The gastrointestinal epithelium and its autochthonous bacterial flora.

Colonization of the gastrointestinal tract by bacteria of the normal flora was followed by bacteriological and special histological techniques in mice from several colonies. These histological techniques were designed to preserve the intimate associations that become established between particular strains of microorganisms and the epithelium of the mucosa of certain areas of the gut. The findings were as follows: 1. The various strains of bacteria of the normal flora became established in the different areas of the guts of infant mice according to a definite time sequence. 2. The first types of bacteria that could be cultured from the gut were lactobacilli and Group N streptococci. Within the first day after birth, these bacteria colonized the entire digestive tract and formed layers on the stratified squamous epithelium of the nonsecreting portion of the stomach and of the distal esophagus. 3. The bacterial types that appeared next were coliforms and enterococci. From about the 9th to the 18th day after birth, these bacteria could be cultured in extremely high numbers from the cecum and the colon. Histological sections of those organs taken during the first 2 or 3 days of that interval revealed microcolonies of Gram-positive cocci in pairs and tiny Gram-negative rods embedded in the mucous layer of the epithelium. The microcolonies were well separated from the mixture of digesta and bacteria that occupied the center of the lumen; they may have consisted of the coliforms and enterococci mentioned above; but this possibility remains to be proved. 4. Histological sections also revealed that, at about the 12th day after birth, long, thin Gram-variable rods with tapering ends were present, side by side, with the small Gram-negative rods and Gram-positive cocci in the mucous layer. By the 15th day after birth, the fusiform bacteria formed thick layers in the mucus, and seemed to be the only bacteria remaining in that location. It has not yet been possible to enumerate these tapered rods by culture methods, but as judged by visual appearances in the histological sections, they seemed to outnumber all other bacteria in the cecum and the colon by a factor of as much as 1000. It must be stressed that these bacterial layers are readily disrupted and even washed away by conventional histological techniques; their discovery was largely due to the use of the special histological techniques described in the text. The bacteriological and histological findings described here constitute further evidence for the hypothesis that symbiotic associations exist between microorganisms and animals, and that a very large percentage of the bacteria in the gastrointestinal tract constitutes a true autochthonous flora. The constant occurrence of several distinct associations of bacteria with the special histological structures of the animal host renders obsolete the notion that the intestine constitutes a chemostat in which the bacterial populations are randomly mixed. For a full understanding of the ecology of the normal microflora, it is necessary to think of body surfaces as distinct microenvironments in which virtually pure cultures of a few species of microorganisms interact with their host and the adjacent microbial populations. Experiments based on this hypothesis are admittedly difficult to design, but on the other hand studies based on the assumption that microorganisms exist as mixtures in the gastrointestinal tract will be only of limited value and may often be misleading.

Lasting biological effects of early environmental influences. I. Conditioning of adult size by prenatal and postnatal nutrition.

Newborn specific-pathogen-free mice (SPF) were separated from their mothers shortly after birth and immediately reallocated at random to foster mothers, each of which received eight young. Under these conditions, the growth rate and adult size of the young were profoundly and lastingly conditioned by some unidentified influence exerted by the foster mother. In SPF mice nursed by their own mothers, the diet of the latter during gestation and lactation, or during lactation alone, conditioned the weight of the young at weaning time, and throughout their whole life span. Lasting depression of growth has been achieved by minor alterations of the dam's diet, for example by lowering its content in magnesium, or in lysine and threonine. The growth-depressing effect so achieved persisted throughout the whole life-span of the young, even though they were given at weaning time and constantly thereafter unlimited amounts of an optimum diet. In contrast, the weight-depressing effect of a diet deficient in lysine and threonine administered to adult animals was completely and rapidly reversible when a complete diet was later substituted for the deficient one. Depression of growth resulting from nutritional experiences during gestation or lactation did not seem to affect adversely the health of the young, or to decrease their longevity. In fact, the results of two experiments in which the animals nursed by mothers on different diets, were kept undisturbed and on optimum diets throughout their whole life span, suggest that the smaller animals had a greater average life expectancy than the larger ones.

The relationship of polymorphonuclear leukocytes to infertility in uteri containing foreign bodies.

A chronic infiltration of polymorphonuclear leukocytes was invariably found in the infertile regions of uteri containing foreign bodies in conventional rats, germfree rats, mice, and rabbits. Polymorphonuclear leukocytes were never found in the fertile regions of these uteri. A foreign body in the uterus of the rat, and probably also the mouse, was associated with a bacterial infection which spread the inflammatory response throughout the horn containing the foreign body, and in the mouse occasionally into the control horn as well. No bacteria could be cultured from the rabbit uterine horn containing a foreign body. In the germfree rat, both the infiltration of polymorphonuclear leukocytes into the uterus and fertility were significantly different from that observed in the conventional rat. Whereas in the conventional rat the inflammation and infertility extended along the entire length of the uterine horn containing a small foreign body, in the germfree rat the inflammation and infertility were closely correlated to the position of the foreign body. As judged by measurements of lysozyme in the uterine lumens of rats and rabbits, polymorphonuclear leukocytes released their contents into solution in the uterine lumen. It is concluded that some substance derived from polymorphonuclear leukocytes may exert toxic effects on fertilized ova or on spermatozoa and thus be responsible for the infertility of uteri containing foreign bodies.

Importance of aliphatic amines in uremia.

Five main aspects were addressed: 1)The demonstration that creatinine is an endogenous precursor of dimethylamine (DMA) in chronic renal failure. 2) The size of the body amine pool measured in transplant patients suggests sequestration in some intracellular compartment. This illustrates the possible error in directly relating serum concentrations to neurological toxicity. 3) Bacterial overgrowth and increased generation of duodenal DMA in the small intestine becomes apparent at a serum creatinine above 8 mg/dl. Two cases show that bacterial overgrowth preceded the increased duodenal DMA. 4)Clinical toxicity is demonstrated by i) correlation of abnormal neurobehavioral parameters with serum amine levels, and ii) by improvement with administration of nonabsorbable broad spectrum antibiotics. Results with adsorption agents are inconclusive. 5) Preliminary tests of behavior modification in a rat model by direct instillation of amines into the brain are positive for TMA but negative for DMA, but no DMA entry into brain cells is demonstrated in the latter. The generation of aliphatic amines represents only one part of a spectrum of alteration induced by proximal intestinal bacterial enzyme action that occurs in renal failure. It is possible that some bacterial activity is beneficial and that the net clinical result is a balance between the "good" and the "evil" bacterial effects.

Effect of cyclacillin and ampicillin on the gut flora of mice.

The oral administration of cyclacillin, a semisynthetic aminoalicyclic penicillin, results in the elimination of the lactobacilli from the gastrointestinal tract of mice. Although cyclacillin has a broad spectrum of activity similar to that of ampicillin, it does not, like ampicillin, affect the other flora of the gastrointestinal tract. Cyclacillin, unlike ampicillin, is rapidly absorbed from the stomach and upper small intestine, so that only small amounts reach the large bowel. Study of the dynamics of the lactobacillus population in the gut shows that in the mouse the prime site of localization and multiplication of lactobacilli is the stomach. The lactobacilli are transient in the small and large intestine, arriving there only after having been shed from the stomach.

Multigenic control of resistance to Yersinia enterocolitica in inbred strains of mice.

By using recombinant inbred mice derived from strains genetically resistant or susceptible to Yersinia enterocolitica, we demonstrated a tentative linkage of resistance with the Es-1 locus on murine chromosome 8. No correlation with resistance and genes associated with immune regulation was evident. In addition, resistance appeared to be multigenic.

A possible role for granulocytes in the action of intrauterine contraceptive devices.

PIP: The roles of uterine inflammation and infection in creating an environment hostile to fertilized eggs or spermatozoa which would explain the contraceptive action of IUDs were investigated in the rat, rabbit, and mouse. Inflammation, as evidenced by the appearance of polymorphonuclear leucocytes, was always present along with a foreign body, and inflamed areas corresponded to regions of known infertility: the entire length of the horn containing the foreign body in the rat uterus, the corresponding horn as well as part of the control horn in the mouse, and only the tissue in contact with the foreign body in the rabbit. Acute inflammatory response (polymorphonuclear stage) persisted indefinitely after insertion of the IUD. Lysozyme measurements in rat and rabbit uteri indicated that polymorphonuclear leucocytes released their contents into the lumen in infertile regions. Cultured rat uteri containing foreign bodies were found to contain over 100 million bacteria of mixed species (compared to none in controls), while rabbit uteri appeared bacteria free. The effects of inflammation were separable from those of infection, however, with the use of germ-free rats. The presence of a foreign body in these rats was associated with a very localized inflammation, with lysozyme only in the segment in contact with the foreign body. Fertilized eggs entered and implanted in the noninflamed region when the foreign body was in the cervical end of the horn; when it was in the ovarian end, all ova were killed, indicating that the antifertility effect of the IUD was possible in absence of bacteria. It is concluded that some component of the polymorphonuclear leucocyte may kill fertilized eggs and thus be responsible for the infertility of uteri containing IUDs.^ieng

Yersinia enterocolitica infection in resistant and susceptible strains of mice.

We investigated natural resistance in mice to Yersinia enterocolitica, an enteric bacterial pathogen of humans, with a view to determine host genetic factors that are important in resistance. Most mouse strains studied (C3H/HeN, BALB/c, BALB.B, DBA/2, A, Swiss, and SWR) were highly susceptible to infection (50% lethal dose [LD50], 2 X 10(2) to 6 X 10(2) Y. enterocolitica administered intravenously [i.v.]). In contrast, C57BL/6 mice were highly resistant (LD50, 2 X 10(5) Y. enterocolitica administered i.v.). Resistance to i.v. Yersinia infection did not appear to be related to the Ity locus (which codes for resistance to Salmonella typhimurium and other pathogens) because Ityr mice (C3H/HeN, DBA/2, A, and SWR) were more susceptible to Y. enterocolitica than were Itys (C57BL/6) mice. In addition, because BALB.B mice (congenic to C57BL/6 mice at the H-2 locus) were susceptible, resistance was probably not H-2 linked. BALB/c X C57BL/6 F1 mice were intermediate in their resistance to Y. enterocolitica infection (LD50, 3 X 10(4) organisms administered i.v.), suggesting that resistance to Y. enterocolitica depends on a gene dosage affect or a resistance gene(s) interaction between susceptible and resistant parents. Further studies with C57BL/6 and BALB/c mice as prototype resistant and susceptible strains were undertaken. A time course study of Y. enterocolitica growth in various organs following i.v. infection revealed no strain difference in bacterial growth during the first 48 h of infection. Thereafter, however, C57BL/6 mice were capable of restricting Y. enterocolitica growth in all tissues (liver, lung, spleen, kidneys), whereas extensive bacterial proliferation occurred in BALB/c mice tissues. BALB/c mice were also more susceptible to oral Y. enterocolitica infection than were C57BL/6 mice, demonstrating increased mortality and greater numbers of bacteria in the Peyer's patches. Finally, whereas thymus-bearing C57BL/6 X BALB/c F1 mice were resistant to infection, athymic (nude) C57BL/6 X BALB/c F1 mice were susceptible. These studies provide a model to investigate natural immunity to enteric pathogens at mucosal surfaces, as well as provide the basis for clarifying the role of host genotype in Y. enterocolitica resistance.

Induction of creatininase activity in chronic renal failure: timing of creatinine degradation and effect of antibiotics.

Creatinine degradation was prospectively studied in four healthy subjects and 35 patients with varying degrees of chronic renal failure by measuring creatininase activity in stool isolates. Patients were subdivided into those with serum creatinine above and below 6 mg/dL. Creatinine degradation in the former group of patients who had not taken antibiotics in the previous 3 months was significantly greater than the latter (64% v 26%; P < 0.001), which was similar to healthy controls. This degradation was abolished when antibiotics were added directly to the patient's stool during incubation (P < 0.002). In a subset of five patients, duodenal intubation demonstrated small bowel bacterial overgrowth associated with high concentrations of toxic methylamines generated there from and increased stool creatinine consumption. We conclude that retained creatinine in advanced chronic renal failure induces bacterial creatininase activity throughout the bowel, causing creatinine degradation and subsequent potential loss of creatinine to the creatinine pool. The modifying effects of antibiotics on creatinine degradation has important clinical implications for the interpretation of serum creatinine measurements in renal failure.

Bacterial populations of the small intestine in uremia.

The small intestinal bacterial flora of 15 patients with chronic renal insufficiency was compared with that of subjects with blind loop synDROME. 9 patients were on regular hemodialysis with high protein intake and 6 (serum creatinine 7.5 to 12.5 mg/dl) were maintained on low protein diet. The chronic renal patients harbored a greatly increased microbial flora of both anaerobes and aerobes in the duodenum and jejunum, quantitatively comparable to those in blind loop subjects. The composition did not differ significantly in the two groups. Some organisms may have the potential to metabolize substrates which reach the intestinal lumen from the diet and bile, and perhaps to generate toxic metabolites that could contribute to uremic toxicity or malabsorption.

Medical applications of dust-free rooms. 3. Use in an animal care laboratory.

Bacterial air sampling in an animal care laboratory showed that dense aerosols are generated during cage changing and cage cleaning. Reyniers and Andersen sampling showed that the airborne bacteria numbered 50 to 200 colony-forming units (CFU)/ft(3) of air. Of the viable particles collected by Andersen samplers, 78.5% were larger than 5.5 mum. A low velocity laminar air flow system composed of high-efficiency particulate air (HEPA) filters and a ceiling distribution system maintained the number of airborne viable particles at low levels, generally less than 2 CFU/ft(3). Vertical air flow of 15 ft/min significantly reduced the rate of airborne infection by a strain of Proteus mirabilis. Other factors shown to influence airborne infection included type of cage utilized, the use of bedding, the distance between cages, and the number of animals per cage.

Amine metabolism and the small bowel in uraemia.

Intestinal intubation was carried out in 21 subjects: 9 with end-stage renal failure, 2 with early renal insufficiency, 7 untreated patients with blind-loop syndrome, and 3 normal volunteers. All 9 patients with uraemia had significantly raised duodenal dimethylamine (D.M.A.) concentrations compared with the other groups tested. Alteration of the intestinal bacterial flora with antibiotics markedly reduced serum D.M.A. and trimethylamine concentrations and modified the symptoms. Potentially toxic metabolites in the small bowel might have significant nutritional and toxic sequelae in uraemia and these findings suggest that current therapeutic approaches to the treatment of end-stage kidney disease should be re-examined in relation to bowel flora.