THE JOURNAL OF INFECTIOUS DISEASES. VOL. 137. NO.5. ]\IAY 1978 © 1978 by the University of Chicago. 0022-1l:l99I7H/3705-0007$OO.75
Possible Effects of Foreign DNA on Pathogenic Potential and Intestinal Proliferation of Escherichia coli Rolf Freter
From the Department of Microbiology, The University of Michigan Medical School, Ann Arbor, Michigan
quired for pathogenicity into the recipient bacterium. A relevant example that illustrates the chain of events necessary to produce pathogenicity is the case of enteropathogenic E. coli strains where distinct virulence factors are necessary in order to cause diarrheal disease. One such factor, an enterotoxin, is the agent which induces the diarrhea. However, when a strain of E. coli K 12 carried the gene coding [or this toxin, it was still unable to produce diarrheal disease in the gut of animals [2]. Obviously, this strain lacked other equally important virulence factors that would have enabled it to survive in the intestine. It is important to note that E. coli strains used in recombinant D~A research are all derivatives of this Kl2 strain. Descri ptions of virulence mechanisms do not adequately emphasize the fact that the myriad of metabolic functions and structural features every bacterial cell must have in order to survive in nature are also required for the multiplication of a pathogen in the human body. The term "virulence [actor" is usually applied only to those characteristics that distinguish pathogenic from nonpathogenic bacteria. It is obvious, however, that the characteristics which a bacterium must possess in order to cause disease are by no means restricted to only those virulence factors. This is a most important consideration in the evaluation of the enfeebled EK2 and EK:i strains of E. coli which are to be used in most recombinant DNA research. Enfeebled EK2 and EK3 strains have been deliberately deprived of nutritional and structural characteristics that are important for microbial survival in nature as well as in human or animal hosts. Therefore, it appears that if an inadvertent introduction of genes coding for only one-or even for a few-virulence factors were to occur by recombinant DNA methods, it would not be sufficient to restore the entire complement of trai ts necessary for E. coli to develop a hi therto
Creation of E. coli Strains with Novel Pathogenic and Epidemiologic Potential
It is recognized that a given bacterium must possess a large number of attributes before it can colonize the human intestinal tract and cause disease. For example, a pathogen must be able to survive in the environment, attach to and multiply on the body surface, spread within the body, resist the numerous defense mechanisms of mammalian tissue, and produce a toxin or interfere with the host's physiology in some other way. If only one of these links is missing, the chain will be broken. The bacterium's progress will stop, and it will not be able to cause disease. Therefore, if a relatively small amount of DNA is inserted into a deliberately "enfeebled" microorganism, it is is highly improbable that this procedure will introduce every single gene reThis research was supported by grant no. Al 07328 and contract no. NOI-AI-62518 from the National Institute of Allergy and Infectious Diseases. Please address requests {or reprints to Dr. Rolf Freter, The University of Michigan Medical School, 6643 Medical Science Building II, Ann Arbor, Michigan 48109.
624
Downloaded from http://jid.oxfordjournals.org/ at East Carolina University on July 13, 2015
Serious questions have been raised regarding the possible hazards inherent in recombinant DNA research. Among the various speculative dangers mentioned have been the following. (1) Are new, highly pathogenic bacteria likely to be created by this new technology and (2) could a microorganism, such as Escherichia coli, carrying foreign DNA become implanted in the human gut and eventually transfer this DNA to humans? These questions have been dealt with in some detail in another publication [I] which was made available to participants in this workshop in the form of a preprint. For this reason I will simply summarize some of the arguments pertaining to the first question and emphasize a few new developments which are relevant to the second problem.
DNA and Pathogenic Potential
Problems of Controlling Intestinal Flora
Control of the bacterial flora inhabiting the large intestine of humans is not possible in any practical or useful way. One reason is that techniques for the cultivation and identification of many of these organisms have only been recently developed. Moreover, several investigators [3] have shown that there are still microorganisms, although identifiable by electron microscopy, which cannot be grown in vitro; hence, one cannot determine their functions and characteristics. Another major difficulty is the delineation of the biochemical and physical parameters affecting the environment in the intestine and especially of the substrates available for bacterial growth. Almost nothing is known about this subject. Several authors have speculated that intestinal bacteria, such as E. coli, live a'Teast or famine" existence caused by the periodic food uptake of their hosts. Their habitat, suddenly swamped with nutrients, is depleted soon after a meal has been digested and absorbed. It has, however, been observed for some years that enterobacteria, including E. coli, may actually reach higher populations in a stressed or starved host [1, 5]. One wonders, therefore, whether and to what extent intestinal E. coli and other bacteria actually depend on diet-derived substrates or on a more or less steady flow of host-derived sub-
stances such as effete epithelial cells, secreted mucus, enzymes, etc. Because of the antagonistic action of other indigenous bacteria, Enterobacteriaceae such as E. coli, as well as the classical pathogens in this family, are kept at low population densities in the normal large intestine. Much experimental work has been done to determine the mechanisms by which the intestinal flora control pathogens, E. coli, or other bacteria. Those mechanisms which have been identified include the following: Eh (oxidation-reduction potential), pH, numerous inhibitors (such as fatty acids, bile salts, hydrogen sulfide), competition for substrates or adhesion sites, and local immunity. However, very little is known concerning in vivo function and interaction of these mechanisms. There are three apparent reasons for this lack of knowledge. First, bacterial interactions are quite specific for a given environment. Since we cannot precisely define the physical and chemical characteristics of the microenvironment at any particular site of the intestine, we cannot construct an in vitro experiment that matches the specific conditions affecting the bacterial flora. Second, there are redundancies among the control mechanisms; if one fails another mechanism takes over. Although an experimenter can inactivate a particular mechanism, the outcome of the experiment remains unchanged because another mechanism will simply come into play. This situation makes the isolation of a given mechanism very difficult. Finally, there is the problem of interactions among the control mechanisms. For instance, local immunity can be shown to be operative; however, other bacterial or host defense mechanisms, acting synergistically with local immunity, are likely to be required before that immunity can have a significant effect [fi]. Again, this makes the study of anyone of the whole system of control mechanisms quite difficult. Problems of Implantation
One must conclude, in view of the above discussion, that nothing definite is known about the attributes that a bacterium must possess in order to be easily implanted into the intestinal tract. Consequently, there is no generally accepted test procedure by which one could determine or quan-
Downloaded from http://jid.oxfordjournals.org/ at East Carolina University on July 13, 2015
unknown pathogenic potential. (This is especially true with respect to the enfeebled E. coli strains.) The above considerations do not rule out the possibility that the introduction of a limited amount of genetic material into E. coli could augment those disease-causing properties already present in the bacteria. However, diarrhea and other diseases caused by E. coli do not give rise to widespread epidemics in countries with adequate sanitation procedures. Only the respiratory route of transmission of infectious agents would be difficult to control in countries like the United States. Fortunately, however, strains of E. coli that are pathogenic for humans do not become airborne. Therefore, it is exceedingly unlikely that life-threatening epidemics might be caused by E. coli strains that carry small segments of foreign DNA.
625
626
Physiological State of Bacteria and Implantation
We have recently tested this hypothesis by attempting to implant a wild-type E. coli strain (C25) and a K 12 strain (X-1666) into mice in two different ways: (1) by introducing the bacteria directly into the stomach of conventional mice in a buffered vehicle and (2) by first monoassociating germ-free mice with the E. coli strain
and then conventionalizing these animals by caging them with conventional mice. In the latter design, the E. coli reach very high population levels (- 5 X IOn per mouse) in the germ-free animals. After caging with conventional mice, the monoassociated animals become implanted with a succession of indigcnous intestinal bacteria until about two weeks later when their gut flora attains the composition found in conventional mice [3]. We speculate that during these two weeks of conventionalization the E. coli strain adapts itself to the constantly changing environment in the mouse gut and therefore has a better chance to remain implanted along with the newly devcloping gut flora. The data obtained in representative experiments are shown in figures 1 and 2. Both the wild-type strain C25 and the Kl2 strain X1666 were eliminated [rom the gut when fed to conventional mice, and elimination was more ra pid wi th the K 12 strai n. This finding corresponds with results reported by others in human feeding experiments. E. coli strain C25 established itself in about 5C;~-10~~ of the 144 mice tested to date and persisted in very low numbers in their fecal Hora for at least several months. Strain X-1666 persisted beyond seven days in only one of a total of 144 conventional mice fed this bacterium. In this one animal, bacteria still persisted in the stool at 90 days after feeding. The results obtained wi th the second method were, however, strikingly different. Neither of the E. coli strains was eliminated from the gut of the conventionalized animals; rather, the strains established themselves in the indigenous flora for the length of the experiments (up to 85 days) at population levels normally assumed by E. coli in the conventional mouse. Data from representative experiments are also shown in figures 1 and 2. Differences in the population levels of E. coli C25 and X-1666 shown in these figures are not significant. In replicate experiments both strains populated conventionalized mice at levels within the range shown in the figures. In subsequent control experiments, strain X1666 was reisolated from mice in which it had become established and then fed to conventional mice. Under these circumstances, the reisolated strain was quickly eliminated from the animals. Hence, establishmen t of X- I666 in the conven-
Downloaded from http://jid.oxfordjournals.org/ at East Carolina University on July 13, 2015
titate the ability of E. coli or any other microorganisms to establish themselves in the intestine. The recent literature does report a number of attempts to implant E. coli K12 into the intestines of volunteers by feeding them large doses of these microorganisms [7, 8]. Such data regularly show that it is almost impossible to implant E. coli K12 in this manner. The inoculum usually survives for only a few days in the host's intestine. To the casual observer, this kind of evidence may appear to be pertinent and conclusive. This, unfortunately, is not the case. Such experiments tell us little about the ability of bacteria to grow in the human intestine. It has been known for some time that the feeding of bacteria, especially those grown under the usual laboratory conditions, rarely results in implantation because the normal intestinal flora is antagonistic to the growth of invaders [9-11]. Published evidence suggests that feeding experiments do not usually result in implantation simply because the bacterial inoculum is not in the proper physiologic state. Improved implantation would result if the inoculum had been grown under conditions (of substrate, pH, Eh, etc.) resembling those found in the intestine. Two laboratories [12, 13] have shown that exclusion, by bacterial antagonism, of invading microorganisms from an established flora may fail to occur when the invading bacteria come from an environment similar to the one which they are about to enter. Depending, therefore, on the physiologic state of the invading bacteria, antagonism exerted by an indigenous flora may prevent implantation of other bacteria or, conversely, may have little or no effect. This evidence suggests that an E. coli strain may have less difficulty in spreading among people once it has succeeded in adapting itself to the intestinal environment of at least one individual.
Freter
DNA and Pathogenic Potential
627
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en
/Mice Monoassociated with E. Coli XI666 and Conventionalized on Day 0 (3)
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Possible Effects of Foreign DNA on Pathogenic Potential and Intestinal Proliferation of Escherichia coli Rolf Freter
From the Department of Microbiology, The University of Michigan Medical School, Ann Arbor, Michigan
quired for pathogenicity into the recipient bacterium. A relevant example that illustrates the chain of events necessary to produce pathogenicity is the case of enteropathogenic E. coli strains where distinct virulence factors are necessary in order to cause diarrheal disease. One such factor, an enterotoxin, is the agent which induces the diarrhea. However, when a strain of E. coli K 12 carried the gene coding [or this toxin, it was still unable to produce diarrheal disease in the gut of animals [2]. Obviously, this strain lacked other equally important virulence factors that would have enabled it to survive in the intestine. It is important to note that E. coli strains used in recombinant D~A research are all derivatives of this Kl2 strain. Descri ptions of virulence mechanisms do not adequately emphasize the fact that the myriad of metabolic functions and structural features every bacterial cell must have in order to survive in nature are also required for the multiplication of a pathogen in the human body. The term "virulence [actor" is usually applied only to those characteristics that distinguish pathogenic from nonpathogenic bacteria. It is obvious, however, that the characteristics which a bacterium must possess in order to cause disease are by no means restricted to only those virulence factors. This is a most important consideration in the evaluation of the enfeebled EK2 and EK:i strains of E. coli which are to be used in most recombinant DNA research. Enfeebled EK2 and EK3 strains have been deliberately deprived of nutritional and structural characteristics that are important for microbial survival in nature as well as in human or animal hosts. Therefore, it appears that if an inadvertent introduction of genes coding for only one-or even for a few-virulence factors were to occur by recombinant DNA methods, it would not be sufficient to restore the entire complement of trai ts necessary for E. coli to develop a hi therto
Creation of E. coli Strains with Novel Pathogenic and Epidemiologic Potential
It is recognized that a given bacterium must possess a large number of attributes before it can colonize the human intestinal tract and cause disease. For example, a pathogen must be able to survive in the environment, attach to and multiply on the body surface, spread within the body, resist the numerous defense mechanisms of mammalian tissue, and produce a toxin or interfere with the host's physiology in some other way. If only one of these links is missing, the chain will be broken. The bacterium's progress will stop, and it will not be able to cause disease. Therefore, if a relatively small amount of DNA is inserted into a deliberately "enfeebled" microorganism, it is is highly improbable that this procedure will introduce every single gene reThis research was supported by grant no. Al 07328 and contract no. NOI-AI-62518 from the National Institute of Allergy and Infectious Diseases. Please address requests {or reprints to Dr. Rolf Freter, The University of Michigan Medical School, 6643 Medical Science Building II, Ann Arbor, Michigan 48109.
624
Downloaded from http://jid.oxfordjournals.org/ at East Carolina University on July 13, 2015
Serious questions have been raised regarding the possible hazards inherent in recombinant DNA research. Among the various speculative dangers mentioned have been the following. (1) Are new, highly pathogenic bacteria likely to be created by this new technology and (2) could a microorganism, such as Escherichia coli, carrying foreign DNA become implanted in the human gut and eventually transfer this DNA to humans? These questions have been dealt with in some detail in another publication [I] which was made available to participants in this workshop in the form of a preprint. For this reason I will simply summarize some of the arguments pertaining to the first question and emphasize a few new developments which are relevant to the second problem.
DNA and Pathogenic Potential
Problems of Controlling Intestinal Flora
Control of the bacterial flora inhabiting the large intestine of humans is not possible in any practical or useful way. One reason is that techniques for the cultivation and identification of many of these organisms have only been recently developed. Moreover, several investigators [3] have shown that there are still microorganisms, although identifiable by electron microscopy, which cannot be grown in vitro; hence, one cannot determine their functions and characteristics. Another major difficulty is the delineation of the biochemical and physical parameters affecting the environment in the intestine and especially of the substrates available for bacterial growth. Almost nothing is known about this subject. Several authors have speculated that intestinal bacteria, such as E. coli, live a'Teast or famine" existence caused by the periodic food uptake of their hosts. Their habitat, suddenly swamped with nutrients, is depleted soon after a meal has been digested and absorbed. It has, however, been observed for some years that enterobacteria, including E. coli, may actually reach higher populations in a stressed or starved host [1, 5]. One wonders, therefore, whether and to what extent intestinal E. coli and other bacteria actually depend on diet-derived substrates or on a more or less steady flow of host-derived sub-
stances such as effete epithelial cells, secreted mucus, enzymes, etc. Because of the antagonistic action of other indigenous bacteria, Enterobacteriaceae such as E. coli, as well as the classical pathogens in this family, are kept at low population densities in the normal large intestine. Much experimental work has been done to determine the mechanisms by which the intestinal flora control pathogens, E. coli, or other bacteria. Those mechanisms which have been identified include the following: Eh (oxidation-reduction potential), pH, numerous inhibitors (such as fatty acids, bile salts, hydrogen sulfide), competition for substrates or adhesion sites, and local immunity. However, very little is known concerning in vivo function and interaction of these mechanisms. There are three apparent reasons for this lack of knowledge. First, bacterial interactions are quite specific for a given environment. Since we cannot precisely define the physical and chemical characteristics of the microenvironment at any particular site of the intestine, we cannot construct an in vitro experiment that matches the specific conditions affecting the bacterial flora. Second, there are redundancies among the control mechanisms; if one fails another mechanism takes over. Although an experimenter can inactivate a particular mechanism, the outcome of the experiment remains unchanged because another mechanism will simply come into play. This situation makes the isolation of a given mechanism very difficult. Finally, there is the problem of interactions among the control mechanisms. For instance, local immunity can be shown to be operative; however, other bacterial or host defense mechanisms, acting synergistically with local immunity, are likely to be required before that immunity can have a significant effect [fi]. Again, this makes the study of anyone of the whole system of control mechanisms quite difficult. Problems of Implantation
One must conclude, in view of the above discussion, that nothing definite is known about the attributes that a bacterium must possess in order to be easily implanted into the intestinal tract. Consequently, there is no generally accepted test procedure by which one could determine or quan-
Downloaded from http://jid.oxfordjournals.org/ at East Carolina University on July 13, 2015
unknown pathogenic potential. (This is especially true with respect to the enfeebled E. coli strains.) The above considerations do not rule out the possibility that the introduction of a limited amount of genetic material into E. coli could augment those disease-causing properties already present in the bacteria. However, diarrhea and other diseases caused by E. coli do not give rise to widespread epidemics in countries with adequate sanitation procedures. Only the respiratory route of transmission of infectious agents would be difficult to control in countries like the United States. Fortunately, however, strains of E. coli that are pathogenic for humans do not become airborne. Therefore, it is exceedingly unlikely that life-threatening epidemics might be caused by E. coli strains that carry small segments of foreign DNA.
625
626
Physiological State of Bacteria and Implantation
We have recently tested this hypothesis by attempting to implant a wild-type E. coli strain (C25) and a K 12 strain (X-1666) into mice in two different ways: (1) by introducing the bacteria directly into the stomach of conventional mice in a buffered vehicle and (2) by first monoassociating germ-free mice with the E. coli strain
and then conventionalizing these animals by caging them with conventional mice. In the latter design, the E. coli reach very high population levels (- 5 X IOn per mouse) in the germ-free animals. After caging with conventional mice, the monoassociated animals become implanted with a succession of indigcnous intestinal bacteria until about two weeks later when their gut flora attains the composition found in conventional mice [3]. We speculate that during these two weeks of conventionalization the E. coli strain adapts itself to the constantly changing environment in the mouse gut and therefore has a better chance to remain implanted along with the newly devcloping gut flora. The data obtained in representative experiments are shown in figures 1 and 2. Both the wild-type strain C25 and the Kl2 strain X1666 were eliminated [rom the gut when fed to conventional mice, and elimination was more ra pid wi th the K 12 strai n. This finding corresponds with results reported by others in human feeding experiments. E. coli strain C25 established itself in about 5C;~-10~~ of the 144 mice tested to date and persisted in very low numbers in their fecal Hora for at least several months. Strain X-1666 persisted beyond seven days in only one of a total of 144 conventional mice fed this bacterium. In this one animal, bacteria still persisted in the stool at 90 days after feeding. The results obtained wi th the second method were, however, strikingly different. Neither of the E. coli strains was eliminated from the gut of the conventionalized animals; rather, the strains established themselves in the indigenous flora for the length of the experiments (up to 85 days) at population levels normally assumed by E. coli in the conventional mouse. Data from representative experiments are also shown in figures 1 and 2. Differences in the population levels of E. coli C25 and X-1666 shown in these figures are not significant. In replicate experiments both strains populated conventionalized mice at levels within the range shown in the figures. In subsequent control experiments, strain X1666 was reisolated from mice in which it had become established and then fed to conventional mice. Under these circumstances, the reisolated strain was quickly eliminated from the animals. Hence, establishmen t of X- I666 in the conven-
Downloaded from http://jid.oxfordjournals.org/ at East Carolina University on July 13, 2015
titate the ability of E. coli or any other microorganisms to establish themselves in the intestine. The recent literature does report a number of attempts to implant E. coli K12 into the intestines of volunteers by feeding them large doses of these microorganisms [7, 8]. Such data regularly show that it is almost impossible to implant E. coli K12 in this manner. The inoculum usually survives for only a few days in the host's intestine. To the casual observer, this kind of evidence may appear to be pertinent and conclusive. This, unfortunately, is not the case. Such experiments tell us little about the ability of bacteria to grow in the human intestine. It has been known for some time that the feeding of bacteria, especially those grown under the usual laboratory conditions, rarely results in implantation because the normal intestinal flora is antagonistic to the growth of invaders [9-11]. Published evidence suggests that feeding experiments do not usually result in implantation simply because the bacterial inoculum is not in the proper physiologic state. Improved implantation would result if the inoculum had been grown under conditions (of substrate, pH, Eh, etc.) resembling those found in the intestine. Two laboratories [12, 13] have shown that exclusion, by bacterial antagonism, of invading microorganisms from an established flora may fail to occur when the invading bacteria come from an environment similar to the one which they are about to enter. Depending, therefore, on the physiologic state of the invading bacteria, antagonism exerted by an indigenous flora may prevent implantation of other bacteria or, conversely, may have little or no effect. This evidence suggests that an E. coli strain may have less difficulty in spreading among people once it has succeeded in adapting itself to the intestinal environment of at least one individual.
Freter
DNA and Pathogenic Potential
627
Q)
en
/Mice Monoassociated with E. Coli XI666 and Conventionalized on Day 0 (3)
;:g ~
Q) C Q.C
Q)
W::E6 W
WU
>
