THE JOURNAL 01- INFECTIOUS DISEASES. VOL. 137, NO.5. MAY 1978 © 1978 by the University of Chicago. 0022-1899/78/37U5-0018$00.88

Recombinant DNA: An Infectious Disease Perspective From the Department of Medicine, Tufts-New England Medical Center Hospital, Tufts University School of Medicine, Boston, Massachusetts

Sherwood L. Gorbach

Please address requests for reprints to Dr. Sherwood L. Gorbach, Department of Medicine, Tufts-New England Medical Ccn tel' Hospital, 171 Harrison Avenue, Boston, Massachusetts 02111.

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evolution of the guidelines for recombinant DNA research. Despite the political implications and external pressures, the meeting was a forum in which reason and science prevailed over philosophy and polemics. Defining the Risks

Discussions of risk assessment are often confounded by the onus to prove the absolute zero possibility of hazard, a task which in scientific terms is virtually impossible. It is equally impossible to deal with all conjectural risks, especially those that have no foundation in scientific observations. On the other hand, there remains a significant middle ground: legitimate areas of potential risk in recombinant DNA research which deserve to be approached with the highest degree of caution and restraint. These potential hazards can be grouped into three categories. (1) An organism such as Escherichia coli Kl2 may be converted, by the transfer of foreign DNA, into a pathogenic strain that resembles other pathogens already familiar to us. By increasing its virulence, the recipient strain could cause disease in a single person or be propagated in the environment, thereby spreading to other individuals. This mechanism implies that the organism has gained the ability to colonize the susceptible host, to produce disease, and to survive in nature. (2) The foreign D~A insert may be transferred from the recipient bacterium to other microorganisms or to somatic cells in higher organisms. In the case of E. coli K12, even if the organism docs not survive in the bowel, it may transfer its genes to other bacteria in the microflora. It has been postulated, particularly with viral inserts, that the foreign DNA may pass the mucosal barrier to the somatic cells of the host. (3) The foreign DNA may encode for injurious products. Such products include toxins, hormones, or even proteins which could induce

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The title of this meeting implies areas of common ground among the partici pants which formed the basis of our discussions. First, is that there are potential risks associated with recombinant DNA experimentation. The level of risk and its application to specific experiments remain highly controversial, but all prudent scientists would recognize that certain experiments cannot be exonerated of unexpected, and even serious, misadventures. The second area of commonality is that such risks can be assessed, measured, quantitated, and subjected to the same scientific scrutiny that characterizes the nature of the research endeavor itself. The major focus of concern over recombinant DNA has been whether disease will be produced in laboratory workers or innocent bystanders, particularly the public at large. This conference has been an attempt, perhaps the first of its kind, to bring molecular biologists together with scientists interested in the pathogenesis of disease. Diverse disci plines were represented: microbiology, epidemiology, gastroenterology, and endocrinology. One of the avowed purposes of the meeting was to widen the scope of discussion to include researchers interested in mechanisms of disease. I t is ironic that infectious disease experts have, in general, remained at the sidelines during this great debate, not engaging in the discussions of potential epidemics and unique virulence factors, areas about which they are particularly knowledgeable. Another reason for convening the meeting was to develop, through interdisciplinary discussions, experimental protocols which could assess the risk of recombinant DNA research. A series of experiments were designed in model systems to produce data that would permit rational

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E. coli in the Natural Environment

The natural habitat of E. coli is the intestinal tract of humans and animals [I]. The organisms can be found in soil and water under circumstances of fecal contamination. The major locus within the body is the large intestine where populations are stabilized at I06-1()H/g of intestinal contents [2, 3]. The upper intestinal tract, including the stomach and small intestine, has a relatively sparse flora with only transient colonization by coliforms, usually in concentrations of < IO:{ / ml. The lower ileum is a transitional zone between the meager flora of the upper intestine and the luxurious coliform populations of the large bowel [4, 5]. The intestinal microflora is extremely complex, and E. coli occupies only a small portion of this ecological niche. Obligate anaerobic bacteria are the major components, with concentrations in the large intestine of 101LI012/ g, nearly the number of bacteria which can be accommodated in that given mass [6, 7]. Thus, anaerobic organisms, of which there are over 500 species in the intestine of humans, outnumber facultative organisms, such as E. coli, by I,OOO-fold. The indigenous microflora is confined to the intestinal lumen and the mucosal surface [8, 9J. Active penetration through the epithelium is an abnormal event. "Microorganisms that possess invasive characteristics are pathogenic, i.e., Shigella, Salmonella, and certain penetrating strains of E. coli. A number of control mechanisms protect the upper intestinal tract from contamination by coliforms carried in food and drink [10]. Particularly important is gastric acid at the portal of entry [II]. Most of the enteric bacilli that contaminate food are destroyed in the stomach. Bile

has antibacterial activity and is thought to have some impact on control of the upper intestinal flora. Propulsive motility or peristalsis moves microorganisms progressively down the small bowel. There are also mucosal factors and immunoglobulins that protect the upper intestine from colonization and invasion. The large intestine has additional control mechanisms which maintain coliform populations at relatively constant levels. Most important among these mechanisms is the metabolic activity of the normal indigenous microHora. An important point to emphasize is that these control mechanisms are overlapping and redundant so that a failure of one system may not affect the total microenvironment significantly. E. coli appears to be a lifelong companion, being acquired in the intestinal tract within one day of birth [3]. Among ] 63 known 0 serotypes of E. coli, only a relatively small number are found with any frequncy in the intestinal tract of humans [1J. An individual is likely to harbor between five and 10 serotypes at anyone time [12, 13]. It should be recognized, however, that sampling for specific serotypes becomes a formidable task so that the exact number of serotypes in the gut can never be determined. The stability of E. coli populations within the intestinal tract has been the subject of many investigations. Certain strains may persist within the same individual for months [14]. On the other hand, there are natural fluctuations in coliform populations which cause new strains to be introduced. Hospitalized patients, for example, are known to acquire specific serotypes associated with that institution within a few days of admission [13]. In England, it has been found that serotypes of E. coli may cluster within patients on specific wards in the hospital [15]. The hospital food was found to be contaminated with E. coli, and it was postulated that the E. coli in the intestinal flora of patients were im planted by exposure to hospital food and medications [16]. Artificial attempts to implant E. coli in volunteers have met with varying success, depending upon the strain employed and the size of the inoculum. Using relatively small numbers of organisms, Sears et al. [17, 18] found that the ingested strains either disappeared or were recovered from the feces for only a few days. Even with larger

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an autoimmune disease. This mechanism implies translation of the specific substance by the microorganism, as well as implantation of the microorganism in the intestinal tract. These potential hazards can best be understood within the general framework of microbial pathogenesis. This involves specific mechanisms for causing disease, colonization of body surfaces, and communicability within the environment.

Gorbach

Infectious Disease View of Recombinant DNA

Characteristics of Pathogenic Microorganisms

A critical constellation of virulence factors is required by a microorganism in order to produce disease: (1) survival in the environment so that it can spread from animal to animal, (2) some mechanism for penetrating the skin or a mucosal surface such as the bowel, genitourinary tract, or oropharynx, (3) multiplication within the host, (4) systemic spread within the host, (5) resistance to host defense mechanisms, and (6) production of toxin or some other mechanism to damage the host to cause those symptoms associated with "disease." Freter has emphasized that the absence of anyone of these characteristics will break the chain of events, rendering the microorganism avirulent [10]. Parenthetically, it should be noted that E. coli Kl2 is intrinsically impaired in most, if not all, of these properties. The surface antigens of E. coli are important determinants in the virulence of the particular strain [22]. 0rskov et al. have noted that both the polysaccharide and the protein surface antigens playa role in pathogenicity [23, 24]. The 0 antigens render the organism resistant to phagocytosis and bacteriocidal forces. Specific 0 serotypes are associated with infantile diarrhea, while others have been related to enterotoxigenic diarrhea, dysentery-like disease, or urinary tract infections [25]. The presence of Kl capsular antigen is highly correlated with neonatal meningitis and other systemic E. coli infections [24]. The fimbriae antigens or pili have been associated

with adhesive and colonization properties [26, 27]. No single surface antigen determines pathogenicity, but organisms that are lacking these surface structures are less virulent. E. coli Kl2 is defective in production of 0 antigen lipopolysaccharide and does not make capsular antigens [21, 28]. The prefix "K" causes some ambiguity in this nomenclature [23]. The capsular polysaccharide antigens, such as Kl, are designated by this letter, taken from the German word for capsule. The protein antigens involving the fimbriae surface structures are also termed "K," i.e., K88. To add to the confusion, the "K" of E. coli K12 is not related to either of these surface antigens, but is a historical appellation assigned to the classic strain used in genetic research. (There are also wild-type strains of E. coli causing urinary tract infections which carry a "KI2" capsular polysaccharide antigen; these strains are not related to the traditional E. coli Kl2 used in genetics experiments.) Epidemiology of E. coli Infection

The diseases caused by E. coli are divided into two broad groups, intestinal and extraintestinal infections. In the United States, E. coli intestinal disease, generally manifesting itself by mild diarrhea, is relatively uncommon [29, 30]. The organisms are transmitted by the fecal-oral route. Person-to-person spread is rather unlikely, and for this reason secondary transmission of E. coli from the index case to another person is rarely observed. These epidemiologic characteristics are based on the requirement for a large oral inoculum of E. coli to initiate disease, an inoculum estimated to be at least IOLI010 organisms [29, 31]. Under natural circumstances, only highly contaminated sources such as food and water can serve as vehicles of transmission. The extraintestinal diseases include urinary tract infections, septicemia, meningitis, pneumonia, and wound infections. The E. coli strains responsible for these infections are different from those causing intestinal disease [24, 32, 33]. E. coli is a leading cause of community-acquired infections that lead to hospitalization [29]. The urinary tract is the major site. In communityacquired infections of the urinary tract, approxi-

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doses (Ion), Smith reported that none of the administered strains of E. coli persisted in the intestine for more than 10 days [19]. On the other hand, Cooke et al. demonstrated regular colonization when large numbers of E. coli were fed to volunteers. Similarly, Formal and Hornick implanted the HS strain of E. coli for as long as three months in some volunteers following a single feeding [20]. The source of the E. coli strain is related to its ability to implant in humans. Strains derived from animal sources were found by Smith to colonize poorly in humans [19]. Hettiaratchy confirmed this result but noted that some colonization occurred even with strains of animal origin [21].

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E. coli Intestinal Infections

E. coli are important pathogens in diarrheal diseases of young animals. In the developing countries of the world, E. coli causes serious diarrhea in young children [30]. This organism also is the leading cause of the diarrhea of travelers [34, 35]. In the United States and England, however, E. coli diarrhea is relatively uncommon. The organisms responsible for intestinal infections in humans fall into certain 0 serogroups. Among the toxigenic organisms, at least two virulence factors, genetically controlled by different plasmids, are required for a fully pathogenic strain [26]. The organism must produce an enterotoxin, either a heat-labile (L 1') or heatstable (ST) variety. In addition, the organism must be capable of colonizing in the small intestine, a property that is related to a pilus or protein antigen. These K antigens are host-specific; thus, the K88 type is associated with diarrhea in piglets, the K99 in calves, and a different type, as yet unclassified, is specific for humans [27]. The invasive strains of E. coli fall within another group of serotypes and possess different mechanisms of pathogenicity. Behaving like shigella organisms, the invasive E. coli are capable of penetrating the intestinal mucosa of the large bowel and multiplying within the epithelial cells; they cause a disease similar to bacillary dysentery [31]. Again, implantation in the bowel is the critical first step in the pathogenic events. Extraintestinal Infections Caused by E. coli

E. coli strains that are isolated from patients with septicemia, uri nary tract infections, pneumonia, and wound infections possess certain characteristics more frequently than do strains isolated from the normal flora. As reported by Minshew

et aI., pathogenic strains exhibited hemolysin production, biosynthesis of colicin V, hemagglutination of human erythrocytes, and the ability to kill 13-day-old chick embryos [33]. E. coli Kl2 was found to be deficient in all of these characteristics. Urinary tract infections caused by E. coli have been the subject of extensive epidemiologic and laboratory study. The 0 serotypes isolated from infected urine are restricted to relatively few groups which may show some variation in different parts of the world [36]. The most prevalent serotypes are 01, 02, 04, 06, 050, and 075. These serogroups are also the most frequent in the intestinal microflora, although there are some anomalies such as 06, which is more frequently associated with infection than with bowel carriage. Similarly, there is a relatively restricted group of K surface antigens associated with urinary tract infections: KI, K2, K3, K5, K12, and K13 [23]. Montgomerie and his colleagues have described virulence factors in E. coli that promote the development of pyelonephritis [32]. These features are resistance to phagocytosis and serum bactericidal activity, presence of K antigen, dulcitol fermentation, and the ability to multiply in urine or in minimal medium. Of this list, E. coli K12 possesses only dulcitol fermentation as a regular character. The pathogenesis of community-acquired urinary tract infection is by the ascending route, from the urethra, to the bladder, and upward to the kidneys. Stamey et al. have shown that the initiating event is colonization of the vaginal introitus and periurethral mucosa by coliforms which are derived from the intestinal flora [37, 38]. Women who have never experienced urinary tract infections rarely have colonization of this site. On the other hand, prospective studies have demonstrated colonization of the vaginal introitus prior to development of overt urinary tract infection. There are two schools of thought regarding the nature of E. coli associated with urinary tract infections. One view asserts that the pathogens, being in the intestinal microflora, are "in the right place at the right time." The other view suggests that the urinary tract pathogens are endowed with "special properties" to produce infection.

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mately 80~'6 are caused by E. coli. Hospitalized patients are also prone to E. coli infection. Indeed, this organism is the leading cause of nosocomial infections, constituting approximately 20c;~ of the total. Again, the urinary tract is the major site of hospital-acquired disease. Indwelling urinary catheters or operative procedures are the predisposing causes in the majority of E. coli infections related to hospitalization.

Gorbach

Infectious Disease Vieio of Recombinant DNA

Studies of E. coli K12

The pedigree of this classic strain starts with an isolation at Stanford in 1922 from the feces of a patient with diphtheria. The strain was maintained for many years in laboratory culture until its use in genetic studies by Lederberg and Tatum in 1947. Since that time, it has been passaged on numerous agar slants and widely employed in a variety of experimental situations. Actually, there are a vast number of progeny of the original K12 strain which possess different genotypic and phenotypic characteristics. The pedigrees of these derivatives have been summarized [39]. All of them, however, are "rough" mutants with no detectable a-antigen specificity [40]. Their features are related to a block in the synthesis of a-specific chains of lipopolysaccharide due to a mutation in the his (histidine)linked rIb region of the chromosome [41J. Serological and phage studies show that the lipopolysaccharide core of E. coli K12 is different from all known core types of wild-type E. coli and Salmonella [41, 12]. The lVT antigens (cholanic acid), which are common to most Enterobacteriaceae, are elaborated by the K12 strain; this antigen is responsible for the mucoid character of K12 organisms growing on agar plates [2·1J. Since colonization of the intestine is felt to be an initial event in many pathologic states involving E. coli, it is natural that this feature has been the subject of several investigations. It is fair to

state that there have been no instances in which the ingested strain of E. coli Kl2 has been implanted in the human intestine. Smith fed eight different E. coli K12 strains, containing various transmissible plasmids, to a volunteer at high doses (IOn) [43]. Some strains could not be isolated at all from the feces, while others persisted in progressively reduced counts for a period of up to four days. The experiment was subsequently repeated, and there was again no persistence beyond four days. To increase the likelihood of implanting E. coli Kl2 in the gut, Smith then used a Kl2 strain that had inserted in it the colicin V (CoIV) plasmid of a wild-type E. coli [44]. (CoIV promotes the survival of wild-type E. coli in the intestinal tract.) When a volunteer consumed the K 12 strains with and without the ColV plasmid, the strains were eliminated from the feces in an equal time frame, none persisting more than four days. Anderson attempted similar implantations with E. coli Kl2 strains in eight volunteers who received doses of up to 1010 organisms [45]. The maximal period of fecal excretion of the test strains was six days, with a mean of three days. These studies were repeated, again using eight volunteers, and the same findings were observed. Gorbach reported at this meeting unsuccessful attempts at implanting E. coli Kl2 in two patients with defective bacterial clearing mechanisms in the small bowel. In one case, the patient had a stricture in the ileum, and the other patient had severe diarrhea due to cholera. The Kl2 strain was eliminated from the small bowel and feces within 24 hr in both patients. A number of investigators have tried in vain to implant E. coli Kl2 in the intestinal tract of laboratory animals [10,46]. Negative results have been noted in mice, rats, chickens, pigs, and calves. E. coli K12 has been able to colonize the stomach of starved sheep. However, these animals possess a complex stomach consisting of a rumen; Smith has referred to this situation as a "test-tube" experiment. Freter has told us at this meeting of his success in implanting E. coli Kl2 into a germ-free mouse [10]. The germ-free animal will accept virtually any enteric organism for colonization even when the strain cannot implant in animals with a conventional microftora. On the other hand, Freter noted that the fur-

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On the basis of current evidence, it appears that both formulations are relevant. There is no doubt that the organisms causing such infection must be implanted in the intestinal microflora in order to colonize the periurethral mucosa subsequently. This explains the high correlation between serotypes in the urinary tract and those in the feces. There are, however, a multiplicity of serotypes in the intestinal flora, whereas only a single serotype, or on rare occasions two, causes infection at any given time. Hence, there is selectivity operating, which can be further demonstrated by the virulence factors associated with urinary tract pathogens. The evidence strongly suggests that urinary tract pathogens must be capable of colonizing the bowel of the host, as well as possessing certain special properties.

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Gorbach

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Attempts to Augment Virulence in E. coli K12

A number of virulence genes have been identified in wild-type strains of E. coli that are associated with either enteric or nonenteric disease. Using a model of intestinal infection, Smith transferred the plasmids for K99 and the enterotoxin into E. coli Kl2 [44J. These strains were fed to four colostrum-deprived calves, animals that are highly susceptible to the wild-type toxigenic strains. One animal developed slight diarrhea, but all of the calves remained well. When they were sacrificed, at 30 hr there was no proliferation of the Kl2 strains in the small or large intestine. The fully virulent, wild-type strain produced severe diarrhea in these animals and proliferated in large numbers within the small intestine. Minshew et al. transferred two virulence properties, hemolysin and colicin V, into E. coli K12 strains [33]. When tested in their 13-day-old chick embryo model, the recipient Kl2 strains still failed to show virulence. Smith performed a similar experiment but used a different animal test system, inoculation into chickens [44]. The Kl2 strain that had received ColV plasmids from wild-type strains of E. coli did demonstrate increased lethality in chickens. However, the dose required to produce death with the Kl2 strain

containing ColV was very large, as compared with a relatively small dose needed for the wild-type ColV strains. Several attempts have been made by Formal and his coworkers to transfer genetic material from virulent shigella strains into E. coli K12 [47]. The primary focus of these studies was to produce vaccines with the Kl2 strain that could colonize the bowel and possess enough shigella antigen to evoke a local immune response. Despite transfer of all of the recognized virulence genes identified with Shigella into E. coli K12, there has been no success in producing a recipient strain that could colonize the bowel or penetrate the intestinal mucosa. When these strains were administered to volunteers, there were no clinical signs of diarrhea. The recipient Kl2 strains were eliminated within six days or less, results similar to those reported by Anderson with other E. coli feeding experiments. Similar studies have been performed with Salmonella. Formal has concluded that the invasive property is determined by a multiplicity of genes; he has stated, "We consider it unlikely that the random insertion of foreign DNA into the E. coli Kl2 genome could supply all of the genetic information necessary to convert this organism into an invasive enteric pathogen" [20]. The Lack of Epidemic Potential of E. coli K12

On the basis of the available evidence, the participants in the Workshop agreed to the consensus statement that E. coli Kl2 could not be converted into an epidemic pathogen. In the first instance, colonization of the intestine by E. coli Kl2 has proven difficult, if not impossible, in conventional animals and in human volunteers. Transfer of virulence genes into E. coli Kl2 has not produced a fully pathogenic strain, although the ColV experiments of Smith suggest that some increase in pathogenic potential could be achieved. Finally, communicability and spread of this organism from person to person is considered extremely unlikely because of its fragility in nature and the high degree of sanitation and public health procedures in this country. Thus, the three tenets for pathogenicity-colonization, the capacity to produce disease, and communicability-arc blocked, and it is not felt

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the I' debilitated Kl2 strain, X1776, failed to implant even in the germ-free mouse. This has been ascribed to the sensitivity of this strain to bile salts that are present in the intestinal lumen. A number of variables are known to influence the colonization of organisms in the intestinal tract. Implantation can be altered by antibiotic administration, starvation, the type of diet, reduction in gastric acid, and antimotility drugs. It is clear that more implantation experiments need to be performed with attention to these variables, many of which may be found in laboratory workers exposed to these organisms. It is important to note that once E. coli Kl2 has been established in the germ-free animal, the addition of wild-type strains to the flora fails to dislodge it. This finding may have implications for feeding experiments, particularly those involving subjects receiving antibiotics to which the specific Kl2 may be resistant.

Infectious Disease View of Recombinant DNA

Transmission of DNA from E. coli K12 to Other Organisms

A potential risk In recombinant DNA research is the transmission of the cloned DNA insert from the E. coli K12 vector to other organisms within the intestinal flora. This concern has prompted a series of investigations into the transmissibility of plasmid DNA in E. coli K12. Smith fed l O? E. coli K12 organisms to a normal volunteer [44]. He used several strains which contained self-transmissible plasmids of the F, I, or A2 transfer groups. These strains could transfer in vi tro the tetracycline resistance plasmids to an E. coli K12 recipient and to resident E.

coli from the normal flora of the volunteer. When the strains were fed to the volunteer, however, they were eliminated from the feces within four days, and there was no evidence of in vivo plasmid transfer to resident strains or to susceptible K12 and H123 E. coli strains fed in the same ingested sample. This experiment was repeated, and again there was failure to transfer in vivo the tetracycline resistance plasmid. Anderson fed large numbers of E. coli Kl2 organisms which contained a nonconjugative plasmid to eight volunteers [49]. In no instance was plasmid transfer to normal flora demonstrated in vivo. However, in vitro studies showed that E. coli strains from the normal flora of three of eight subjects carried transfer plasmids which could mobilize one of the non transmissible plasmids, but not the other. Anderson suggested that "transfer would therefore be possible if a suitable conjugative plasmid entered a strain carrying a nonautotransferring hybrid plasmid." Curtiss described in vitro transfer experiments in which he measured the mobilization of a series of recently developed, nonconjugative plasmids under optimal laboratory condi tions [46]. He estimated that the maximal probability for transmission of such plasmid vectors from an E. coli K12 host is 10-16 per surviving bacteria per day in the intestinal tract of warm-blooded animals. He emphasized that the chance of transfer is even less since other factors, not taken into account, would reduce transfer in the intestinal tract. The in vivo deterrents include the following factors. (1) Diminished bacterial metabolic activity leads to decreased conjugation. In the test tube, the generation time of E. coli is 20-40 min, but it is 4-6 hr in the intestinal tract. (2) Conjugation is inhibited by fatty acids, bile, and other constituents of the gut. (3) Conjugation is inefficient at the pH and Eh (oxidationreduction potential) of the intestine. It was the consensus of the participants that the transmissibility studies, while given some comfort by their negative findings, are not sufficient in number or in scope to exclude the potential risk in this area. In addition, several discussants raised the possibility of transfer of genetic material to indigenous flora and to somatic cells of the host by unknown mechanisms. Within the complex milieu of the intestinal tract, it is possible

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possible to convert the Kl2 strain into an epidemic pathogen. Whether the organism could be converted to a pathogen even in terms of an individual laboratory worker is still moot. It is recognized that a large inoculum would be required to induce intestinal colonization or infection. Most of the infectious diseases caused by E. coli are associated with colonization of body surfaces and mucous membranes, particularly in the intestine. The factors influencing colonization and those responsible for disease production are controlled by a number of genetic elements. Even the surface antigens have to have certain antigenic structures in order to render the strain pathogenic. The other virulence factors constitute a diverse list controlled by chromosomal and extrachromosomal genes. Hence, it is difficult to conceive that the multiple deficiencies of E. coli K12 could be corrected by a random, or even designed, insertion of foreign DNA. Pike has recently analyzed 3,921 cases of laboratory-associated infections [47]. Only two instances were caused by E. coli. In the 30 years that E. coli K12 has been used in genetics research, there have been no reported cases of laboratory-acquired infections due to this organism. Monitoring the fecal flora of laboratory workers over a two-year period in a P-l type of facility failed to reveal the Kl2 organisms (marked by nalidixic acid resistance) or the self-transmissible plasmids used in the experiments [48]. Nevertheless, the issue of pathogenicity and colonization in an individual subject requires further investigation.

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that such transfer proceeds in ways not discernible in the test tube or in artificial laboratory conditions. More animal and human feeding experiments should be performed to confirm the previous observations and to provide further assurance that in vivo transfer of these plasmids would not occur. Additional experiments need to be designed for study of viral inserts since this represents another area of concern. (A special conference has been convened to deal with the problems of viruses and oncogenic gene segments.) As an initial step, the participants have developed a series of experimental protocols for risk assessment experiments. Such experiments would evaluate colonization, acquisition of virulence, and promiscuous transmission of foreign DNA inserts. This process will allow a rational evolution of laboratory guidelines for conducting recombinant DNA experimentation with E. coli K12.

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Infectious Disease View of Recombinant DNA

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30. Sack, R. B. Human diarrheal disease caused by entcrotoxigenic Escherichia coli. Annu. Rev. MicrobioI. 29:333-353, 1975. 31. DuPont, H. L., Formal, S. B., Hornick, R. B., Snyder, M. J., Liboriat i, J. P., Sheahan, D. G., LaBree, E. H., Kalas, J. P. Pathogenesis of Escherichia coli diarrhea. 1\'. Eng-I..1. Med. 285:1-9,1971. 32. Montgomerie, J. Z. Factors affecting virulence in Escherichia coli urinary tract infections. J. Infect. Dis. 137:64:)--{)17,1978. 33. Minshew, B. H., jorgensen, J., Swanstrum, M., GrootesReuvecamp, G. A., Falkow, S. Some characteristics of Escherichia coli strains isolated from extraintestinal infections of humans. J. Infect. Dis. 137:648654,1978. 34. Gorbach, S. L., Kean, B. H., Evans, D. G., Evans, D. J., Jr., Bessudo, D. Travelers' diarrhea and toxigenic Escherichia coli. x. Engl. J. Med. 292:933-936, 1975. 35. Merson, M. H., Morris, G. K., Sack, D. A., Wells, J. G., Feeley,.J. C., Sack, R. B., Creech, W. B., Kapikian, A. Z., Gangarosa, E. J. Travelers' diarrhea in Mexico. X. Engl. J. Med. 294: 1299-1305,1976. 36. Crunebcrg, R. I\., Leigh, D. A, Brumfitt, W. E. coli serotypes in urinary tract infection. Studies in domiciliary ante-natal and hospital practice. In F. O'Grady and W. Brumfitt [ed.]. Urinary tract infection. Oxford University Press. London, 1968, p. 68-79. 37. Stamey, T. A., Timothy, M., Millar, M., Mihara, G. Recurrent urinary infections in adult women. Calif. Med. 115:1-19, 1971. 38. Stamey, T. A., Sexton, C. C. The role of vaginal colonization with Enterobacteriaceae in recurrent urinary infections. J. Urol. 113:214-217, 1975. 39. Bachman, B. J. Pedigrees of some mutant strains of

Recombinant DNA: an infectious disease perspective.

THE JOURNAL 01- INFECTIOUS DISEASES. VOL. 137, NO.5. MAY 1978 © 1978 by the University of Chicago. 0022-1899/78/37U5-0018$00.88 Recombinant DNA: An I...
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