Bacterial Load of Fresh Vegetables and Their Resistance to the Currently Used Antibiotics in Saudi Arabia

Abstract

This study was carried out to describe the bacterial load and the occurrence of some disease-causing enteric bacteria on raw vegetables sold in Saudi markets. The study further aimed to analyze antibiotic resistance rates, production of extended-spectrum beta lactamase, and plasmid carriage among bacterial population of raw vegetables. Results revealed that none of them contained Bacillus cereus, Salmonella, and Escherichia coli O157:H7. However, Staphylococcus aureus and Shigella were detected in 11.8% and 4.4% of the samples, respectively. The bacterial loads ranged from 3 to 8 log₁₀ CFUg⁻¹ for aerobic bacteria and 1 to 4 log₁₀ CFUg⁻¹ for coliforms as well as Enterobacteriaceae. The isolates exhibited resistance in decreasing order for ampicillin (76.5%), cephalothin (69.5%), trimethoprime-sulfamethoxazole (36.7%), aminoglycosides (21.9%), tetracycline (17.2%), fluoroquinolones (17.2%), amoxycillin-clavulanic acid (13.3%), and chloramphenicol (7.8%). Maximum resistance to extended-spectrum beta-lactam antibiotics occurred in 14.8% of isolates and the production of extended-spectrum beta-lactamase was achieved by 2.3% of isolates. Multiple resistances to four or more antimicrobial agents along with plasmid with varied sizes were documented. These investigations indicate the occurrence of antibiotic resistance and plasmid carriage among bacterial isolates populating raw vegetables.

Introduction

R aw and minimally processed vegetables are essential to the human diet. In Saudi Arabia, there is an increased demand in consumption of fresh vegetables (Anonymous, 1993). Contamination of fresh produce with microbial pathogens and antibiotic-resistant bacteria is of special concern, because it is likely consumed raw, without any type of cleansing or processing. Outbreaks of illness associated with consumption of raw vegetables (Beuchat et al., 2001; Mukherjee et al., 2006) along with the prevalence and transmission of antibiotic resistance among bacteria associated with food from animals and raw produce have been well documented, which occurred with increased frequency worldwide (Threlfall et al., 2000; Van den Bogaard and Stobberingh, 2000; Aarestrup et al., 2002; Hayes et al., 2003; Schroeder et al., 2004).

Contamination of raw produce with pathogenic and nonpathogenic microorganisms can occur at any point during production to consumption (De Roever, 1998). Antibiotic-resistant bacteria could colonize vegetables because of the direct use of antibiotics during cultivation and use of contaminated fertilizers or irrigated water to croplands (Witte, 1998). All these could result in spread of resistance to indigenous soil bacteria through horizontal transfer, which could in turn transfer resistance back to animals or humans via crops (Witte, 1998; Nwosu, 2001; Sengeløv et al., 2002).

Recently, a great attention has been paid to the bacteria that can carry and disseminate resistance to the extended-spectrum beta-lactam drugs. Beta-lactamases are one of the mechanisms by which bacteria exhibit antibiotic resistance to beta-lactam drugs. Strains carrying extended-spectrum beta-lactamase (ESBLs) had been detected in food animals (Blanc et al., 2006; Mesa et al., 2006; Johnson et al., 2007). Data from the Arabian Peninsula, including Saudi Arabia, suggested that extended-spectrum beta-lactam–resistant bacteria constitute a major problem in nosocomial and community-acquired infections (Rafay et al., 2007; Al-Zarouni et al., 2008; Mokaddas et al., 2008; Al-Agamy et al., 2009). However, there is only little information describing the presence of antibiotic resistance bacteria, including those that are extended-spectrum beta-lactams resistant, in fresh vegetables. Therefore, this study was carried out to describe the bacterial population of fresh vegetables in Saudi Arabia and the presence of antimicrobial resistance, including extended-spectrum beta-lactam–resistant isolates among them.

Materials and Methods

Sample collection and preparation

Fresh vegetable samples were purchased from various local markets and street vendors in Taif city, Saudi Arabia. Composite samples of every commodity were obtained from each location (replicate of three samples) and the sample sizes varied according to the type of vegetables, but generally those purchased at the same time were considered as single sample (Table 1). The samples of each commodity were collected on different days and stored under refrigeration. On the basis of manner of consumption, samples were trimmed of spoiled parts, their outer leaves were removed, and the exterior surfaces were rinsed with distilled water. Leafy vegetables were peeled off aseptically and cucumbers were sliced, whereas watermelons and cantaloupes were cut in small cubes. Analyses were carried out within 24 h of purchase.

Table 1.

Average Mean Counts (Log₁₀ CFU g ⁻¹ ) of Microbial Items Analyzed in Fresh Vegetables Commercially Available in Taif (Western Region of Saudi Arabia)

Vegetable	Total (n=68)	Aerobic bacteria	Coliform bacteria	E. coli	Enterobacteriaceae
Cantaloupe	5	7.5	3.01	2	3.1
Watermelon	2	4.2	1.01	—	2.02
Spinach	11	8.5	4.01	5	3.31
Tomatoes	4	3.6	1.8	—	1.01
Lettuce	11	8.4	3.01	3	4.1
Coriander	4	4.4	1.48	—	1.5
Cucumber	4	5.6	1.2	—	2.32
Green onion	6	5.7	3.01	—	3.3
Celery	8	6.6	3.01	1	3.3
Parsley	8	7.4	3.01	1	2.7
Cabbage	3	5.7	3.4	—	4.10
Cauliflower	2	4.2	1.5	—	2.9

n, number of samples for each vegetable type.

Bacteriological analysis

Twenty-five grams of each sample was aseptically weighed into sterile stomacher bags and homogenized with 225 mL of buffered peptone water (Oxoid) at low speed for 1 min in Lab-Blender. These were serially 10-fold diluted and analyzed for total microbial count with direct plating methods. Standard plate counts were done on plate count agar (Scharlau) using the spread plate technique. Total coliform count and other Enterobacteriaceae were determined on violet red bile agar (Oxoid) using the pour plate technique. All microbial counts were calculated as CFU g⁻¹ and then transferred to log₁₀ CFU g⁻¹. Presence of fecal Escherichia coli (thermotolerant) was determined using eosin methylene blue agar (Scharlau) after incubation at 44.5°C for 24 h. Staphylococcus aureus was isolated on Baird-Parker agar containing egg yolk emulsion (Hi-Media). Cetrimide agar (BioLife) was used for the isolation of Pseudomonas aeruginosa. For Bacillus cereus, Kim & Goepfert (KG) agar (Kim and Goepfert, 1971) was used. Shigella was recovered on Salmonella-Shigella agar (Hi-Media). The methods used were of the Association of Official Analytical Chemists (AOAC, 1980) and in the Compendium of Methods for the Microbiological Examination of Foods (Downes and Ito, 2001). Isolates were identified and confirmed using oxidase, catalase, and potassium hydroxide test (KOH) and by commercially available biochemical test (API tests; bioMèrieux). The API strips were analyzed according to the manufacturer's instructions. Reactions were recorded and identified using APILab plus software (bioMèrieux).

Polymerase chain reaction of pathogenic bacteria

The polymerase chain reaction (PCR) assay was used for the simultaneous detection of Salmonella and E. coli O157:H7. Preenrichment steps were done. Homogenized samples in buffered peptone water were grown under shaking conditions (160 rpm) at 37°C for 24 h and subsequently enriched in Rappaport-Vassilidis broth (Oxoid) for Salmonella and tryptone soya broth (Difco) supplemented with 20 mg L⁻¹ novobiocin (Sigma) for E. coli O157:H7. The DNA extraction from food sample enrichments as well as positive control bacterial strains and the PCR conditions were performed as previously described (Wang et al., 1997). Sequences targeting the invasion invA gene (Sal-3 and Sal-4) in Salmonella spp. (Rahn et al., 1992) and enterolysin hlyA gene (O157-3 and O157-4) in E. coli O157:H7 virulence determinant (Wang et al., 1997) were indicated.

The PCR was conducted in a Thermal Cycler PXE-0.5 (THERMO; Electron Corporation) and the amplified product (10 μL) was electrophoresed on a 1.5% agarose gel in Tris-Borate-EDTA buffer at 80 V. The gels were stained with ethidium bromide and bands were visualized under a ultraviolet transluminator and photographed. The control positive PCR strains were Salmonella (invA gene positive) (obtained from Microbiology department, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt) and reference E. coli O157:H7 strain (Bundesst. bakt. serol. Untersuchngsanstalt, National Reference Laboratory for EHEC, Innsbruck, Austria).

Antibiotic sensitivity testing

The antibiotic resistance behavior of the isolated strains was determined on cation-adjusted Mueller-Hinton agar (Hi-Media) using Kirby-Bauer disk diffusion method according to the standards and interpretive criteria described by NCCLS (2006). The used antibiotics were ampicillin (AMP 25), amoxycillin-clavulanic acid (AMC 20/10), kanamycin (KAN 30), nalidixic acid (NAL 30), streptomycin (STR 10), ciprofloxacin (CIP 10), chloramphenicol (CHL 30), cefoxitin (FOX 30), cefotaxime (CTX 30), ceftazidime (CAZ 30), cephalothin (CEF 30), gentamicin (GEN 10), tetracycline (TET 30), and trimethoprim-sulfamethoxazole (SXT 1.25/23.75). All disks were purchased from Hi-Media and Mast-Diagnostics (Mast Group Ltd.) except for tetracycline and trimethoprim-sulfamethoxazole, which were purchased from Bioanalyse Ltd., and the results were recorded on the bases of the zone-size interpretative chart supplied by the manufacturers. The quality control was performed to check the quality of medium and potency of antibiotic disks before use against some sensitive ATCC reference strains, including S. aureus ATCC 29213, E. coli ATCC 25922 (beta-lactamase negative), E. coli ATCC 35218 (beta-lactamase positive), and P. aeruginosa ATCC 27853 (MicroTrol Discs; BD Diagnostics).

Phenotypic detection of ESBL

The first step of ESBL detection criteria was resistance or reduced susceptibility to cefotaxime and/or ceftazidime. Phenotypic confirmation of ESBL production was performed by double disk diffusion synergy test (Jarlier et al., 1988).

Plasmid detection and sizing

An alkaline lysis protocol (Sambrook et al., 1989) was used to recover extrachromosomal DNA from 1.5 mL of overnight Luria-Burtani broth of preselected multiantibiotic-resistant strains. Extracted contents were electrophoresed for 2 h on 1.0% agarose gels in 1× TAE buffer. The size estimates of the isolated plasmids were obtained by comparing their motilities on agarose gel with standard molecular DNA marker (Lambda DNA-HindIII digest) and Shigella flexneri 49 containing different plasmids (supplied by Dr. B. El-Deeb, Faculty of Science, Taif University, Saudi Arabia).

Results

Bacteriological quality

The bacteriological quality of the fresh vegetables with regard to the population size of aerobic bacteria (aerobic plate count [APC]), coliform, and Enterobacteriaceae is presented in Table 1. Plate counts ranged from 3 to 8 log₁₀ CFU g⁻¹ for aerobic bacteria and from 1 to 4 log₁₀ CFU g⁻¹ for coliform and Enterobacteriaceae, depending on the sample type. ON the basis of the results of bacterial isolates analyzed, we observed two main groups. The first group comprised raw whole vegetables (not leafy) and the second group comprised leafy vegetables. As indicated, the average values of aerobic bacteria in both groups were 5.5 versus 7.1 log₁₀ CFU g⁻¹, respectively. A total of 128 bacterial isolates could be recovered from 68 screened fresh vegetable samples based on the selective isolation on different culture media (Table 2). Bacteriological identification of randomly selected colonies revealed isolation of 10 genera, namely P. aeruginosa (33.8%), Enterobacter spp. (23.5%), Citrobacter spp. (30.9%), Serratia spp. (26.5%), Providencia spp.(20.6%), Edwardseilla spp. (13.2%), Klebsiella spp. (5.9%), Shigella spp. (4.4%), E. coli (9.4%), and S. aureus (11.8%).

Table 2.

Recovery of Bacterial Species from Fresh Vegetables Sampled in Taif, Saudi Arabia

Vegetables (n=68)	Escherichia coli	Enterobacter spp.	Citrobacter spp.	Providencia spp.	Edwardsiella spp.	Klebsiella spp.	Serratia spp.	Shigella spp.	Staphylococcus aureus	Pseudomonas aeruginosa
Cantaloupe	2	1	0	0	0	1	1	0	0	0
Watermelon	0	0	0	0	0	0	0	0	1	0
Spinach	5	5	8	4	2	1	2	2	1	6
Tomatoes	0	1	2	0	0	1	0	0	1	1
Lettuce	3	4	6	3	3	0	5	1	2	2
Coriander	0	1	2	0	1	0	2	0	0	3
Cucumber	0	0	1	0	0	0	1	0	0	1
Green onion	0	0	0	1	0	0	2	0	1	2
Celery	1	2	1	2	1	0	0	0	0	3
Parsley	1	0	0	1	0	0	2	0	0	1
Cabbage	0	2	1	2	2	0	3	0	2	3
Cauliflower	0	0	0	1	0	1	0	0	0	1
Total (%)	12 (9.4)	16 (23.5)	21 (30.9)	14 (20.6)	9 (13.2)	4 (5.9)	18 (26.5)	3 (4.4)	8 (11.8)	23 (33.8)

The PCR results for vegetable samples enrichment culture revealed that none of them positively reacted with the primers coding specific regions of Salmonella and E. coli O157:H7.

Antibiotic resistance

The bacterial isolates from various vegetables were evaluated for their antibiotic resistance (Table 3). Overall, 76.5% (n=98) of the 128 randomly selected isolates showed a phenotypic resistance to at least one of the antimicrobial agents analyzed. All these isolates exhibited resistance phenotypes against ampicillin (76.5%). Antibiotic resistance in a decreasing order was observed to cephalothin (69.5%), trimethoprime-sulfamethoxazole (36.7%), aminoglycosides (21.9%), tetracycline (17.2%), fluoroquinolones (17.2%), extended-spectrum beta-lactam antibiotics (14.8%), amoxycillin-clavulanic acid (13.3%), and chloramphenicol (7.8%). Plasmid DNA analysis in a preselected multidrug-resistant isolates revealed plasmids. The plasmids varied in mass from 3.6 to 52 kbp and in number from 1 to 3 per bacterial cell (Table 4).

Table 3.

Antibiotic Resistance Phenotype in Bacterial Population of Fresh Vegetables

	Beta-lactams						Aminoglycosides			Fluoroquinolones		Sulfonamides	Phenicoles	Tetracycline
Bacterial species	AMP	AMC	FOX	CAZ	CTX	CEF	KAN	GEN	STR	NAL	CIP	SXT	CHL	TET
E. coli	10	3	4	3	4	10	2	0	5	2	2	4	1	2
Enterobacter spp.	14	0	2	1	3	11	0	2	2	4	0	2	1	3
Citrobacter spp.	18	0	2	1	3	14	0	0	0	0	0	4	0	2
Providencia spp.	10	3	0	0	0	11	0	1	0	2	0	6	0	4
Edwardsiella spp.	5	2	0	0	2	5	0	0	0	1	0	5	0	1
Serratia spp.	13	0	1	0	0	11	0	0	2	0	0	2	2	2
Klebsiella spp.	2	1	0	0	0	3	1	0	0	0	0	1	1	2
Shigella spp.	2	1	0	0	0	3	0	0	0	0	0	0	0	0
P. aeruginosa	19	3	4	3	6	18	17	3	0	13	3	16	5	0
S. aureus	5	4	1	0	1	3	0	0	0	0	4	7	0	6
Total, n=128 (%)	98 (76.5)	17 (13.3)	14 (11)	8 (6.2)	19 (14.8)	89 (69.5)	20 (15.6)	6 (4.7)	9 (7)	22 (17.2)	9 (7)	47 (36.7)	10 (7.8)	22 (17.2)

AMP, ampicillin; AMC, amoxyicillin-clavulanic acid; KAN, kanamycin; NAL, nalidixic acid; CHL, chloramphenicol; CIP, ciprofloxacin; FOX, cefoxitin; CTX, cefotaxime; CAZ, ceftazidime; GEN, gentamicin; TET, tetracycline; SXT, trimethoprim-sulphamethoxyzol; STR, streptomycin; CEF, cephalothin.

Table 4.

Antibiotic Resistance and Plasmid Profile of Preselected Multidrug-Resistant Isolates Recovered from Fresh Vegetables

Bacterial species	Origin	Resistance profiles	Plasmids size (kbp)
Enterobacter aerogenes	Lettuce	AMP, FOX, NAL, SXT, CTX, CEF	3.6, 12.6
Enterobacter agglomerans	Cabbage	AMP, KAN, STR, TET, CHL	5.1
Klebsiella pneumonia	Tomato	AMP, TET, CHL, SXT, CEF	4.7, 12.6
E. coli	Spinach	AMP, STR, CIP, CTX, CAZ, STX, NAL, TET	3.6, 5.1, 23
	Lettuce	AMP, FOX, CTX, CEF	3.6
S. aureus	Lettuce	NAL, STR, GEN,CIP, CEF	4.6, 12.6
P. aeruginosa	Spinach	AMP, CEF, KAN, NAL, CIP, STR, CTX, CAZ, SXT	4.7, 12.6, 52
	Parsley	AMP, FOX, AMC, CEF	4.7, 12.6
Shigella flexneri	Spinach	AMP, STR, TET, CEF	3.6, 6.6

By ESBL selective screening, 3 (2.3%) of 19 bacterial isolates (2 E. coli and 1 Enterobacter spp.) were found to be ESBL producers.

Discussion

Bacteriological quality

The results of hygienic quality parameter analysis demonstrated that the population size of aerobic bacteria had a varied count depending on sample type. Similar reports of varied aerobic mesophilic bacteria in raw vegetables have been documented (Saddik et al., 1985; Badosa et al., 2008; Erkan et al., 2008). The microbial quality of nonleafy vegetables was better than that of leafy ones. About 51.5% of samples examined in this study had APC higher than 7 log₁₀ CFU g⁻¹ and were most commonly leafy vegetables. Our results are consistent with those reported for other fresh leafy vegetables (Viswanathan and Kaur, 2001; Badosa et al., 2008).

Contamination with coliforms and Enterobacteriaceae denotes unhygienic conditions. The bacterial populations of coliforms and Enterobacteriaceae were detected at a low as well as high limit among samples. Similar population sizes have been reported in other studies. However, higher coliform counts (>4 log₁₀ CFU g⁻¹) have been reported by other workers (Al-Mohizea, 1996; Viswanathan and Kaur, 2001).

Fresh vegetables are good sources of nutrients; however, they could pose a health risk if consumed raw (USFDA, 2001). The results of this study indicated that Salmonella, E. coli O157:H7, and B. cereus were not detected. This finding is consistent with other published studies in which these foodborne pathogens were not detected (Sagoo et al., 2003; Johnston et al., 2005; Warminśka-Radyko et al., 2007) or could be at low prevalence (Al-Mohizea, 1996; Abadias et al., 2008; Badosa et al., 2008), while in Sweden, Söderström et al. (2005) suggested a link between EHEC 0157 outbreaks and the consumption of locally produced lettuce. However, in our study, eight samples were found to be positive for S. aureus and three samples were positive for Shigella. Our results agreed with previous studies that detected S. aureus (Viswanathan and Kaur, 2001; Erkan et al., 2008) in vegetable samples. In contrast, other studies did not detect Shigella or S. aureus in vegetable samples surveyed (Johnston et al., 2006; Warminśka-Radyko et al., 2007). The possible source of raw vegetable contamination by S. aureus and Shigella could be the hand of sellers, washing water, or insects at retails. Further, thermotolerant E. coli was detected in 9.4% of samples, mostly from leafy vegetables. Similarly, Mukherjee et al. (2006) detected E. coli contamination in vegetable samples and was mostly of leafy greens, lettuces, cabbage. This may be owed to the large surface area of these leafy vegetables, which could encounter contaminated water (irrigation and sprinkling) or other possible contaminants, including feces, flies, and dust. Higher prevalence of fecal E. coli has been demonstrated by others (Viswanathan and Kaur, 2001). In addition to E. coli, other bacteria found on samples belonged mostly to Enterobacter spp., Citrobacter spp., Providencia spp., Serratia spp., Klebsiella spp., and P. aeruginosa. Recovery of these enteric bacteria and Pseudomonas has been previously reported (Duncan and Razzell, 1972; Viswanathan and Kaur, 2001).

Antimicrobial resistance

The broad use of antimicrobials in agriculture selects for resistant bacteria, which may enter the food chain and potentially to humans (Khachatourians, 1998). Among the most common antimicrobial agents are drugs that are either identical or related to those administered to humans. In this study, 76.5% of bacterial population from vegetables at retail level showed resistance phenotypes to at least one antibiotic. This is in agreement with previously published data that found high resistance rates in bacterial populations from vegetables (Oesterblad et al., 1999; Hamilton-Miller and Shah, 2001; Boehme et al., 2004). All of the isolates in this study displayed a multidrug resistance phenotype against ampicillin and mostly to cephalothin (69%–76%), followed less frequently (4%–36%) by resistance to other antimicrobial agents analyzed. Similar multiresistance phenotypes of bacteria populating fresh vegetables have been reported worldwide (Oesterblad et al., 1999; Hamilton-Miller and Shah, 2001; Viswanathan and Kaur, 2001; Boehme et al., 2004).

The resistance to extended-spectrum beta-lactam and beta-lactamase inhibitors is of great clinical significance. Resistance to beta-lactam antibiotics is primarily mediated by beta-lactamases production. Many different beta-lactamases had been described (Livermore and Woodford, 2006). In this study, maximum resistance rate to the extended-spectrum beta-lactam antibiotics was expressed by 14.8% of isolates, likewise 2.3% was shown to be ESBL producers. Other previous studies reported occurrence of resistance to the second and third generation of cephalosporins among bacterial population of vegetables (Viswanathan and Kaur, 2001; Boehme et al., 2004). However, Oesterblad and coworkers (1999) did not observe any resistance to these drugs. In one other study, similar resistance rates to third-generation cephalosporins were noticed in isolates from fresh vegetables (Shahid et al., 2009); however, a high rate was displayed by isolates from cheese (Amador et al., 2009). A CTX resistance most found in Enterobacteriaceae was recently identified in P. aeruginosa strains. Similarly, other researchers identified this resistance trait in P. aeruginosa and other bacterial species (Viswanathan and Kaur, 2001; Boehme et al., 2004). The association of CTX β-lactamase–encoding genes with mobile elements (Eckert et al., 2006) may facilitate the spread of bla _CTX-M genes among bacteria. The CTX-resistant P. aeruginosa in this study may be due to horizontal resistance transfer or loss of membrane permeability.

Other possible mechanisms, such as a non–beta-lactamase-mediated resistance to beta-lactam drugs, could be also encountered. Non–beta-lactamase mechanisms included loss or deficiency of specific porins (Benz, 1994), penicillin-binding proteins (PBPs) alteration (Wilke et al., 2005), and increased efflux pumps (Poole, 2004; Fisher et al., 2005).

Despite the resistance to narrow and extended-spectrum beta-lactam drugs, we detected resistance to fluoroquinolones including ciprofloxacin (7% of isolates) and nalidixic acid (17.2% of isolates). Resistance to quinolones is chiefly mediated through chromosomal mutation in DNA gyrase, topoisomerase, and plasmid-mediated quinolone. Plasmid-mediated quinolone resistance is of great concern, because these resistance determinants are potentially spread among bacteria due to plasmid mobility (Robicsek et al., 2006a). More recently, another new mechanism of plasmid-associated quinolone resistance that involves ciprofloxacin-modifying aminoglycoside acetyltransferase gene has been discovered (Robicsek et al., 2006b). Further, resistance to aminoglycosides was observed most commonly against kanamycin (15.6%) and less against gentamicin (4.7%), and streptomycin (7%). The ciprofloxacin and gentamicin resistance in bacterial population isolated from vegetables has been previously reported (Viswanathan and Kaur, 2001; Boehme et al., 2004, Shahid et al., 2009). In contrast, other workers did not observe any resistance to this fluoroquinolone drug and gentamicin (Oesterblad et al., 1999). In contrary, Al-Tawfiq (2007) reported that gentamicin demonstrated the highest resistance among all tested aminoglycosides during a retrospective analysis performed in Saudi Arabia. Appearance of fluoroquinolone, gentamicin, and cephalosporins resistance in bacteria identified in our study suggested animal or human sources, because these classes are not used in plant agriculture, or plant-associated bacteria through horizontal transfer could be also supposed.

A comparative data on the resistance patterns of the bacterial species described in our study with those of bacterial species originating from clinical settings in Saudi Arabia and Arabic Gulf region revealed a correlation. In Saudi Arabia, E. coli isolated from chicken intestine was found to be resistant to many antibiotics (Al-Ghamdi et al., 1999). In line with this result, Altalhi et al. (2010) observed high rates of resistance among E. coli isolates from chicken meat against sulfonamides, nalidixic acid, gentamicin, chloramphenicol, and streptomycin. Further, a gradual increase in resistance rates against fluoroquinolones, aminoglycosides, and trimethoprime-sulfamethoxazole and multidrug resistance among bacteria had been reported in many retrospective analysis (Kader and Kumar, 2005; Al-Tawfiq, 2006, 2007; Al-Harbi and Al-Fifi, 2008). In recent years, data from Arabian Gulf region show high occurrence of ESBL-producing isolates, with rates as high as 31.7% in Kuwait, 41% in United Arab Emirates, and 55% in Saudi Arabia (Al-Zarouni et al., 2008; Mokaddas et al., 2008; Al-Agamy et al., 2009).

Plasmid detection

The ability of bacteria to acquire, maintain, and disseminate exogenous genes through mobile genetic elements such as plasmids, transposons, and integrons has been the important factor in increasing resistance in the nature (Mundy et al., 2000). The plasmid DNA analysis of the preselected multiresistant strains in this study showed that the size of the plasmid DNA varied. Similarly, Bezanson et al. (2008) showed the presence of plasmids with size ranging from 3.3 to 58 kbp in bacteria that populated raw vegetable salads. In the same environment, Altalhi (2009) isolated small and large plasmids from bacterial communities associated with the plant grapevine in Taif region of Saudi Arabia.

In summary, the present study indicates that the bacterial populations in fresh vegetables are common in the animal and human environments and constitute a part of natural microflora, and their occurrence can affect the products quality and shelf life. Further, the occurrence of antimicrobial resistance and plasmid carriage in bacterial populations in fresh vegetables at retails may constitute threats to consumers, possibly via resistance transfer.

Footnotes

Acknowledgments

The authors are grateful to the anonymous reviewers for their suggestions to improve the manuscript. This work was supported by a grant from the Taif University (Project 1/430/402). The authors thank Abo-Bakr Al-Aedarous for skillful technical assistance.

Disclosure Statement

No competing financial interests exist.

References

Aarestrup

, Hasman

, Jensen

, Moreno

, Herrero

, Domínguez

, Finn

, Franklin

. Antimicrobial resistance among Enterococci from pigs in three European countries. Appl Environ Microbiol, 2002; 68:4127–4129.

Abadias

, Usall

, Anguera

, Solsona

, Vinas

. Microbiological quality of fresh, minimally-processed fruit and vegetables and sprout from retail establishment. Int Food Microbiol, 2008; 123:121–129.

Al-Agamy

, Shibl

, Tawfic

. Prevalence and molecular characterization of extended-spectrum beta-lactamase producing Klebsiella pneumoniae in Riyadh, Saudi Arabia. Ann Saudi Med, 2009; 29:253–257.

Al-Ghamdi

, EI-Morsy

, Al-Mustafa

, Al-Ramadhan

, Hanif

. Antibiotic resistance of Escherichia coli isolated from poultry workers, patients and chicken in the eastern province of Saudi Arabia. Trop Med Int Health, 1999; 4:278–283.

Al-Harbi

, Al-Fifi

. Antibiotic resistance pattern and empirical therapy for urinary tract infection in children. Saudi Med J, 2008; 29:854–858.

Al-Mohizea

. Microbiological studies on some salad vegetable in local markets. J King Saud Univ (Agric Sci), 1996; 8:99–106.

Altalhi

. Plasmids profiles, antibiotic and heavy metal resistance incidence of endophytic bacteria isolated from grapevine (Vitis vinifera L.) Afr J Biotechnol, 2009; 8:5873–5882.

Altalhi

, Gherbawy

, Hassan

. Antibiotic resistance in Escherichia coli isolated from retail raw chicken meat in Taif, Saudi Arabia. Foodborne Pathog Dis, 2010; 7:281–285.

Al-Tawfiq

. Increasing antibiotic resistance among isolates of Escherichia coli recovered from inpatients and outpatients in a Saudi Arabian Hospital. Infect Control Hosp Epidemiol, 2006; 27:748–753.

10.

Al-Tawfiq

. Occurrence and antimicrobial resistance pattern of inpatient and outpatient isolates of Pseudomonas aeruginosa in a Saudi Arabian hospital: 1998–2003. Int J Infect Dis, 2007; 11:109–114.

11.

Al-Zarouni

, Senok

, Rashid

, Al-Jesmi

, Panigrahi

. Prevalence and antimicrobial susceptibility pattern of extended-spectrum beta-lactamase producing Enterobacteriaceae in the United Arab Emirates. Med Princ Pract, 2008; 17:32–36.

12.

Amador

, Fernandes

, Prudêncio

, Brito

. Resistance to β-lactams in bacteria isolated from different types of Portuguese cheese. Int J Mol Sci, 2009; 10:1538–1551.

13.

Anonymous. The nutritional assessment of the people of Saudi Arabia. The final report, King Abdulaziz city for science and technology: Riyadh, 1993.

14.

[AOAC] Association Of Official Analytical Chemists. Official Methods of Analysis, 13th. Washington, DC: Association of Official Analytical Chemists, 1980; 376–384.

15.

Badosa

, Trias

, Pares

, Pla

, Montesinos

. Microbiological quality of fresh fruit and vegetable products in Catalonia (Spain) using normalized plate-counting methods and real time polymerase chain reaction (QPCR) J Sci Food Agric, 2008; 88:605–611.

16.

Benz

. Uptake of solutes through bacterial outer membranes. Bacterial Cell Walls. Ghuysen

J-M

, Hakenbeck

. Amsterdam: Elsevier, 1994; 397–424.

17.

Beuchat

, Farber

, Garrett

, Harris

, Parish

, Suslow

, Busta

. Standardization of a method to determine the efficacy of sanitizers in inactivating human pathogenic microorganisms on raw fruits and vegetables. J Food Prot, 2001; 64:1079–1084.

18.

Bezanson

, MacInnis

, Potter

, Hughes

. Presence and potential for horizontal transfer of antibiotic resistance in oxidase-positive bacteria populating raw salad vegetables. Int J Food Microbiol, 2008; 127:37–42.

19.

Blanc

, Mesa

, Saco

, Lavilla

, Prats

, Miro

, Navarro

, Cortes

, LIagostera

. Extended spectrum and plasmidic class C beta-lactamase producing Escherichia coli strains isolated from poultry, pig, rabbit farms. Vet Microbiol, 2006; 118:299–304.

20.

Boehme

, Werner

, Klare

, Reissbrodt

, Witte

. Occurrence of antibiotic resistant enterobacteria in agricultural foodstuffs. Mol Nutri Food Res, 2004; 48:522–531.

21.

De Roever

. Microbiological safety evaluations and recommendations on fresh produce. Food Control, 1998; 9:321–347.

22.

Downes

, Ito

. Compendium of Methods for the Microbiological Examination of Foods, 4th. Washington, DC: American Public Health Association, 2001; 20001–3710.

23.

Duncan

, Razzell

. Klebsiella biotypes among coliforms isolated from forest environments and farm produce. Appl Microbiol, 1972; 24:933–938.

24.

Eckert

, Gautier

, Arlet

. DNA sequence analysis of the genetic environment of various bla_CTX-M genes. J Antimicrob Chemother, 2006; 57:14–23.

25.

Erkan

, Vural

, Oezekinci

. Investigating the presence of Staphylococcus aureus and coagulase negative Staphylococci (CNS) in some leafy green vegetables. Res Biol Sci, 2008; 3:930–933.

26.

Fisher

, Meroueh

, Mobashery

. Bacterial resistance to beta-lactam antibiotics: compelling opportunism, compelling opportunity. Chem Rev, 2005; 105:395–424.

27.

Hamilton-Miller

JMT

, Shah

. Identity and antibiotic susceptibility of enterobacterial flora of salad vegetables. Int J Antimicrob Agents, 2001; 18:81–83.

28.

Hayes

, English

, Carter

, Proescholdt

, Lee

, Wagner

, White

. Prevalence and antimicrobial resistance of Enterococcus species isolated from retail meats. Appl Environ Microbiol, 2003; 69:7153–7160.

29.

Jarlier

, Nicholas

, Fournier

, Philippon

. Extended-spectrum beta-lactamase conferring transmissible resistance to newer beta-lactams agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev Infect Dis, 1988; 10:867–878.

30.

Johnson

, Sannes

, Croy

, Johnston

, Clabots

, Kuskowski

, Bender

, Smith

, Winokur

, Belongia

. Antimicrobial drug-resistant Escherichia coli from humans and poultry products, Minnesota and Wisconsin, 2002–2004. Emerg Infect Dis, 2007; 13:838–846.

31.

Johnston

, Jaykus

, Anciso

, Mora

, Moe

. A field study on the microbiological quality of fresh produce of domestic and Mexican origin. Int J Food Microbiol, 2006; 112:83–95.

32.

Johnston

, Jaykus

, Moll

, Martinez

, Anciso

, Mora

, Moe

. A field study on the microbiological quality of fresh produce. J Food Prot, 2005; 68:1840–1847.

33.

Kader

, Kumar

. Prevalence and antimicrobial susceptibility of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumonia in a general hospital. Ann Saudi Med, 2005; 25:239–242.

34.

Khachatourians

. Agricultural use of antibiotics and the evolution and transfer of antibiotic-resistant bacteria. CMAJ, 1998; 159:1129–1136.

35.

Kim

, Goepfert

. Enumeration and identification of Bacillus cereus in foods. Appl Microbiol, 1971; 22:581–587.

36.

Livermore

, Woodford

. The b-lactamase threat in Enterobacteriaceae, Pseudomonas and Acinetobacter. Trends Microbiol, 2006; 14:413–420.

37.

Mesa

, Blanc

, Blanch

, Cortés

, González

, Lavilla

, Miro

, Muniesa

, Saco

, Tortola

, Mirelis

, Coll

, LIagostera

, Prats

, Navarro

. Extended-Spectrum-β-lactamse-producing Enterobacteriaceae in different environments (human, food, animal farms and sewage) J Antimicrob Chemother, 2006; 58:211–215.

38.

Mokaddas

, Abdulla

, Shati

, Rotimi

. The technical aspects and clinical significance of detecting extended-spectrum beta-lactamase producing Enterobacteriaceae at a tertiary-care hospital in Kuwait. J Chemother, 2008; 20:445–451.

39.

Mukherjee

, Speh

, Jones

, Buesing

, Diez-Gonzalez

. Longitudinal microbiological survey of fresh produce grown by farmers in the upper Midwest. J Food Prot, 2006; 69:1928–1936.

40.

Mundy

, Sahm

, Gilmore

. Relationships between enterococcal virulence and antimicrobial resistance. Clin Microbiol Rev, 2000; 13:513–522.

41.

[NCCLS] National Committee For Clinical Laboratory Approved Standard. Performance Standard for Antimicrobial Susceptibility Testing, 16th. Villanova, PA: NCCLS, 2006.

42.

Nwosu

. Antibiotic resistance with particular reference to soil microorganisms. Res Microbiol, 2001; 152:421–430.

43.

Oesterblad

, Pensala

, Peterzéns

, Heleniusc

, Huovinen

. Antimicrobial susceptibility of Enterobacteriaceae isolated from vegetables. J Antimicrob Chemother, 1999; 43:503–509.

44.

Poole

. Resistance to beta-lactam antibiotics. Cell Mol Life Sci, 2004; 61:2200–2223.

45.

Rafay

, Al-Muharrmi

, Toki

. Prevalence of extended-spectrum beta-lactamases producing isolates over 1-year period at a university hospital in Oman. Saudi Med J, 2007; 28:22–27.

46.

Rahn

, De Grandis

, Clarke

, Mcewen

, Galán

, Ginocchio

, Curtiss

, Gyles

. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol Cell Probes, 1992; 6:271–279.

47.

Robicsek

, Jacoby

, Hooper

. The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infect Dis, 2006a. 6:629–640.

48.

Robicsek

, Strahilevitz

, Jacoby

, Macielag

, Abbanat

, Park

, Bush

, Hooper

. Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med, 2006b. 12:83–88.

49.

Saddik

, El-Sherbeeny

, Bryan

. Microbiological profiles of Egyptian raw vegetables and salads. J Food Prot, 1985; 48:883–886.

50.

Sagoo

, Little

, Ward

, Gillespie

, Mitchell

. Microbiological study of ready-to-eat vegetables from retail establishments uncovers a national outbreak of salmonellosis. J Food Prot, 2003; 66:403–409.

51.

Sambrook

, Fritsch

, Maniatis

. Molecular Cloning—A Laboratory Manual, 1–1 2nd. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989; 1.21–1.52.

52.

Schroeder

, White

, Meng

. Retail meat and poultry as a reservoir of antimicrobial resistant Escherichia coli. Food Microbiol, 2004; 21:249–255.

53.

Sengeløv

, Yvonne

, Halling-Sørenson

, Baloda

, Andersen

, Jensen

. Bacterial antibiotic resistance levels in Danish farmland as a result of treatment with pig manure slurry. Environ Int, 2002; 953:1–9.

54.

Shahid

, Malik

, Adil

, Jahan

, Malik

. Comparison of beta-lactamase genes in clinical and food bacterial isolates in India. J Infect Dev Ctries, 2009; 3:593–598.

55.

Söderström

, Lindberg

, Andersson

. EHEC O157 outbreak in Sweden from locally produced lettuce. Euro Surveill, 2005; 10:2794.

56.

Threlfall

, Ward

, Frost

, Willshaw

. The emergence and spread of antibiotic resistance in food-borne bacteria. Int J Food Microbiol, 2000; 62:1–5.

57.

[USFDA] U.S. Food And Drug Administration. Outbreaks associated with fresh and fresh-cut produce. incidence, growth, and survival of pathogens in fresh and fresh-cut produce, chapter IV, analysis and evaluation of preventive control measurers for the control and reduction of microbiological hazards on fresh and fresh-cut produce, Center for Food Safety and Applied Nutrition, USFDA. 2001. www.fda.gov/Food/ScienceResearch/ResearchAreas/SafePracticesforFoodProcesses(Online.)

58.

Van Den Bogaard

, Stobberingh

. Epidemiology of resistance to antibiotics links between animals and humans. Int J Antimicrob Agents, 2000; 14:327–335.

59.

Viswanathan

, Kaur

. Prevalence and growth of pathogens on salad vegetables, fruits and sprouts. Int J Environ Health, 2001; 203:205–213.

60.

Wang

, Cao

, Cerniglia

. A universal protocol for PCR detection of 13 species of foodborne pathogens in food. J Appl Microbiol, 1997; 83:727–736.

61.

Warminśka-Radyko

, Laniewska-Trakenheim

, Mikš

. Microbiological contamination of vegetable salads. Pol J Nat Sci, 2007; 22:733–741.

62.

Wilke

, Lovering

, Strynadka

. Beta-lactam antibiotic resistance: a current structural perspective. Curr Opin Microbiol, 2005; 8:525–533.

63.

Witte

. Medical consequences of antibiotic use in agriculture. Science, 1998; 279:996–997.