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Identity and antibiotic susceptibility of enterobacterial flora of salad vegetables
International Journal of Antimicrobial Agents 18 (2001) 81 – 83 www.ischemo.org
Short communication
Identity and antibiotic susceptibility of enterobacterial flora of salad vegetables J.M.T. Hamilton-Miller *, Saroj Shah Department of Medical Microbiology, Royal Free and Uni6ersity College Hospital Medical School, London NW 3 2PF, UK Received 31 January 2001; accepted 27 February 2001
Abstract The enterobacterial flora from carrots (organic and non-organic) and salad vegetables has been identified and antibiotic susceptibilities determined. Pseudomonas fluorescens and Pantoea (formerly Enterobacter) agglomerans were the species most commonly found; the former was usually resistant to at least six of the antibiotics under test. Rahnella aquatilis (often producing b-lactamase) was also found in carrots. There were no clear differences in flora from organic and non-organic carrots. Thus, uncooked vegetables are a potential source of highly resistant opportunistic pathogens. © 2001 Published by Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Salad vegetables; Antibiotic resistance; Pathogens
1. Introduction Although it is well established that food may be a source of pathogenic bacteria, relatively little is known of the ‘normal flora’ of vegetables and, in particular, their resistance to antibiotics, as was recently related in a report to the UK Government [1]. We have investigated antibiotic resistance patterns of epiphytic enterobacterial flora from salad vegetables (i.e. those likely to be eaten uncooked) and have also compared the flora of ordinary carrots with those cultivated under organic conditions.
2. Definitions Organic farming avoids the use of artificial fertilizers and pesticides and aims to provide soil full of micro-or
Some of these results were presented as a Poster at the 40th ICAAC, Toronto, September 2000. * Corresponding author. Tel.: + 44-207-794-0500; fax: + 44-207435-9694. E-mail address: [email protected] (J.M.T. HamiltonMiller).
ganisms and earthworms. Production of organic food is subject to EU and UK regulations and is usually certified in the UK by the Soil Association. The term ‘enterobacteria’ used here means Gramnegative bacilli that grow in the presence of bile.
3. Materials and methods
3.1. Vegetables These were mainly purchased in supermarkets; a few samples were home grown or from retail greengrocers shops. Organic carrots conformed to the Soil Association’s ‘UK Standard 5’.
3.2. Microbiological methods Vegetables were sampled with a moistened cotton wool swab that was then plated on MacConkey agar. The bacteria isolated were identified by correlating colonial appearance, Gram stain, oxidase reaction, biochemical reactions in the appropriate API strip and pigment production in Kings A and B media [2]. An-
0924-8579/01/$20 © 2001 Published by Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 1 ) 0 0 3 5 3 - 3
82
J.M.T. Hamilton-Miller, S. Shah / International Journal of Antimicrobial Agents 18 (2001) 81–83
tibiotic susceptibility was determined by disk diffusion and b-lactamase production was detected using nitrocefin.
4. Results
4.1. Organic and non-organic carrots Eleven batches were tested, five organic and six nonorganic. Between two and four separate bacterial species (mode three) were isolated from each batch, 33 strains in total. The vast majority of the isolates were Pantoea agglomerans, Pseudomonas fluorescens and Rahnella aquatilis (Table 1). There were no qualitative or quantitative differences between the flora of the organic and non-organic carrots. More than 70% of the strains were resistant to ampicillin and first and second generation cephalosporins and one-third to co-amoxiclav, cefotaxime or trimethoprim. Resistance to the other antibiotics tested was unusual or not seen (Table 2). Again, there were no overall differences between strains from organic and non-organic carrots. Pseudomonas spp. was usually resistant to six antibiotics and R. aquatilis to three, while most of the P.agglomerans were fully sensitive. Betalactamase activity was detected in 12 strains (six of which were R. aquatilis) of 27 tested.
4.2. Salad 6egetables The following were tested: lettuce (three samples), radish (two), chicory (two) and one each of cress, celery, water cress, salad cress, sweet pepper, cucumber, mint, salad onion, rocket and chives. All but three (lettuce, rocket and chives) yielded Gram-negative bacilli, usually more than one species (range 0– 4; mode 3). The 40 strains isolated are shown in Table 1. There was a wider variety of species than had been grown from carrots, but again P. fluorescens and P. agglomerans predominated, although in contrast with carrot flora, R. aquatilis was rarely found.
Sensitivity patterns of these strains were similar to those of organisms from carrots (Table 2) in that resistances to ciprofloxacin, gentamicin, sulphonamide, imipenem or ceftazidime were rare. Pseudomonas spp. from carrots was resistant to six or seven antibiotics and Pantoea spp. was generally sensitive to all antibiotics tested. Beta-lactamase production was demonstrated in ten strains (six of which were Pseudomonas spp.) of the 29 tested.
5. Discussion We did not set out to investigate the total epiphytic flora. The organisms that we considered to be of potential importance were those capable of surviving in the human intestine, i.e. enterobacteria, as defined above. Considering the variety of root and leaf vegetables tested, the bacterial flora was remarkably consistent. It was notable that we did not isolate Escherichia coli or enterococci from any specimen, indicating a lack of contamination with human or animal faeces. The most frequently isolated species, P. fluorescens, was usually resistant to six antibiotics (range 5–8). This species, a recognized rare opportunistic pathogen [3], has been associated with blood products [4]. The second most commonly found species, P. agglomerans, is still regarded by clinical microbiologists as a member of the genus Enterobacter [5]. Most strains isolated were fully sensitive (range resistant to 0–4 antibiotics). P. agglomerans may cause opportunistic infections [6] and the genus Enterobacter has been nominated as ‘emerging pathogen of the millennium’ [7]. R. aquatilis was more often isolated from carrots than from other vegetables; most strains produced blactamase. The natural habitat of this species is water, although it may also cause opportunistic infections [8]. We found that washing (and in the case of carrots, peeling) did not remove bacteria from vegetables. Several of the products were labelled as ‘prewashed’ and it is interesting to note that a popular authority on cook-
Table 1 Bacterial species isolated from carrots and salad vegetables Pantoea spp.
Pseudomonas spp.
Rahnella aquatilis
Others
Carrots (n =33)
12 (36%): all P. agglomerans
11 (33%): 10 P. fluorescens; 1 P. luteola
9 (27%)
1 Ewingella americana
Other vegetables (n= 40)
13 (33%): 11 P. agglomerans; 2 P. dispersa
19 (48%) 17 P. fluorescens; 1 P. oryzihabitans; 1 P. luteola
1
7: 2 Stenotrophomonas maltophilia; 2 Acinetobacter spp.; 1 Sphingobacter multi6orum; 1 Enterobacter cloacae; 1 Escherichia 6ulneris
J.M.T. Hamilton-Miller, S. Shah / International Journal of Antimicrobial Agents 18 (2001) 81–83
83
Table 2 Resistance to 11 antibiotics in bacteria isolated from carrots (11 specimens) and other salad vegetables (15 specimens) Antibiotic
Carrots
Others
No. resistant (n= 33)
% Resistant
No. resistant (n =40)
% Resistant
Ceftazidime Gentamicin Ciprofloxacin
0 0 0
0 0 0
5 1 0
12 2 0
Imipenem Sulphonamide Co-amoxiclav Cefotaxime Trimethoprim
2 4 10 11 11
6 12 30 33 33
7 1 20 21 23
17 2 50 52 57
Ampicillin Cefuroxime Cefaclor
24 24 27
73 73 82
25 29 36
62 72 90
ing recommends that lettuce should not be washed before use [9]. The bacterial flora of organic and non-organic carrots did not differ despite the contrasting husbandry practices. A literature search revealed few other studies similar to this: as cited by Berlau et al. [10] heavy bacterial colonization of mustard and cress in green salads prepared in a hospital kitchen has been reported, involving Acinetobacter spp. (among others), and Klebsiella spp. have been isolated from various foods. Our findings, although somewhat different from these, demonstrate that uncooked vegetables are a potential source of nosocomial infection in vulnerable patients and more importantly, that multiple antibiotic resistance is common in epiphytic bacteria.
[3]
[4]
[5]
[6]
[7]
[8]
References [1] Advisory Committee on the Microbiological Safety of Food. Report on Microbial Antibiotic Resistance in Relation to Food Safety— Synopsis, Chapter 4, Paragraph 68. London: The Stationery Office, 1999. [2] Barrow GI, Feltham RKA, editors. Cowan and Steel’s Manual
.
[9] [10]
for the Identification of Medical Bacteria. 3rd ed. Cambridge: Cambridge University Press, 1993. Von Graevenitz A, Weinstein J. Pathogenic significance of Pseudomonas fluorescens and Pseudomonas putida. Yale J Biol Med 1971;44:265 – 73. Lazarus HM, Magalhaes-Silverman M, Fox RM, Creger RJ, Jacobs M. Contamination during in vitro processing of bone marrow for transplantation: clinical significance. Bone Marrow Transpl 1991;7:241 – 6. Farmer JJ. Enterobacteriaceae: introduction and identification. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. Manual of Clinical Microbiology, 7th ed. Washington: American Society for Microbiology, 1999:442 – 58. De Champs C, Le Seaux S, Dubost JJ, Boisgard S, Sauvezie B, Sirot J. Isolation of Pantoea agglomerans in two cases of septic monoarthritis after plant thorn and wood sliver injuries. J Clin Microbiol 2000;38:460 – 1. Sanders WE, Sanders CC. Enterobacter spp.: pathogens poised to flourish at the turn of the century. Clin Microbiol Rev 1997;10:220 – 41. Chang CL, Jeong J, Shin JH, Lee EY, Son HC. Rahnella aquatilis sepsis in an immunocompetent adult. J Clin Microbiol 1999;37:4161 – 2. Smith D. How to Cook, Book II. London: BBC Worldwide, 1999. Berlau J, Aucken HM, Houang E, Pitt TL. Isolation of Acinetobacter spp. including A. baumannii from vegetables: implications for hospital-acquired infections. J Hosp Inf 1999;42:201 –4.
Short communication
Identity and antibiotic susceptibility of enterobacterial flora of salad vegetables J.M.T. Hamilton-Miller *, Saroj Shah Department of Medical Microbiology, Royal Free and Uni6ersity College Hospital Medical School, London NW 3 2PF, UK Received 31 January 2001; accepted 27 February 2001
Abstract The enterobacterial flora from carrots (organic and non-organic) and salad vegetables has been identified and antibiotic susceptibilities determined. Pseudomonas fluorescens and Pantoea (formerly Enterobacter) agglomerans were the species most commonly found; the former was usually resistant to at least six of the antibiotics under test. Rahnella aquatilis (often producing b-lactamase) was also found in carrots. There were no clear differences in flora from organic and non-organic carrots. Thus, uncooked vegetables are a potential source of highly resistant opportunistic pathogens. © 2001 Published by Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Salad vegetables; Antibiotic resistance; Pathogens
1. Introduction Although it is well established that food may be a source of pathogenic bacteria, relatively little is known of the ‘normal flora’ of vegetables and, in particular, their resistance to antibiotics, as was recently related in a report to the UK Government [1]. We have investigated antibiotic resistance patterns of epiphytic enterobacterial flora from salad vegetables (i.e. those likely to be eaten uncooked) and have also compared the flora of ordinary carrots with those cultivated under organic conditions.
2. Definitions Organic farming avoids the use of artificial fertilizers and pesticides and aims to provide soil full of micro-or
Some of these results were presented as a Poster at the 40th ICAAC, Toronto, September 2000. * Corresponding author. Tel.: + 44-207-794-0500; fax: + 44-207435-9694. E-mail address: [email protected] (J.M.T. HamiltonMiller).
ganisms and earthworms. Production of organic food is subject to EU and UK regulations and is usually certified in the UK by the Soil Association. The term ‘enterobacteria’ used here means Gramnegative bacilli that grow in the presence of bile.
3. Materials and methods
3.1. Vegetables These were mainly purchased in supermarkets; a few samples were home grown or from retail greengrocers shops. Organic carrots conformed to the Soil Association’s ‘UK Standard 5’.
3.2. Microbiological methods Vegetables were sampled with a moistened cotton wool swab that was then plated on MacConkey agar. The bacteria isolated were identified by correlating colonial appearance, Gram stain, oxidase reaction, biochemical reactions in the appropriate API strip and pigment production in Kings A and B media [2]. An-
0924-8579/01/$20 © 2001 Published by Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 1 ) 0 0 3 5 3 - 3
82
J.M.T. Hamilton-Miller, S. Shah / International Journal of Antimicrobial Agents 18 (2001) 81–83
tibiotic susceptibility was determined by disk diffusion and b-lactamase production was detected using nitrocefin.
4. Results
4.1. Organic and non-organic carrots Eleven batches were tested, five organic and six nonorganic. Between two and four separate bacterial species (mode three) were isolated from each batch, 33 strains in total. The vast majority of the isolates were Pantoea agglomerans, Pseudomonas fluorescens and Rahnella aquatilis (Table 1). There were no qualitative or quantitative differences between the flora of the organic and non-organic carrots. More than 70% of the strains were resistant to ampicillin and first and second generation cephalosporins and one-third to co-amoxiclav, cefotaxime or trimethoprim. Resistance to the other antibiotics tested was unusual or not seen (Table 2). Again, there were no overall differences between strains from organic and non-organic carrots. Pseudomonas spp. was usually resistant to six antibiotics and R. aquatilis to three, while most of the P.agglomerans were fully sensitive. Betalactamase activity was detected in 12 strains (six of which were R. aquatilis) of 27 tested.
4.2. Salad 6egetables The following were tested: lettuce (three samples), radish (two), chicory (two) and one each of cress, celery, water cress, salad cress, sweet pepper, cucumber, mint, salad onion, rocket and chives. All but three (lettuce, rocket and chives) yielded Gram-negative bacilli, usually more than one species (range 0– 4; mode 3). The 40 strains isolated are shown in Table 1. There was a wider variety of species than had been grown from carrots, but again P. fluorescens and P. agglomerans predominated, although in contrast with carrot flora, R. aquatilis was rarely found.
Sensitivity patterns of these strains were similar to those of organisms from carrots (Table 2) in that resistances to ciprofloxacin, gentamicin, sulphonamide, imipenem or ceftazidime were rare. Pseudomonas spp. from carrots was resistant to six or seven antibiotics and Pantoea spp. was generally sensitive to all antibiotics tested. Beta-lactamase production was demonstrated in ten strains (six of which were Pseudomonas spp.) of the 29 tested.
5. Discussion We did not set out to investigate the total epiphytic flora. The organisms that we considered to be of potential importance were those capable of surviving in the human intestine, i.e. enterobacteria, as defined above. Considering the variety of root and leaf vegetables tested, the bacterial flora was remarkably consistent. It was notable that we did not isolate Escherichia coli or enterococci from any specimen, indicating a lack of contamination with human or animal faeces. The most frequently isolated species, P. fluorescens, was usually resistant to six antibiotics (range 5–8). This species, a recognized rare opportunistic pathogen [3], has been associated with blood products [4]. The second most commonly found species, P. agglomerans, is still regarded by clinical microbiologists as a member of the genus Enterobacter [5]. Most strains isolated were fully sensitive (range resistant to 0–4 antibiotics). P. agglomerans may cause opportunistic infections [6] and the genus Enterobacter has been nominated as ‘emerging pathogen of the millennium’ [7]. R. aquatilis was more often isolated from carrots than from other vegetables; most strains produced blactamase. The natural habitat of this species is water, although it may also cause opportunistic infections [8]. We found that washing (and in the case of carrots, peeling) did not remove bacteria from vegetables. Several of the products were labelled as ‘prewashed’ and it is interesting to note that a popular authority on cook-
Table 1 Bacterial species isolated from carrots and salad vegetables Pantoea spp.
Pseudomonas spp.
Rahnella aquatilis
Others
Carrots (n =33)
12 (36%): all P. agglomerans
11 (33%): 10 P. fluorescens; 1 P. luteola
9 (27%)
1 Ewingella americana
Other vegetables (n= 40)
13 (33%): 11 P. agglomerans; 2 P. dispersa
19 (48%) 17 P. fluorescens; 1 P. oryzihabitans; 1 P. luteola
1
7: 2 Stenotrophomonas maltophilia; 2 Acinetobacter spp.; 1 Sphingobacter multi6orum; 1 Enterobacter cloacae; 1 Escherichia 6ulneris
J.M.T. Hamilton-Miller, S. Shah / International Journal of Antimicrobial Agents 18 (2001) 81–83
83
Table 2 Resistance to 11 antibiotics in bacteria isolated from carrots (11 specimens) and other salad vegetables (15 specimens) Antibiotic
Carrots
Others
No. resistant (n= 33)
% Resistant
No. resistant (n =40)
% Resistant
Ceftazidime Gentamicin Ciprofloxacin
0 0 0
0 0 0
5 1 0
12 2 0
Imipenem Sulphonamide Co-amoxiclav Cefotaxime Trimethoprim
2 4 10 11 11
6 12 30 33 33
7 1 20 21 23
17 2 50 52 57
Ampicillin Cefuroxime Cefaclor
24 24 27
73 73 82
25 29 36
62 72 90
ing recommends that lettuce should not be washed before use [9]. The bacterial flora of organic and non-organic carrots did not differ despite the contrasting husbandry practices. A literature search revealed few other studies similar to this: as cited by Berlau et al. [10] heavy bacterial colonization of mustard and cress in green salads prepared in a hospital kitchen has been reported, involving Acinetobacter spp. (among others), and Klebsiella spp. have been isolated from various foods. Our findings, although somewhat different from these, demonstrate that uncooked vegetables are a potential source of nosocomial infection in vulnerable patients and more importantly, that multiple antibiotic resistance is common in epiphytic bacteria.
[3]
[4]
[5]
[6]
[7]
[8]
References [1] Advisory Committee on the Microbiological Safety of Food. Report on Microbial Antibiotic Resistance in Relation to Food Safety— Synopsis, Chapter 4, Paragraph 68. London: The Stationery Office, 1999. [2] Barrow GI, Feltham RKA, editors. Cowan and Steel’s Manual
.
[9] [10]
for the Identification of Medical Bacteria. 3rd ed. Cambridge: Cambridge University Press, 1993. Von Graevenitz A, Weinstein J. Pathogenic significance of Pseudomonas fluorescens and Pseudomonas putida. Yale J Biol Med 1971;44:265 – 73. Lazarus HM, Magalhaes-Silverman M, Fox RM, Creger RJ, Jacobs M. Contamination during in vitro processing of bone marrow for transplantation: clinical significance. Bone Marrow Transpl 1991;7:241 – 6. Farmer JJ. Enterobacteriaceae: introduction and identification. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. Manual of Clinical Microbiology, 7th ed. Washington: American Society for Microbiology, 1999:442 – 58. De Champs C, Le Seaux S, Dubost JJ, Boisgard S, Sauvezie B, Sirot J. Isolation of Pantoea agglomerans in two cases of septic monoarthritis after plant thorn and wood sliver injuries. J Clin Microbiol 2000;38:460 – 1. Sanders WE, Sanders CC. Enterobacter spp.: pathogens poised to flourish at the turn of the century. Clin Microbiol Rev 1997;10:220 – 41. Chang CL, Jeong J, Shin JH, Lee EY, Son HC. Rahnella aquatilis sepsis in an immunocompetent adult. J Clin Microbiol 1999;37:4161 – 2. Smith D. How to Cook, Book II. London: BBC Worldwide, 1999. Berlau J, Aucken HM, Houang E, Pitt TL. Isolation of Acinetobacter spp. including A. baumannii from vegetables: implications for hospital-acquired infections. J Hosp Inf 1999;42:201 –4.