antibiotics may enter the environment having been excreted in the faeces and/ or urine of treated animals. Antibiotic run off from plant applications isalso possible. Animals have also been shown to acquire antibiotic tissueresidues from contact with an environment in which other animals have beentreated with sulphadimidine and furazolidone. Similarly, oxilinic acid hasbeen detected in crabs and mussels in the vicinity of fish farms for up to 13days after treatment (Coyne et al (1997) and antibiotic resistant bacteria havealso been recovered from sediment from fish farms (Kerry et al 1994,Depaola et al 1995).
Bacterial gene transfer is now thought to occur not only in the human andanimal intestine but throughout the biosphere, especially in nutrient-rich sitessuch as aquatic systems, sediments, soils, in the vicinity of plant roots, and inthe sludge of the biological sewage treatment systems. Resistant bacteria canbe isolated from all of these sites. Resistance may also be spread frombacteria borne onplants and vegetables treated with antimicrobials orfertilised with wastes containing animal or human faecal residues or derivedfrom fish farms. Resistance should therefore be taken as a phenomenon ofglobal genetic ecology.
A number of reservoirs and habitats may be sites for the emergence andmaintenance of antimicrobial resistant microorganisms. These includehospitals, farms, aquaculture, human or animal commensal bacteria, andhabitats where faeces and urine from humans and animals are found. Sewagefrom humans and livestock given antimicrobials is a mechanism of spreadingresistance genes. Antimicrobials excreted by humans and animals are foundin sewage water and may degrade slowly and exert a continuous selectionpressure. In a similar way, antimicrobials used in plant protection arewashed into the soil and ground water where they may select resistantbacteria, so favouring the dissemination of resistance genes. The US EPArecognises that there are deficiencies in the present knowledge of theenvironmental fate and ecological effect of streptomycin (EPA Pesticide FactSheet, 1988, updated). No information is currently available on itsbreakdown in soil and water. Also the Agency is unable to assess thepotential for oxytetracycline to contaminate groundwater because theenvironmental fate of oytetracycline has not been characterised, and neitheris it able to assess the ecological effects of oxytetracycline on terrestrial oraquatic wildlife, again because no data are available.
Common factors between the four ecological compartments (humans,animals, plants and soil-water) are the antimicrobials, the bacteria and thegenes that code for resistance. The genes move between the bacteria in eachcompartment, and the bacteria may move between the compartments. Antibiotic resistance marker genes in genetically modified plants
Genetic modification of plants usually involves two steps where selectablemarkers are being used: 1) engineering of the gene construct which is used totransform the plant; this will normally be done in E. coli; 2) transformationof the plant and selection of transformants. Markers are used for selecting the
desired transformants among the non-transformed individuals. Some of theantibiotic resistance marker genes encode resistance to: ampicillin,chloramphenicol, kanamycin, streptomycin, amikacin, tetracycline,hygromycin, gentamicin and phleomycin resistance markers. 3.5.1. The fate of plant DNA in the gastro-intestinal tract
Consumed as a component of e.g. fresh fruit or vegetable, antibioticresistance marker genes are treated in the gastro-intestinal (GI) tract in asimilar way to any other gene present in food of plant or animal origin. During movement in the GI tract, plant DNA is rapidly degraded into smallfragments. According to an estimate, 1 g of maize tissue will yield no morethan 100 µg of genomic DNA in the gut, and estimated 50 pg intact markergene in transgenic maize. About 1-2% of orally ingested M13 DNA persiststransiently as fragments between 1 and 7 h after feeding in the gut and faecesof mice (Schubber et al., 1994). The bulk of these fragments are 100-400 bpin size (the size of the gene which codes for TEM-1 beta-lactamase is <900bp). The small intestine contains about 2.2-0.7% (1-8 h after feeding), thececum 2.4-1.1% (2-18 h), the large intestine 0.2-1.7% (2-8 h) of the DNAorally administered. A few percent (<5%) of the ingested DNA (up to 1700bp) may be excreted as fragments in the faeces (Schubbert et al., 1994,1997). 3.5.2. The potential for integration of DNA from food into intestinal
Transformation of intestinal bacteria with food-derived plant DNA has neverbeen demonstrated and attempts to transform competent E. coli bacteria withplant DNA in vitro have been unsuccessful. In nature, the processes ofintegration, heterologous transcription and translation, and not DNA flux, arelikely to be the limiting factors in functional gene exchange. Recombinationis probably the most serious barrier to functional inter-specific gene transfer. Because of this, gene transfer events mediated by natural transformation aremost likely to occur between members of the same or closely related species. It is important to note that most transgenic plants have pUC 18 plasmid,which does not have homology to most bacterial genomes, and no transferfunctions. Thus it seems unlikely that pUC18 DNA could sucessfullytransform bacteria pathogenic to man. 3.5.3. The probability of expression of an integrated gene in intestinal
The antibiotic resistance marker genes used for selection of planttransformants have regulatory sequences that may not function in gutmicroorganisms; in those cases, recombination would have to occur torestore functionality. Complicated rearrangements, especially under selectivepressure, may bring a prokaryotic promoter in front of the marker gene,leading to its expression. The expression of antibiotic resistance markergenes which serve to facilitate the selection of transformants is under thecontrol of regulatory sequences. In principle, these regulatory sequencesmight allow the marker genes to be expressed at least in some types ofintestinal bacteria. However, promoters of genes from one phylogenetic
group of bacteria usually do not work in a member of another phylogeneticgroup (Salyers 1997). A ‘silent’ resistance can become activated by insertionof an insertion sequence in the promoter region or mutations in the promoterregion that cause it to become active. Only if a selective pressure is present,which gives an advantage to the recipient of the gene, will the gene transferevent have any concrete consequences. 3.5.4. The protein encoded by antibiotic resistance gene in plant as a
A special concern with respect to antibiotic resistance genes is the theoreticalpossibility that clinical therapy could be compromised due to inactivation ofan oral dose of antibiotic as a result of consumption of food derived from thetransgenic plant. Any such risk arising as a result of the protein shouldcorrelate with the amount of enzyme remaining functionally active in the GItract. This, on the other hand, depends on a) the estimated daily intake (EDI)and thus the level of the active protein in food, and b) stability of the proteinin the GI tract. FDA calculated that the EDI of APH(3’)II (kanamycinresistance gene) was 480 µg/person/day (FDA 1994). Proteins present infood and entering the GI tract are broken down to smaller peptides andamino acid constituents by digestive enzymes. The purified APH(3’)-IIprotein was degraded in 10 seconds in in vitro assays developed to simulatethe human GI environment. Tomato extract and non-fat milk, added todetermine whether the presence of additional food-source proteins mightslow the proteolytic degradation of the enzyme, did not prevent the effectivedegradation of the protein.
It has been calculated that, under conditions where the maximum amount ofkanamycin could be inactivated by the marker protein in ingested tomatoes,the loss of antibiotic efficacy would be 1.5% of a 1 g dose of neomycin(Redenbaugh et al. 1993, 1994), and only after oral administration. 3.5.5. Impact on human health, on animal health and animal production
Most, if not all, of the antibiotic resistance genes ingested (in theform of plants) will be degraded in the gastrointestinal tract beforereaching the critical areas where potential transformation ofmicroorganisms take place.
Even if intact DNA is present, the probability that competentmicroorganisms will be naturally transformed by this exogenousDNA in humans, animals or environment is very low.
The probability that gene transfer between plant and microorganisms occursand creates health problems seems to be extremely low and needs to beconsidered only in special cases where the antibiotic is administered by theoral route and there is also heavy selection pressure. The nature of the geneand its expression product as well as the conditions in the GI tract willdetermine whether or not a food safety problem exists. Aspects to considerare the importance of the substrate antibiotics in human and animal therapy
and whether there are alternatives (e. g. vancomycin, other glycopeptides,fluoroquinolones, tetracycline, gentamicin, newer derivatives of beta-lactamantibiotics), frequency of use (probability of selection pressure) and route ofadministration. The substrate profile of enzymes should be carefullyanalysed to see whether there is any chance that they may catalyse theinactivation of an important antibiotic used in human therapy. Use of theantibiotic in the environment may cause additional selection pressure; e.g. streptomycin and oxytetracycline are used as pesticides.
Hence, although the risk of gene transfer is extremely small, each plantcontaining antibiotic resistance genes should be evaluated on a case-by-casebasis. The main emphasis should be on the evaluation of the selection actingon the bacterial recipients after possible horizontal gene transfer. Assessmentof potential for transfer of antibiotic resistance marker genes from plant intothe bacterial community should be examined more closely.
Red Cell Typing Methods Method 2.1. Slide Test for with the manufacturer’s instructions. Determination of ABO Type of Red Cells Procedure Principle Place 1 drop of anti-A,B on a third slide, Specimen if parallel tests are to be performed withthis reagent, or on a single clean, labeledThe reagent manufacturer’s instructions mustslide if this is the only test performed. be