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Sowing diseases, new and old
Genetic engineering and the world health crisis

The world is heading for a major crisis in public health as outbreaks of new and re-emerging infectious diseases continue to occur with increasing frequency. The current strains of many pathogens are resistant to known treatments, some to nearly all known drugs and antibiotics. There can be little doubt that it is the transfer of genes across unrelated species of animals and plants (i.e., horizontal gene transfer) that is responsble for the development of drug and antibiotic resistances. The phenomenal increase in virulent infections and antibiotic resistance coincides with the commercialisation of genetic engineering biotechnology. Genetic engineering is inherently hazardous because it depends precisely on designing gene transfer vectors (carriers) to cross wide species barriers. The urgent question which needs to be addressed is the extent to which genetic engineering biotechnology, by facilitating horizontal gene transfer and recombination, is contributing to the emergence and resurgence of infectious, drug-resistant diseases. An enquiry into the possible contribution of genetic engineering biotechnology to the etiology of infectious diseases is all the more pressing in the light of other recent relevant findings indicating that microorganisms genetically engineered for 'contained use' may not be effectively contained.


World health crisis

THE world is heading for a public health crisis. At least 30 new diseases, such as AIDS, ebola, several kinds of hepatitis and other deadly viruses have emerged over the past 20 years (see Table 1) (compiled from WHO Report 1996 and other sources), while old infectious diseases such as tuberculosis, cholera, malaria and diphtheria are coming back worldwide. Practically all the pathogens are resistant to drug treatment, many to multiple antibiotics.

One-third of the 52 million deaths from all causes in the world in 1995 were due to infectious diseases; over half of these in young children. The top killers were tuberculosis (3.1 million, mostly adults), malaria (2.1 million, including 1 million children), hepatitis B (1.1 million) and AIDS (more than 1 million). 'The optimism of a relatively few years ago that many of these diseases could easily be brought under control has led to a fatal complacency among the international community,' says the Director-General of the World Health Organisation (WHO), Dr Hiroshi Nakajima, in a press release. Emerging diseases have become a major public health concern during the 1990s.

Have infectious diseases and drug resistance gone up recently?

Precise epidemiological data are not yet available, but the signs are that both the incidence and severity of outbreaks of multi-drug-resistant pathogens may have experienced a sharp upturn within the past 10 to 15 years. For example, Salmonella infections have gone up 20-fold in some countries in Europe since 1980. Similar increases are reported for haemorrhagic E. coli 0157 food poisoning: between 1986 and 1996, the frequency of infection increased 10- fold in England and Wales, and 100- fold in Scotland.

The first widely used anti-malarial drug, chloroquine, came into use during World War II, and resistance did not appear until the early 1960s. By contrast, the new drug, mefloquine, released in 1985, became useless in 60% of malaria cases within five years. Comparable accelerations in the development of antibiotic resistances have taken place in the same period.

Table 1. New diseases within the past 20 years
(incomplete list)

1977:    Legionella pneumophila, potentially fatal Legionnaires' disease
1977:    Ebola virus, haemorrhagic fever - fatal in up to 80% of cases.
1977:    Hantaan virus, potentially fatal haemorrhagic fever with renal syndrome.
1977:    Campylobacter jejuni, diarrhoea.
1980:    Human T-lymphotropic virus I (HTLV-1), T-cell lymphoma-leukaemia
1981:    Staphylococcus aureus, bacteria, toxic shock syndrome
1982:    Escherichia coli O157:H7, bacteria, bloody diarrhoea
1982:    HTLV-2 virus, hairy cell leukaemia
1983:    Helicobacter pylori, bacteria, peptic ulcer disease and stomach cancer.
1983:    Human immunodeficiency virus (HIV), AIDS.
1985:    Enterocytozoon bieneusi, parasite, persistent diarrhoea
1986:    Cyclospora cayetanensis, parasite, persistent diarrhoea
1988:    Hepatitis E virus, epidemics of jaundice in hot climates
1988:    Human herpesvirus 6, fever and rash
1988:    Salmonella typimurium, bacteria, diarrhoea
1989:    Hepatitis C virus, which causes liver cancer as well as liver disease.
1989:    Ehrlichia chafeensis, bacteria, infection
1991:    Guanarito virus, Venezuelan haemorrhagic fever.
1991:    Encephalitozoon hellem, parasite, conjunctivitis
1991:    New species of Babesia, parasite, infection
1992:    New strain Vibrio cholerae O139, epidemic cholera.
1992:    Bartonella henselae, bacteria, cat-scratch disease, bacillus angiomatosis
1993:    Sin Nombre virus, adult respiratory distress syndrome
1993:    Encephalitozoon cuniculi, parasite, disseminated disease
1994:    Sabia virus, which causes Brazilian haemorrhagic fever
1994:    Equine morbilivirus, respiratory illness
1995:    Human herpesvirus 8, Kaposiþs sarcoma in AIDS patients.
1995:    New monkey pox virus, human-to-human transmission, potentially fatal
1996:    New rabies in Australia 1996: New hantavirus, human-to-human transmission

Antibiotics were introduced in the early 1940s to treat infectious diseases, and resistance did not appear until the early 1950s. Resistance to penicillin, ampicillin and antipseudomonas penicillins in Staphylococcus aureus went from almost 0% in 1952 to more than 95% in 1992. By the 1980s, S. aureus had also developed high levels of resistance to the synthetic penicillin, methicillin and all other b-lactams. The new fluoroquinolone antimicrobial, ciprofloxacin, was introduced in the mid-1980s, but resistance to it had reached more than 80% by 1992. A study carried out by the Centers for Disease Control showed that ciprofloxacin resistance in S. aureus went from less than 5% to greater than 80% within one year. By 1990, nearly every common pathogenic bacterial species had developed varying degrees of antibiotic resistance, often multiple resistances. These include, besides Staphylococcus aureus (toxic shock syndrome, postoperative infections), Streptococcus aureus (toxic shock-like syndrome), S. pneumoniae (pneumonia), S. pyrogenes (rheumatic fever), Haemophilus influenzae (meningitis), Mycobacterium leprae (leprosy), Neisseria gonorrhoeae (gonorrhoea), Shigella dysenteriae (dysentery) and several other species of microbes that infect the human gut: E. coli, Klebsiella, Proteus, Salmonella, Serratia marcescens, Pseudomonas, Enterococcus faecium, Enterobacteriaceae and Vibrio cholerae (cholera). A strain of Staphylococcus isolated in Australia was resistant to 31 different drugs, including cadmium, penicillin, kanamycin, neomycin, streptomycin, tetracycline and trimethyloprim. The various resistance capabilities were due to genes carried on different plasmids (independently replicating units of genetic material) that could be separately passed on from one bacterium to another. Two strains of E. coli isolated in a transplant ward outside Cambridge were resistant to 21 out of 22 common antibiotics, including imipenen, cefotaxime, ceftazdime, ciprofloxacin, gentamicin, ampicillin, azlocillin, coamoxiclav, timentin, cephalexin, cefuroxime, cefamandole, streptomycin, neomycin, kanamycin, tobramycin, trimethoprim, sulfamethoxazole, chloramphenicol and nitrofurantoin. These multi-resistant strains of bacteria are rapidly becoming totally invulnerable to treatment. Scientists in Japan have already isolated a strain of Staphylococcus aureus that is resistant even to the last-resort antibiotic, vancomycin. Recent data showed that vancomycin resistance in Enterococci grew from 3% in 1993 to 95% in 1997 in hospitals in San Francisco. In Italy, erythromycin resistance in Streptococcus increased 20- fold just between 1993 and 1995.

Factors contributing to the recent resurgence of infectious diseases

The precise reasons for the resurgence of infectious diseases since the 1980s are not known. Many contributing factors have been suggested, among them:

  • population growth combined with rapid urbanisation,

  • wars, civil turmoil and natural disasters,

  • rapid increases in international air travel and the growing traffic in trade,

  • expanding areas of human habitation and the consequent environmental destruction,

  • social changes, including the clustering of young children in day-care centres and growing numbers of the elderly in nursing homes,

  • complacency towards infectious diseases in the public health sector and collapse of public health systems due to economic or social crises,

  • the overuse and abuse of antibiotics in intensive farming and medicine. One factor which has not yet been considered is genetic engineering biotechnology.

Gene technology and gene ecology

Gene ecology is an emerging discipline. It is prompted by recent findings that the genetic material - DNA or in some instances, RNA - can transfer horizontally from one organism to another through the external environment, instead of vertically by reproduction. Gene ecology is the totality of how genes function, mutate, move, transfer and recombine subject to feedback regulation from the interconnected levels of the genome, the physiology of the organism and the external ecological conditions.

Gene technology, or genetic engineering, can profoundly disturb the ecology of genes. The past 15-20 years witnessed the rise of genetic engineering biotechnology on a commercial scale. It is a technology for manipulating and transferring genes horizontally between species that do not normally interbreed. It is designed to break down species barriers and, increasingly, to overcome the species' defence mechanisms which degrade or inactivate foreign genes.

For the purpose of manipulating, replicating and transferring genes, a range of artificial vectors have been constructed. Natural vectors for horizontal gene transfer comprise replicating, often mobile, units of genetic material: viruses, plasmids and transposons. However, other pieces of DNA can also be taken up by cells and hence act as agents for horizontal gene transfer. Viruses are infectious particles consisting of genetic material wrapped in a protein coat. Animal and plant viruses cause many diseases, including cancer; viruses that attack bacteria are bacteriophages or phages for short. Plasmids are replicating units (replicons) of genetic material outside the chromosome. Transposons are mobile replicating units (jumping genes) that can move from one site to another in the same or different chromosome, or from chromosome to plasmid and vice versa. Plasmids and transposons typically carry virulence genes and genes for antibiotic resistance.

Artificial vectors

Artificial vectors are made by joining together parts of the most infectious natural vectors in order to enhance horizontal gene transfer. The artificial vectors are 'crippled', which means that most, if not all, the genes causing disease and infectivity are removed. But it does not mean those functions cannot be supplied by, or regained from, other viruses and parasitic genetic agents that are always present in the environment and in the cells of all organisms. The gene to be transferred is, as a rule, integrated into the genetic material of the vector; viruses, however, can also transfer genes that are not integrated, but merely packaged within the protein coat .

To make a transgenic organism, the vector carrying the foreign gene to be transferred - transgene - is allowed to infect the cells of the organism. Once inside the cell, the vector can either multiply many copies of itself or become integrated into the genome of the cell . Most artifical vectors possess one or more antibiotic resistance marker genes to track the movement of the gene(s) transferred, so they can be selected with the appropriate antibiotic(s). There are three main routes for horizontal gene transfer: infection with viruses (transduction); through pieces of genetic material taken up into the cell from the environment (transformation); or by unusual mating taking place between unrelated species (conjugation). As the entire orientation of genetic engineering is to facilitate horizontal gene transfer, it is to be expected that antibiotic resistance genes as well as virulence genes will inadvertently spread and recombine to generate new, drug- and antibiotic- resistant pathogens (see box below).

Genetic engineering biotechnology breaks down species barriers

ONE main contributing factor to the recent increase in the scope and frequency of horizontal gene transfer may be the deliberate acts of genetic engineers to break down species barriers. They do so by constructing a range of chimaeric vectors for cloning and transferring genes. These artificial vectors have the following important characteristics that enhance horizontal gene transfer.

  • They are already derived from elements that mediate horizontal gene transfer most effectively.
  • Their chimaeric nature means that they possess sequence homologies to DNA from widely different species and their viral pathogens,plasmids and trans-posons, thus facilitating successful horizontal transfer and recombination.
  • They routinely contain antibiotic resistance marker genes enhancing their successful transfer in the presence of antibiotics, either intentionally applied or as xenobiotics in the environment.
  • They often have origins of replication and transfer sequences, all of which facilitate horizontal gene transfer and recombination. In this context, the fact that they are 'crippled', so that genes for mobility and/or virulence are removed, is irrelevant, as helper functions can be supplied by other viruses, plasmids and mobile genetic elements present in the donor, recipient or a third strain of bacteria. And virulence genes can be regained by recombination.
  • It is well-known that chimaeric plasmids and viral vectors are subject to structural instabilities which make them more prone to recombine. Vector instability is a continuing problem for genetic engineers and the biotech industry as far as the stability of the transferred genes is concerned. It also increases the probability and scope for unintended, secondary horizontal gene transfer.
  • The now routine incorporation of strong promoters and enhancers in vectors to boost expression of transgenes is one main cause of structural instability, which is in addition to the instability arising from the attendant metabolic stress to the organism that, again, may increase unintended horizontal gene transfer.
  • Finally, vectors are designed to escape restriction thereby also enhancing the probability of successful horizontal gene transfer.

The construction of artificial vectors is fundamental to genetic engineering. All classes of genetic elements that mediate horizontal gene transfer have been used in constructing vectors: plasmids, phages, transposons, plus a range of pathogenic plant and animal viruses. As stated in a standard textbook on genetic manipulation, 'Many animal viruses have been subjugated as vectors. Virtually every virus that has been studied in any detail and that has a DNA genome or a DNA stage in its replication cycle has been manipulated in this way.'

Although different classes of vectors are distinguishable on the basis of the main framework sequence, practically every one of them is chimaeric. Important chimaeric vectors are the shuttle vectors which enable genes to be cloned (multiplied) in E. coli and transferred (transfected) into unrelated species in every Kingdom. Similarly, vectors used in manipulating plants and animals typically contain sequences from a range of plant and animal viral pathogens, as well as antibiotic resistance genes, often originating from promiscuous resistance plasmids and transposons. Phage vectors and phasmid vectors (hybrid of phage and plasmid) are also extensively used, and may have special relevance for the evolution of pathogenicity islands in bacterial pathogens.

Thus, genetic engineering biotechnology has effectively opened up highways for horizontal gene transfer and recombination, where previously, there was only restricted access through narrow, tortuous footpaths. These gene transfer highways connect species in every Domain and Kingdom with the microbial populations via the universal mixing vessel, E. coli. We review further circumstantial evidence that artificial gene transfer vectors increase the scope and frequency of horizontal gene transfer.

Indeed, the evolution of virulence and the spread of drug and antibiotic resistances are now linked to the extensive horizontal gene transfer and recombination events among bacteria and viruses, many of which may have occurred in recent years. For example, horizontal gene transfer and subsequent genetic recombination have given rise to the new bacterial strains responsible for the cholera outbreak in India in 1992, and for the Streptococcus epidemic in Tayside in 1993. The E. coli 0157:07 strain involved in the recent outbreaks in Scotland is believed to have originated from horizontal gene transfer from the pathogen, Shigella. Many unrelated bacterial pathogens, causing diseases from bubonic plague to tree blight, are now found to share an entire set of genes for invading cells, which have spread widely by horizontal gene transfer. Similarly, genes for antibiotic resistances have been transferred horizontally and have recombined with one another to generate multiple antibiotic resistances throughout the bacterial populations. Antibiotic resistance genes spread readily between human beings, and from bacteria inhabiting the gut of farm animals to those in human beings. Antibiotic-resistant strains of pathogens have been endemic in many hospitals for years. In the USA, up to 60% of hospital-acquired infections are antibiotic resistant. A special form of multiple resistance, which confers resistance to a wide range of chemically unrelated drugs, made its appearance among pathogens in the early 1990s, although it has been produced in E. coli in the laboratory since the 1980s. Even more disturbing is the finding that antibiotics can increase the frequency of horizontal gene transfer 10-10,000-fold. Thus, antibiotics create the very conditions facilitating the spread of antibiotic resistance. The evolution and spread of antibiotic resistance predate genetic engineering, and are largely due to the profligate use of antibiotics in intensive farming, and in medicine. However, the abuse of antibiotics per se does not account for the emergence of new viruses and new virulent strains of bacteria. Fifty new viruses have been identified just between 1988 and 1996. The urgent question which needs to be addressed is the extent to which genetic engineering biotechnology, by facilitating horizontal gene transfer and recombination, is contributing to the resurgence of infectious, drug-resistant diseases, and will continue to do so if allowed to proceed unchecked (See box below).

Artificial gene transfer vectors increase horizontal gene transfer

IT is not easy to transfer genes successfully between species as there are barriers to horizontal gene transfers. That is why, apart from transposons which are promiscuous, such events were relatively rare in our evolutionary past. For example, analyses of 145 nonvertebrate globin gene sequences showed that there were probably two cases of horizontal gene transfer, one from the common ancestor of ciliates and the green algae to the ancestor of cyanobacteria, and the other, from the ancestor of the yeasts to the ancestor of bacteria. Natural gene transfer vectors - viruses, plasmids and transposons - have probably always existed, especially in the microbial species; but they were, to varying degrees, host- specific, so that the frequency of conjugal transfers was higher between the same species than with other species. Naturally isolated vectors - even those that carry antibiotic resistance genes and have therefore already undergone evolution by horizontal gene transfer - have not exhibited the same wide range of transfer capabilities.

Horizontal gene transfers have been directly demonstrated between bacteria in the marine environment, in the freshwater environment and in the soil. Again, in all the experiments, horizontal gene transfers were mediated by specially constructed hybrid plasmid vectors, of the sort used in genetic engineering. Horizontal gene transfer occurs preferentially in interfaces between air and water and in the sediment, and especially under nutrient depletion conditions, thus refuting the claim that nutrient-rich media are necessary to support horizontal gene transfer. Horizontal gene transfer of antibiotic resistances has even been demonstrated in wastewater treatment ponds, the effluent from which is increasingly being used for irrigation in developing countries. In fact, frequencies of horizontal gene transfer may be greater under natural conditions than in the laboratory.

Horizontal gene transfer is not limited to the external environment. It has been demonstrated between gut bacteria in mice and chickens and in the gut as well as urogenital and respiratory tract of human beings.

Stephenson and Warnes wrote, 'The threat of horizontal gene transfer from recombinant organisms to indigenous ones is...very real and mechanisms exist whereby, at least theoretically, any genetically engineered trait can be transferred to any prokaryotic organism and many eukaryotic ones.'

A year later, another molecular geneticist, who works on transgenic plants, admitted that '...the potential for horizontal [gene] transfer may be greater than thought previously.'

Recent findings

This question is all the more important in the light of yet other recent findings indicating that microorganisms genetically engineered for 'contained use' may not be effectively contained, while our regulators are contemplating further relaxations of the guidelines on contained use. Thus, biologically 'crippled' strains of bacteria have actually been found to survive in the environment to exchange genes with other species.

Naked viral DNA may be more infectious than the virus itself. DNA released from cells is not readily broken down in the environment, thereby retaining the ability to transform organisms; and routine chemical treatments for inactivating pathogenic microorganisms and viruses, before they are discharged into the environment, may be ineffective, leaving a large percentage of pathogens in an active, infectious state.

Even if chemical inactivation methods are 100% effective, large amounts of recombinant DNA have already been, and continue to be, routinely released into the environment in one form or another. Many kinds of dangerous recombinant DNA, containing pathogenic sequences, cancer-causing viruses, as well as antibiotic resistance genes, can almost certainly transform bacteria in the environment and further recombine, spreading not only antibiotic and virulence genes in bacteria, but also generating new viral pathogens.

There is an urgent need to re-assess the safety regulation, not only of deliberate releases, but also of contained use. Instead our regulators are contemplating further relaxations.

Conclusion

The totality of evidence is sufficiently compelling, especially in view of the precautionary principle, to warrant, at the very least, an independent, full public enquiry into genetic engineering biotechnology and the etiology of infectious diseases. In addition, we desperately need research directed at understanding general mechanisms for horizontal gene transfer, which aims to strengthen the barriers against the transfer of recombinant DNA, or which can form the basis for scientific risk assessment. Such research must be carried out by independent research groups dedicated to the task, and not left in the hands of those who are involved in commercial exploitation of genetic engineering biotechnology. (Third World Resurgence No. 92, April 1998)

Some recent scientific findings

 

  • Biologically 'crippled' laboratory strains of bacteria can often survive in the environment to exchange genes with other organisms
  • Routine chemical inactivation methods may leave up to 10% of viruses and other pathogens in an infective state
  • Legal limits of 'tolerated releases' from contained use vastly exceed the minimum infective dose of some pathogens: 10,000 colony- forming units/ml in air or water versus a minimum infective dose of 50 bacteria for E. coli 0157:H7
  • DNA released from dead and living cells persists in the environment and transfers to other organisms
  • Naked viral and vector DNA may be more infectious, and have a wider host range than the virus
  • Vector DNA resists digestion in the gut of mice, enters the blood stream to infect white blood cells, spleen and liver cells, and integrates into the mouse cell genome

This article is an extract from a report entitled 'Gene Technology and Gene Ecology of Infectious Disease', which is being published by the Third World Network and The Ecologist.

The report was prepared by Mae-Wan Ho, Biology Dept., Open University, Walton Hall, Milton Keynes, MK7 6AA, UK; Terje Traavik and Orjan Olsvik, Depts. of Virology and Microbial Genetics, Inst. of Medical Biology, University of Tromso, Norway; Tore Midtvedt, Medical Microbial Ecology, Karolinska Institute, Doktorsringen 4A, 1 tr. Box 60400, S-104 01, Stockholm, Sweden; Beatrix Tappeser, Inst. for Applied Ecology, Postfach 6226, D-79038 Freiburg, Germany; C. Vyvyan Howard, Foetal Infant Toxipathology, University of Liverpool, Liverpool L69 3BX, UK; Christine von Weizsacker, Postfach 130165 53061 Bonn, Germany; and George C McGavin, Asst. Curator of Entomology, Museum of Natural History, Oxford University, Parks Road, Oxford OX1 3PW, UK.

 

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