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APPENDIX D IMPACT OF ANTEMICROBIALS ON THE MICROBIAL ECOLOGY OF THE GUT Dwayne C. Savage1 Much evidence has been published on the influence of anti- biotics on population levels and antimicrobial resistance in Escherichia colt. E. cold and some of its close relatives, such as salmonellae, are pervasive pathogens. Possibly because E. cold is easy to culture and manipulate In vitro, it has also become the major bacterial tool of molecular biologists. Thus, there is great interest concerning its resistance to antimicrobial agents. As documented below, it can be recognized as a member of the "normal gut floras" of many species of animals. It is usually a minority member of such floras. Studies of the antimicrobial resistance of E. cold have been reported in depth (see, for example, Food and Drug Administration, 1978~. However, the findings of these studies may be inadequate to demonstrate how predominant flora may interact with antimicro- bial drugs. Consequently, I have chosen to minimize discussion of E. cold and to emphasize findings and concepts concerning the types of bacteria that predominate in the "normal gut flora." "NORMAL" GUT FLORA The Gastrointestinal Ecosystem In my opinion, the term "normal gut flora" is confusing and probably obsolete (Savage, 1977~. The confusion begins with the word "gut," which usually means "intestine." Much evidence supports the hypothesis that most higher animals, including cattle, swine, and chickens (possibly even humans), have "gastrointestinal micro- biota" composed of indigenous microbes colonizing specific habitats located throughout the gastrointestinal tract, not just in the gut. Some of this evidence is presented later in this paper. The words "normal flora" also are confusing. "Normal flora" is usually used collectively to describe various microbial spe- cies found by cultures or microscopy to be on the skin and mucous membranes and in certain body cavities of both healthy and sick animals. The term is also used as a synonym for "indigenous micro- biota" meaning, collectively, those autochthonous microbial resi Department of Microbiology, University of Illinois, Urbana. 130
131 dents of habitats on certain body surfaces or in particular body cavities of normal animals. These definitions do not necessarily describe the same microorganisms. The first suggests that all microbial types found on or in, or cultured from, certain sur- faces or cavities are normal residents of habitats in those sites. However, much recent evidence supports the concept that many microbial types that can be isolated at any given time from an open ecosystem such as the gastrointestinal tract cannot be identified as indigenous to the system and must be regarded as transients. Transients can be transported to a habitat in a gastroin- testinal ecosystem in food and other materials (including feces in coprophagous animals such as chickens and pigs) or even by passing down from habitats above the one being sampled. Certain transients, some of which may be pathogens, may temporarily colonize niches in habitats in perturbed ecosystems. Systems may be perturbed by antimicrobial drugs (as shall be amplified), by starvation or other forms of malnutrition, and perhaps by certain environmental conditions such as hyperbaric atmospheres and cir- cumstances generating fear and other stresses. Such conditions influence the factors that regulate the population levels and localization of indigenous microorganisms in the ecosystem (Savage, 1977~. These factors are discussed later in this paper. Anatomy of the Gastrointestinal Tract As already noted, microbial habitats can be found in various locations in the gastrointestinal tracts of animals of different species. The gastrointestinal tracts of mammals and birds have five major sections: esophagus, stomach, small intestine, cecum, and large intestine. Depending upon the animal species, any of these sections may be further compartmentalized or divided into subsections. In mammals, there are three basic variations on this overall theme: the ruminant, cecal, and "straight tube" systems. In the ruminant, the stomach is ramified into compart- ments (Hungate, 1966~. In mammals with a cecum, the cecum is a blind pouch extending laterally from the distal end of the small intestine and the proximal end of the large bowel (McBee, 1977~. In chickens, the "stomach" consists of a storage compartment (crop), proventriculus, and gizzard (Fuller and Turvey, 1971~; two ceca are present (Bauchop, 1977; McBee, 1977~. Depending upon the species of animal, any or all of these areas may contain habitats for indigenous microorganisms. Such habitats may include
132 the contents of the lumen, the epithelial surface, or even pits in the mucosa called Crypts of Lieberkuhn. The epithelial and cryptal habitats may be particularly important. In mammals and birds, the esophagus is lined with a stratified squamous epithelium that may or may not be keratinized (Savage, 1977~. Some gastric" compartments, such as the crop in chickens (Fuller and Turvey, 1971), part of the stomach in rodents (Savage, 1977), and the rumens of cattle and sheep (McCowan et al., 1978), are lined with a stratified squamous epithelium that is usually keratinized. In chickens and mammals that have been ex- amined (including humans) gastric compartments not lined with a squamous epithelium and the small and large intestines (including the cecum) are lined with a single layer of columnar cells. In the small intestine, the mucosa is organized so that the epithe- lium covers finger- or leaf-shaped villi that protrude into the lumen. Villi are not found in the stomach or large intestine, although the mucosa in both areas may fold when the lumen is empty. Columnar epithelium also lines the Crypts of Lieberkuhn, which are located at the bases of the villi in the small bowel and are spaced periodically in the mucosa of the stomach and large bowel (Savage, 1977~. Depending upon the animal species, crypts and epithelial surfaces may provide habitats for microbial communities throughout the gastrointestinal tract. Evidence that epithelial, cryptal, and luminal habitats exist for indigenous microorganisms in all areas of the gastroin- testinal tract has been provided primarily by studies of labora- tory rodents (Savage, 1977~. The indigenous microbiotas of most mammalian and avian species have not been defined as well as they have for rodents. Nevertheless, some evidence on the microbiotas of calves, swine, and chickens supports a hypothesis that the concepts d~scussed above apply to those species as they do to laboratory rodents. In the discussion to follow, that point is amplified for swine and chickens, and some information on humans is included to provide perspective. The calf is treated separately because it is an ungulate with an enormous complex biota in its rumen. However, the biota in the rumen is similar to that in the large intestines of monogastric animals such as humans, pigs, and chickens. The Microbiota of the Stomach Microorganisms of many types have been isolated from the contents of the stomachs of humans and swine (Savage, 1977) and
133 from the crops of chickens (Fuller and Turvey, 1971~. Most of the types isolated should probably be regarded as transients since the stomachs of most animals undoubtedly empty more rapidly than microorganisms can multiply. Thus, microbes in the lumen pass out of the stomach with the contents (Savage, 1977~. Never- theless, certain types may be regarded as autochthonous to habi- tat~ in the area. Lactobacillus spp. at high population levels (10 organisms per gram of mucosa) can be cultured from and ob- served microscopically on the squamous epithelium of the crops of chickens ~ Fuller and Turvey , 197 1) . Likewise, Lactobac~llus spp. and Candida spp. can be cultured at comparable population levels from the squamous epithelium in the pars oesophagia of swine (Fuller_ al., 1978; Savage, 1977~. Although such organisms are usually found in the stomachs of humans as well (Savage, 1977) , much more research is needed to test the hypothesis that humans have an indigenous gastric microbiota. The Microbiota of the Small Intestine . . . . The small intestines of humans and chickens (and undoubtedly also swine and calves) also yield many microbial species (Dickman _ al., 1976; Savage, 1977~. Most of the organisms are probably transients, especially in the upper two-thirds of the bowel where peristalsis moves luminal content much more rapidly than microbes can multiply (Savage, 1977~. Microbes of types found in the large bowel (see below) may be identified, occasionally at high popula- tion levels, in cultures from the lower third of the gastrointestinal tract, where the content moves somewhat sluggishly and may not move at all for a tome. Such organisms may be indigenous to the region or may be contaminants from the large bowel that have crossed the ileo-cecal valve into the area. Neither of these hypotheses can be set aside on the basis of evidence that is available at this time. In chickens, however, microbes seen adhering to the epithelium of the small intestine (Fuller and Turvey, 1971) may be indigenous to that area (Savage, 1977~. These organisms are filamentous prokaryotes with Gram-negative ultrastructure (Savage, 1977~. Their population levels are unknown (they have not been cultured In vitro), but are probably quite high. Similar organisms are recognized as indigenous inhabitants of the epithelial surfaces of the small bowels of labora- tory rodents (Savage, 1977~.
134 The Microbiota of the Large Intestine The large bowels of humans and the coca and colons of swine and chickens contain enormous populations of microorganisms (more than 1 x 1011 microbes per gram dry weight of content). The con- tents of those regions move sluggishly and allow ample time for microbial multiplication (Savage, 1977~. The populations are com- posed primarily of Gram-positive and Gram-negative bacteria that cannot multiply in atmospheres containing oxygen (Table 1~. Indeed, many of the species are intolerant of oxygen and are killed by ex- posure to it or to growth media or diluting fluids with oxidation- reduction potentials above certain negative levels. Human feces yield up to 400 species in as many as 40 microbial genera (Drasar and Hill, 1974; Holdeman _ al., 1976; Moore and Holdeman, 1974~. The vast majority of the species are oxygen-intolerant anaerobic bacteria. More than 99% of the total microbial population obli- gately gains its energy through anaerobic processes. In the gastrointestinal ecosystem of humans, facultative bacteria (i.e., able to use both aerobic and anaerobic processes to generate energy) such as _ cold are usually outnumbered by the anaerobes by as much as 1,000 to 1. The systems of swine and chickens are undoubtedly similar (Table 1~. Some of the microbial species in the ecosystems adhere to or colonize secretions in the epitheli,~m of the ceca or colons (Savage, 1977~. In swine, spirochetes and a variety of other microbial spe- cies have been found in epithelial habitats (Allison et al., 1979; Savage, 1977~. In chickens, both Gram-positive and Gram-negative bacteria can be observed on the colonic surface (Fuller and Turvey, 1971~. In humans, bacteria have been seen microscopically on the surface, but have not been characterized well (Savage, 1977~. Since many such microbial species have not been cultured In vitro (Savage, 1977), they are not listed in Table 1. Nevertheless, they cannot be ignored as components of the ecosystem. The Microbiota of the Rumen The biota in the rumen of the adult bovine animal is also highly complex, consisting of protozoa and bacteria at enormous population levels (total levels greater than l x 1011 organisms per gram dry weight of content) (Bauchop, 1977; Hungate, 1966~. Most of the bacterial species in the rumen belong to anaerobic genera. Some of them are similar to those found in the large bowels of monogastric animals (Table 1), but others are undoubtedly unique to the ecosystem of the rumen (Hungate, 1966~. The population levels of facultative
135 TARLF~ 1 Principal Bacterial Genera Reported to be Present in the Feces or Content of the Large Bowels of Swine, Chickens, or Humans . Predominant Genera . . Swine Chickens Humans . Eubacterium Peptostreptococcus Clostridium Lactobacillus - Propionibacterium Streptococcus Peptococcus Megasphaera . Minor Genera . Swine Bacteroides Bifidobacterium , Treponema Veil lone lla Escherichia Eubacterium Bacteroides Fusobacterium Peptostreptococcus Bifidobacterium Gemminger - Clostridium . Lactobacillus Propionibacterium . . Chi ckens Staphylococcus Streptococcus Escherichia Eubacterium Bacteroides Fusobacterium Peptostreptococcus Ruminococcus Coprococcus Bifidobacterium - Gemminger Clostridium Lactobacillus Humans Acidaminococcus Staphvlococcus Propionibacterium Peptococcus Desulfomonas Succinivibrio S treptococcus Es cherichia Swine: Fuller _ al., 107~; Hackman and TJilkir~s , 1975; Kinyon and Harris, 1979; Kolacz et al., 1971; tiorish~ta and Ogata, 1470; Ogata and FIorishita, 1969; ~ussell, 1979; Terarla et al., 1976. Chickens: Psarnes and Impey, 196SS, 1970; Fuller and Turvey, 1971; Gilliland et al., 1975; Ochi et al., 1964; Salanitro et al., 1974, 1477, 1978; Timms, 1968. Flumans: Akama and Otani, 1970; Dickman et al., 1976; Drasar and Tlill, 1974; Gilliland _ al. , 1975; Holdeman et al., 1976; Mitsuoka,_ 1969; )litsuoka and Ohno, 1977; Moore and Hol~leman, 1974. Populat ion levels of many species Oexceed 1 x 109 organisms per gram of co-ntent. Most exceed 1 x 101 organisms per gram. Population levels less than 1 x 109 organisms ger gram of content. Many s pecles have levels of less than 1 x 1~) organisms per gra~n.
136 organisms such as E. cold are usually nonexistent or quite low. Certain bacterial types, including some facultat~ve ones, are believed to adhere to the epithelium of the rumen (McCowan et al., 1978~. The biota of the remainder of the bovine intestinal tract has not been characterized. Summary There is no doubt that all mammalian and avian species have microbial floras that are indigenous to their gastrointestinal tractse In humans, calves, swine, and chickens, climax floras, such as might be found in a healthy adult, contain primarily anaerobic bacteria in most habitats of the tract. Under normal conditions those anaerobes vastly outnumber facultative microbes such as E. colt. In fact, in normal, unperturbed systems the anaerobes undoubtedly function to restrict the population levels of E. cold and its relatives. Unfortunately, as noted earlier, information on the anaerobes with pertinence to this report is far less well developed than it.is for E. colt. This problem com- plicates the answers to most of the questions raised in the following paragraphs. ANTIBIOTIC-RESISTANT STRAINS IN NORMAL FLORA Investigators interested in E. colt, primarily as a potential pathogen, have provided considerable data on antibiotic-resistant strains in normal flora. Strains of E. cold with resistance to numerous antibiotics, many carrying transferable plasmids coding for such resistance, can be isolated from calves, swine, and poul- try being fed (Table 2) or treated (Table 3) with antimicrobial drugs. Such strains can also be isolated from animals ostensibly not fed or treated with the drugs, but with much less frequency than from animals receiving them (Franklin and Glatthard, 1977; Petrocheilou et al., 1976) (Tables 1, 2~. Most investigators do not provide reassurance, however, that these controls have not been in contact with antibiotics, for example, through association with parental animals treated with drugs. Reliable information of the type available for E. cold is vir- tually unavailable for the bacterial species predominating in the gastrointestinal ecosystem. Anaerobic bacteria of many genera can develop resistance to antimicrobial drugs. This has been demon- strated for organisms from the rumen (Ful~hum et al., 1968; Wang et _ ., 1969), from feces of humans (Anderson and Sykes, 1973; Burt and
137 TABLE 2 Some Reports Containing Evidence that Resistant Strains of . . · . . . Escher ooZz can be Isolated More Frequently from Animals Fed Diets ,, Containing Certain Antibiotics than from Animals Fed Drug-Free Diets Animal Antibiotic Reference Swine Tetracyc lines Penic ill in Vi rginiamycin mixture of chlor- te tracyc line, peni- cillin, and sulfamethazine Fuller et al., 196 0 Smith. 1968 Mercer et al., 197 1 Siegel et al., 19 74 Lin ton et al. , 19 7 5 1978 Lan~lois et al., 197 SS Ahart et al., Fuller et al., 1960 fiercer et al., 19 71 Siegel et al., 1974 Langlois _ al., 1978 fiercer_ al., 1971 A mixture of Virginia- Pohl et al., 1977 mycin, tylosin, Furoxone, and sulfa guan idine Calves Tetracyclines Edwards, 1962 Loken_ al., 1971 Siegel et al., 1974 Linton et al., 1975 Ahart et al., 1978 Penic illin Siegel et al., 1974 Chickens Te tracycline Smith, 1968 Turke ys Tetracycline Baldwin _ al., 197 6 aIsolated resistant strains of E. cold were resistant to one or more antimicrobial drugs, often not only to the drug used in the feed but also to one or more other compounds. b Present at subtherapeutic levels in the feed.
138 TABLE 3 l Some Reports Containing Evidence that Resistant Strains of Esoheriahia ooLican be Isolated More Frequently from Animals being Treated with Certain Antibiotics than from Untreated Animals Animal Antibiotic Reference Swine Many dif forest types Larsen and Nielsen, 197 5 Cows Penicillin plus dilly- Rollins et al., 19 74 dros trep tomycin Chicke ns Te tracyc lines Chopr a et al., 19 63 Howe et al., 1976 Humans Tetracyc lines Schmidt et al., 197 3 Flirsh et al.. 1973 Bar tlett _ al., 197 5 Miller_ al., 1977 a Isolated resistant strains of _ cold were resistant to one or Pl() C2 ant imicrobial drugs, of ten not only to the drug used in tree tment but also to one or more other compounds. bused prophylactically or to treat a particular disease.
139 Woods, 1975; Finegold, 1970, 1977) and swine (Rood et al., 1978), and from the ceca of chickens (Barnes and Goldberg, 1962). More- over, investigators have demonstrated that plasmids code for resist- ance to several antibiotics in certain species of Streptococcus (Malke, 1979; Van Embden et al., 1977), Lactobacillus (Klacnhammer _ . et al., 1979), and Bacteroides (Guiney and Davis, 1978; Onderdonk et al., 1979; Welch et al., 1979), and in Clostridium perfringens (Sebald and Brefort, 1975), all bacterial species recognized to be members of the large bowel flora of mammals and birds (Table 1~. Plasmids coding for resistance can be transferred In vitro from donor to recipient strains of Clostridium perfringens (Sebald and Brefort, 1975~. Such plasmids may also transfer from donor to re- cipient strains of Bacteroides In vitro (Welch et al., 1979) and in viva in formerly germfree rodents (Onderdonk et al., 1979) and from donor Bacteroides to E. cold in vitro (Mancini and Behme, 1977~. _ _ Strong evidence supports findings that there is a conjugative trans- fer of an R plasmid from a strain of Bacteroides ochraceus isolated from the human mouth to a strain of E. cold (Guiney and Davis, 1978~. Burt and Woods (1976) reported that "A-factor" plasmids from E. cold can be transferred in vitro to strains of B. fragilis, other species Of Bacteroides, and some species of Fusobacterium, if the recipient strains are heated before being exposed to the donor. Most such information has been gained in studies conducted within the last 4 or 5 years. Indeed, many recent reports on the transmissibility of plasmids in anaerobes appear only as abstracts in the literature. Much of the work concerns anaerobic bacteria as pathogens rather than as members of the indigenous microbiota. How- ever, these efforts provide good indications of gene transmission among the anaerobic members of the gastrointestinal ecosystem. Un- fortunately, little of the evidence has been gained in such a way that the proportion of antibiotic-resistant strains in "normal" flora can be determined. CHANGES INDUCED BY SUBTHERAPEUTIC LEVELS OF ANTIBIOTICS IN FEED The Proportion of Resistant Strains in Gastrointestinal Microbiota . As suggested above, reliable information on the proportion of resistant strains in gastrointestinal microbiota is almost non- existent for the major components of the indigenous biota. Ample evidence supports observations that strains of E. cold with resist- ance to penicillins, tetracyclines, and other antibiotics can be isolated much more frequently from animals fed subtherapeutic doses of tetracycline and penicillin in their diets (Table 2) than from
140 animals eating diets free of the drugs. Indeed, after animals have consumed the diets containing drugs for just a few days, more than 90% of the E. cold strains isolated are resistant to the drugs used in the diets and to other compounds. By contrast, less than 10% of the strains isolated from animals not fed the drugs are resistant to antibiotics. Some efforts have been made to develop such information for certain other types of bacteria that can be cultured from the gastrointestinal tracts of animals. For example, dietary chlortetracycline was found to induce re- sistant strains of Streptococcus faecalis that predominate over sensitive strains in ceca of chickens (Elliott and Barnes, 1959~. One such resistant strain predominated in the animals 5 months after the antibiotic had been removed from the diet. Similarly, 90% of the strains of lactobacilli or streptococci isolated from the feces of swine were commonly resistant to peni- cillin or to chlortetracycline if isolated from animals fed diets containing that drug. By contrast, more than 90% of the same strains isolated from animals not fed the drugs were sensitive to them (Fuller et al., 1960~. Some other studies (Aha-r-t et al., 1978) in which "anaerobes" were isolated and found to be resistant to antibiotics cannot be evaluated. The methods used by those in- vestigators provide no clues to the types of bacteria involved. Potential Pathogens in the Gastrointestinal Microbiota . Virtually no information is available on the influence of antimicrobial drugs on the relative proportions and absolute num- bers of potential pathogens in the indigenous biotas except for _. cold and its relatives. Actually, "pathogen" is difficult to define in reference to the biota. In swine, Treponema hyodysen- teriae can cause dysentery only when acting with other anaerobic components of the indigenous biota, none of which are known to be pathogens (Kinyon and Harris, 1979~. Likewise, many other indige- nous species have the capacity to cause disease under the right circumstances. In several species of animal, including pigs and chickens, Clostridium perfringens can cause diarrhea! disease under certain conditions (Finegold, 1977; Rood et al., 1978~. In humans and other animals, certain Bacteroides spp. induce abscesses in normally sterile tissues, often in association with facultative bacterial species (Finegold, 1977~. Many other species of anaer- obic bacteria can cause disease under certain conditions (Finegold, 1977~. Most of them are normally present in the gastrointestinal tracts of animals at extremely high population levels. Thus, the
141 question of whether or not antibiotics tend to increase the rela- tive proportions and absolute numbers of potential pathogens is moot. Flora of Areas of the Body Other Than the Skin Information pertaining to the effects of antibiotics on flora at sites other than the skin is sparse. Strains of bacteria with resistance to antibiotics have been isolated from the vaginal secretions of cows (Panangala and Barnum, 1978~. However, these studies were conducted with cows that had been treated with anti- biotics but not necessarily given feed containing subtherapeutic doses of the drugs. Moreover, there was no evidence that the bac- teria were members of the indigenous flora of the bovine vagina. Virtually nothing of which I am aware has been published on the composition of the skin flora of chickens and mammals other than humans (McBride et al., 1977~. The skin and noses of pigs and chickens fed diets containing tetracyclines yield abnormally high proportions of strains of Staphylococcus aureus with resist- ance to the drug (Smith and Crabb, 1960~. However, S. aureus may or may not be a member of the indigenous skin and nasal biota of such animals. Persistence of Changes when Selection Pressure is Removed The duration of resistance when selection pressure is removed cannot be determined satisfactorily for most species found in the gastrointestinal tract. E. cold strains bearing drug resistance plasmids may persist in the biotas of animals for months after the selection pressure is removed (Hartley and Richmond, 1975; Smith, 1975~. Similarly, as already noted, Streptococcus strains with resistance to chlortetracycline may persist in the ceca of chickens for months after the drug is removed from their diets (Elliott and Barnes, 1969~. Whether or not such findings apply to the majority components of the biota is not known. CHANGES IN FLORA RESULTING FROM THE THERAPEUTIC USE OF ANTIBIOTICS There are few definitive data concerning changes in the anaerobic components of flora resulting from the therapeutic use of antibiotics. Evidence does substantiate that therapeutic doses of certain antibiotics enable resistant strains of E. cold
142 to proliferate in the gastrointestinal tracts of several species of animals (Table 3) and to transfer plasmids coding for such resistance in the gastrointestinal tracts of humans (Anderson et al., 1973~. Likewise, many streptococcal strains isolated from the mouths and blood of humans treated with penicillin and some other antibiotics may be resistant to the drugs used (Phillips _ al., 1976~. Moreover, there is no doubt that antimicrobial therapy allows strains of other types of pathogenic anaerobic bacteria to grow in and be isolated from lesions of diseases in humans (Finegold, 1977~. Strains of such organisms may persist for a time in the human gut (Finegold, 1977~; however, there are no data pertaining to the development and persistence of resist- ance in the major components of the indigenous biota of the gas- trointestinal tract. CHANGES IN FLORA RESULTING FROM LEVELS OF ANTIBIOTICS USED TO PROMOTE GROWTH There is no reliable information pertaining to the action of antibiotics used as growth promoters on the major components of the indigenous microbiota In viva. As discussed above, both penicillin and tetracycline can induce resistance in anaerobes of many species. FACTORS AFFECTING ABILITY TO COLONIZE . Many factors influence the population levels and localization of microorganisms in the gastrointestinal ecosystem (Tables 4 and 5~. Some of the forces are exerted by the host, its environment, and diet (the allogenic factors). Some are exerted by the micro- organisms themselves (the autogenic factors). The microbiota can alter the forces generated by the host, making them either more or less effective in controlling the Bogota. Thus, the processes in- fluencing a microbe's ability to colonize a niche in a habitat in the gastrointestinal tract are enormously complex. It is known, however, that the strictly anaerobic bacteria are the most impor- tant components of the biota involved in stabilizing established communities in adult animals. The processes involved in maintaining such stability undoubt- edly work most effectively in adult animals with unperturbed indi- genous biotas. Most importantly, these processes prevent transient microbial species from colonizing niches in the system (Savage, 1977~. Such transients (many of which may be potential microbial
143 TABLE 4 Some Allogenic Factors Known to Inf luence the Composition of the Indigenous Microbiota in Certain Regions of the Gastrointestinal Tracta - Factor Pos sib le Region of Normal Features that Influence Inf luence Composition of Microbiota Tempe rature Stomach Body tempe ratur e ~ 37 °C Small int es tine Large intes tine pH Stomach Ac idic Small intestine Neutral to alkaline Large intestine Neutral to alkaline Stasis Stomachs Periodic Small int estine Periodic in lower part of region Large intestine Prolonged (residence time) ~ includi ng cecum) es peci ally in cecum Oxygen Large intestines Concentration low, if present at all Oxidation- Large intestine Low potential, especially when reduct ion microbes are present pot ent i al Enzymes Seal 1 intestines Pancreatic enzymes present Bile acids Stomach Low concentration Smal 1 intestine Conjugated bile acids present Large intestine Deconjugated bile acids present Epitlaelial All regions of Sloughing of cells necessitates turnover tract replacement of attached cells but the cel Is may provide micro bial nut rient s Urea Large intestines Can be carbon and nitrogen source to r certain microbial s pe cies
144 TABLE 4 ( Continued) Factor Mucin Diet Drugs Phago cyt ic cel Is Antibodies Poss ible Reg lo n of Inf luence All regions of tract All r eg ions of tra ct All regions of tra ct Crypts of Leiberkuhn All reg -ions of tra ct Adapted from Savage , 19 77; see also Booth et al., and van Route, 1975. No real Featur es the t Inf luence Composition of Microbiota Mucin contr ibutes to the vi scos- ity of the environment and may act as a microbial nutrient The diet provides microbial nu- tr lent s and af fe ct s the nature of habitats in the tract Inf luence i s characteris t ic of the drunks) Microbiota may be phagocyto sed ~ select ively) Precise ef feet on biota not known. Antibody protein may be ut ilized nutritionally by microorganisms 19 7 7, and Gib bons Evidence is insufficient to suggest that the factor may operate in other reg ions .
145 TABLE 5 Some Autogenic Factors Known to Influence the Composition of the T-A; ~~..e 1~; army; r~= in C=rt=;n Rev; And:: of the (~tr~int~.c:tinn1 Trams Factor Volatile fatty acids Bacteriocins Nutritional competition Hydrogen sulfide Adherence to epitI~elium Possible Region of Influence Any Sari ta t Any habitat Any habitat Probably in jarge int estine Any habi tat Adapted from Savage, 1977; see also Booth et al. and van Route, 197 5. No real Featur es tea t In f lue nce Composition of Microbiota Ef Sect dependent upon oxidation- reduct ion pot enti al F.f :fe ct s uncertain Ef fects uncertain Lay depress population levels of some facultative s pecies of bac- te ria Allows microorganisms to colonize areas of the tract where peristal- t ic rat e moves content s more rap- idlv than bacteria can multiply , 1977, and GiLhons providence is insufficient to suggest that the factor may operate in other reg ions .
146 pathogens constantly enter the system via food, water, and other ingesta. They usually do not persist long in the unperturbed system, but may colonize a niche in a perturbed system. As mentioned above, a system may be perturbed by antimicro- bial drugs (Decuypere et al., 1973; Dubos et al., 1963; Savage and Dubos, 1968; Savage and McAllister, 1971; Van der Waaij and Berghuis, 1974), starvation and other forms of nutritional stress (Savage, 1977), and even by some less drastic changes in an ani- mal's lifestyle (Holdeman et al., 1976~. In infants, in whom the climax gastrointestinal microbiota characteristic of the adult has not yet been established, the biota may also be regarded as perturbed. During this developmental period of animals, the population levels of E. cold may be quite high (Savage, 1977~. Once the anaerobic components of the biota are established, however, the levels drop to the normally low levels of adults. As long as the communities of anaerobes remain intact in adult animals, the population levels of E. cold remain low. If the communities of anaerobes are perturbed (e.g., by antimicrobial drugs), the levels of E. col-i may rise dramatically. Such evi- dence has been considered strong support for the hypothesis that the anaerobes control the levels of E. cold in the ecosystems (Morishita and Mitsuoka, 1976~. However, the mechanisms of such control are not well understood. In vitro, anaerobes may also inhibit it-factor transfer among strains of E. cold (Anderson, 1975~. Because antimicrobial drugs perturb the gastrointestinal ecosystem, they must upset the balance of forces that regulate the biota. The precise factors that are perturbed, especially those affected by subtherapeutic doses of drugs, can only be conjectured. No doubt exists, however, that subtherapeutic doses of both peni- cillin and tetracycline in animal feeds somehow tip the balance towards resistant strains in the biota of the animal fed the diets. Interestingly, when certain antibiotics are administered, some microbial strains may not themselves have to be resistant to the drugs to survive because resistance in another organism may enable them to do so (Hackman and Wilkins, 1975~. Humans associating with animals fed subtherapeutic doses of antibiotics may temporarily carry resistant strains of E. cold that are present in the animals. Such bacterial strains may transfer from animals to humans, however, whether or not the animals are fed antibiotics (Hirsh and Wiger, 1977, 1978; Hirsh et al., 1974; Linton _ al., 1977~.
147 When antibiotics are removed from an ecosystem, the mainte- nance of resistance me y become an undue physiological burden for some strains. Thus, they may fail to compete with unhandicapped strains of the same species and eventually decline in prevalence and disappear from the system (Anderson, 1974~. At this time, however, this hypothesis can be neither supported nor rejected by evidence provided by studies of the major components of the indigenous microbiota--the strictly anaerobic bacteria (Finegold, 1970~. SUMMARY Antibacterial drugs such as penicillin and the tetracyclines, when incorporated as growth promotants into the feed of animals, provide a selective environment in the gastrointestinal tract favoring the proliferation of resistant strains of Escherichia colt, Streptococcus spp., and at least some of the major (strictly anaerobic) bacterial components of the indigenous microbiota. As with _ colt, some strains of strict anaerobes carry genetic information for resistance on plasmids. For a few such bacterial species, the plasmids can be transferred to recipient strains of the same species. Certain strains of Bacteroides may even be able to transfer their plasmids to recipient strains of E. cold and vice versa. Such information provides limited evidence that the mechanisms of antibiotic resistance in some strains of anaerobic bacteria in the gastrointestinal ecosystem are similar to the mechanisms of such resistance in E. colt. However, it does not reveal anything about the proportion of resistant anaerobic strains that reside in animals receiving drugs in feed. Most importantly, perhaps, it reveals nothing about whether or not such resistance is transferred between microbial species in the gastrointestinal tract and whether or not resistance is maintained in the tract of an animal not being fed or treated with drugs. Information pertaining to these questions is insufficient for the major components of the biota, especially as they interact with each other and their host. Microorganisms in the gastrointestinal ecosystem interact biochemically and genetically with each other and biochemically with their animal host. Such interactions are complex mechanistically and not well understood. Much more evidence is needed before the impact of antibiotics on the system can be understood.
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