga toxin producing E. coli strain (Griffin and Tauxe, 1991).Serogroup O20 obtained in this study is a well -known human enteric pathogen and has been isolated most frequently from the water in Coimbatore (Grover, 2006).
Conventional culture techniques are universally recognized as gold standard method for diagnosis of E. coli but this process is rather lengthy and may last 5-10 days or more.Many foods with short shelf lives would have been consumed by the time the result of analysis becomes available.Hence, the development of simple and rapid assays would enable identification of E. coli in contaminated foodstuffs in a timelier manner.Therefore, an attempt was made to detect E. coli in vegetable washings using E. coli mdh gene primer with minimum amount of time.The PCR amplification of mdh gene gave positive result in all 43 isolates and this was in conformity with the findings of Ibenyassine et al. (2007).Hsu and Tsen (2001) also used mdh gene for direct detection of E. coli from milk and water samples.The enzyme malate dehydrogenase from E. coli catalyzes the interconversion of malate and oxaloacetate, a critical step in carbohydrate metabolism (Wright and Viola, 2001) and thus, detection of its gene can specifically be used for direct detection of E. coli in vegetable washings (Ibenyassine et al., 2007).
In India, isolation of STEC has been reported from cattle (Pal et al., 1999), sheep (Wani et al., 2004;Bhat et al., 2008), fish (Kumar et al., 2001), human faeces (Khan and Ner, 2002) and piglets (Dutta et al., 2010).But the information related to the prevalence of STEC in vegetables is rare.Hence, in the present study, the isolates were screened for presence of stx genes to establish their STEC nature.The stx1 gene was detected in two isolates both of serogroup O65, however, stx2 gene was demonstrated in 12 isolates that were representative of O69 ( 9) and rough strains (3).None of the isolates showed the amplification of both stx1 and stx2 genes.However, Dutta et al. (2010) reported the presence of stx1 as well as stx2 gene in O60 and O138 serogroups isolated from an organized pig farm in Kolkata.Toma et al. (2003) have reported stx gene in the serogroups O15, O28, O111, O121, O145 and O157 and placed them in STEC category.The STEC strains have been characterized by production of either one or both of the two major types of Stx proteins designated as shiga like toxin-1 (stx1toxin) and shiga like toxin-2 (stx2 toxin) (Rangel and Sparling, 2005) and the production of the later is associated with increased risk of developing Haemolytic Uraemic Syndrome (Boerlin et al., 1999).STEC strains have also been isolated from a number of environmental sources (Rangel and Sparling, 2005) that may result in contamination of vegetables.Detection of stx1 and stx2 genes only in two and nine isolates, respectively in present study may also be attributed to loss of virulence genes during subculture as reported by Karch et al. (1992).

Table 2 :
Toni et al. (2006)ssociated with raw vegetables In a study,Toma et al. (2003)compared the efficacy of simplex and multiplex PCR to categories ETEC strains by targeting elt and est genes.The present study indicated that ETEC strains belonged to the serogroup O26, O27, O126 and O128.Both the genes were also detected in six untypable strains which were in conformation with our findings.Toni et al. (2006)reported presence of elt gene in O149 and est gene in O8, O20, O64, O101 and O149 serogroups.Presence of these genes indicate the enterotoxigenic nature of these isolates and such ETEC isolates were reported to have the ability to cause profuse, watery diarrhea by release of LT, ST or both enterotoxins