Antimicrobial susceptibility of Enterobacteriaceae isolated from vegetables

Monica Österblada,*, Olli Pensalab, Margareta Peterzénsb, Hans Heleniuscc and Pentti Huovinena

a Antimicrobial Research Laboratory, National Public Health Institute, PO Box 57, FIN-20521, Turku b Finnish Customs Laboratory, Tekniikantie 14, FIN-02150 Espoo c The Department of Biostatistics, University of Turku, Lemminkäisenkatu 1, 20520 Turku, Finland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is potential for the normal faecal flora of humans to be augmented by resistant strains of bacteria, acquired from food. The frequency of resistance in the aerobic Gram-negative faecal flora is often very high. The purpose of this study was to find out whether food strains contribute to this resistance. One hundred and thirty-seven vegetable samples were studied, 48 of Finnish origin, and 89 imported. From these samples, 535 different strains of bacteria belonging to the family Enterobacteriaceae were isolated. Enterobacter spp. were most frequent, Escherichia coli was rare. Sensitivity testing was undertaken only for isolates with different biotypes and antibiograms. No resistance was found to cefotaxime, aztreonam, imipenem, gentamicin, nalidixic acid or ciprofloxacin. The frequency of trimethoprim resistance was 0.2%, sulphamethoxazole resistance 1.3%, and tetracycline resistance 5.5%. These frequencies were much lower than those found in faecal flora. Chloramphenicol and cefuroxime resistance was found in 12% and 14% of isolates, respectively. The only statistically significant differences between the Finnish and imported strains were for these two; the Finnish isolates were more resistant to cefuroxime, whereas the imported ones were more resistant to chloramphenicol. Consequently, bacteria from vegetables are not responsible for the high prevalence of resistant Enterobacteriaceae in faecal flora in Finland; they are in fact unusually susceptible to the antibiotics studied. Multiresistance profiles, typical of strains associated with human activities, were not identified in these isolates.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A high prevalence of antibiotic resistance is often encountered in Enterobacteriaceae of human origin, 1,2 whether derived from normal flora or clinical specimens, the flora of animals in contact with human refuse 7 or exposed to antibiotics, 8,9 or in sewage. 10 The high levels of resistance found in the faecal flora of healthy people, even in the absence of antimicrobial selection, 5 cannot yet be satisfactorily explained. Too little is known of resistance in populations outside the clinical environment to understand fully the dynamics of the spread of antimicrobial resistance.

The Enterobacteriaceae are not only pathogens and commensals of the mammalian gastrointestinal tract; 11 they are also found in abundance in almost any moist environment, notably soil, water, and the domestic environment. 12 Some, like Escherichia coli, are always considered to be of faecal origin, and exist only transiently in other environments. Others are natural environmental strains and can be found among the flora associated with growing vegetables. 13 It is possible that these environmental bacteria contribute to normal flora via food. Raw meat and vegetables are particularly likely to carry large numbers of bacteria. The same E. coli and Klebsiella spp. serotypes have been found in food and in the patients who consumed it. 14,15 A sterile diet was shown to lower the number of E. coli serotypes found in the faeces of test persons. 16 Bacteria escaping alive through the digestive tract to the colon are often transient, 17,18 the resident flora having a protective effect against intruders. Transfer of resistance within the gastrointestinal tract is still possible; thus, if our food contains substantial numbers of resistant bacteria, it could be an important source of resistance in faecal flora.

Vegetables were selected for this study, since they are often eaten raw. Resistant bacteria could colonize vegetables for a number of reasons: the direct use of antibiotics during cultivation, the use of contaminated fertilizers or irrigation water, 19 or some unknown non-human selection pressure. To our knowledge only two previous studies have addressed resistance frequencies among Enterobacteriaceae on vegetables. 20,21

We decided to screen the predominant types of Enterobacteriaceae found on common vegetables, both Finnish and imported, in shops and supermarkets in southwestern Finland. The aim of the study was to determine the contribution of these bacteria to the relatively high resistance levels found in faecal flora.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Origin of vegetable samples

Forty-eight fresh vegetable specimens of Finnish origin and 53 imported specimens were bought, mainly from three large supermarkets in the town of Turku (Table I). The locally grown vegetables were bought during August- December 1996 (a season when they dominate the market), and the foreign produce during January- April 1997 (since the supply of locally grown vegetables decreases in the spring). The origin of the imported vegetables was not always identifiable, but those for which the source was known came from Italy (cauliflower) and The Netherlands (Savoy cabbage, endive). Deep-frozen imported vegetables were also studied. These were obtained from the Finnish Customs Laboratory, and consisted of 36 samples of lots that had been discarded, because of high counts of faecal coliforms. These originated from Hungary (n= 28), Poland (n= 5), The Netherlands (n= 2), and Portugal (n= 1). The frozen samples had been processed in the normal manner for food of this type: diced or chopped, and some briefly heated, in their country of origin (paprika, leek and onion are not heated).


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Table I. Description of vegetable samples
 
Sampling of vegetables

If the products were not already plastic wrapped, they were picked from the stall with plastic bags, without the sampler touching the vegetable, to avoid possible contamination with laboratory strains from the hands of the sampler. They were stored at +4°C until processed (maximum interval 2 days). Using a knife, tray, and disposable gloves wiped with 70% ethanol after each sample, the vegetables were trimmed of spoiled parts, had their outer leaves removed, and were cut into pieces. The frozen vegetables were stored at -20°C. They were then spooned, with ethanol-washed spoons, from their plastic bags, and weighed and processed in the same manner as the fresh samples. Thirty-five-gram portions were put into Erlenmeyer flasks, and 150 mL of Brain Heart Infusion broth (Difco Laboratories, Detroit, MI, USA) was added. They were incubated at 37°C overnight in a shaker. Decimal dilutions were made in physiological saline, and plated on to MacConkey agar plates (Oxoid, Unipath Ltd, Basingstoke, UK). The plates were incubated overnight at 35°C. After incubation, each different colony type was streaked for purity, and frozen at -20°C in Tryptone Soya (Oxoid) broth with 20% glycerol, if not processed immediately.

Identification of isolates

All isolates were Gram-stained and tested for oxidase, catalase activity, and fermentation of glucose, by standard methods. Glucose-fermenting, Gram-negative, oxidase-negative, catalase-positive strains were considered to belong to the family Enterobacteriaceae, and only these were included in further testing. The isolates were identified at least to genus level by 22 biochemical tests, using BBI (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA), in-house and Oxoid media, diagnostic discs (Rosco, Taastrup, Denmark), and chemicals from Sigma (Sigma Chemical Co., St Louis, MO, USA). The tests were performed as described in the manufacturers's manuals and the Clinical Microbiology Procedures Handbook. 22

The results were compared with a database generated by combining the databases for Enterobacteriaceae in Bergey's s Manual 23 and the Manual of Clinical Microbiology, 24 using the Excel 5.0 spreadsheet program (Microsoft, Redmond, WA, USA). Unclear cases, with the genus determined at a probability <65%, were retested, and in some instances were tested with the API-E test (bioMérieux, Lyon, France).

MIC determinations

MIC were determined using a standard agar dilution method on Mueller- Hinton II medium (BBL). The antimicrobials tested (with concentration ranges) were: ampicillin (0.25- 256 mg/L), amoxycillin/clavulanic acid (0.5/0.25- 64/32 mg/L), cephalothin (0.25- 64 mg/L), cefuroxime (0.06- 64 mg/L), cefotaxime (0.06- 32 mg/L), aztreonam (0.25- 64 mg/L), imipenem (0.25- 64 mg/L), gentamicin (0.25- 64 mg/L), tetracycline (0.12- 64 mg/L), nalidixic acid (0.5- 128 mg/L), ciprofloxacin (0.06- 8 mg/L) trimethoprim (0.12- 1024 mg/L), sulphamethoxazole (0.5- 1024 mg/L), chloramphenicol (2- 128 mg/L). Co-amoxiclav was obtained from SmithKline Beecham Pharmaceuticals (Rixensart, Belgium), aztreonam from Bristol- Myers Squibb (Italy), imipenem from Merck Sharp & Dohme (Westpoint, PA, USA), and ciprofloxacin from Bayer (Leverkusen, Germany). All other antimicrobials were from Sigma. NCCLS breakpoints were used. 25 Intermediately resistant isolates were grouped with the resistant isolates.

Statistical methods

The significance of differences in resistance frequencies between the three groups were tested using logistic regression analysis. The differences were quantified with odds ratios (OR), applying 95% confidence intervals (95% CI). 26 Because the frequencies were small, exact tests and confidence intervals were used. The computations were performed with the LogXact-Turbo program (Cytel Software Corporation, Cambridge, MA, USA). 27


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Number of vegetable isolates

From each sample, only isolates with differing biotypes or antibiograms were included in the analysis. There were 182 isolates from the Finnish vegetables (mean [SD]: 4.2 [2.7] isolates per sample), 192 from the fresh foreign (4.1 [2.3]), and 162 from the frozen (4.7 [2.3]). Of the fresh vegetables, 10 yielded no growth of Enterobacteriaceae (five from each group). This group included a variety of vegetables, among them iceberg lettuce. Usually when bacterial counts are determined from vegetables the samples are blended, but this has been reported to free inhibiting substances from some plants, e.g. onion, 21 cauliflower and radishes. 28 The method used here seemed to avoid this problem, as growth was obtained from several onion samples. The method has the disadvantage that total bacterial counts cannot be calculated, but in this study the diversity of the flora was the main interest, not the hygienic quality of the vegetables.

Identity of vegetable isolates

Enterobacter spp. were the most common isolates in all three sample groups (Table II). The numbers in plain type in Table II represent the totals identified to species level, while the numbers in bold type following the genus names are the total numbers of isolates identified to genus level.


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Table II. Identity of Enterobacteriaceae isolates from vegetable samples
 
Antimicrobial resistance

Of the 14 antimicrobials tested, no resistance at all was found to cefotaxime, aztreonam, imipenem, gentamicin, nalidixic acid and ciprofloxacin. Decreased susceptibility to ciprofloxacin (MIC >=0.25- 1 mg/L) was found in three isolates from Finnish vegetables (Enterobacter aerogenes, Enterobacter amnigenus, Rahnella aquatilis), one from the fresh imported (Serratia liquefaciens), and one from the frozen (Enterobacter dissolvens).

Trimethoprim and sulphamethoxazole resistance was practically non-existent, appearing only in a Finnish Escherichia hermannii isolate (sul R), a fresh imported Kluyvera cryocrescens isolate (sul R, trmp R), and four E. coliisolates (sul R) from three frozen samples (Table III). Tetracycline and amoxycillin / clavulanic acid resistance was more common (around 5% in all groups), as was chloramphenicol, cefuroxime (12- 15%), and ampicillin resistance (30%) (Table III). Intrinsically resistant species were excluded from the calculations; thus: all enterobacteria with a functional AmpC ß-lactamase (most of the species found in this study, except E. coli and Klebsiella spp.) are omitted from the ampicillin, cephalothin, and co-amoxiclav results; 29 Klebsiella spp. are omitted from the ampicillin results, since they have chromosomal class A ß-lactamases; 29 and Serratia spp. are omitted from the tetracycline results.


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Table III. Resistance frequencies among all isolates (n = 535)
 
The MIC profiles for the resistant strains indicated an absence of multiresistance plasmids typically found in isolates from human sources. The following multiresistance profiles were found: of the 44 isolates resistant or intermediately resistant to tetracycline, only six had other resistances; four were also resistant to cefuroxime, one to chloramphenicol, and one to both. The others were susceptible or intrinsically resistant. Eighteen Serratia spp. isolates and one Enterobacter cloacae isolate were resistant to both cefuroxime and chloramphenicol. Cefuroxime resistance in species with a chromosomal AmpC ß-lactamase is often caused by mutations in regulatory genes; 29 this is not transferable.

In Table IV, the resistance of vegetables of different origin is compared within the four biggest genus groups. The odds ratio that the two `imported' groups would differ from the locally grown group was calculated, along with a P value, to determine whether any of the differences between the three vegetable groups were significant. For clarity, only odds ratios of significant differences are included. The isolates from the Finnish vegetables were more resistant to cephalosporins, and even for those species where the differences were not significant the trend was the same. The isolates from the imported vegetables were more resistant to chloramphenicol.


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Table IV. Statistically significant differences in resistance between strains of the four most commonly isolated genera isolated from Finnish, imported fresh, and imported deep-frozen vegetables
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, a very low frequency of antimicrobial resistance in Enterobacteriaceae isolated from vegetables was found. This contrasts with previous studies, which have found high resistance levels. One of these studies 21 did not take into account the ubiquitous intrinsic resistance of Enterobacteriaceae to ampicillin and cephalothin; this probably explains the finding that 30- 90% of colonies were resistant to >=2 antibiotics. Höiby et al. found a number of rare resistance factors, including ciprofloxacin and gentamicin, among strains isolated from iceberg lettuce. 30 The lettuce had apparently been subject to faecal contamination, since it was identified as the vehicle for an outbreak of shigella infection. The species distribution in our material, with very few E. coli, the dominant aerobic rod in faecal flora, suggests that faecal contamination is rare. The material in this study thus gives a more reliable picture of the resistance levels that can be expected in most Enterobacteriaceae on vegetables in Europe.

The most striking finding in this study was the nearly complete absence of resistance among the vegetable strains to trimethoprim and sulphamethoxazole: 0.2% and 1.3%, respectively, in all isolates. These resistance factors are usually spread on plasmids or transposons, often linked to other resistance genes. 31 In Finnish clinical strains, high levels are usually found: trimethoprim resistance is typically 15- 20%. 31,32 In a previous study we have determined resistance levels in faecal flora. Direct comparison with this study is difficult, because a different methodology, utilizing replica plating and breakpoint testing, was employed. 5 Resistance was defined as the breakpoint concentration at which >=1% of colonies from a sample grew. We have previously shown that the sensitivity of that method is comparable in sensitivity to the one described in this study. 33 The rates of trimethoprim and sulphamethoxazole resistance per sample in this study were 0.8% (n= 1/127) and 4% (n= 5/127), respectively. In faecal flora, the corresponding percentages were 14% and 19%. 5

Chloramphenicol and tetracycline resistance are two traits that are often linked to trimethoprim and sulphonamide resistance, and are often co-transferred by conjugation from faecal Enterobacteriaceae. 5 The rate of tetracycline resistance in this study was 5.5%, whereas in clinical isolates it can be around 16%. 32 Chloramphenicol resistance was higher (12%), but the MIC values were all in the range 16- 32 mg/L. The majority of resistant strains from faecal samples have MICs of >=128 mg/L; such MICs are often seen in transferable resistance (M. Österblad, unpublished data). This, together with the findings discussed in the previous paragraph, points to the absence, or at least rarity, of the most common multiresistance plasmids in the vegetable Enterobacteriaceae. This finding is similar to that of Krumperman, 34 who discovered that multiresistant E. coli were found in very much larger numbers in humans, intensively reared animals and contacts with these than in wild and grazing animals and their environment. The use of antibiotics is certainly one of the major differences between these populations, but other selective forces can not be excluded. Tolerance to organic solvents (which can be used as disinfectants) has been implicated in the emergence of fluoroquinolone-resistant mutants among clinical E. coli isolates. 35 A more positive hypothesis from the human viewpoint would be that there is a negative selection against multiresistance plasmids in the agricultural environment.

On the basis of previous resistance data, higher levels of resistance might have been expected in imported strains; clinical strains from southern Europe are more resistant than Scandinavian strains. 32,36 Higher quantities of antibiotics are used in agriculture and animal husbandry in other parts of Europe than in Scandinavia. 37 Chloramphenicol and cefuroxime were the only antibiotics in which differences between isolates from locally grown and imported samples were found and, since the Finnish isolates were more resistant to cefuroxime, and the imported ones to chloramphenicol, neither of them can be said to have a greater degree of resistance. This also indicates that the vegetable and the human enterobacterial populations are separate.

In conclusion, we found no evidence that Enterobacteriaceae from vegetables contribute substantially to the high levels of resistance in faecal flora. In future studies, molecular typing methods might usefully be employed to explore the interrelationship of human and environmental populations of the Enterobacteriaceae.


    Acknowledgments
 
Many thanks to the laboratory technicians Minna Lamppu and Tarja Laustola for their competent assistance; to Heikki Arvilommi and Raija Manninen for valuable comments on the manuscript; to Katriina Lager for helping with the data processing. Financial support was provided by the Maud Kuistila Memorial Foundation, the Oskar Öflund Foundation, and Svenska Kulturfonden i Finland.


    Notes
 
* Corresponding author. Tel: +358-2-2519 255; Fax: +358-2-251 Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 23 June 1998; returned 29 September 1998; revised 26 October 1998; accepted 23 November 1998