Close contact of vegetables with soil, polluted water, and animal manure and unsanitary conditions during processing of restaurant salads led us to study the distribution of virulence factors, O-serogroups, and antibiotic resistance properties in Shiga toxigenic Escherichia coli (STEC) isolated from vegetables and salads. Samples of vegetables and salad (n =420) were collected and evaluated for the presence of E. coli using culture and a PCR assay. Total prevalence of E. coli in studied samples was 49.5%. E. coli was found in 49.6% of vegetable samples and 49% of salad samples. Leek and traditional salad had the highest incidence of E. coli. Significant differences in the incidence of E. coli were found between the hot and cold seasons. Of the 149 E. coli isolates from vegetable samples, 130 (87%) were STEC, and of the 59 E. coli isolates from salad samples, 50 (84%) were STEC. The most commonly detected virulence factors were stx1 and eaeA. A significant difference was found between the frequency of the attaching and effacing and the enterohemorrhagic E. coli subtypes. Serogroups O26 (46% of isolates), O157 (14%), O121 (10%), and O128 (9%) were the most commonly detected serogroups among the STEC strains. The tetA, sul1, aac(3)-IV, dfrA1, blaSHV, and CITM antibiotic resistance genes were found in 96, 47.7, 90, 51, 27, and 93% of isolates, respectively. The highest levels of resistance were found against ampicillin (96.6% of isolates), tetracycline (87%), and gentamicin (90%). This study shows the importance of vegetables and salads as potential sources of E. coli infection.

According to the Centers for Disease Control and Prevention (5), an estimated 76 million people become sick, more than 325,000 are hospitalized, and 5,000 die from foodborne illness each year in the United States. Foodborne illness costs the nation $10 billion to $83 billion each year in pain and suffering, lost efficiency, and medical expenditures (5). Some outbreaks of foodborne disease in recent years have been associated with consumption of contaminated vegetables and restaurant salads (5, 18). Consumption of vegetables and salads contaminated with Escherichia coli may lead to outbreaks of severe foodborne diseases and serious clinical complications (10, 24).

Vegetables are in close contact with soil and animal manure and are sometimes irrigated with polluted water. Because polluted water (12), soil (17), human feces (8), and animal manure (22, 27) are the main sources for E. coli, vegetables can easily become contaminated in the field. Cross-contamination can also occur during harvest and processing.

Shiga toxigenic Escherichia coli (STEC) infections can result in intensive clinical complications such as lethal hemolytic uremic syndrome and hemorrhagic colitis (13). Outbreaks of foodborne diseases and their complications are associated with certain STEC O-serogroups such as O26, O103, O113, O157, O121, O128, O145, O91, O45, and O111 and with untypeable strains (19, 20).

To appraise the pathogenicity of STEC strains, latent virulence factors must be assessed. The genes most frequently associated with STEC infections are those for Shiga toxins (stx1 and stx2), intimin (eaeA), and hemolysin (hlyA) (21).

In addition to O-serogroups and virulence factors, drug resistance is an important epidemiologic characteristic of STEC strains because therapeutic options are limited in cases of infection with multidrug resistant strains (7, 19–21). Antibiotic-resistant STEC strains are known to harbor antibiotic resistance genes and can cause more severe diseases in humans and animals (7, 15, 20).

Because of increasing consumption of vegetables and salad and the uncertain microbiological status of these products in Iran, the present study was conducted to investigate the prevalence of E. coli in various types of vegetables and restaurant salads and the distribution of O-serogroups and virulence factors and the antibiotic resistance properties of these isolates.

Samples and E. coli isolation.

From September 2012 to September 2013, 300 samples of leek (n = 50), radish (n = 50), basil (n = 50), parsley (n = 50), spinach (n = 50), and lettuce (n = 50) and 120 samples of commercial and traditional whole salad were collected from supermarkets and groceries in various areas of Khozestan Province, Iran. All samples were immediately transported to the Food Hygiene and Public Health Research Center (Islamic Azad University, Shahrekord Branch, Iran).

Each 10-g sample of crushed vegetable or salad was homogenized for 2 min in 90 ml of peptone water (Merck, Darmstadt, Germany), and cultures were grown on 5% sheep blood and MacConkey agar (Merck) for 18 to 24 h at 37°C. Colonies with the typical color and appearance of E. coli were picked and streaked again on blood agar plates and then restreaked on EMB agar (Merck). Colonies with a green metallic sheen were presumed to be E. coli. These colonies were then subjected to biochemical tests for growth on triple sugar iron agar and lysine iron agar, oxidative and fermentative degradation of glucose, citrate utilization, urease production, indol fermentation, tryptophan degradation, glucose degradation (methyl red test), and motility. Colony identity was confirmed using a PCR assay based on the detection of the E. coli 16S rRNA gene described by Sabat et al. (25).

DNA extraction.

Bacterial strains were subcultured overnight in Luria-Bertani broth (Merck), and genomic DNA was extracted using a DNA extraction kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions.

Detection of O-serogroups, virulence factors, and antibiotic resistance genes in STEC isolates.

Table 1 shows the primers used for detection of serogroups, virulence genes, and antimicrobial resistant genes in the STEC isolates and the PCR programs and conditions. A DNA thermocycler (FlexCycler 2, Eppendorf, Hamburg, Germany) was used for all PCRs. The PCR amplification products (10 μl) were subjected to electrophoresis in a 1% agarose gel in 1× Tris-EDTA buffer at 80 V for 30 min and then stained with ethidium bromide. All runs included PCR grade water as a negative DNA control and E. coli strains O157:K88ac:H19, CAPM 5933 and O159:H20, CAPM 6006 as positive controls.

TABLE 1.

Oligonucleotide primers and PCR programs used for amplification of O-serogroups, virulence factors, and antibiotic resistance genes of Escherichia coli isolates from vegetables and salads

Oligonucleotide primers and PCR programs used for amplification of O-serogroups, virulence factors, and antibiotic resistance genes of Escherichia coli isolates from vegetables and salads
Oligonucleotide primers and PCR programs used for amplification of O-serogroups, virulence factors, and antibiotic resistance genes of Escherichia coli isolates from vegetables and salads

Antimicrobial susceptibility testing.

The Kirby-Bauer disk diffusion method using Mueller-Hinton agar (Hi Media Laboratories, Mumbai, India) was used to determine antimicrobial susceptibility according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (6). After incubating the inoculated plate at 37°C for 18 to 24 h in an aerobic atmosphere, the susceptibility of the E. coli isolates to each antimicrobial agent was determined, and the results were interpreted in accordance with interpretive criteria provided by CLSI (6). E. coli ATCC 25922 was used as a quality control strain for antimicrobial susceptibility determinations.

Statistical analysis.

Data for serogroups, virulence factors, and antibiotics resistance properties of E. coli isolates from vegetable and salad samples were analyzed for significant relationships using SPSS/16.0 software (SPSS, IBM, Armonk, NY). Differences were considered significant at P < 0.05.

Of the 420 samples tested, 208 (49.5%) were positive for E. coli: 149 (49.6%) of 300 vegetable samples and 59 (49%) of 120 restaurant salad samples. The most commonly contaminated samples were leek (66% of samples) and traditional salad (70% of samples). A significant difference (P = 0.008) in the incidence of E. coli was found between traditional and commercial salad samples. Vegetable samples were more commonly contaminated in spring (59% of samples) followed by summer (21%) and autumn (13%), whereas salads more commonly contaminated in summer (71% of samples) followed by spring (15%) and autumn (10%) (Table 2). Significant differences (P =0.037) in the incidence of E. coli were also found between the hot and cold seasons.

TABLE 2.

Seasonal distribution of Escherichia coli in vegetables and salads samples

Seasonal distribution of Escherichia coli in vegetables and salads samples
Seasonal distribution of Escherichia coli in vegetables and salads samples

The attaching and effacing E. coli (AEEC) STEC subtype was commonly detected in both vegetables (69.2% of samples) and salads (60% of samples) (Table 3), and a significant difference was found between the frequency of AEEC and enterohemorrhagic E. coli (EHEC) STEC subtypes (P = 0.047). Of the 149 E. coli isolates from vegetable samples, 130 (87.2%) were STEC, and of the 59 E. coli isolates from salad samples, 50 (84.7%) were STEC. All of the EHEC isolates from vegetables and salads harbored the stx1, eaeA, and ehly virulence genes; stx1 was the most commonly detected virulence factors followed by eaeA. We found significant associations between the incidence of stx1 and ehly (P = 0.014), eaeA and ehly (P = 0.015), and eaeA and stx2 (P = 0.016).

TABLE 3.

Distribution of virulence factors in Shiga toxin-producing Escherichia coli subgroups isolated from vegetable and salad samples

Distribution of virulence factors in Shiga toxin-producing Escherichia coli subgroups isolated from vegetable and salad samples
Distribution of virulence factors in Shiga toxin-producing Escherichia coli subgroups isolated from vegetable and salad samples

The most commonly detected serogroups among the STEC strains were O26 (46% of isolates), O157 (14%), O121 (10%), and O128 (9%) (Table 4). Statistical analyses indicated a significant association between the incidence of serogroups O157 and O26 (P = 0.045) and O157 and O103 (P = 0.027) in vegetables and between serogroups O26 and O157 (P =0.012) and O26 and O103 (P =0.020) in salads.

TABLE 4.

Distribution of O-serogroups in STEC strains of vegetables and salads

Distribution of O-serogroups in STEC strains of vegetables and salads
Distribution of O-serogroups in STEC strains of vegetables and salads

Antibiotic resistance patterns and the distribution of antibiotic resistance genes in STEC isolates from vegetable and salad samples are shown in Table 5. The genes encoding resistance against tetracycline (tetA), sulfonamide (sul1), gentamicin (aac(3)-IV), trimethoprim (dfrA1), cephalothin (blaSHV), and ampicillin (CITM) were found in 96, 47.7, 90, 51, 27, and 93% of STEC isolates, respectively (Table 5). The highest levels of resistance were found against ampicillin (96.6% of isolates), tetracycline (87%), and gentamicin (90%) (Table 5). Significant differences were found between the incidences of tetA and tetB (P = 0.037), sul1, cat1, and cmlA (P =0.028), and CITM, cat1, and cmlA (P = 0.030). We also found significant differences in the incidence of resistance to tetracycline and imipenem (P = 0.007), ampicillin and chloramphenicol (P = 0.009), gentamicin and ciprofloxacin (P = 0.014), tetracycline and nitrofurantoin (P =0.017), and ampicillin and lincomycin (P = 0.018).

TABLE 5.

Antimicrobial resistance properties in Shiga toxin–producing Escherichia coli isolated from vegetables and salads

Antimicrobial resistance properties in Shiga toxin–producing Escherichia coli isolated from vegetables and salads
Antimicrobial resistance properties in Shiga toxin–producing Escherichia coli isolated from vegetables and salads

The high prevalence of E. coli in vegetables (50% of samples) and salads (49% of samples) in Iran and the high prevalence of the STEC O157, O145, O103, O26, O111, O91, O128, O121, O113, and O45 serogroups, which are associated with virulence factors and resistance to commonly used antibiotics, indicate a public health issue facing consumers.

Among various types of vegetables, lettuce has been considered an important source of STEC (30, 31, 35). One possible reason for the high prevalence of E. coli isolates in leek, radish, and lettuce is that cultivation of these vegetables requires large amounts of animal manure, which is one of the main sources of STEC. The shape and morphological characteristics of these vegetables also may promote the accumulation and survival of bacteria. The high incidence of STEC in restaurant salads can be attributed to primary contamination of vegetables used for salad preparation and cross-contamination during processing.

Molecular evaluation of STEC O-serogroups in salads and vegetables revealed that the most common O-serogroups were O26 (46% of isolates), O157 (14%), O121 (10%), and O128 (9%). The prevalence of the O157 serogroup was higher in the vegetable samples than in the salad samples. STEC O157 strains are of animal origin, and animal manure and polluted water are the most common vehicles for contamination of vegetables. Previous outbreaks of food-borne illness due to consumption of vegetables were also caused by the STEC O157 isolates (9, 28). STEC O26, O103, O111, O121, and O128 strains are associated with approximately 75% of total non-O157 STEC illnesses in the United States annually (4, 34). Dehkordi et al. (7) found the prevalence of STEC O157, O26, O103, O111, O121, and O128 to be 26, 12, 6, 6, 4, and 8%, respectively. Tzschoppe et al. (33) found STEC O26, O103, O111, O121, O145, and O157 in ready-to-eat vegetables, and Momtaz et al. (19) found that these serogroups were the most common STEC strains in meat samples.

Contaminated water used in the processing of restaurant salads can infect people working in restaurants and food service areas. Use of contaminated equipment and poor public and personal hygiene are also factors that can explain the high prevalence of STEC strains in restaurant salads.

Seasonal distribution of STEC strains in salad and vegetable samples in our study could indicate seasonal variation in sources of contamination or growth opportunities. The high incidence of STEC strains in summer salad samples (71%) could be related to poor personal hygiene in this season, whereas climate variables such as heat, rain, and variations in barometric pressure may have contributed to the high incidence of STEC strains in the spring vegetable samples (60%). Alternatively, the higher prevalence of STEC strains may be related to faster bacterial growth in hot seasons. Results of other studies (1, 2, 37) have indicated a seasonal distribution for E. coli, with the highest numbers of infection cases occurring during the warmer months (1, 2).

The c7ommon presence of STEC virulence factors has been reported in other investigations (19–21). In our study, the stx1 and eaeA genes were found in the majority of STEC isolates from vegetables and salads. Momtaz et al. (19) found these genes in 91% of vegetable samples and 89% of salad samples. The stx genes are most commonly associated with diarrhea or asymptomatic excretions (3, 11). However, STEC strains containing stx and eae genes can be associated with severe clinical illness, hemolytic uremic syndrome, and hemorrhagic colitis (3, 11). In our study, 100% of EHEC strains were positive for the stx1, eaeA, and ehly genes and 12 to 14% of AEEC strains were positive for the stx1, stx2, and eaeA genes. Kilic et al. (14) reported that 48% of E. coli isolates from food samples contained the eaeA gene. Lukášová et al. (16) found that of 22 STEC isolates from food stuffs, 9 (41%) were positive for the stx1, stx2, eaeA, and ehxA virulence genes. The results reported by Momtaz and others (19–21) are consistent with our findings.

The STEC isolates found in our study were resistant to cephalothin, trimethoprim, ampicillin, tetracycline, ciprofloxacin, gentamicin, and streptomycin, all antibiotics commonly used in veterinary and human medicine. Among the STEC isolates from vegetables and salads, the highest incidence of resistance genes were for those encoding resistance to ampicillin (CITM, 93% of isolates), tetracycline (tetA, 96%), and gentamicin (aac(3)-IV, 90%). Similar results have been reported by Dehkordi et al. (7), Srinivasan et al. (32), Momtaz et al. (21), and Rao et al. (23). In a study in Kenya, STEC strains of food stuffs had the highest levels of resistance to tetracycline (76%), which was lower than the resistance found in our samples (91%). Prevalence of resistance to ampicillin, ciprofloxacin, tetracycline, and gentamicin in the reported by Dehkordi et al. (7) and Momtaz et al. (19) were 30, 6, 80, and 55% and 38, 14, 84, and 34%, respectively, which were similar to our data. The prevalence of resistance to chloramphenicol and nitrofurantoin in our study was 9 and 18%, respectively. High levels of resistance among STEC strains to several antibiotics, especially chloramphenicol and nitrofurantoin, was reported by Walsh et al. (36), Solomakos et al. (29), and Schroeder et al. (26).

Vegetables and salads harbor STEC strains with different seasonal distributions and incidences of main O-serogroups. Our results indicate that vegetables are more likely to be the source of E. coli O157, whereas restaurant salads are more likely to be the source of E. coli O26, O121, and O128. Vegetables and restaurant salads in Iranian markets harbored multidrug resistant and virulent strains of E. coli. Therefore, consumption of these foods may put consumers at increased risk for severe gastrointestinal disorders that cannot be treated using common antimicrobial agents.

The authors thank Prof. A. Akhondzadeh Basti (Department of Food Hygiene, College of Veterinary Medicine, University of Tehran) and Prof. F. Safarpoor Dehkordi (Department of Food Hygiene and Quality Control, University of Tehran) for their important technical and clinical support. This work has been supported by the Islamic Azad University grant AD799105/2014.

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