Prohiotics, Other Nutritional Factors, jwd Intestinal Microflora, edited by Lars A. Hanson and Robert H. Yolken, Nestle Nutrition Workshop Series, Vol. 42, Nestec Ltd.. Vevey/Lippineott-Raven Publishers, Philadelphia © 1999.

Microbial of the Intestinal Microflora: Influence of Interactions with the Host

Dirk van der Waaij

Retired Professor of Medical Microbiology, University of Groningen, Groningen, The Netherlands

As a medical doctor specializing in medical microbiology, my scientific interest is the patient-related importance of the gastrointestinal microflora. This concerns the defense function of the microflora, the conditions that lead to distortion of this "win-win" arrangement, the medical consequences of such distortions, and a sim- plified classification of that can cause infectious disease. The current terminology used to identify the various fractions and stages of the intestinal microflora has been reviewed by Rusch (1). The normal or indigenous microflora of humans is known to consist of a rather static resident (autochthonous) part and a transient part. Under normal circumstances, the microflora has a dynami- cally changing composition. The turnover of the transient part depends on the compo- sition of the autochthonous microflora, the gut of the host, and the influx of new microorganisms. This influx is determined by the degree of contamination of ingested food and beverages, which relates to the hygienic condition of the envi- ronment. As will be outlined later, the net result of a contamination with foreign is determined by the dose (the number of microbes ingested) and the compo- sition of the endogenous ecosystem. Another point of practical importance concerns . Since antimicrobial treatment is rapidly losing ground because of the development of multiple resistance, including resistance to new antibiotics brought on the market, there is a need for new antimicrobial strategies. The problem of multiple resistance of microorganisms is worldwide (2,3). An international study group of biomedical scientists has under- taken to try to find a solution to this problem. This group, named the International Study Group on New Antimicrobial Strategies (ISGNAS) (4), has indicated that an important key to a new strategy could be the modulation of gastrointestinal tract microflora and its many complex interactions with the host defense system. For successful modulation of gut microflora, however, more research is needed on these interactions. 2 OF INTESTINAL MICROFLORA

Insight into the development of miraculously stable bacterial such as our intestinal microflora and into the role of the autochthonous microflora in the defense system may be gained from information on the development of these systems during evolution. This information may also help in understanding the development of resistance to antibiotics by microorganisms each time a new becomes available.

GROUPING OF BACTERIA ACCORDING TO THEIR DEGREE OF PATHOGENICITY For practical (medical) purposes, all existing bacteria can be placed in one of three dominant groups, each group of different (from 1 -3 increasing) pathogenicity. The composition of these groups may differ slightly between man and animals and among animal species. In particular, the first two groups may differ among animal species.

Nonpathogenic By far the largest group is not pathogenic at all, or only in extremely compromised individuals. Bacteria belonging to this group can be found in the digestive tract and on the skin of all healthy human subjects, as well as on the skin of animals and . These bacteria live in peaceful coexistence with the defense (immune) system of the host. They may permanently induce or maintain suppression of the so-called gut-associated lymphoid tissue (GALT) and in this way avoid inducing a chronic inflammatory response (see below).

Potentially Pathogenic A second and smaller group is potentially pathogenic. In healthy subjects, these bacteria are kept well under control by the immune system of the digestive tract, the GALT, as well as some nonspecific defense factors. We all experience daily the fact that this control occurs without causing any signs or symptoms of disease. Representatives of this group, which are also called opportunistic, can be found in practically every healthy human subject, in every animal, and on many plants. In compromised subjects, however, they may cause infections.

Pathogenic The third group, a small one, comprises the pathogenic . Following contamination in sufficient numbers, these microorganisms can cause disease in noncompromised healthy subjects. In unvaccinated individuals, these bacteria are not readily controlled by the immune system. In contrast to representatives of groups 1 and 2, bacteria of group 3 are not normally cleared without signs of disease (an inflammatory response) and are not found in healthy subjects. If they are present, the host organism is called a carrier and is regarded as infectious. MICROBIAL ECOLOGY OF INTESTINAL MICROFLORA 3

DEVELOPMENT OF THE MICROFLORA AND A DEFENSE SYSTEM IN EVOLUTION First Ecosystem on Earth: Ancestor of Our Microflora The earliest life on earth may have consisted of a photosynthesizing species of auxotrophic bacteria. During the subsequent millions of years, more and more of these species will have developed. With the appearance of these first bacterial spe- cies, our planet may have provided a spectacular sight from space, with purple-, red-, and later green-colored islands. Land may have been covered by a dense bacterial population, but the sea may also have had a rich bacterial life (5). When one realizes that many bacterial species may divide for reproduction every 20 minutes, one may question why our planet has not been entirely taken over by bacteria. An important reason for restricted growth has been, and still is, for nutrients. Even in niches with an adequate nutrient supply, bacteria have to compete with each other for practically every morsel. An important source of nutrients for the great majority of more recently developed nonauxotrophic bacteria is other organisms, both living and dead. Bacterium have only a restricted enzymatic capacity for digestion, how- ever, regardless of the species, and they cannot digest the larger and more complex molecules by themselves. Thus, bacteria must cooperate with other bacterial species to digest the more complex molecules. Only in concert with other species, for exam- ple, in ecosystems, can they form the enzymatic pattern required for the breakdown and digestion of dead organisms and other complex nutrients (the process of decom- position).

Nucleated Cells Developed from Bacterial Ecosystems A major change occurred in the first eons when certain bacteria developed that produced oxygen. The increasing presence of oxygen in the atmosphere made a change from anaerobic to aerobic metabolism possible. The emergence of aerobes changed life on earth completely, as it made possible the development of nucleated cells, presumably from bacterial aggregates (6). Later on, the development of plants and animals could follow from these cells.

Maintenance of Bacterial Ecosystems by Antibiosis and Development of Resistance Most bacterial populations in nature exist in "open systems" with continuous influx of new (foreign) bacteria along many different routes. Therefore, in ecosys- tems, indigenous bacteria have to protect the nutrient sources of their . The numerous bacterial ecosystems that were formed during evolution have devel- oped the means to defend their communities by antibiosis. Antibiosis means the production of substances that are either "static" (growth inhibiting) or "cidal" (toxic and directly killing) to most, and preferably all, incoming bacteria and fungi that are foreign to the ecosystem and do not contribute to it in any way. All permanent 4 M1CR0BIAL ECOLOGY OF INTESTINAL MICROFLORA members of the ecosystem are obviously resistant to these static/cidal substances. Conversely, newly arrived microorganisms that produce antibiotic-like substances that are effective against members of the ecosystem may cause relatively little harm to the system. These usually enter the ecosystem in relatively small numbers and consequently release relatively little antibiotic substance. In addition, their antibiotic may rapidly become inactivated. This inactivation occurs either by chemical binding to organic material in the system or, after an adaptation period, by enzymatic inactiva- tion. This is an assumption based on observations in the human gut microflora (7).

Development of a Defense System to Microorganisms Small animals with a short life span were the first that developed from nucleated cells. They could live with a primitive nonspecific defense system, that is, microbial adherence and subsequent phagocytosis and killing. In higher, longer living animals and humans, additional means for protection against bacterial invasion developed. An increasingly complex defense system, known as the immune system (8), was formed stepwise. With the increasing life span of animals, the complexity of the immune system became enhanced, and a "learning system" was needed and devel- oped. This learning system enabled the host to distinguish between bacteria of their own ecosystem and newly ingested foreign (potentially invasive) bacteria. This makes it plausible that host organisms play an important role in maintaining the composition of their microflora (ecosystem).

MICROBIAL ECOSYSTEMS ASSOCIATED WITH HUMANS AND ANIMALS Contamination of all external and internal surfaces with microorganisms in hu- mans and animals begins at birth and results, after several weeks or months, in the establishment of a stable microflora. This microflora is called autochthonous. This point is discussed in greater detail elsewhere in this volume (see chapter by T. Midtvedt, p. 79). Three basic, inherited host- and microflora-related mechanisms have developed in evolution. These will now be discussed in greater detail. They comprise (i) the need for metabolic cooperation between nonauxotrophic microbes, (ii) the role of antibiosis and resistance development (adaptation), and (iii) the development of the specialized immune system that can learn to distinguish between autochthonous and foreign microbes.

Need for Cooperation between Microorganisms and Host During human and animal life, the symbiotic relationship with intestinal microor- ganisms represents a fruitful win-win arrangement for both microbes and the colo- nized host organism. Both members of this symbiotic system, host and microflora, provide something essential for mutual benefit. The following are of particular im- portance with regard to between host and microflora: MICROB1AL ECOLOGY OF INTESTINAL MICROFLORA 5

1. Symbiosis between bacteria and host with its microflora is defined as the method by which a beneficial (win—win) balance is maintained between a wide array of microbial species and the host. The word balance is intentional, as the systems involved may be unstable, nonlinear dynamic systems, that is, systems based on many continuously changing interactions that in concert maintain a balance. 2. The evolutionary conservation of this microbe-host cell symbiosis *, of immense value for good health and extended life of both the microbial symbiont and the host organism. Any of this symbiosis threatens the welfare of micro- bial symbionts and the host. Symbiosis between the autochthonous microflora and the host exists in the gut. The basis of the intestinal ecosystem is the bacteria that can live in the mucus covering epithelial linings, where they form the autochthonous microflora of the host or- ganism. The existence of a stable microflora in the digestive tract is guaranteed by a continuous supply of nutrients of constant composition provided by the living host. Since each bacterium has a limited enzymatic capacity, concerted action with other bacteria is necessary for the digestion of complex molecules such as those presented in the intestinal mucus and saliva. Saliva in the mouth and intestinal mucus form the specific nutritional sources for the bacteria of the autochthonous microflora; the mucus overlying the epithelium forms their home layer. In conclusion, the stability of the gut ecosystem depends on interbacterial cooperation and the availability from the host of a source of nutrients that is constant in both composition and amount.

Modulation of the Endogenous Intestinal Microflora by Daily Oral Intake From the home layer, the autochthonous bacteria are released into the gut contents. This enables them to mix continuously with food debris and its microbial contami- nants ingested by the host; in this way, the intestinal microflora is formed: the net result of mixing of autochthonous and foreign microbes. In the ingested food, autochthonous bacteria will find many substances that differ from those released by the host in the secretions; some may flourish on these, and others may starve. Autochthonous bacteria, which live in large numbers (estimated 1010/g) in the intes- tinal mucus, are at a considerable advantage over the relatively small numbers of microbes ingested in hygienically prepared food and beverages, although the autoch- thonous microflora in the home layer will be modulated by variations in the composi- tion of ingested nutrients. However, under normal circumstances, the modulated autochthonous ecosystem is hostile to ingested bacteria in a degree compatible with its in the gut contents. This hostility is directed at most, if not all, the newly ingested bacteria, although to a different degree in different species.

Antibiosis in the Intestinal Ecosystem From the moment bacteria enter the mouth, they encounter the gastrointestinal ecosystem. As outlined above, in the home layer, autochthonous bacteria will hinder 6 M1CROBIAL ECOLOGY OF INTESTINAL MICROFLORA proliferation of newcomers or kill them by producing toxic substances (e.g., antibiot- ics) or by competing for nutrients. The defense capacity of the autochthonous part of the intestinal microflora is maximal in the home layer. Substances with antibiotic activity are ubiquitous in nature. In medical reports, these are mostly referred to as antibiotics or bactericines. Antibiotics produced by the autochthonous microflora in vivo can be regarded as part of an innate ("primitive;" ready for direct action) defense system. Although they may reach the highest concentrations in the home layer, their concentration in the intestinal contents may still be capable of suppressing newly ingested germs. Such primitive (antibiosis- based) defense systems can be found in different bacterial and fungal ecosystems and, as recently described, in vertebrate cells of several animal species including humans (9,10).

Colonization Resistance of the Digestive Tract A hostile reception for newcomers to the gut ecosystem is provided by an interac- tive defense network formed by cooperation between the host and the host's mi- croflora. This complex interactive system is called the colonization resistance of the digestive tract (11-13). It is nowadays generally accepted that the autochthonous microflora plays a major role in the establishment of the colonization resistance. However, it still is not yet clear which bacteria (or groups of bacteria) in the microflora and which host factors are involved. Only some of the biochemical factors and mechanisms that contribute to colonization resistance are known at present. Factors that may play a role include competition for nutrients, bactericines, short-chain fatty acids, and extracellular en- zymes.

Adaptation of Microorganisms to Noxious Environmental Factors such as Antimicrobial Drugs Although bacteria strongly outnumber all other organisms on Earth, their first appearance and their persistence in numerous different species have only been possi- ble because of their capacity to adapt to ever changing environmental conditions. Adaptation occurs by mutation and, perhaps of equal importance, by selection. Selec- tion is determined by the composition of the direct environment. When the environ- ment becomes harmful, the only bacteria that can survive are those that have (by coincidence) the necessary survival tools to cope with the altered environment. In addition, after landing in foreign ecosystems, newcomers must have the right en- zymes, or develop them in time, to make use of the nutrients available in the new niche. Otherwise, they will starve and die. In addition, newcomers must be resistant to locally produced antibiotics and should not produce an antibiotic that affects nutritionally important members of the ecosystem. These general principles apply also to the intestinal ecosystem. M1CROBIAL ECOLOGY OF INTESTINAL MICROFLORA 7

Host Factors Involved in Colonization Resistance The host factors contributing to colonization resistance are the following: 1. The innate defense mechanisms such as mechnical propulsive movement (swal- lowing, peristaltic movement), secretion of saliva and mucus (feeder layer), epi- thelial cell desquamation, nonspecific blocking factors for bacterial adherence, and presumably antibiotics. 2. Adaptive mechanisms, such as those of the GALT (14), that produce or secrete immunoglobulin A (IgA), as well as the systemic immune system. As mentioned earlier, the GALT plays a role in learning which bacteria are autochthonous in the gut contents and which are not. Several of these innate and adaptive host factors are modulated by the autochthonous microflora and vice versa (15).

SUMMARY OF THE NETWORK OF INTERACTIONS MAINTAINING THE STABILITY OF THE INTESTINAL MICROFLORA Our present knowledge makes the following likely: 1. The human ecosystem is composed mainly of bacteria that must cooperate closely with one another, as well as with their host organism. Only in a healthy host can the home layer, that is, the digestive tract microflora, be adequately nourished and remain constant in composition. Diseased individuals often secrete qualita- tively and quantitatively altered saliva and mucus. 2. Suppression of ingested infectious agents by colonization resistance in a host organism depends on the quality of the home layer, as well as on the composition and stability of the intestinal ecosystem. 3. Although we are beginning to understand and appreciate the existence of this win-win-oriented network between host and microflora, details of the factors involved are still sparse. It is generally accepted, however, that the network is dynamic in composition and function. Although the autochthonous flora in the home layer is stable, its numbers fluctuate in the gut contents, and this may affect the defense capacity (i.e., the colonization resistance). In healthy subjects, the mean suppressive capacity of the colonization resistance and its standard devia- tion are well above the limit required to maintain normal health.

SOME CLINICALLY IMPORTANT DEFENSE-RELATED ASPECTS OF AUTOCHTHONOUS MICROFLORA The phases following colonization of the gut are adhesion to the mucosa, transloca- tion, and either adhesion to phagocytic host cells and subsequent ingestion followed by killing or in case of defense failure the opposite, that is, avoidance of adhesion and multiplication in the interstitium. Central in these events is success or failure of adhesion. Therefore, blocking or enhancement of adhesion is of great clinical relevance. M1CR0BIAL ECOLOGY OF INTESTINAL M1CROFLORA

Bacterial Attachment to Host Cells by Lectins Carbohydrate molecules of varying complexity are ubiquitous on the surfaces of intestinal epithelial cells and are named collectively glycocalyx (16). So-called ad- hesins enable the bacteria to react physically and chemically with the glycocalyx substrate of the host cells. However, their binding may also involve other bacteria and form bacterial aggregates in the colonic microflora. Aggregate formation may be beneficial for metabolic reasons and be part of the win-win situation. Glycoproteins (lectins), as well as extracellular polysaccharides (d-galactose, d- mannose, and iV-acetylneuraminic acid), are important receptors of host cells but also of bacteria. This may be an example of an early (primitive) mechanism for enhancement of binding that precedes phagocytosis. Lectins are ubiquitous and show specific and reversible carbohydrate-binding activity. A specific lectin is commonly effective at very low concentrations, in the millimolar range or lower. Although lectins are similar to antibodies in their ability to agglutinate cells, they differ in that lectins are part of the innate (nonspecific) immune system; their structures are diverse, and their specificity is restricted to carbohydrates (17,18). If they become available for blocking of host cell receptors, bacteria (probiotics) or bacterial lectins should mimic opportunistic (potentially pathogenic) bacterial species. This could be a beneficial and useful property of probiotics. The concept emerging from studies of bacterial adherence to mucosal surfaces is that in most cases carbohydrates on the mucosal cell function as receptors for bacterial lectins and thus for attachment (19-21). The host organism welcomes the autochthonous microflora by providing anchorage sites.

Translocation of Intestinal Microorganisms The close association between bacteria and the intestinal epithelial surface in- volves colonization of the mucosal epithelial cells (22) and, from there, translocation from time to time (23). Translocation can be defined as the active or passive penetra- tion of microorganisms through the epithelial lining into the lamina propria. Nor- mally, it involves small numbers of endogenous (autochthonous and ingested) intes- tinal bacteria. Under normal circumstances, these are apparently efficiently cleared in the lamina propria by the host defense system. This rapid clearance may be due both to the small numbers of endogenous bacteria involved and to induction of specific hyporesponsiveness of the adaptive part of the immune system in Peyer's patches.

Interindividual Differences in Composition of the Autochthonous Microflora The autochthonous microflora in the home layer differs not only among different animal species, including humans, but also among subjects of the same species (13). These differences in composition of the bacterial flora exist among normal, healthy M1CROB1AL ECOLOGY OF INTESTINAL MICROFLORA 9 individuals and consequently also among sick individuals. The latter may have in addition a changed mucus layer and thus a changed home layer. In diseased individu- als, the condition of the microflora may have consequences for the defensive ability (the colonization resistance) of their ecosystems. It is of practical importance there- fore to have a means of measuring colonization resistance. This will be discussed further below.

Practical Implications and Consequences for Treatment In patient care, medical interest in bacteria and their antibiotic-sensitivity patterns is normally confined to bacteria isolated from infection sites. This has led to the abundant and uncontrolled use of antibiotics and hence to multiple-antibiotic resis- tance (24). The practical implications of this for the bacterial ecosystems are only slowly being recognized. Most clinicians are still unaware that ecosystems are pres- ent, active, and essential components in the defense system of their patients. The discovery of antibiotics greatly reduced their interest in the microbial colonization patterns of their patients and the consequences for treatment.

CONSEQUENCES OF DISTURBING THE INTESTINAL SYMBIOSIS Disturbance of Intestinal Symbiosis A basic tenet of infectious disease pathogenesis involves the balance between the virulence of contaminating microbes and of the defense capacity of the invaded host. There are good reasons for stating that clinically manifested infections in hospital inpatients usually develop as a result of a transitory breakdown of the host defense mechanisms. Such infections are caused by opportunistic microorganisms (group 2) rather than by pathogenic organisms (group 3), which possess specific disease- producing factors for healthy subjects. The symbiosis between autochthonous bacteria can be rapidly and seriously al- tered by many factors, for example: • Antimicrobial treatment; • Primary and secondary immune deficiencies (e.g., immunological dysfunction as- sociated with underlying hematoproliferative diseases); • Surgery that breaks down natural anatomic barriers or leaves behind foreign bodies in the form of nonabsorbable sutures; • Malnutrition; • Physical or emotional stresses of varying kinds. Disruption of the symbiotic state (involving the host and the host's autochthonous microflora) by any of these factors rapidly leads to a "lose-lose" situation for both the microflora and the host, and therewith to loss of colonization resistance. This means that the host becomes more susceptible to mucosal colonization by opportunis- tic and more readily translocating microorganisms. These may then occupy the niche 10 MICROBIAL ECOLOGY OF INTESTINAL MICROFLORA of the affected autochthonous microflora. In addition, weakened host defenses allow opportunistic members of the normal microflora to penetrate beyond the lamina propria and cause systemic infection.

Adaptation to Microflora-disrupting Factors The adaptive processes of most microbial species have assured their survival despite a plethora of naturally occurring antimicrobial products over eons of time. Against this background it is not surprising that following the discovery of penicillin, the widespread clinical abuse of antimicrobial agents has resulted in drug resistance in ever greater numbers of bacterial species and strains.

MEASURING/MONITORING SIGNIFICANT CHANGES IN THE COMPOSITION OF THE INTESTINAL MICROFLORA As the intestinal microflora appears to be of key importance in the defense system of the host, measuring fluctuations in its composition, and thereby the capacity for colonization resistance, becomes clinically important. If we could maintain the microflora and its stimulating effect on the host defense within normal limits during the course of disease and its treatment, we might be able to prevent infections. A simple method for measuring the microflora is therefore required. To indirectly monitor the composition of the feces, Midtvedt (11) developed sev- eral microflora-associated characteristics (MACs). My coworkers have also tried to develop a technique that could be used for measuring and thence for monitoring colonization resistance (25). Another approach for studying colonization resistance concerns analysis of bacteria in suspensions. This involves fecal suspensions ana- lyzed both by flow cytometry (26) (which appears to be a fast and economic method) and by computer processing of microscopic images of bacteria (27) (a method that provides more detailed information). These methods have been found to be both reproducible and reliable. The latter requires one technician-day for the analysis of three to four fecal samples for the presence of specific antibacterial antibodies of the three major isotypes (IgM, IgG, and IgA); the former permits the same kind of analysis on 10 to 12 samples per day. In both cases, fecal bacteria can be labeled with either fluorescent antibodies (as in the case of determination of the binding isotypes) or with 16S ribosomal RNA (rRNA)-targeted probes to analyze the compo- sition of the microflora (28,29).

Computer Processing of Microscopic Images of Fecal Bacteria Measuring the intestinal microflora can be achieved by preparing a suspension of fecal samples from healthy subjects or patients. The washed suspensions are placed on a microscope slide and examined by phase contrast microscopy and a digital image-processing system (IPS). The IPS measures in real time the exact morphological dimensions of at least 700 bacteria per fecal sample in several micro- M1CROB1AL ECOLOGY OF INTESTINAL M1CROFLORA II scopic views per slide to analyze statistically (and thus characterize) the microbial populations in the fecal samples. The bacteria involved in such analysis represent the highly concentrated (predominantly anaerobic) bacterial population of the gut. The IPS pattern of the bacteria in a fecal sample may change significantly when the anaerobic flora is seriously affected by an antimicrobial drug. The numbers of the various different images that characterize the bacterial population change in such cases. To measure these changes, it is necessary to characterize the situation numerically. The characteristics of a fecal population appear to be largely determined by the degree of chaos (entropy) of two "morphology factors."—factor 1, which correlates mainly with the length of the objects; and factor 2, which correlates mainly with their width (30). In a recent study, the entropy of the morphology of all images measured in each sample was used for comparison of the bacterial population in subsequent samples in each patient (31).

Criteria Used to Determine the Degree of Disturbance of the Intestinal Microflora Because a particular group of patients may differ from other patients and from healthy subjects, each group should be regarded as a separate population. This im- plies that what is found in a certain group of patients does not necessarily apply to all patients who are treated with the same antibiotic. Therefore, patients should act as their own control. Likewise, normal values, and their 99% confidence limits (99% CL), should be calculated from the mean log concentration of the bacteria in the fecal microflora collected in the first 2 or 3 days after admission (control samples). Criteria for serious microflora disruption are as follows:

1. The percentage of samples collected during and after treatment that deviate above or below the 99% CL level of the mean value found in the control samples. 2. Significant changes in the entropy of the IPS pattern of the fecal flora during or shortly after treatment, in comparison with pretreatment values and those in sam- ples collected later than 1 week after treatment. 3. yS-aspartylpeptidase activity in the feces can be measured as an MAC like several other biochemical variables (see "Microbial Functional Activities," by Mid- tvedt).

Comparison of Variables Used for Determining Microflora Disruption by Antibiotic or Other Home Layer-modifying Treatment To illustrate these techniques, the results obtained in 10 patients are shown in Table 1 and the entropy of their IPS pattern in Figure 1. In this particular study, patients were treated soon after admission with a new oral antibiotic for 10 days. Some patients, however, had been treated beforehand at home with a different antibi- otic. 12 MICROBIAL ECOLOGY OF INTESTINAL MICROFLORA

TABLE 1. Correlation image-processing system (IPS) pattern and microflora-associated characteristics (MAC) IPS entropya /3-Aspartylpeptidase6 Patient changed during/after decreased during/after 1 1/0 -/- 2 8/0 +/- 3 8/0 +/- 4 9/0 nd 5 7/0 +/ + 6 9/0 +/nd 7 3/nd +/ + 8 9/1 +/+c 9 2/0 -/- 10 6/0 + /- +, exceeds the 99% confidence limit; -, less than the 99% confidence limit; not done (in most cases, because too little material was available so that priority was given to other tests). a IPS entropy in day 0 and day 1 samples: mean 4.23; 99% confidence limit ±0.08. Presented in the body of the table is the number of days at which the IPS entropy was <4.23-0.08 = 4.15. b Only /3-aspartylpeptidase values <30% of initial value are included. c No match between MAC and IPS.

Figure 1 shows the course of entropy of the microscopic images in the fecal suspensions of three patients: numbers 2, 9, and 10. These three patients were se- lected because the entropy changes of their microflora nicely illustrate what can be seen during treatment with an antibiotic that reaches the gut contents in suppressive amounts. In patient 10, a dramatic decrease of the entropy was followed by a rapid repair; in patient 2, the lower confidence limit was reached only on day 6, while in the third patient (no. 9) only a small degree of suppression and thus decrease in entropy occurred. Note that in all three patients the entropy (degree of diversity) returned to near normal values during treatment! Some adaptive mechanism was already functioning from the onset. This could be explained by the fact that most of the patients had been treated with another antibiotic before admission to the hospital. Table 1 shows evidence that none of the patients responded identically. Further- more, the two indices of microflora disruption by antibiotic treatment were shown to be quite well correlated: /3-aspartylpeptidase activity (MAC) values correlated with IPS entropy in eight of the nine patients studied by this MAC index.

Tentative Conclusions On the basis of the results reported, the following tentative conclusions can be drawn: 1. The antibiotic may have been inactivated by chemical binding and/or enzymes in the intestinal contents of many (if not most) of the participating patients. Antibiotic inactivation has been found in in vitro studies (7). MICROB1AL ECOLOGY OF INTESTINAL MICRO FLORA 13

ENTROPY OF MICROMORPHOLOGY faecal microflora

4.50 99% confidence limit

4.20 A T \ & j —'— pat.2 \ \ / \ \ / \ -•K / < h 3.90 \ \ / \ a ' A \ / \ ' ^A ' ' // \\ O \ - - A- - pat.9 V 1A \tk \ \ /A'V \ C CD 1/ \ \ // 3.60 \ 1 \ / ~«~ pat. 10 \\ / \ / \ 99% i • • • \' confidence limit \ / V 3.30 \ / \! 3.00 1 23456789 10 time in days FIG. 1. Entropy of bacteria in fecal suspensions determined by computer processing of micro- scopic images. The results of sampling are shown for three representative patients with lower respiratory tract infection treated with the same oral antibiotic.

2. Regarding the tests employed to study the degree of flora disruption by the antibi- otic investigated, the IPS entropy score was not, in this small study, significantly different from the /3-aspartylpeptidase activity. The IPS method does, however, provide more detailed information, and recent development of the method sug- gests that rRNA probe labeling provides a much more detailed result, although at higher cost. For routine use (daily monitoring) of patients, the micromorphometric determination of the entropy of microscopic images with flow cytometry appears the most practical method and thus is probably optimal under these circumstances.

ACKNOWLEDGMENT I would like to acknowledge the assistance of Dr. H. Bootsma and the skillful sample analysis in the patient study by Mrs. Jaqueline de Boer and Dr G.W. Welling. 14 MICROB1AL ECOLOGY OF INTESTINAL MICROFLORA

REFERENCES

1. Rusch VC. The concept of symbiosis: a survey of terminology used in description of associations of dissimilarly named organisms. Microecol Ther 1989; 19: 33-59. 2. Zajicek G. Antibiotic resistance and the intestinal flora. Cancer J 1996; 9: 214-5. 3. Zajicek G. Microbial resistance to antibiotics and the wisdom of the body. Cancer J 1994; 7:168-9. 4. Araneo BA, Cebra JJ, Beuth J, el al. International Study Group on New Antimicrobial Strategies writing committee. Z Bakteriol Hyg 1995; 283: 431-65. 5. Knoll AH. The early evolution of eukaryotes: a geological perspective. Science 1992; 256: 622-7. 6. Margulis L. Serial endosymbiosis theory: symbiosis in cell evolution; microbial communities in the archean and protozoic forms. New York: WH Freeman and Co, 1993: 1-14. 7. Jansen G, Weissing F, De Vries-Hospers HG, Tonk R, van der Waaij D. The non-enzymatic inactiva- tion of 13 beta-lactam antibiotics in human feces. Infection 1992; 20:53-7. 8. Sima P, Vetvicka V, eds. Evolution of immune reactions. Boca Raton, FL: CRC Press, 1990: 1-36. 9. Boman HG. Peptide antibiotics and their role in innate immunity. Annu Rev Immunol 1995; 13: 61-92. 10. Zasloff M. Antimicriobial molecules from frogs, sharks and man. In: Hoffmann JA, Janeway CA, Natori S, eds. Phylogenetic perspectives in immunity: the host defense. Austin, TX: Landes Co, 1994:31-41. 11. Midtvedt T. Microflora-associated characteristics (MACs) and germfree animal characteristics (GACs) in man and animal. Microecol Ther 1985; 15: 295-302. 12. Van der Waaij D. The ecology of the human intestine and its consequences for overgrowth by such as Clostridium difficile. Annu Rev Microbiol 1989; 43: 69-87. 13. Van der Waaij D, van der Waaij BD. The colonization resistance of the digestive tract in different animal species and in man: a comparative study. Epidemiol Infect 1990; 105: 237-43. 14. Kagnoff MF. Immunology of the digestive system. In: Johnson LR, ed. Physiology of the digestive tract, 2nd ed. New York: Raven Press, 1987: 1699-1714. 15. Sneller MC, Strober W. M cells and host defense. J Infect Dis 1986; 154: 737-41. 16. Rhodes JM. Colonic mucus and mucosal glycoproteins: the key to colitis and cancer? Gut 1989; 30: 1660-6. 17. Beuth J, Ko HL, Uhlenbruck G, Pulverer G. Lectin-mediated bacterial adhesion to human tissue. Eur J Clin Microbiol 1987; 6: 591-3. 18. Beuth J, Ko HL, Roszkowski W, Roszkowski K, Ohshima Y. Lectins: mediators of adhesion for bacteria in infectious diseases and tumor cells in metastasis. Z Bakteriol 1990; 274: 350-8. 19. Sharon N. Bacterial lectins, cell recognition and infectious disease. FEBS Lett 1987; 217: 145-57. 20. Beuth J, Ko HL, Schroten H, Solter J, Uhlenbruck G, Pulverer G. Lectin-mediated adhesion of Streptococcus pneumoniae and its specific inhibition in vitro and in vivo. Z Bakteriol Hyg A 1987; 265: 160-8. 21. Steuer MK, Beuth J, Pulverer G, Steuer M. Experimental and clinical studies on microbial lectin blocking: new therapeutic aspects. Z Bakteriol 1994; 283 (suppl 25): 112-7. 22. Freter R. Prospects for preventing the association of harmful bacteria with host mucosal surfaces. In: Beachy EH, ed. Bacterial adherence. London: Chapman and Hall, 1980: 439-58. 23. Wells CL, Maddaus MA, Simmons RL. Proposed mechanisms for the translocation of intestinal bacteria. Rev Infect Dis 1988; 10: 958-79. 24. Tomas A. Multiple antibiotic-resistant pathogenic bacteria. N Engl J Med 1994; 330: 1247-51. 25. Welling GW, Meijer-Severs GJ, Helmus G, et al. The effect of ceftriaxone on anaerobic bacterial flora and the bacterial enzymatic activity in the intestinal tract. Infection 1991; 19: 313-6. 26. Van der Waaij LA, Mesander G, Limburg PC, van der Waaij D. Direct flow cytometry of anaerobic bacteria in human feces. Cytometry 1994; 16: 270-9. 27. Meijer BC, Kootstra GJ, Wilkinson MHF. A theoretical and practical investigation into the characteri- zation of bacterial species by image analysis. Binary 1990; 2: 21-31. 28. Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA. Combination of 16S rRNA- targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 1990; 56: 1919-25. 29. Wilkinson MHF, Fansen GJ, van der Waaij D. Computer processing of microscopic images of bacteria: morphometry and fluorimetry. Trends Microbiol 1994; 2: 484-89. 30. Meijer BC, Kootstra GJ, Wilkinson MHF. Morphometrical parameters of gut microflora in human volunteers. Epidemiol Infect 1991; 107: 383-91. M1CROBIAL ECOLOGY OF INTESTINAL M1CR0FL0RA 15

31. MeijerBC, Kootstra GJ. Geertsma DG, Wilkinson MHF. Effects of ceftriaxone on fecal flora: analysis by micromorphometry. Epidemiol Infect 1991; 106: 513-21.

DISCUSSION Dr. Yolken: I'd like to congratulate you on being brave enough to want to computerize stool samples. I would like to ask specifically whether your system can distinguish aerobic from anaerobic bacteria, Gram-positive from Gram-negative. Can you really distinguish the different types of bacteria that may be present in the stool? Dr. van der Waaij: The computer cannot distinguish between Gram-positive and Gram- negative bacteria because it cannot measure the edges as accurately as phase contrast micros- copy. However, with the new DNA probe techniques, lactobacilli and bifidobacteria can be distinguished, although Gram-positive organisms are more difficult than Gram-negative be- cause the probes do not enter the Gram-positive so easily. Regarding aerobes versus anaerobes, we can distinguish Enterobacteriaceae from bacteroides, but we cannot yet identify oxygen usage. Thus, if you can identify a family as being predominantly aerobic or anaerobic, then the answer is yes: they can usually be distinguished, though it is not yet possible to distinguish anaerobic streptococci from aerobic streptococci. Dr. Black: You talked about opportunistic infection, and you also talked about truly patho- genic infection, where it does not matter what you've got in your gut, you're going to be infected. Can you tell us how often opportunistic infection occurs—that is, the type you demonstrated with your irradiated mice—as compared to truly pathogenic infection, and do you believe that one can decrease the incidence of so-called opportunistic infection through the use of probiotic materials? Dr. van der Waaij: Studies from the United States and Europe indicate that personnel working in intensive-care departments can be colonized with the same bacteria that cause infections in the patients but are completely unharmed by them (1). So they can apparently coexist with those bacteria, although they continue to excrete them for weeks. I will therefore stick to my definition of pathogenic: pathogenic organisms are bacteria that are capable of causing infections in otherwise healthy individuals (e.g., typhoid, diphtheria, or pertussis). Can we improve the resistance of an individual by giving probiotics? I think this is for the future. I do not know of any study that has shown it so far. I think it will remain very difficult to achieve, so long as we have no means of determining the colonization resistance (CR) of an individual. Thus, in individuals with a high CR, probiotics may have no effect at all, or no measurable effect, while in subjects with a low CR there may be a large effect. Dr. Klish: I had the opportunity in the early 1970s to study probably the one and only gnotobiotic child born into a sterile environment—David "Bubbleboy," who became very famous in the United States. We attempted some colonization experiments on that child. He had X-linked combined immune deficiency, so he had no inherent immunity, though he did have a little bit of background colonization, primarily by anaerobes (it was impossible to eradicate spore formers from his food source, even though we sterilized and irradiated the food). We tried initially to colonize him with lactobacilli and could not. We gave him 1012 organisms multiple times on a daily basis for over 2 or 3 weeks, and we only achieved transient colonization. It became obvious to us that colonization also requires sequences of bacteria, that the bacteria interrelate with each other, and that you cannot colonize with one species unless you have another to provide cofactors for growth. That was particularly true for the lactobacilli. Dr. van der Waaij: Did the lactobacilli come from the family environment of the boy? 16 MICROBIAL ECOLOGY OF INTESTINAL MICROFLORA

Dr. Klish: At that time, we were too naive to recognize that there were multiple strains of lactobacilli. What we used was just a typical lactobacillus such as you find in yogurt. Dr. Fuller: You showed in your decontaminated mice that the Escherichia coli challenge was translocated. Was there any evidence that the normal flora organisms were also being translocated at that stage? Dr. van der Waaij: In one group of the animals, there was a normal flora, and in the other group not. In the ones with the normal flora in the first 4 or 5 days, we certainly have evidence of translocation after a dose as high as 109 organisms. However, that ceases by day 4. Therefore, we killed the animals at 4 days to have the best chance of showing the difference between the animals with a normal flora and those without. There were two differences: (i) the presence and the absence of flora and (ii) the dose difference (the dose in the decontami- nated animals was between 50 and 100 bacteria, and in the others it was 109). Regardless of the enormous dose difference, there was also an enormous difference in the colonization pattern and in the occurrence of translocation.

REFERENCE

1. Chambers ST, Steele Ch, Kunin CM. Enteric colonization with resistant bacteria in nurses working in intensive care units. J Antimicrob Chemother 1987; 19: 685-93.