Abstract
Fresh produce is known to carry nonpathogenic epiphytic microorganisms. During agricultural production and harvesting, leafy greens can become contaminated with antibiotic-resistant pathogens or commensals from animal and human sources. As lettuce does not undergo any inactivation or preservation treatment during processing, consumers may be exposed directly to all of the (resistant) bacteria present. In this study, we investigated whether lettuce or its production environment (irrigation water, soil) is able to act as a vector or reservoir of antimicrobial-resistant Escherichia coli. Over a 1-year period, eight lettuce farms were visited multiple times and 738 samples, including lettuce seedlings (leaves and soil), soil, irrigation water, and lettuce leaves were collected. From these samples, 473 isolates of Escherichia coli were obtained and tested for resistance to 14 antimicrobials. Fifty-four isolates (11.4%) were resistant to one or more antimicrobials. The highest resistance rate was observed for ampicillin (7%), followed by cephalothin, amoxicillin-clavulanic acid, tetracycline, trimethoprim, and streptomycin, with resistance rates between 4.4 and 3.6%. No resistance to amikacin, ciprofloxacin, gentamicin, or kanamycin was observed. One isolate was resistant to cefotaxime. Among the multiresistant isolates (n = 37), ampicillin and cephalothin showed the highest resistance rates, at 76 and 52%, respectively. E. coli isolates from lettuce showed higher resistance rates than E. coli isolates obtained from soil or irrigation water samples. When the presence of resistance in E. coli isolates from lettuce production sites and their resistance patterns were compared with the profiles of animal-derived E. coli strains, they were found to be the most comparable with what is found in the cattle reservoir. This may suggest that cattle are a potential reservoir of antimicrobial-resistant E. coli strains in plant primary production.
INTRODUCTION
The emergence, propagation, accumulation, and maintenance of strains of antimicrobial-resistant (AR) pathogenic bacteria have become a worldwide health concern in human and veterinary medicine (1, 2). The rise of antimicrobial resistance is due to the use of antimicrobials for the treatment of human, animal, and plant diseases (3–5). The use of antimicrobials selects for resistance not only in pathogenic bacteria but also in commensal bacteria (6). As a result, the commensal population of an individual gives a good reflection of the selective pressure exerted by the use of antimicrobial agents in that population's environment (7). By using indicator bacteria, misjudgment or overestimation of resistance levels may be minimized (7, 8). Moreover, these commensal bacteria may serve as a source of resistance genes that can easily be transferred to pathogens. Therefore, the level of antimicrobial resistance in Escherichia coli is a useful indicator of resistance levels expected in pathogenic bacteria (6, 9).
AR bacteria and antimicrobial resistance genes can be exchanged between the animal reservoir and the human reservoir (7, 10, 11). This can be a consequence of direct contact with animals or their environment or through indirect contact through the food chain (12).
Many studies of the presence of antimicrobial resistance in several animal-producing environments such as sheep, cattle, swine, or broiler farms have been conducted (13–21). However, only a few publications are available about studies of whether vegetables or the environment where they are produced can act as a carrier or reservoir of antimicrobial resistance (22, 23).
Fresh vegetables normally carry natural nonpathogenic epiphytic microorganisms, but during growth and harvesting, produce can be contaminated with pathogens or commensals from animal and human sources (24–26). Contamination of produce can occur in the field by contaminated soil, by exposure to contaminated water (e.g., by crop irrigation, application of pesticides, or flooding) or by deposition of feces by livestock or wild animals (27–32). Fecal bacteria are able to survive for extended periods in soils (33), manure (34–36), and water (25, 37) and thereby provide a potential inoculum for contamination.
As lettuce is grown close to the soil, it has more of a chance to get contaminated than vegetables that grow above the soil (38). Furthermore, as lettuce does not undergo any inactivation or preservation treatment during processing, consumers may be exposed directly to all of the (resistant) bacteria present. Therefore, the aim of this study was to check whether lettuce or the environment where it is produced is able to act as a vector or reservoir of AR bacteria.
MATERIALS AND METHODS
Collection of samples.
Between April 2011 and September 2012, three different crop production cycles of lettuce (Lactuca sativa L. var. capitata) were followed in four open-air and four greenhouse production systems in Belgium. At none of the farms was animal production present. Within each crop production cycle (ca. 5 to 14 weeks), the production system was sampled four times. The first sampling took place at the start, during the planting of lettuce seedlings. Samples of seedlings (n = 9) and field soil (n = 9, 200 g each) were taken at that time point. The leaves of the nine seedling were separated from the peat soil and pooled as one sample of seedling leaves. The nine peat soil samples were separated into sets of three for analysis. The other three sampling times were during the cultivation of the lettuce and approximately 2 weeks before, 1 week before, and during harvesting of the lettuce crop. At each of these sampling times, lettuce leaves (n = 9), the surrounding soil (n = 9 200-g samples), the source of the irrigation water (n = 1 1-liter sample), and if possible, the irrigation water itself (at the tap) were sampled (n = 1 1-liter sample). Six farms used collected rainwater stored in an open well, and two farms used bore hole water. Lettuce (n = 9 plants) and the surrounding soil (n = 9 200-g samples) were collected with disinfected gloves, and the nine samples were transferred directly into sterile bags. Water samples were collected in accordance with ISO 19458:2006 (39). All samples were stored and transported to the laboratory at 4°C, where further handling and isolation of E. coli were conducted within 4 to 24 h.
Enumeration or detection, isolation, and identification of E. coli.
Before analysis, nine lettuce plants and the surrounding soil were randomly separated into sets of three to obtain three pooled samples for analysis of E. coli. For the enumeration of E. coli bacteria in the pooled soil (and pooled peat soil) samples or pooled lettuce leaves (and seedling leaves), 10 g of soil or lettuce leaves was weighed in a stomacher bag and homogenized for 1 min in 90 ml of peptone. RAPID'E. coli 2 Agar (Bio-Rad, Hercules, CA), a selective chromogenic medium, was used for the enumeration of E. coli (incubation at 44°C for 24 h) (40). In parallel, 25 g of the pooled sample was also weighed in a stomacher bag and homogenized for 1 min in 225 ml of buffered peptone water and incubated at 37°C for 20 h for the enrichment of E. coli.
The E. coli bacteria in 100 ml of each irrigation water sample collected (at the source or at a tap) were enumerated according to ISO 9308-1:2000 (i.e., membrane filtration of 100 ml), with the exception that the more selective RAPID'E. coli 2 Agar chromogenic medium was used instead of Tergitol 7 medium (40, 41). The ISO 9308-1:2000 standard Tergitol 7 medium used in preliminary trials was not selective enough to suppress competitive flora in the (surface) water, which interfered with reliable enumeration of typical E. coli colonies. At the same time, 1 liter of water was filtered and the filters were incubated in 100 ml of buffered peptone water and incubated at 37°C for 20 h for the enrichment of E. coli.
The incubated RAPID'E. coli 2 Agar chromogenic medium was investigated for typical E. coli colonies, and a maximum of three colonies of each sample were selected from the plate. The isolates were tested by indole and oxidase tests for biochemical confirmation of E. coli. Only if no typical colonies were detected by the enumeration method (limit of detection, 10 CFU/g), E. coli colonies were isolated from the buffered peptone water enrichment by means of four-by-four streaking (20 μl) on RAPID'E. coli 2 Agar chromogenic medium.
The collected E. coli colonies (346 isolates obtained by the enumeration method, 253 colonies obtained by the detection method) were stored on Trypticase soy agar slants at 4°C until they were tested for antimicrobial susceptibility.
Antimicrobial susceptibility testing.
Antimicrobial susceptibility testing was carried out by the Kirby-Bauer disk diffusion method on Mueller-Hinton agar plates as recommended by the Clinical and Laboratory Standards Institute (42). The antimicrobials tested were (abbreviations and amounts are in parentheses) amoxicillin-clavulanic acid (AMC; 20/10 μg), ampicillin (AMP; 30 μg), cefotaxime (CEF; 30 μg), cephalothin (CEP; 30 μg), chloramphenicol (CLR; 30 μg), ciprofloxacin (CIP; 5 μg), gentamicin (GEN; 10 μg), kanamycin (KAN; 30 μg), nalidixic acid (NAL; 30 μg), streptomycin (STR; 10 μg), sulfonamides (SULFA; 240 μg), tetracycline (TET; 30 μg), and trimethoprim (TRIM; 5 μg). After 18 h of incubation, the E. coli isolates were classified as susceptible or resistant according to the clinical interpretative criteria recommended by CLSI (43). In order to dichotomize the antimicrobial resistance results, isolates with intermediate susceptibility were considered resistant. A total of 599 E. coli colonies were tested for antimicrobial susceptibility. When E. coli isolates from the enrichment sample were included for antimicrobial susceptibility testing (only when no E. coli bacteria could be enumerated), the isolated E. coli colonies could have originated from the same strain. In that case, only E. coli isolates with different antimicrobial resistance patterns were included in the results (44). This resulted in a total of 473 colonies (Table 1).
Table 1.
Prevalence of E. coli in the lettuce primary-production environment
| Site and farm no. or parameter | No. of samples, isolates |
||||
|---|---|---|---|---|---|
| Seedling peat soil | Lettuce soil | Lettuce | Seedlings | Water | |
| Greenhouse | |||||
| 1 | 7, 9 | 36, 17 | 36, 10 | 3, 0 | 18, 18 |
| 2 | 7, 8 | 36, 15 | 36, 6 | 3, 0 | 18, 24 |
| 3 | 7, 11 | 36, 24 | 36, 5 | 3, 0 | 18, 12 |
| 4 | 7, 8 | 36, 27 | 36, 5 | 3, 0 | 17, 6 |
| Open air | |||||
| 5 | 2, 3 | 24, 16 | 18, 3 | 3, 0 | 9, 3 |
| 6 | 9, 9 | 36, 27 | 36, 21 | 3, 0 | 15, 37 |
| 7 | 9, 9 | 36, 23 | 36, 12 | 3, 0 | 12, 31 |
| 8 | 9, 9 | 36, 22 | 30, 10 | 3, 3 | 10, 30 |
| Total (n = 473) | 57, 66 | 276, 171 | 264, 72 | 24, 3 | 117, 161 |
| % Enumerable E. coli samplesa | 96.5 | 36.6 | 5.3 | 4.3 | 59.2 |
| Range (min-max) | <0.7–3.9 | <0.7–3.2 | <0.7–2.0 | <0.7–1.4 | <0–3.6 |
Proportion of samples exceeding the detection limit for enumeration of E. coli (50 CFU/g or 0.7 log CFU/g for solid samples and 1 CFU/100 ml or 0 log CFU/100 ml for water samples).
SPSS statistics 20 and Microsoft Excel were used for statistical analysis. The χ2 test was used to analyze differences in the frequency of resistance between isolates obtained from greenhouses, those from open-air farms, those from different farms, and those from different samples. Because of the low prevalence of antimicrobial resistance among the E. coli isolates, logistic regression did not provide any additional output.
RESULTS
Prevalence of E coli.
From April 2011 to December 2012, 740 samples were collected, 57 seedling peat soil samples, 23 seedling leaf samples, 264 lettuce samples (792 samples separated into sets of three), 276 soil samples (828 samples separated into sets of three), and 120 irrigation water samples (Table 1). E. coli bacteria were enumerable in almost all of the samples (56/57) of seedling peat soil, whereas E. coli was present at levels of >10 CFU/g in approximately 37% of the soil samples. E. coli bacteria were enumerated (>1 CFU/100 ml) in 60% of the irrigation water samples (11.1% [4/36] of the groundwater and 79.8% [67/84] of the open-well water samples). The lowest proportions of samples exceeding 10 CFU/g E. coli were found in the lettuce leaves (5%) and seedling leaf samples (4%). Thus, overall, significantly more samples for E. coli enumeration were found among the environmental samples (soil and water) than among the food crop samples (lettuce leaves and seedlings) (P < 0.05).
This resulted in a total of 473 E. coli isolates obtained from the 738 samples gathered during the sampling period, with 66 isolates from seedling peat soil, only 3 isolates from seedling leaves, 171 isolates from surrounding soil, 161 isolates from irrigation water, and 72 isolates from lettuce leaves (Table 1).
Antimicrobial susceptibility.
The results of in vitro susceptibility testing of all of the E. coli isolates from different sources are shown in Table 2. No distinction is made between the different sources of water because of the low prevalence of resistance (open-well water, 8.4%; groundwater, 5.6%). Fifty-four (11.4%) of 473 isolates were found to be resistant to one or more of the antimicrobial agents tested. Only 1 of the 473 isolates was resistant to NAL, and another 1 was resistant to CEF. Resistance to AMP was more common (7% in all of the groups). The prevalence of AMC, CEP, TET, and TRIM resistance was between 4 and 4.5%, followed by the prevalence of STR (3.6%), SULFA (3.0%), and CLR (1.9%) resistance. There were no E. coli isolates with reduced susceptibility to the aminoglycosides amikacin (AMI), KAN, and GEN and the quinolone CIP.
Table 2.
Prevalence of 473 AR E. coli isolates in 264 lettuce, 276 soil, 57 seedling peat soil, and 120 irrigation water samples
| Antimicrobial(s) | Amt(s)/disc (μg) | No. (%) of resistant isolates from: |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| All lettuce samples (n = 72) | Greenhouse lettuce (n = 26) | Open-air lettuce (n = 46) | All soil samples (n = 171) | Greenhouse soil (n = 83) | Open-air soil (n = 88) | Seedling soil (n = 66) | Water (n = 161) | Total (n = 473) | ||
| AMI | 30 | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| AMC | 20/10 | 8 (11.1) | 5 (19.2) | 3 (6.5) | 8 (4.7) | 3 (3.6) | 4 (4.5) | 1 (1.5) | 3 (1.9) | 20 (4.2) |
| AMP | 30 | 11 (15.3) | 6 (23.1) | 5 (10.9) | 12 (7.0) | 8 (9.6) | 4 (4.5) | 3 (4.5) | 7 (4.3) | 33 (7.0) |
| CEF | 30 | 0 (0.0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (1.5) | 0 (0) | 1 (0.2) |
| CEP | 30 | 10 (13.9) | 6 (23.1) | 4 (8.7) | 6 (3.5) | 3 (3.6) | 3 (3.4) | 2 (3.0) | 3 (1.9) | 21 (4.4) |
| CLR | 30 | 1 (1.4) | 0 (0) | 1 (2.1) | 2 (1.2) | 0 (0) | 2 (2.3) | 2 (3.0) | 4 (2.5) | 9 (1.9) |
| CIP | 5 | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| GEN | 10 | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| KAN | 30 | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| NAL | 30 | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (0.6) | 1 (0.2) |
| STR | 10 | 4 (5.6) | 0 (0) | 4 (8.7) | 3 (1.8) | 2 (2.4) | 1 (1.1) | 5 (7.6) | 5 (3.1) | 17 (3.6) |
| SULFA | 240 | 4 (5.6) | 1 (3.8) | 3 (6.5) | 3 (1.8) | 2 (2.4) | 1 (1.1) | 2 (3.0) | 5 (3.1) | 14 (3.0) |
| TET | 30 | 5 (6.9) | 2 (7.7) | 3 (6.5) | 3 (1.8) | 2 (2.4) | 1 (1.1) | 4 (6.1) | 8 (5.0) | 20 (4.2) |
| TRIM | 5 | 5 (6.9) | 1 (3.8) | 4 (8.7) | 5 (2.9) | 3 (3.6) | 2 (2.3) | 4 (6.1) | 5 (3.1) | 19 (4.0) |
Of the 54 AR E. coli isolates, 37 were found to be multiresistant (Table 3). AMP resistance was present in approximately 75% of the multiresistant isolates. AMC and CEP accounted for approximately 50%. The most frequently observed pattern found in the multiresistant isolates was the combination of AMC, AMP, and CEP resistance (n = 9) (Table 3). Four isolates were resistant to seven antimicrobial agents.
Table 3.
Antimicrobial resistance patterns of E. coli isolates from lettuce, soil, and irrigation water samples from eight lettuce production farms
| No. of antimicrobials to which isolates were resistant | No. (%) of isolates | Most frequent pattern (no. of isolates)a |
|---|---|---|
| 0 | 419 (88.6) | |
| 1 | 17 (3.6) | AMP10 (4) |
| 2 | 5 (1.1) | STR10-TRIM5 (2), AMP10-CEP30 (2) |
| 3 | 20 (4.2) | AMC30-AMP10-CEP30 (9) |
| 4 | 3 (0.6) | AMP10-SULFA-TET30-TRIM5 (2) |
| 5 | 2 (0.4) | AMC30-AMP10-STR10-SULFA-TRIM5 (1), AMP10-STR10-SULFA-TET30-TRIM5 (1) |
| 6 | 3 (0.6) | AMP10-CLR30-STR10-SULFA-TET30-TRIM5 (3) |
| 7 | 4 (0.8) | AMC30-AMP10-CEP30-STR10-SULFA-TET30-TRIM5 (2), AMP10-CEP30-CLR30-STR10-SULFA-TET30-TRIM5 (2) |
| All isolates | 473 (100) |
Each abbreviation includes the antimicrobial amount on the disc (micrograms) to which the bacteria were resistant.
A significant difference in antimicrobial resistance was observed between E. coli isolates obtained from soil, water, and lettuce in greenhouse farms and those obtained from open-air farms (percentages of resistant isolates, 13 and 8.9% in greenhouse and open-air farms, respectively; P < 0.05). A difference was also observed in the prevalence of antimicrobial resistance in seedling peat soil E. coli isolates from greenhouse farms (27.8%) and those from open-air farms (3.3%). The prevalence of resistant isolates was significantly higher on lettuce leaves (22.2%) than in the surrounding soil (8.8%, P < 0.05) or in irrigation water (7.5%, P < 0.05).
DISCUSSION
In the present study, E. coli was isolated from both environmental (water and soil) samples and food samples (lettuce leaves) taken at the primary-production stage during multiple farm visits at various points in the lettuce crop cycle throughout the year. This is in contrast to the majority of studies on antimicrobial resistance in E. coli that have used isolates obtained from fecal samples at animal production facilities or from food of animal origin. The presence of elevated numbers of E. coli bacteria in vegetable food products is an indicator of improper sanitary treatment and fecal contamination with an increased probability of having zoonotic pathogens present (45, 46). Lettuce farms try to restrict the presence of E. coli in irrigation water, in planting soil, and on the marketable lettuce crop by using “good agricultural practices” (47, 48). This might explain why no E. coli bacteria could be enumerated in the majority of the samples (67% of all samples) and in 95% of the lettuce samples (<10 CFU/g or <1 CFU/100 ml). This is in agreement with other publications reporting E. coli enumeration prevalences of only 0.3% (49), 1.6% (50), and 8.2% (51) on all kinds of different fresh vegetables, such as lettuce, tomatoes, and onions.
Significantly more samples with higher E. coli levels (>10 CFU/g) were obtained from open-air farms than from greenhouse farms. This could be expected, as greenhouses are more isolated from environmental influences that can cause contamination. In particular, if vegetables are grown in open fields next to an animal-rearing operation, there is the possibility that produce will be contaminated by animals (52). Possible fecal (and thus E. coli) contamination is further enhanced by precipitation and runoff water from neighboring (pasture) fields along with possible flooding of the field with contaminated surface water (53–55). Notwithstanding the more frequent isolation of enumerable E. coli in samples from open-air farms, the antimicrobial resistance rate was significantly lower in E. coli isolates from open-air samples (17%) than in those from greenhouse samples (30%) (P < 0.05). This could be attributed to the finding that the number of resistant E. coli isolates was significantly higher in the seedling peat soil used in the greenhouses (P < 0.05). Almost 40% of the E. coli isolates from the seedling peat soil samples used to set up the lettuce crop production in the greenhouses was shown to carry antimicrobial resistance genes, compared to only 3% of the E. coli isolates from the peat soil of seedlings being used for open-air farms (P < 0.05). The reason for this increased percentage of resistant E. coli isolates is unclear, as no significant difference in the enumeration of E. coli between the different nurseries that produced the seedlings (peat soil) was observed (P > 0.05). The most plausible reason could be the use of a different fertilizer. Fertilizer is known to contain large amounts of antimicrobial resistance genes (56), and manure management influences the environmental fate of the resistance (57). For example, high-intensity management (amending, watering, and turning) is more effective in reducing the prevalence of antimicrobial resistance genes than low-intensity management (no amending, watering, or turning) (58).
No difference in antimicrobial resistance rates between greenhouses and open-air farms was observed in E. coli isolates obtained from soil and irrigation water samples (P > 0.05). A significantly higher number of AR E. coli isolates was retrieved from lettuce leaves than from soil or irrigation water (P < 0.05). It is remarkable that although lettuce leaves had far fewer isolates than either soil or water samples, the proportion of resistant E. coli was much higher. A potential explanation might be that the susceptible isolates from soil and water were all clonal and diluted the percentage of resistant isolates. Further research on this topic would be very valuable, as it might help to find out which processing steps and/or environmental factors have a major effect on the prevalence of resistant bacteria.
The E. coli contamination of a lettuce primary-production environment is complex, as there is usually no direct contact with farm animals (there were no farm animals present on the lettuce production farms sampled in the present study). E. coli is established as a fecal contaminant of zoonotic origin in the lettuce primary-production environment, and it is known that overall cattle, swine, broilers, and wild animals show higher prevalences of (multiple-)AR E. coli strains (Table 4 and Fig. 1) than the overall low prevalence of AR E. coli isolates obtained in the present study (11.4%). This lower level of resistance may be the result of reversibility of antimicrobial resistance or negative selection toward multiresistant plasmids in the absence of the antimicrobial pressure or a dilution (15, 44, 59–61).
Table 4.
Percentages of AR E. coli isolates obtained from lettuce primary-production environments versus animal primary-production environments and surface water in Belgium
| Isolate source | % of isolates resistant to: |
Reference | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| AMI | AMC | AMP | CEF | CEP | CLR | CIP | GEN | KAN | NAL | STR | SULFA | TET | TRIM | SXT | ||
| Lettuce production | 0.0 | 4.2 | 7.0 | 0.2 | 4.4 | 1.9 | 0.2 | 0.0 | 0.0 | 0.2 | 3.6 | 3.0 | 4.2 | 4.0 | ||
| Pigs | 51.0 | 4.5 | 26.8 | 14.0 | 4.5 | 3.2 | 12.7 | 43.3 | 58.6 | 56.7 | 50.3 | 65 | ||||
| Pigs | 12.3 | 50.8 | 13.8 | 27.7 | 76.9 | 72.3 | 53.8 | 66 | ||||||||
| Poultry | 29.5 | 74.4 | 9.0 | 52.6 | 69.2 | 74.4 | 50.0 | 66 | ||||||||
| Poultry | 85.0 | 19.1 | 24.3 | 62.9 | 5.0 | 6.9 | 63.1 | 60.1 | 74.3 | 64.8 | 63.1 | 65 | ||||
| Poultry | 0.1 | 80.2 | 18.7 | 0.1 | 61.2 | 41.0 | 61.1 | 49.8 | 60 | |||||||
| Dairy cattle | 14.1 | 10.3 | 5.1 | 10.4 | 34.6 | 34.6 | 0.0 | 66 | ||||||||
| Cattle | 26.6 | 4.5 | 14.3 | 11.0 | 3.9 | 5.2 | 12.3 | 23.4 | 28.6 | 19.5 | 19.5 | 65 | ||||
| Veal calves | 70.6 | 0.0 | 50.0 | 41.2 | 20.6 | 29.4 | 41.2 | 52.9 | 79.4 | 73.5 | 70.6 | 65 | ||||
| Hares | 4.5 | 3.0 | 1.5 | 3.0 | 13.6 | 16.7 | 1.5 | 66 | ||||||||
| Surface water | 13.0 | 63.0 | 6.5 | 26.1 | 65.2 | 71.7 | 8.7 | 66 | ||||||||
Fig 1.
Comparison of multiple resistance patterns of E. coli isolates obtained from various animal species, surface water, and lettuce primary production facilities (65, 66).
The present study confirmed previous results stating that AR bacteria may be present on vegetables. However, in most cases, a direct comparison of studies is difficult because of the different types of samples involved, the different scopes of bacterial species targeted, the different methods of strain isolation used, or the different antimicrobials tested (12, 44, 49, 62–64). The results of the present study can only be compared to the few other studies that focused on antimicrobial resistance testing of Enterobacteriaceae isolates from fresh vegetables (44, 49, 63). For example, Osterblad et al. (44) and Schwaiger et al. (49) found, in agreement with the present study, no AMI, GEN, or CIP resistance in Enterobacteriaceae isolates from fresh vegetables. In this study also, only one isolate was resistant to NAL, which corresponds to the 100% susceptibility detected by Osterblad et al. (44). Hassan et al. (63) also observed low resistance to KAN in E. coli isolates from fresh vegetables. In this study, the highest antimicrobial resistance rates were observed for AMP, AMC, CEP, STR, TET, TRIM, and SULFA. This corresponds to other studies that showed, in general, the highest levels (if tested) of resistance to those antimicrobials. Only with sulfamethoxazole-trimethoprim (SXT) and STR were highly variable resistance results obtained; the resistance rate was quite high (50%) in some cases and low (0%) in others (44, 49, 63). In recent studies, a high prevalence of resistance to the third-generation cephalosporin CEF (49, 63) was observed, in contrast to this study, where only one isolate showed resistance.
The results of the present study on the antimicrobial resistance of E. coli isolates obtained from the plant primary-production side were compared to other Belgian data available for antimicrobial resistance rates of E. coli isolates from animal production sources such as poultry, cattle, pig, and hares, as well some available data from E. coli isolates obtained from surface water (Table 4) (60, 65, 66). Antimicrobial resistance varied greatly among the different sources of E. coli isolates. The antimicrobial resistance of commensal E. coli is the highest in poultry, followed by pigs and cattle. Veal calves showed remarkably higher resistance than cattle, with resistance percentages similar to those of pigs and for some comparable to those of poultry. The resistance pattern of the plant production environment was closest to that of cattle isolates, as the highest antimicrobial resistance rates were also noted for AMP, STR, TET, TRIM, and SULFA. In comparison to the E. coli strains isolated from animal production facilities, E. coli strains isolated from the vegetable production chain had a lower overall rate of resistance to almost all of the antimicrobials investigated, which has also been noted by others (44, 49).
Because of the EU ban in 1994, CLR has not been used in nearly 20 years in EU member states, including Belgium (49, 67, 68). However, the percentages of E. coli isolates from both animal production (14 to 50%) and plant production facilities, as established in the present study (2%), that showed CLR resistance were still remarkable. Also, the study of Osterblad et al. (44) noted CLR resistance in 12% of the Enterobacteriaceae isolates obtained from vegetables.
The percentage of resistance to multiple antibiotics occurring among E. coli isolates was lower than in other publications involving E. coli isolates from a variety of animal species. Furthermore, depending on the animal species, a different multiresistance pattern was observed (15, 18, 60, 65, 66, 69) (Fig. 1). When the resistance patterns were compared with animal profiles, the closest resistance pattern found was that of cattle. This may suggest that cattle are a potential reservoir of AR E. coli strains in plant primary production.
Raw foods of animal origin, often studied and known to carry antibiotic-resistant commensal E. coli or zoonotic pathogenic bacteria, are most of the time heat treated or subjected to an equivalent processing technique to inactivate microorganisms before consumption. The transfer of AR bacteria in those types of foods to humans is probably minimal (70). Exposure is still possible because of cross-contamination in the kitchen (71). However, the present study and also other studies have revealed an antibiotic resistance pool in food-borne commensal bacteria in many ready-to-consume food products (23, 49, 72). Because many of these foods (including fresh produce such as lettuce) are directly consumed without further processing, AR bacteria can be directly transmitted to humans through daily food intake (70). As there is a potential to pass resistance to other bacteria, including those normally present in the human gastrointestinal tract, ready-to-eat foods such as fresh produce may serve as a vehicle for expanding the pool of antibiotic resistances available to (pathogenic or other) bacteria inhabiting humans (72). If these resistance genes are transferred to human-pathogenic bacteria, infections may become more difficult to treat. Although assessments of exposure to AR E. coli through the consumption of meat (via undercooking or cross-contamination) have been published (71, 73), transmission via vegetable consumption has not been considered because of a lack of data on the prevalence of resistant E. coli in vegetable production. The present study can fill this data gap.
Conclusion.
This study determined the presence of AR E. coli on vegetables and in a vegetable production environment, illustrated by the case study of lettuce. It proved that vegetables may act as a reservoir and vector of antimicrobial resistance. It can be highlighted that E. coli isolates from the lettuce crop showed a higher antimicrobial resistance rate than those from soil or irrigation water samples. The percentage of multiresistance to antibiotics among the E. coli isolates in the present study is lower than that in other publications involving E. coli isolates from a variety of animal species. The antimicrobial resistance patterns suggest cattle as the main source of commensal resistant E. coli contamination, as the presence of resistance and resistance patterns with animal profiles are the most comparable to what is found in cattle. Because fresh produce such as lettuce is directly consumed without further microbial inactivation treatment, it may contribute directly to human exposure to AR bacteria. This study emphasizes the need for “good agricultural practices” to keep fecal contamination and E. coli levels low, thus reducing the probability not only of exposure to human-pathogenic zoonotic bacteria but also of exposure to AR commensal E. coli.
ACKNOWLEDGMENT
This research leading to these results has been facilitated by the European Community's Seventh Framework Program (FP7) under grant agreement 244994 (project VEG-i-TRADE).
Footnotes
Published ahead of print 23 August 2013
REFERENCES
- 1. Anderson RM. 1999. The pandemic of antibiotic resistance. Nat. Med. 5:147–149 [DOI] [PubMed] [Google Scholar]
- 2. Levy SB, Marshall B. 2004. Antibacterial resistance worldwide: causes, challenges and responses. Nat. Med. 10:S122–S129 [DOI] [PubMed] [Google Scholar]
- 3. Stockwell VO, Duffy B. 2012. Use of antibiotics in plant agriculture. Rev. Sci. Tech. 31:199–210 [DOI] [PubMed] [Google Scholar]
- 4. McManus PS, Stockwell VO, Sundin GW, Jones AL. 2002. Antibiotic use in plant agriculture. Annu. Rev. Phytopathol. 40:443–465 [DOI] [PubMed] [Google Scholar]
- 5. Gustafson RH, Bowen RE. 1997. Antibiotic use in animal agriculture. J. Appl. Microbiol. 83:531–541 [DOI] [PubMed] [Google Scholar]
- 6. van den Bogaard AE, Stobberingh EE. 2000. Epidemiology of resistance to antibiotics—links between animals and humans. Int. J. Antimicrob. Agents 14:327–335 [DOI] [PubMed] [Google Scholar]
- 7. van den Bogaard AE, Stobberingh EE. 1999. Antibiotic usage in animals—impact on bacterial resistance and public health. Drugs 58:589–607 [DOI] [PubMed] [Google Scholar]
- 8. Lester SC, Pla MD, Fu W, Schael IP, Jiang H, Obrien TF. 1990. The carriage of Escherichia coli resistant to antimicrobial agents by healthy children in Boston, in Caracas, Venezuela, and in Qin-Pu, China. N. Engl. J. Med. 323:285–289 [DOI] [PubMed] [Google Scholar]
- 9. Sáenz Y, Zarazaga M, Brinas L, Lantero M, Ruiz-Larrea F, Torres C. 2001. Antibiotic resistance in Escherichia coli isolates obtained from animals, foods and humans in Spain. Int. J. Antimicrob. Agents 18:353–358 [DOI] [PubMed] [Google Scholar]
- 10. Aarestrup FM, Wegener HC, Collignon P. 2008. Resistance in bacteria of the food chain: epidemiology and control strategies. Expert Rev. Anti Infect. Ther. 6:733–750 [DOI] [PubMed] [Google Scholar]
- 11. Wooldridge M. 2012. Evidence for the circulation of antimicrobial-resistant strains and genes in nature and especially between humans and animals. Rev. Sci. Tech. 31:231–247 [DOI] [PubMed] [Google Scholar]
- 12. McGowan LL, Jackson CR, Barrett JB, Hiott LM, Fedorca-Cray PJ. 2006. Prevalence and antimicrobial resistance of enterococci isolated from retail fruits, vegetables, and meats. J. Food Prot. 69:2976–2982 [DOI] [PubMed] [Google Scholar]
- 13. Dai L, Lu LM, Wu CM, Li BB, Huang SY, Wang SC, Qi YH, Shen JZ. 2008. Characterization of antimicrobial resistance among Escherichia coli isolates from chickens in China between 2001 and 2006. FEMS Microbiol. Lett. 286:178–183 [DOI] [PubMed] [Google Scholar]
- 14. Enne VI, Cassar C, Sprigings K, Woodward MJ, Bennett PM. 2008. A high prevalence of antimicrobial resistant Escherichia coli isolated from pigs and a low prevalence of antimicrobial resistant E. coli from cattle and sheep in Great Britain at slaughter. FEMS Microbiol. Lett. 278:193–199 [DOI] [PubMed] [Google Scholar]
- 15. Knezevic P, Petrovic O. 2008. Antibiotic resistance of commensal Escherichia coli of food-producing animals from three Vojvodinian farms, Serbia. Int. J. Antimicrob. Agents 31:360–363 [DOI] [PubMed] [Google Scholar]
- 16. Lanz R, Kuhnert P, Boerlin P. 2003. Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland. Vet. Microbiol. 91:73–84 [DOI] [PubMed] [Google Scholar]
- 17. Persoons D, Dewulf J, Smet A, Herman L, Heyndrickx M, Martel A, Catry B, Butaye P, Haesebrouck F. 2012. Antimicrobial use in Belgian broiler production. Prev. Vet. Med. 105:320–325 [DOI] [PubMed] [Google Scholar]
- 18. Lim SK, Lee HS, Nam HM, Cho YS, Kim JM, Song SW, Park YH, Jung SC. 2007. Antimicrobial resistance observed in Escherichia coli strains isolated from fecal samples of cattle and pigs in Korea during 2003-2004. Int. J. Food Microbiol. 116:283–286 [DOI] [PubMed] [Google Scholar]
- 19. Persoons D, Bollaerts K, Smet A, Herman L, Heyndrickx M, Martel A, Butaye P, Catry B, Haesebrouck F, Dewulf J. 2011. The importance of sample size in the determination of a flock-level antimicrobial resistance profile for Escherichia coli in broilers. Microb. Drug Resist. 17:513–519 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Varga C, Rajic A, McFall ME, Avery BP, Reid-Smith RJ, Deckert A, Checkley SL, McEwen SA. 2008. Antimicrobial resistance in generic Escherichia coli isolated from swine fecal samples in 90 Alberta finishing farms. Can. J. Vet. Res. 72:175–180 [PMC free article] [PubMed] [Google Scholar]
- 21. Dewulf J, Catry B, Timmerman T, Opsomer G, de Kruif A, Maes D. 2007. Tetracycline-resistance in lactose-positive enteric coliforms originating from Belgian fattening pigs: degree of resistance, multiple resistance and risk factors. Prev. Vet. Med. 78:339–351 [DOI] [PubMed] [Google Scholar]
- 22. Duffy EA, Lucia LM, Kells JM, Castillo A, Pillai SD, Acuff GR. 2005. Concentrations of Escherichia coli and genetic diversity and antibiotic resistance profiling of Salmonella isolated from irrigation water, packing shed equipment, and fresh produce in Texas. J. Food Prot. 68:70–79 [DOI] [PubMed] [Google Scholar]
- 23. Ruimy R, Brisabois A, Bernede C, Skurnik D, Barnat S, Arlet G, Momcilovic S, Elbaz S, Moury F, Vibet MA, Courvalin P, Guillemot D, Andremont A. 2010. Organic and conventional fruits and vegetables contain equivalent counts of Gram-negative bacteria expressing resistance to antibacterial agents. Environ. Microbiol. 12:608–615 [DOI] [PubMed] [Google Scholar]
- 24. Beuchat LR. 2006. Vectors and condition for pre-harvest contamination of fruits and vegetables with pathogens capable of causing enteric diseases. Br. Food J. 108:38–53 [Google Scholar]
- 25. Steele M, Odumeru J. 2004. Irrigation water as source of foodborne pathogens on fruit and vegetables. J. Food Prot. 67:2839–2849 [DOI] [PubMed] [Google Scholar]
- 26. James J. 2006. Microbial hazard identification in fresh fruit and vegetables. John Wiley & Sons, Hoboken, NJ [Google Scholar]
- 27. Ingham SC, Fanslau MA, Engel RA, Breuer JR, Breuer JE, Wright TH, Reith-Rozelle JK, Zhu J. 2005. Evaluation of fertilization-to-planting and fertilization-to-harvest intervals for safe use of noncomposted bovine manure in Wisconsin vegetable production. J. Food Prot. 68:1134–1142 [DOI] [PubMed] [Google Scholar]
- 28. Brackett RE. 1999. Incidence, contributing factors, and control of bacterial pathogens in produce. Postharvest Biol. Technol. 15:305–311 [Google Scholar]
- 29. Tournas VH. 2005. Moulds and yeasts in fresh and minimally processed vegetables and sprouts. Int. J. Food Microbiol. 99:71–77 [DOI] [PubMed] [Google Scholar]
- 30. Johannessen GS, Bengtsson GB, Heier BT, Bredholt S, Wasteson Y, Rorvik LM. 2005. Potential uptake of Escherichia coli O157:H7 from organic manure into crisphead lettuce. Appl. Environ. Microbiol. 71:2221–2225 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31. Solomon EB, Yaron S, Matthews KR. 2002. Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization. Appl. Environ. Microbiol. 68:397–400 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32. Brandl MT. 2006. Fitness of human enteric pathogens on plants and implications for food safety. Annu. Rev. Phytopathol. 44:367–392 [DOI] [PubMed] [Google Scholar]
- 33. Islam M, Morgan J, Doyle MP, Phatak SC, Millner P, Jiang X. 2004. Persistence of Salmonella enterica serovar Typhimurium on lettuce and parsley and in soils on which they were grown in fields treated with contaminated manure composts or irrigation water. Foodborne Pathog. Dis. 1:27–35 [DOI] [PubMed] [Google Scholar]
- 34. Kudva IT, Blanch K, Hovde CJ. 1998. Analysis of Escherichia coli O157:H7 survival in ovine or bovine manure and manure slurry. Appl. Environ. Microbiol. 64:3166–3174 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35. Nicholson FA, Groves SJ, Chambers BJ. 2005. Pathogen survival during livestock manure storage and following land application. Bioresour. Technol. 96:135–143 [DOI] [PubMed] [Google Scholar]
- 36. Forshell LP, Ekesbo I. 1993. Survival of Salmonellas in composted and not composted solid animal manure. Zentralbl. Veterinaermed. B 40:654–658 [DOI] [PubMed] [Google Scholar]
- 37. Chalmers RM, Aird H, Bolton FJ. 2000. Waterborne Escherichia coli O157. Symp. Ser. Soc. Appl. Microbiol. 2000:124S–132S [DOI] [PubMed] [Google Scholar]
- 38. Doyle MP, Erickson MC. 2008. Summer meeting 2007—the problems with fresh produce: an overview. J. Appl. Microbiol. 105:317–330 [DOI] [PubMed] [Google Scholar]
- 39.International Organization for Standardization 2006. Water quality—sampling for microbiological analysis. ISO 19458:2006 International Organization for Standardization, Geneva, Switzerland [Google Scholar]
- 40.Association Française de Normalisation 2004. RAPID'E. coli 2 validated for the enumeration of coliforms at 37°C. BRD-07/08-12/04 Association Française de Normalisation, Saint-Denis, France [Google Scholar]
- 41.International Organization for Standardization 2000. Water quality—detection and enumeration of Escherichia coli and coliform bacteria. Part 1: membrane filtration method. ISO 9308-1:2000 International Organization for Standardization, Geneva, Switzerland [Google Scholar]
- 42.Clinical and Laboratory Standards Institute 2008. Performance standards for antimicrobial disc susceptibility tests for bacteria isolated from animals. Approved standard (M31-A3), 3rd ed. Clinical and Laboratory Standards Institute, Wayne, PA [Google Scholar]
- 43.Eucast and CLSI potency NEO-Sensitabs 2011. Zone diameter interpretative criteria and MIC breakpoints according to CLSI. Rosco Diagnostica, Taastrup, Denmark: http://www.rosco.dk [Google Scholar]
- 44. Osterblad M, Pensala O, Peterzens M, Heleniuse H, Huovinen P. 1999. Antimicrobial susceptibility of Enterobacteriaceae isolated from vegetables. J. Antimicrob. Chemother. 43:503–509 [DOI] [PubMed] [Google Scholar]
- 45. Savichtcheva O, Okabe S. 2006. Alternative indicators of fecal pollution: relations with pathogens and conventional indicators, current methodologies for direct pathogen monitoring and future application perspectives. Water Res. 40:2463–2476 [DOI] [PubMed] [Google Scholar]
- 46. Gerba CP. 2009. The role of water and water testing in produce safety, p 129–142 In Fan X, Niemira BA, Doona CJ, Feeherry FE, Gravani RB. (ed), Microbial safety of fresh produce. Wiley-Blackwell, Oxford, United Kingdom [Google Scholar]
- 47.World Health Organization 2003. Codex alimentarius: code of hygienic practices for fresh fruits and vegetables (CAC/RCP 53-2003). World Health Organization, Geneva, Switzerland [Google Scholar]
- 48.GLOBALG.A.P 2012. Control points and compliance criteria: integrated farm assurance. All farm base. GLOBALG.A.P., Cologne, Germany: http://www1.globalgap.org/cms/upload/The_Standard/IFA/Version_4_2011/English/CPCC_TC/101012_GG_IFA_CPCC_TE_ENG_Interim_Final_V4_TC.pdf [Google Scholar]
- 49. Schwaiger K, Helmke K, Holzel CS, Bauer J. 2011. Antibiotic resistance in bacteria isolated from vegetables with regards (sic) to the marketing stage (farm vs. supermarket). Int. J. Food Microbiol. 148:191–196 [DOI] [PubMed] [Google Scholar]
- 50. Mukherjee A, Speh D, Dyck E, Diez-Gonzalez F. 2004. Preharvest evaluation of coliforms, Escherichia coli, Salmonella, and Escherichia coli O157:H7 in organic and conventional produce grown by Minnesota farmers. J. Food Prot. 67:894–900 [DOI] [PubMed] [Google Scholar]
- 51. Bohaychuk VM, Bradbury RW, Dimock R, Fehr M, Gensler GE, King RK, Rieve R, Barrios PR. 2009. A microbiological survey of selected Alberta-grown fresh produce from farmers' markets in Alberta, Canada. J. Food Prot. 72:415–420 [DOI] [PubMed] [Google Scholar]
- 52. Jay MT, Cooley M, Carychao D, Wiscomb GW, Sweitzer RA, Crawford-Miksza L, Farrar JA, Lau DK, O'Connell J, Millington A, Asmundson RV, Atwill ER, Mandrell RE. 2007. Escherichia coli O157:H7 in feral swine near spinach fields and cattle, central California coast. Emerg. Infect. Dis. 13:1908–1911 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53. Casteel MJ, Sobsey MD, Mueller JP. 2006. Fecal contamination of agricultural soils before and after hurricane-associated flooding in North Carolina. J. Environ. Sci. Health A Tox. Hazard. Subst. Environ. Eng. 41:173–184 [DOI] [PubMed] [Google Scholar]
- 54. Rose JB, Epstein PR, Lipp EK, Sherman BH, Bernard SM, Patz JA. 2001. Climate variability and change in the United States: potential impacts on water- and foodborne diseases caused by microbiologic agents. Environ. Health Perspect. 109:211–221 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55. Curriero FC, Patz JA, Rose JB, Lele S. 2001. The association between extreme precipitation and waterborne disease outbreaks in the United States, 1948-1994. Am. J. Public Health 91:1194–1199 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 56. Sengeløv G, Agerso Y, Halling-Sorensen B, Baloda SB, Andersen JS, Jensen LB. 2003. Bacterial antibiotic resistance levels in Danish farmland as a result of treatment with pig manure slurry. Environ. Int. 28:587–595 [DOI] [PubMed] [Google Scholar]
- 57. Baquero F, Martinez JL, Canton R. 2008. Antibiotics and antibiotic resistance in water environments. Curr. Opin. Biotechnol. 19:260–265 [DOI] [PubMed] [Google Scholar]
- 58. Storteboom HN, Kim SC, Doesken KC, Carlson KH, Davis JG, Pruden A. 2007. Response of antibiotics and resistance genes to high-intensity and low-intensity manure management. J. Environ. Qual. 36:1695–1703 [DOI] [PubMed] [Google Scholar]
- 59. Guenther S, Grobbel M, Luebke-Becker A, Goedecke A, Friedrich ND, Wieler LH, Ewers C. 2010. Antimicrobial resistance profiles of Escherichia coli from common European wild bird species. Vet. Microbiol. 144:219–225 [DOI] [PubMed] [Google Scholar]
- 60. Persoons D, Dewulf J, Smet A, Herman L, Heyndrickx M, Martel A, Catry B, Butaye P, Haesebrouck F. 2010. Prevalence and persistence of antimicrobial resistance in broiler indicator bacteria. Microb. Drug Resist. 16:67–74 [DOI] [PubMed] [Google Scholar]
- 61. Andersson DI, Hughes D. 2011. Persistence of antibiotic resistance in bacterial populations. FEMS Microbiol. Rev. 35:901–911 [DOI] [PubMed] [Google Scholar]
- 62. Boehme S, Werner G, Klare I, Reissbrodt R, Witte W. 2004. Occurrence of antibiotic-resistant enterobacteria in agricultural foodstuffs. Mol. Nutr. Food Res. 48:522–531 [DOI] [PubMed] [Google Scholar]
- 63. Hassan SA, Altalhi AD, Gherbawy YA, El-Deeb BA. 2011. Bacterial load of fresh vegetables and their resistance to the currently used antibiotics in Saudi Arabia. Foodborne Pathog. Dis. 8:1011–1018 [DOI] [PubMed] [Google Scholar]
- 64. Johnston LM, Jaykus LA. 2004. Antimicrobial resistance of Enterococcus species isolated from produce. Appl. Environ. Microbiol. 70:3133–3137 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 65. Butaye P. 2013. Report: antimicrobial resistance in commensal E. coli from poultry, pigs, cows and veal calves. CODA-CERVA, Veterinary and Agrochemical Research Centre, Brussels, Belgium [Google Scholar]
- 66. Casteleyn C, Dewulf J, Catry B, de Kruif A, Maes D. 2006. Antibioticumresistentie in Escherichia coli bij landbouwhuisdieren, hazen, septisch materiaal en oppervlaktewater in Vlaanderen. Flemish Vet. J. 75:23–30 [Google Scholar]
- 67.Anonymous 1990. Council regulation (EEC) no. 2377/90 laying down a community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Off. J. Eur. Communities L224 1. http://new.eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:31990R2377&rid=1
- 68.Anonymous 1994. Commission regulation (EC) no. 1430/94 of 22 June 1994 amending annexes I, II, III and IV of council regulation (EEC) no. 2377/90 laying down a community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Off. J. Eur. Communities L156 6. http://new.eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:31994R1430&rid=1
- 69. Jiang HX, Lu DH, Chen ZL, Wang XM, Chen JR, Liu YH, Liao XP, Liu JH, Zeng ZL. 2011. High prevalence and widespread distribution of multi-resistant Escherichia coli isolates in pigs and poultry in China. Vet. J. 187:99–103 [DOI] [PubMed] [Google Scholar]
- 70. Wang H, McEntire JC, Zhang L, Li X, Doyle M. 2012. The transfer of antibiotic resistance from food to humans: facts, implications and future directions. Rev. Sci. Tech. 31:249–260 [DOI] [PubMed] [Google Scholar]
- 71. Depoorter P, Persoons D, Uyttendaele M, Butaye P, De Zutter L, Dierick K, Herman L, Imberechts H, Van Huffel X, Dewulf J. 2012. Assessment of human exposure to 3rd generation cephalosporin resistant E. coli (CREC) through consumption of broiler meat in Belgium. Int. J. Food Microbiol. 159:30–38 [DOI] [PubMed] [Google Scholar]
- 72. Bezanson GS, MacInnis R, Potter G, Hughes T. 2008. Presence and potential for horizontal transfer of antibiotic resistance in oxidase-positive bacteria populating raw salad vegetables. Int. J. Food Microbiol. 127:37–42 [DOI] [PubMed] [Google Scholar]
- 73. Presi P, Stark KDC, Stephan R, Breidenbach E, Frey J, Regula G. 2009. Risk scoring for setting priorities in a monitoring of antimicrobial resistance in meat and meat products. Int. J. Food Microbiol. 130:94–100 [DOI] [PubMed] [Google Scholar]