Frederick R. Falkiner*
Department of Clinical Microbiology, Trinity College, Dublin; Central Pathology Laboratory,
St James’s Hospital, Dublin 8, Ireland
The problem of antibiotic resistance emerging from the use and misuse of antibiotics in veterinary practice was considered as long ago as the 1960s by the famous Swann Committee.1 What seems to be less well understood, and certainly less well documented, is the use of antimicrobials in the world of plant science. Antibiotics have been widely used in the control of contamination of micropropagation and plant tissue culture for 40 years or so2 and for a similar length of time in the topical treatment of bacterial diseases of fruit trees.3 It is probably time to bring this to the attention of medical microbiologists.
The first symposium to consider the problem of bacterial contamination and its control in plant tissue culture was held in Cork, Ireland, in 1987.4 The concept of propagation, on agar, of plant tissues from fast growing tissue such as apical meristems has been well established for many decades. In the same way as virologists incorporate antimicrobials into their tissue culture media to control bacterial and fungal growth, so plant propagators have included such agents in their agar media.
What is relatively new, however, is the massive scale on which plant material is now propagated and the consequent financial importance of the industry in the supply of plants for house, garden and agriculture. When plants are propagated in their hundreds of thousands, the prospect of bacterial contamination of even a small proportion is clearly costly.
Plant tissue is normally prepared for propagation aseptically with sterile instruments in a clean air environment. The plant material is usually decontaminated with hypochlorite or another suitable ‘antiseptic’. The tissue is placed on a special medium (usually that developed by Murashige & Skoog,5 of which there are now many variations) and as it grows and develops it is transplanted on to agars of differing composition until the rooted plant is suitable for hardening off and planting in compost.
All reports of the prophylactic use of antimicrobials by incorporating them in the culture media are prefaced by a statement to the effect that antibiotics should not replace sound aseptic technique. The problem is that the use of antibiotics in this way certainly conflicts with the normal principles of prophylactic use, in that the ‘pathogen’ is unknown and of uncertain susceptibility and the period of administration is prolonged. Plant tissue culture media are fairly hostile, at least to bacteria, particularly because of the high sugar concentration of these media. Under these circumstances the bacteria are reluctant to grow or metabolize and are, therefore, largely resistant to antibiotics; it is likely, therefore, that bacteria will remain as persisters for the duration of the phase of culture. It is well recognized that contamination can become evident during later growth phases in less hostile media, for instance where sugar concentrations are lower. Persistence is also likely to be an explanation for this covert contamination. The other unknown effect is that of the medium itself on the activity of the antibiotic. Plant tissue culture media are complex mixtures of a wide range of compounds including minerals, amino acids, plant hormones and sugars. It is easy to see how the antibiotic might not function under these conditions.
There are several possible sources of contaminating organisms. If the plant material was not adequately decontaminated it may carry plant-associated organisms from the field or organisms of animal or human origin from compost or manure. Another possibility is the emergence of human-associated organisms derived from the staff who undertake the propagation: breakdown of asepsis is more likely to occur at certain times of the day, such as around break times.6 Coagulase-negative staphylococci, diphtheroids and other microorganisms from the skin or respiratory tract may appear as contaminants at a later stage.6,7 The other consideration is the possibility of toxic effects of the antibiotics on the plant tissue. Reports of problems of phytotoxicity are numerous; it has been associated with most antimicrobials, including aminoglycosides and tetracyclines, but reports relate only to specific hosts. The antibiotics least likely to be phytotoxic are those acting at sites such as the bacterial cell wall rather than those which act on the ribosomes or DNA. Mycoplasmas8 and possibly L-forms9 may cause significant contamination, so – lactams are not the whole answer.
At the first symposium in Cork, the opening paper was a thoughtful review of the problems; it was designed to encourage the delegates to consider the possible role of commensal organisms and the relationship between plant and pathogen, commensal or contaminant.1 0 There was a discussion of the difficulties of using antibiotics in this way1 1 and reviews of technology available for the identification of bacterial contaminants.1 2 , 1 3 Many papers described the use of antibiotics in tissue culture and in discussion there were anecdotal reports of very large but unspecified quantities of third-generation cephalosporins being used commercially. Since that meeting, many of the problems have been addressed so it was interesting to attend the second symposium, again held in Cork, in September 1996. It was notable how, in the intervening years, a more rational approach to the use of antibiotics had been adopted and how there had been a move away from ‘prophylaxis’ towards treatment of tissue before propagation. This ‘treatment’ involves immersing the tissue in an ‘appropriate’ antibiotic following confirmation of the presence of a likely pathogen.
There was also a greater awareness that contaminants might not be harmful: two delegates reported beneficial effects of Bacillus subtilis in protecting against further bacterial or fungal contamination.14,15 Also of interest were reports from Eastern Europe16 and North Africa17 of successful use of older and less widely used agents such as neomycin, novobiocin and in discussion, sulphaguanidine and polymyxin, which, with the exception of neomycin, one would hope, would have a less significant impact on the emergence of resistance.
In discussion at this meeting a delegate from the USA alluded to the huge quantities of antibiotics used by spraying to treat fruit trees topically. Reported amounts of antibiotics vary but, at a meeting held at the end of last September at the Royal Society of Medicine, Levy reckoned that some 20–50,000 lb, or as much as 20 tons, of streptomycin and tetracycline are used in the USA for this purpose.3 These drugs have been registered for such use for some 40 years. They used to be sprayed from the air but are now more commonly applied from the ground— presumably by tractor. Antibiotics are also used on a smaller scale in the treatment of potatoes by dusting, again a process that must have certain consequences. It is not surprising that, since the 1950s, antibiotic resistance has emerged amongst the plant pathogens and that the industry has been forced to seek alternative approaches.3 Administration by injection of oxytetracycline to fruiting and ornamental palm trees has been used successfully for some time in Florida.3 One feels that this is perhaps an encouraging development if the fruit are not loaded with oxytetracycline as a consequence. It is fair to point out, though, that monitoring fruit for streptomycin residues since the 1950s has not yet revealed detectable levels.3 No antibiotics are permitted for these purposes in the UK or, as far as I can tell, in Europe.
While a more logical approach seems to have been adopted in micropropagation and plant tissue culture,18–20 the use of antibiotics in the plant world should remain under scrutiny. It must be a matter for concern that antibiotics such as oxytetracycline are in apparently carefree and widespread use because, while the amounts used in this way are small compared with the total amount of antibiotics used in clinical and veterinary practice in the USA (30 million pounds weight), the mode of application is likely to have consequences out of proportion to the quantities used.3 While tetracyclines may not be in widespread clinical use, resistance to tetracylines is often closely tied in with resistance to other antibiotics.21 Streptomycin selects resistant bacteria which are resistant to other antibiotics3 and there is some measure of crossresistance to other aminoglycosides. The relative ease with which certain important human pathogens may become dependent on streptomycin is also a matter for concern.22 It should not go unnoticed that a decrease in the clinical use of streptomycin and tetracycline in Denmark has corresponded with the decline of one strain of methicillinresistant Staphylococcus aureus which was also resistant to these two drugs.23
Plants and humans do not generally share pathogens and there seems to be no term analgous to ‘zoonosis’ for an infection acquired from a plant. However, organisms which are of clinical importance, such as Burkholderia cepacia (a pathogen of onions), are deemed ‘environmental’ and many more are closely related to our hospital pathogens, e.g. other pseudomonads, Xanthomonas and Erwinia spp., so transfer of resistance can be readily envisaged. Perhaps a renewed interest might be shown in pot plants brought in to patients, as a potential source of human pathogens or a source of transmissible resistance, since many of these will be from micropropagated stock.
As a final thought, one can only hope that tractor drivers on fruit farms in the USA are adequately protected and that those who like to talk to their trees are not unduly disappointed if, through the auditory damage of streptomycin, they fail to hear any response!
I would like to thank my various colleagues who read the manuscript at various stages and Professor Alan Cassells of University College, Cork who stimulated my interest in the subject.
1. Joint Committee on the Use of Antibiotics in Animal Husbandry and Veterinary Medicine. (1969). Report. (Chairman: Professor M. M. Swann). HMSO, London.
2. Katznelson, H. & Sutton, M. D. (1951). Inhibition of plant pathogenic bacteria in vitro by antibiotics and quaternary ammonium compounds. Canadian Journal of Botany 29, 270–8.
3. Levy, S. B. (1992). The Antibiotic Paradox—How Miracle Drugs are Destroying the Miracle. Plenum, New York.
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5. Murashige, T. & Skoog F. (1962). A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiologia Plantarum 15, 473–97.
6. Boxus, P. & Terzi, J. M. (1987). Big losses due to bacterial contamination can be avoided in mass propagation schemes. Acta Horticulturae 212, 91–3.
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