Presence of Multidrug-Resistant Shiga Toxin-Producing Escherichia coli,Enteropathogenic E. coli and Enterotoxigenic E. coli,on Raw Nopalitos ( Opuntia ficus-indica L.) and in Nopalitos Salads from Local Retail Markets in Mexico

Abstract

The presence of multidrug-resistant pathogenic bacteria in food is a significant public health concern. Diarrheagenic Escherichia coli pathotypes (DEPs) are foodborne bacteria. In Mexico, DEPs have been associated with diarrheal illness. There is no information about the presence of multidrug-resistant DEPs on fresh vegetables and in cooked vegetable salads in Mexico. “Nopalitos” (Opuntia ficus-indica L.) is a Cactacea extensively used as a fresh green vegetable throughout Mexico. The presence of generic E. coli and multidrug-resistant DEPs on raw whole and cut nopalitos and in nopalitos salad samples was determined. One hundred raw whole nopalitos (without prickles) samples, 100 raw nopalitos cut into small square samples, and 100 cooked nopalitos salad samples were collected from markets. Generic E. coli was determined using the most probable number procedures. DEPs were identified using two multiplex polymerase chain reaction procedures. Susceptibility to 16 antibiotics was tested for the isolated DEP strains by standard test. Of the 100 whole nopalitos samples, 100 cut nopalitos samples, and 100 nopalitos salad samples, generic E. coli and DEPs were identified, respectively, in 80% and 10%, 74% and 10%, and 64% and 8%. Eighty-two DEP strains were isolated from positive nopalitos samples. The identified DEPs included Shiga toxin–producing E. coli (STEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC). All isolated strains exhibited resistance to at least six antibiotics. To the best of our knowledge, this is the first report of the presence of multidrug-resistant and antibiotic resistance profiles of STEC, ETEC, and EPEC on raw nopalitos and in nopalitos salads in Mexico.

Introduction

The tender young pads of the Cactacea Opuntia ficus-indica L. and Nopalea species, known as “nopalitos,” are extensively used as a fresh green vegetable throughout Mexico, Central America, and in some parts of the United States (Russell and Felker, 1987). Opuntia is a succulent plant with tissues specialized for water storage (Russell and Felker, 1987). Nopalitos, as other vegetables, have low protein and fat, but the crude fiber is higher than that in most other vegetables (Hamdi, 2006). In addition, they contain minerals such as P⁺⁵, Fe⁺³, Ca⁺², Mg⁺², Mn⁺², Zn⁺², Na⁺, and K⁺ (Hamdi, 2006; Hernández-Urbiola et al., 2010). Analyses showed that nopalitos contain 17 different amino acids, 9 of which are considered essential. Also the analyses showed different levels of amino acids content related to different maturity stages of the plant (Hernández-Urbiola et al., 2010).

Nopalitos are a major commercial crop in Mexico, accounting for more than 824,602 tons of production in 2014 (SAGARPA, 2015). This Cactacea is commonly consumed in a raw state (e.g., juice, liquefied with other vegetables, and green salads) and in cooked state (e.g., roasted, omelets and salads mixture with raw vegetables among others), both in Mexico and in other countries. However, no nopalitos outbreaks of pathogenic bacteria such as diarrheagenic Escherichia coli pathotypes (DEPs) have been reported. Nevertheless, a recent outbreak in several European countries of foodborne illness from entero-aggregative-hemorrhagic E. coli originating in sprouts (Buchholz et al., 2011) highlights the importance of screening for DEPs in vegetables such as nopalitos. DEPs are foodborne pathogens (Kaper et al., 2004) and the Shiga toxin–producing E. coli (STEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC) are an important cause of diarrhea in developed countries and in visitors from regions where DEPs are not endemic (Nataro and Kaper, 1998; Estrada-García et al., 2005, 2009; Paniagua et al., 2007). In Mexico, DEPs have been associated with diarrheal illness in both native children (Estrada-García et al., 2005, 2009; Paniagua et al., 2007) and visitors to the country (Paredes-Paredes et al., 2011).

The emergence of antibiotic-resistant pathogenic bacteria is associated with the use of antibiotics in animals raised for food. Resistant bacteria can be transmitted to humans through foods, particularly those of animal origin. However, the presence of multidrug-resistant pathogenic bacteria in raw vegetables and minimally processed fresh vegetables is a significant public health concern (EFSA, 2008). No data exist on the presence of multidrug-resistant DEPs on vegetables such as nopalitos in México. The objective of this study was to measure the presence of generic E. coli and multidrug-resistant DEPs (enteroinvasive E. coli [EIEC], STEC, EPEC, ETEC, and enteroagregative E. coli [EAEC]) on raw whole and cut nopalitos, and in cooked nopalitos salads from public markets in Mexico.

Materials and Methods

Nopalitos samples

A total of 100 raw whole nopalitos, 100 raw nopalitos cut into small squares, and 100 cooked nopalitos salad samples were collected during an 8-month study period. Nopalitos samples were purchased from five vegetable retailers in each of four public markets (20 retailers). Samples were purchased every 2 weeks from each retailer, resulting in 20 samples per day. Each raw whole nopalitos sample consisted of four nopalitos without prickles (average weight 200 g for sample [∼50 g per nopalito]) and with similar shape and weight. For cut raw nopalitos and cooked nopalitos salads each sample weighed ∼200 g. Nopalitos salad samples consisted of cooked nopalitos (principal ingredient) cut into small squares mixed with raw cut vegetables (coriander, tomato, and onion), grated fresh cheese, and salt. At the time of purchase, whole nopalitos, cut nopalitos, and nopalitos salads were in plastic containers and sold at room temperature. Samples were placed in sterilized plastic bags and then in a cooler containing frozen gel packs for transport to the laboratory. They were analyzed no more than 1 h after purchase.

Samples preparation

One liter of sterilized Lactose Broth (LB; Bioxon, Mexico City, Mexico) was added to each whole nopalitos sample. For cut nopalitos and nopalitos salad, subsamples (100 g) from each sample were placed in a sterile plastic bag and 900 mL of LB was added to each nopalitos sample. The samples and subsamples were manually rubbed for 2 min while in the sealed bag, and sample and subsample dilutions for microorganism counts were prepared using sterilized peptone water (SPW, 0.1%). Samples and subsamples were analyzed to detect the presence of generic E. coli, STEC, EPEC, ETEC, EIEC, and EAEC as previously described (Gómez-Aldapa et al., 2013; Rangel-Vargas et al., 2015).

Microbiological analyses

Generic E. coli were analyzed by the most probable number (MPN) procedure exactly as described in the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) (US-FDA, 2013). In brief, 1 mL of serial dilutions in 0.1% SPW of each sample or subsample homogenate was inoculated into nine tubes containing LB and into Durham tubes. After incubation at 35°C/48 h, a loopful of positive culture suspension (determined by turbidity and gas production) was transferred to tubes containing Brilliant Green 2% Bile LB (BRILA-Broth) (Bioxon). After incubation at 44.5 ± 0.2°C for 48 h, the tubes that were positive for growth and gas production were used to identify indole formation (US-FDA, 2013). All tubes that were positive for indole and gas production were streaked onto eosin methylene blue agar (EMB; Bioxon). Two to three presumptive E. coli colonies were selected from EMB plates and biochemically characterized by the indole, methyl red, Voges–Proskauer, citrate tests (IMViC; Bioxon). Biochemical confirmation of presumptive E. coli isolates was done with the API 20-E test (bioMérieux, Hazelwood, MO). Generic E. coli levels were calculated using the MPN tables described in BAM (US-FDA, 2013). We isolated one to three E. coli strains from each E. coli-positive tube of BRILA-Broth. It is known that the confirmation of only one colony as E. coli is sufficient to regard that broth tube as positive for E. coli. These positive tubes were used to calculate the E. coli concentrations as described in BAM (US-FDA, 2013). All confirmed E. coli strains were streaked on tryptic soy agar slants, incubated at 37°C for 24 h, and maintained at 3–5°C until they were used for polymerase chain reaction (PCR).

Multiplex PCRs for DEPs locus identification

All E. coli strains isolated and confirmed by biochemical tests were analyzed using two multiplex PCRs to identify several genetic loci associated with diarrheagenic E. coli exactly as previously described (Gómez-Aldapa et al., 2013). In brief, the first multiplex PCR identifies the following loci: heat-stable and heat-labile enterotoxins (st and lt) for ETEC, intimin (eaeA) and bundle-forming pilus (bfp) for EPEC, Shiga toxins 1 and 2 (stx1, stx2) and intimin (eaeA) for STEC, and invasion-associated loci (ial) for EIEC. The second multiplex PCR identifies three EAEC plasmid-borne virulence genes, including the master regulon (aggR), dispersin (aap), and the autotransporter Tol C (aatA). STEC isolates were further characterized by expression of the O157 lipopolysaccharide and H7 flagellar antigens using only the E. coli O157:H7 latex agglutination test kit (RIM^® E. coli O157:H7 Latex Test Kit; Remel, Lenexa, KS). All reagents, nucleoside triphosphates, primers, and Taq polymerase used in PCRs were from Invitrogen (AccesoLab, Guadalajara, México) and a thermal cycler Px2 (Thermo Electron Corporation, Beverly, MA) was used. We used positive control strains (ETEC, EIEC, EPEC, STEC, and EAEC) in all PCR tests, they were isolated from patients and were donated by Dr. Cerna from the Escuela Nacional de Ciencias Biologicas, IPN, México; the negative control strain was E. coli ATCC 25922.

The MPN for STEC, EPEC, and ETEC was calculated following the MPN method in BAM (US-FDA, 2013). As we described previously, PCR confirmation of only one E. coli strain as a DEP was sufficient to regard the original broth tube from which was isolated the E. coli strain analyzed as positive for STEC, EPEC, or ETEC. Thus, we used this information to calculate the DEP concentration using the MPN tables (US-FDA, 2013).

Antibiotic susceptibility testing

All DEP isolates were screened for antibiotic sensitivity against 16 different antibiotics using disk diffusion tests as described by the Clinical and Laboratory Standards Institute (CLSI, 2009, 2014). Antibiotic type and their concentration were as follows: amikacin 30 μg/mL, amoxicillin/clavulanic acid 20/10 μg/mL, ampicillin 10 μg/mL, ceftriaxone 30 μg/mL, chloramphenicol 30 μg/mL, ciprofloxacin 5 μg/mL, colistin 10 μg/mL, erythromycin 15 μg/mL, gentamicin 10 μg/mL, kanamycin 30 μg/mL, nalidixic acid 30 μg/mL, neomycin 10 μg/mL, streptomycin 10 μg/mL, sulfisoxazole 250 μg/mL, tetracycline 30 μg/mL, and trimethoprim-sulfamethoxazole 1.25/23.75 μg/mL. All antibiotics were purchased from Sigma Chemical (St. Louis, MO).

Results and Discussion

Of the three different groups of samples, namely, 100 whole nopalitos, 100 cut nopalitos, and 100 nopalitos salad, generic E. coli and DEPs were identified, respectively, in 80% and 10%, 74% and 10%, and 64% and 8%. In these samples, the minimum, median, and maximum levels of E. coli concentrations were, respectively, <3, 7.4, and 1100 MPN/g; <3, 3, and 240 MPN/g; and <3, 3, and 1100 MPN/g. Generic E. coli was isolated from 218 samples (80%). A total of 820 E. coli strains were isolated from these 218 nopalito samples: 320, 280, and 220 E. coli strains, respectively, from whole nopalito samples, cut nopalito samples, and nopalito salad samples. All these 820 E. coli isolates were analyzed by PCR to identify DEP.

Thus, 82 strains were identified as DEPs from all 820 E. coli isolates. This is because one DEP strain was isolated from each positive tube to DEP obtained by the MPN procedure. Identified DEPs included STEC (36 strains), EPEC (25 strains), and ETEC (21 strains). These were isolated, respectively, from 12%, 8%, and 8% of all nopalito samples (Table 1). In positive samples, DEP concentration ranged from 3.6 to 1100 MPN/g (Table 1). No E. coli O157:H7 was detected in any STEC-positive sample. For the STEC, stx2 locus alone was detected in 17 strains and the stx1 locus was detected in 19 strains. The eaeA locus was not detected in any STEC strain. STEC strains are human pathogens associated with foodborne illness; some can cause hemorrhagic colitis, which in some cases may progress to hemolytic uremic syndrome (HUS) (Nataro and Kaper, 1998). Pathogenesis of STEC is linked to different virulence factors such as Shiga toxins (stx1 and stx2) (Nataro and Kaper, 1998). It has been reported that stx1 and stx2 cause different degrees and types of tissue damage (Louise et al., 1995). Stx2 is more toxic than stx1 to human renal endothelial cells (Louise et al., 1995). Other important virulence determinants in STEC include the locus of gene encoding the outer membrane adhesin, intimin (eae), which mediates tight contact between STEC or EPEC and intestinal epithelial cells (Ito et al., 2007). However, some eaeA-negative strains can cause HUS and occasional outbreaks (Bonnet et al., 1998; Nataro and Kaper, 1998; Paton et al., 1999; Feng et al., 2001; Karch et al., 2005). These eaeA-negative STEC strains are postulated to have other putative adherence and virulence-associated factors (Paton et al., 1999; Ethelberg et al., 2004; Cookson et al., 2007).

Table 1.

Identified Diarrheagenic Escherichia coli Pathotype and Locus, DEP Concentration, and Number of DEP Isolates from Samples of Raw Whole Nopalitos, Raw Cut Nopalitos, and Cooked Nopalitos Salad Samples

Nopalitos type	Code of positive sample	DEP identified	Identified locus	Concentration (MPN/g)	Number of DEP isolates from sample ^a	Antibiotic resistance (number of antibiotics)–resistance pattern
Whole	W1	STEC	stx1	9.2	2	(13)-AMC-AMK-COL-ERY-GEN-K-NEO-STR-CIP-TCY-SOX-AMP-SXT
	W2	STEC	stx1	15	3	(9)-AMC-AMK-COL-ERY-GEN-K-NEO-NAL-TYC
	W3	STEC	stx1	7.4	2	(10)-AMC-AMK-COL-ERY-GEN-K-NEO-STR-AMP-SXT
	W4	STEC	stx2	1100	9	(7)-AMC-AMK-COL-ERY-GEN-K-NEO
	W5	EPEC	eaeA	11	3	(12)-AMC-AMK-COL-ERY-GEN-K-NEO-STR-CIP-SOX-NAL-SXT
	W6	EPEC	eaeA	7.4	2	(8)-AMC-AMK-COL-ERY-GEN-K-NEO-STR
	W7	EPEC	eaeA	93	5	(7)-AMC-AMK-COL-ERY-GEN-K-STR
	W8	ETEC	St	36	6	(7)-AMC-AMK-COL-ERY-GEN-K-STX
	W9	ETEC	St	3.6	1	(11)-AMC-AMK-COL-ERY-GEN-K-NEO-STR-CIP-TCY-SXT
	W10	ETEC	St	43	4	(7)-AMC-AMK-COL-ERY-GEN-K-STX
Cut	C1	STEC	stx1	3.6	1	(13)-AMC-AMK-COL-ERY-GEN-K-NEO-STR-CIP-TCY-SOX-AMP-SXT
	C2	STEC	stx1	3.6	1	(10)-AMC-AMK-COL-ERY-GEN-K-NEO-STR-AMP-SXT
	C3	STEC	stx1	3.6	1	(13)-AMC-AMK-COL-ERY-GEN-K-NEO-STR-CIP-TCY-SOX-AMP-SXT
	C4	STEC	stx2	3.6	1	(9)-AMC-AMK-COL-ERY-GEN-K-NEO-NAL-TYC
	C5	STEC	stx2	240	6	(6)-AMC-AMK-COL-ERY-GEN-K
	C6	EPEC	eaeA	28	5	(8)-AMC-AMK-COL-ERY-GEN-K-NEO-STR
	C7	EPEC	eaeA	3.6	1	(8)-AMC-AMK-COL-ERY-GEN-K-NEO-STR
	C8	ETEC	st	3.6	1	(7)-AMC-AMK-COL-ERY-GEN-K-STX
	C9	ETEC	st	3.6	1	(7)-AMC-AMK-COL-ERY-GEN-K-STX
	C10	ETEC	st	7.4	2	(8) -AMC-AMK-COL-ERY-GEN-K-NEO-CRO
Salad	S1	STEC	stx1	11	3	(6)-AMC-AMK-COL-ERY-GEN-K
	S2	STEC	stx1	150	6	(9)-AMC-AMK-COL-ERY-GEN-K-NEO-NAL-TYC
	S3	STEC	stx2	3.6	1	(7)-AMC-AMK-COL-ERY-GEN-K-NEO
	S4	EPEC	eaeA	3.6	1	(10)-AMC-AMK-COL-ERY-GEN-K-NEO-STR-CRO-TYC
	S5	EPEC	eaeA	240	6	(6)-AMC-AMK-COL-ERY-GEN-K
	S6	EPEC	eaeA	7.4	2	(8)-AMC-AMK-COL-ERY-GEN-K-NEO-STR
	S7	ETEC	st	11	3	(11)-AMC-AMK-COL-ERY-GEN-K-NEO-STR-CIP-TCY-SXT
	S8	ETEC	st	11	3	(11)-AMC-AMK-COL-ERY-GEN-K-NEO-STR-CIP-TCY-SXT

n = 100 samples for each type of nopalito.

Each DEP isolate was obtained from each positive tube of DEP determined by MPN procedure.

AMK, amikacin; AMC, amoxicillin/clavulanic acid; AMP, ampicillin; CIP, ciprofloxacin; COL, colistin; CRO, ceftriaxone; DEP, diarrheagenic E. coli pathotype; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli; ERY, erythromycin; GEN, gentamicin; K, kanamycin; MPN, most probable number; NAL, nalidixic acid; NEO, neomycin; SOX, sulfisoxazole; STEC, Shiga toxin–producing E. coli; STR, streptomycin; SXT, trimethoprim-sulfamethoxazole; TCY, tetracycline.

ETEC represents another major cause of diarrhea in developing countries and visitors from regions where ETEC is not endemic (traveler's diarrhea) (Nataro and Kaper, 1998). ETEC is usually transmitted by contaminated food. The ETEC pathotype has been isolated from mung bean sprouts (Cerna-Cortes et al., 2013) and carrot juice (Torres-Vitela et al., 2013).

EPEC is another cause of diarrhea in developed countries (Trabulsi et al., 2002; Estrada-García et al., 2009). Typical EPEC strains contain both eaeA and bfp, whereas atypical EPEC strains contain only eaeA (Hernandes et al., 2009). Unlike typical EPEC strains, which are found only in humans, atypical EPEC strains have been isolated from a variety of animal species (Cortes et al., 2005).

All the isolated DEP strains exhibited resistance to at least six antibiotics: amoxicillin/clavulanic acid, amikacin, colistin, erythromycin, gentamycin, and kanamycin. In contrast, all were sensitive to chloramphenicol (Tables 1 and 2). Four STEC strains (two isolated from whole nopalitos and two from cut nopalitos samples) were resistant to 13 antibiotics, three EPEC strains (isolated from whole nopalitos samples) were resistant to 12 antibiotics and 7 ETEC strains (1 isolated from whole nopalitos and 6 from nopalitos salad samples) were resistant to 11 antibiotics (Table 1). These results are similar to previous reports of isolation of antibiotic-resistant DEP strains from vegetables, meats, dairy products, fish, and seafood, and human feces (Estrada-García et al., 2005; Van et al., 2008; Solomakos et al., 2009; Canizalez-Roman et al., 2013; Bolton et al., 2014).

Table 2.

Counts of Diarrheagenic E. coli Pathotype Strains Exhibiting Resistance (r), Intermediate Resistance (i), or Susceptible (s) to 16 Antibiotics

Antibiotics	R	I	S
Amoxicillin/clavulanic acid	82^a	0	0
Amikacin	82	0	0
Colistin	82	0	0
Erythromycin	82	0	0
Gentamicin	82	0	0
Kanamycin	82	0	0
Neomycin	50	15	17
Streptomycin	33	15	34
Trimethoprim/sulfamethoxazole	29	13	40
Tetracycline	22	15	45
Ciprofloxacin	14	10	58
Nalidixic acid	13	5	64
Ampicillin	7	20	55
Sulfisoxazole	7	15	60
Ceftriaxone	3	10	62
Chloramphenicol	0	0	82

Strain counts.

All the isolated strains were susceptible to chloramphenicol and 62 to ceftriaxone, whereas most strains were sensitive to nalidixic acid, ciprofloxacin, tetracycline, ampicillin, and sulfisoxazole (Table 2). These results coincide with a study in which DEP strains isolated from different foods including vegetables were sensitive to chloramphenicol, trimethoprim/sulfamethoxazole, and nalidixic acid (Canizalez-Roman et al., 2013). In contrast, chloramphenicol-resistant DEP strains have been isolated from raw meat (beef, pork, and chicken) and shellfish in Vietnam (Van et al., 2008), and chloramphenicol-resistant E. coli O157:H7 from raw bovine, caprine, and ovine milk in Greece (Solomakos et al., 2009).

DEP strains in this study exhibited simultaneous resistance to different antibiotics (Tables 1 and 2). These results coincide with previous studies in which DEP strains isolated from vegetables were resistant to multiple antibiotics (Van et al., 2008; Solomakos et al., 2009).

All antibiotics tested in this study are used in human medicine. In fact, most of these antibiotics are considered to be “critically important” or “highly important” for human medicine by the World Health Organization (WHO, 2012). However, almost all classes of antibiotics available to humans have also been used in animal production (WHO, 2000; Anonymous, 2011), and antimicrobial resistance has been increasingly identified in animal pathogens (Katsuda et al., 2009). Over recent years, there has been mounting evidence that the use of antimicrobials in agriculture is a major factor driving antimicrobial resistance globally (Silbergeld et al., 2008). Nevertheless, although food products of animal origin are considered the principal foods carrying such antimicrobial-resistant bacteria/genes, contamination during preparation, handling, and processing of fresh food of plant origin, such as nopalitos salads, is becoming a current concern.

Like other raw vegetables, nopalitos are potential vectors for pathogens such as multidrug-resistant DEP strains. Pathogenic microorganism sources in the field include soil, water, wild and domestic animals, drift and runoff from adjacent farms, and manure (Islam et al., 2004). Research into environmental sources of multidrug-resistant DEP contamination indicates that water is a principal source, particularly irrigation water containing manure, wildlife feces, or sewage effluents (Islam et al., 2004).

Multiple sources of pathogenic microorganisms can occur during packaging, distribution, and marketing of nopalitos. Once they are incorporated into food preparation, raw nopalitos can act as sources of cross-contamination. In addition, food handler's personal hygiene is also a major important source of contamination.

Prevention and control of health risks from multidrug-resistant DEP strains requires incorporation and a consistent application of good agricultural and manufacturing practices throughout the nopalitos production process, from crop to harvest to retailer. Proper nopalitos handling and processing practices need to be promoted and implemented in both nopalitos growers and consumers.

To the best of our knowledge, this is the first report in the literature of the presence of multidrug-resistant STEC, ETEC, and EPEC in raw whole nopalitos, raw cut nopalitos, and in cooked nopalitos salads in Mexico.

Footnotes

Acknowledgment

This research was funded by the “Fondos Mixtos de Fomento a la Investigación Científica y Tecnológica, Consejo Nacional de Ciencia y Tecnología–Gobierno del Estado de Hidalgo, Mexico” (Grant No. 192649).

Disclosure Statement

No competing financial interests exist.

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