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The bacterial flora in the human colon, although extremely diverse, has a relatively stable composition and non-infectious anaerobic bacteria are dominant. The flora forms a pool of numerous different antigens separated from mucosal immunocompetent cells by just a single layer of epithelial cells. Despite this thin barrier, however, the colonic mucosa is physiologically only mildly inflamed. This study looked at the mucosal humoral immune response against faecal anaerobes. By flow cytometric analysis the in vivo immunoglobulin coating of anaerobic bacteria in faecal samples of 22 healthy human volunteers was determined. In a previous study flow cytometric analysis of faecal bacteria has been found to be a very sensitive method to detect immunoglobulins on faecal bacteria. This technique showed that in vivo many bacteria are coated with IgA (24-74%) and less with IgG and IgM. The presence of many bacteria coated with IgA implies that IgA coating does not result in permanent removal of the species from the colon. The absence of immunoglobulin coating suggests that there is immunological unresponsiveness for anaerobic bacterial antigens. It is concluded that both immunological unresponsiveness and preferential coating with IgA are responsible for the relative absence of colonic mucosal inflammation.

We describe a flow cytometry method for analysis of noncultured anaerobic bacteria present in human fecal suspensions. Nonbacterial fecal compounds, bacterial fragments, and large aggregates could be discriminated from bacteria by staining with propidium iodide (PI) and setting a discriminator on PI fluorescence and by exclusion of events with large forward scatter. Since anaerobic bacteria, which account for over 99.9% of all fecal bacteria, die during sample preparation, a fixation step was not necessary. A second aim of this study was to investigate the technical possibility of measurement of in vivo IgA coating of fecal anaerobic bacteria as well as their bacterial size. Fecal samples of 22 healthy human volunteers were analyzed. The fluorescence distribution of IgA-coated bacteria labeled with fluorescein isothiocyanate (FITC)-anti-Hu-IgA had overlap with noncoated bacteria. However, with match region subtraction, detection of low levels of specific FITC fluorescence on IgA-coated bacteria was achieved. The median bacterial two-dimensional surface area was 1.0 mu m(2). To validate flow cytometry data, all samples were analyzed with an image analysis system as well. With this new method, a rapid evaluation of fecal flora with high sensitivity for specific FITC fluorescence is possible without culturing. (C) 1994 Wiley-Liss, Inc.

SummaryHealthy persons were shown to possess circulating antibodies of both IgA, IgG and IgM isotype directed against the bacteria of their faecal microflora, assessed by immunomorphometry. After removal, by absorption, of the fraction of antibodies directed against the autochthonous faecal bacteria or cross-reacting with allogenous faecal bacteria, there were still antibodies left directed against allogenous faecal bacteria of both the IgA, IgG and IgM isotype. However, relatively more antibodies of the IgA isotype appeared to be directed against allogenous bacteria than against indigenous faecal bacteria. Persons who reacted with specific antibodies to many bacteria of their own flora also tended to react specifically to bacteria in the allogenous microflora of the other volunteers. The patterns of antibodies directed to faecal bacteria of different morphologies (morphotypes) were unique for each individual.

We have investigated the antibiotic inactivating capacity of intestinal contents in vitro in faeces. In the presently reported study the influence of beta-lactamase catalyzed hydrolysis on the antimicrobial activity of 13 commonly used beta-lactam antibiotics was investigated, while the influence of non-specific adsorption of antibiotics to faecal compounds was also taken into account. The following antibiotics were tested: benzylpenicillin, amoxicillin, amoxicillin/clavulanate, cloxacillin, piperacillin, temocillin, cefuroxime, cefamandole, cephradine, cefotaxime, ceftazidime, aztreonam and imipenem. Faecal samples were obtained from 30 healthy volunteers. Six different concentrations of each antibiotic were added to 1 g of faeces. After 24 h of incubation at 37 degrees C the remaining amount of active antibiotic was determined by means of a "growth inhibition assay". The contribution to the test results of non-specific adsorption to macromolecules was calculated by means of a model and the inactivation data were subsequently corrected. The amount of antibiotic non-specifically bound to faecal macromolecules varied from 0% to 80% of the amount of antibiotic initially added to the faeces. A considerable difference was found in the degree of inactivation of several antibiotics. However, in contrast to earlier investigations, the results of this study show that in a normal population the influence of beta-lactamase catalyzed hydrolysis on the activity of beta-lactam antibiotics is apparently very small when compared to the influence of non-specific adsorption of beta-lactam antibiotics to faecal compounds.

The accuracy of an improved quantitative immunofluorescence method to measure the antibody binding capacity of intestinal bacteria was assessed. The improvements comprise: calibration on a non-fading fluorescence standard (uranyl glass), prolonged camera exposure time (over 4 s) and shading correction of the digitized image. This method can measure levels of IgG, IgM and IgA with variation coefficient of 6.1%, 6.4% and 9.8%, respectively. With this accuracy biological alterations in the antibody binding capacity of intestinal bacteria can be monitored at a statistically significant level.

SUMMARYA new technique to study the prevalence of circulating antibodies directed against different morphological groups (‘morphotypes’) of bacteria in fresh faeces is presented. The technique combines quantitative indirect immunofluorescence with digital image analysis. Plasma antibody titres and patterns of IgA, IgG and IgM isotype against morphotypes of faecal bacteria were determined in ten healthy individuals.

The normal flora of the intestinal tract, mainly consisting of anaerobic bacteria, protects the host against colonization by pathogenic microorganisms. Antimicrobial treatment with ceftriaxone may influence the colonic microflora and as a consequence, the protective effect. Ten healthy volunteers received 1 g of ceftriaxone intramuscularly for five days. This resulted in a significant decrease (p

The influence of intestinal flora modulation by oral bacitracin on the recovery of myelopoiesis after Ara-C was studied in C3H/Law mice. Bacitracin resulted in a 3-5 log increase of Gram-negative bacteria and a 10-fold increase of the intestinal endotoxin concentration. Initiation of bacitracin before Ara-C stimulated the initial rebound increase of colony-forming units for granulocytes and macrophages (CFU-GM) from 23.2 +/- 1.3 to 28.4 +/- 1.4 x 10(3) per femur. Starting the bacitracin after Ara-C advanced the second phase of the rebound CFU-GM increase with 6 days. An important role in the recovery of myelopoiesis after cytostatic drugs in C3H/Law mice is suggested for the intestinal Gram-negative microflora, probably mediated by bacterial endotoxin.

The influence of oral treatment with a suspension of non-pathogenic Escherichia coli cells (commercially available as: Symbioflor II) on the morphological composition of the gut microflora and on the systemic humoral immune response (the IgG-, IgA- and IgM-isotype) against the bacterial cells in the Symbioflor II preparation was measured. After a pretreatment period of 21 days, ten healthy human volunteers ingested 1*10(8) cells of E. coli daily for 14 days. Thereafter a follow-up period of 28 days completed the study. The results of this study indicated that no effect of the treatment on the composition of the gut microflora could be observed. However, the immune-fluorescence measurements revealed a significant increase in circulating amounts of IgG directed against the administered E. coli cells. It is concluded that the treatment only resulted in a specific humoral immune response, while the gut microflora is not modulated.

SUMMARYExperimental data suggest a role for the microflora in the disease expression of systemic lupus erythematosus (SLE). In active SLE anti-ds-DNA antibodies are supposed to be pathogenic by forming immune complexes with DNA. Bacteria might induce the production of anti-ds-DNA antibodies. To explore the relation between the host and his microflora in SLE in comparison with healthy controls we studied the prevalence of systemic antibodies to faecal bacteria that were discriminated by their morphology by indirect immunofluorescence.IgM titres against their own faecal microflora were found to be lower both in active and inactive SLE when compared to healthy individuals. IgG-class antibacterial antibodies were increased in inactive SLE but decreased in active SLE compared to inactive SLE and healthy controls, although plasma levels of total IgG were almost doubled in active SLE. The lower IgG antibacterial antibody titres in active SLE might possibly result from sequestration of these IgG antibodies in immune complexes, indicating a possible role for antibacterial antibodies in exacerbations of SLE.

The influence of Enterococcus faecalis on the morphology of the bacterial cells which make up the gut microflora and on the levels of circulating IgG bound to the gut microflora was assessed. After 29 days of pretreatment monitoring, ten healthy human volunteers ingested 10(7) viable cells of E. faecalis three times daily, for 21 days. After this treatment another 21 days of follow-up completed the study. Each volunteer delivered eleven faecal samples during the entire study period of 71 days with a 7 day interval. Before and after the faeces sampling period, blood samples were collected from all volunteers. The influence of the ingestion of E. faecalis on the morphology of the gut microflora was measured by image analysis. In addition, the binding of circulating IgG to intestinal bacteria in all intermediate faecal samples was measured by means of quantitative immunofluorescence. The oral administration of E. faecalis resulted in a significant change of the morphological composition of the gut microflora and in a significant decrease in IgG-binding capacity of the gut microflora.

In vitro and in vivo studies have shown that various strains of ''viridans streptococci'' (nongroupable alpha-hemolytic streptococci) inhabiting the oropharynx suppress the growth of gram-positive and gram-negative microorganisms. We conducted an inventory of the oropharyngeal flora from ambulatory asthma and chronic obstructive pulmonary disease (COPD) patients and a control group to examine the interaction between viridans streptococci and potential pathogens in vivo. In addition, the difference in colonization patterns of these bacteria was studied. Oral washings from 195 patients, 48 asthma (24.6%), 147 COPD (75.4%), and 157 control subjects were examined microbiologically on two occasions with a 2-wk interval, resulting in a total of 384 and 295 oral washings, respectively. All patients were ina stable phase of disease throughout the study. The distribution of low (less than or equal to 10(4)/ml) or high (greater than or equal to 10(5)/ml) concentrations of viridans streptococci did not differ substantially between asthma or COPD patients and control subjects. Potentially pathogenic microorganisms found in a low (less than or equal to 10(4)/ml) or high (greater than or equal to 10(5)/ml) concentration were equally distributed between the two groups. Staphylococcus aureus and beta-hemolytic streptococci were found significantly less often in the asthma and COPD group (p

SummaryThe use of polyclonal antibodies in differentiating between samples of faecal microflora derived from ten healthy volunteers was assessed. The distribution of FITC-labelled antibodies (of the IgA, IgG and IgM isotype) over 144 morphologically distinct subsets of faecal bacteria (‘morphotypes’) was quantified by means of digital morphometry and quantitative immunofluorescence. Furthermore, a new dataprocessing algorithm was developed which makes objective quantitation of the antibody distributions over the faecal morphotypes possible. The results of this study imply that the antibody binding capacity of faecal morphotypes is a unique characteristic of faecal microflora.

On the basis of previously obtained evidence that Gram-negative bacteria may influence the activity of leukaemia, a study of the composition of the flora, the immune stimulation by the Gram-negative bacteria and the endotoxin concentration in faeces was conducted in patients with low-grade malignant B-cell lymphoma as well as in patients with acute leukaemia. In these patients it was investigated whether the number of facultative anaerobic Gram-negative bacteria in the faeces correlated with endotoxin concentration. In addition, IgA coating of Gram-negative bacteria in the faeces was determined and the titre of circulating antibodies to endogenous Enterobacteriaceae species from the faeces of the corresponding patient was studied. No clear difference was found to exist in the percentage of IgA-coated Gram-negative bacteria in the faeces and in the circulating antibody titres to endogenous Enterobacteriaceae between healthy control persons, lymphoma patients and patients with acute leukaemia. Antimicrobial treatment to establish selective decontamination (SD) of Enterobacteriaceae species from the digestive tract did cause a significant decrease in the faecal endotoxin concentration in a subset of patients treated for SD with polymyxin.

The colonization resistance (CR) of the gastrointestinal tract to potential pathogens depends partly on factors within the host but to a greater extent on the normal (anaerobic) gut flora. Its strength varies between individuals. These individual differences in resistance to colonization by pathogenic microorganisms may explain differences in susceptibility to infection. CR is lowered by remission-inducing treatment (radiation and/or chemotherapy) in leukaemia, but more severely by certain antibiotics. Development (by selection or transfer) of resistance to these antibiotics may lead to overgrowth and penetration of the mucosal lining by the overgrowing (potentially) pathogenic bacteria. Other antibiotics however, if sufficiently dosed, have been found to eliminate (potential) pathogens selectively without decreasing CR. This selective decontamination of the gastrointestinal tract has been successfully used prophylactically in neutropenic patients but needs to be monitored bacteriologically. It should perhaps be used more widely in the hospital to control development and spread of antibiotic-resistant strains.

Aztreonam, a minimally absorbable monobactam antibiotic that is active primarily against gram-negative bacilli, was given orally to 10 healthy volunteers in three regimens: 60 mg, 300 mg, and 1,500 mg daily for five days. The regimens were administered at least two weeks apart so that the flora could return to normal. Aztreonam had no effect on oropharyngeal flora cultured every other day. Fecal cultures from volunteers receiving the 300-mg regimen yielded no gram-negative bacilli after an average of 4.4 days of treatment. Administration of the highest dose gave similar results, albeit after a shorter interval (3.0 days). None of the regimens appreciably influenced total microscopic (anaerobic) bacterial counts in the feces. However, during administration of the 1,500-mg regimen, fecal counts of enterococci increased from an average of approximately 10(4)/g to greater than 10(7)/g, and fecal counts of yeasts tended to increase by the end of the five-day treatment period. Fecal concentrations of aztreonam increased as the dose increased in eight volunteers. No aztreonam was recovered from the feces of two volunteers, perhaps because of drug inactivation.

After oral treatment with polymyxin for only 1 day, fecal aerobic gram-negative bacteria were found completely suppressed in C3H/Law mice. Complete suppression of aerobic gram-negative bacteria was accompanied by a reduction of the fecal endotoxin concentration from 100 to 10 micrograms endotoxin per gram of feces as measured with the Limulus amebocyte lysate assay. Oral administration of polymyxin affected hemopoietic stem cell kinetics at different stages. The kinetic behavior of hemopoietic stem cells was determined as the in vivo sensitivity to the S phase specific cytostatic drug hydroxyurea. The hydroxyurea kill of bone marrow spleen colony-forming cells diminished not significantly (p less than 0.10) from 14 to 4% after 2 days of polymyxin treatment. Already after 1 day of treatment the hydroxyurea kill of bone marrow progenitor cells forming granulocyte-macrophage colonies in vitro decreased from 29 to 7% (p less than 0.05). It took 8 days of treatment before the hydroxyurea kill of splenic granulocyte-macrophage colonies was found reduced from 53 to 14% (p less than 0.001). The decreased susceptibility of hemopoietic stem cells to hydroxyurea during polymyxin treatment appears to argue for a role of intestinal aerobic gram-negative bacteria in the regulation of hemopoiesis, probably mediated by endotoxin.

Article de synthese. Etude des divers aspects de l'ecologie microbienne de l'intestin dans des modeles animaux. Developpement de la flore intestinale apres la naissance. Facteurs ecologiques associes a la colonisation de l'intestin par Clostridium difficile

Summary If antimicrobial drugs are used without paying notice to their effect on the digestive tract microflora, resistance, « overgrowthand super-infection may emerge. Regarding their effect on the alimentary tract flora, antimicrobial drugs can be classified in : 1. drugs which do suppress the CR-determining (anaerobic) flora and 2. in antimicrobial agents which leave the CR responsible flora unchanged. The first category of antibiotics will not cause « overgrowthby resistant microorganisms. They can even be successfully applied for infection prophylaxis in neutropenic patients, provided they are sufficiently high dosed to completely suppress all susceptible potential pathogenic microbes which colonise the digestive tract. This application of CR-indifferent drugs is called Selective Decontamination of the digestive tract (SDD). Antibiotics on the other hand which decrease the CR by suppressing CR-responsible (anaerobic) bacteria in the alimentary canal, should become « second choice antibiotics; particularly in a clinical setting. If these drugs are indicated on the basis of the sensitivity pattern of microorganisms involved in an infection, on pharmacokinetic properties, or on kidney function or allergy for CR-indifferent drugs, combination of therapy with SDD should be considered.

SUMMARYThe effect of oral administration of antibiotics on the intestinal flora of conventional mice and their resistance to colonization by orally introducedEscherichia coli, Klebsiella pneumoniaeandPseudomonas aeruginosawas studied. Colonization resistance (CR) was expressed as the log of the oral bacterial dose followed by a persistent take in 50 % of the contaminated animals. The intestinal flora was virtually eliminated by the antibiotics and this elimination was accompanied by a precipitous fall of CR. CR gradually returned to normal values during the period of repopulation of the intestinal tract by the organisms surviving the treatment. Antibiotic treatment resulted in the disappearance of Enterobacteriaceae, enterococci, staphylococci and yeasts and, under appropriate housing conditions, the animals remained free of these organisms indefinitely. Germ-free mice contaminated with the intestinal flora of an antibiotic-treated animal and their offspring housed in a germ-free isolator showed high values of CR. Their intestinal flora consisted of anaerobic bacteria only. Apparently, these anaerobes are responsible for CR in these and in conventional mice.

SUMMARYThe effect of oral administration of neomycin cephalothin or kanamycin cephalothin on the aerobic intestinal bacterial flora, was studied in dogs maintained under isolation conditions in a conventional animal room. The dogs were successfully freed of aerobic bacteria with both combinations within two to seven days after the start of antibiotic treatment, and were maintained bacteria free for up to 21 days. Decontamination was attained more rapidly in dogs that were bathed in hexachlorophene surgical soap before and during the first and third days of antibiotic treatment. There was no evidence of toxicity from either of the antibiotic combinations. These results indicate that, as with mice and monkeys, decontamination of dogs with oral antibiotics is feasible. The technique is of potential value in preventing endogenous bacterial infections in canine experimental studies involving use of immunosuppressive agents.

This paper describes a novel method for detecting immunoglobulin A (IgA) coated potentially pathogenic microorganisms (PPMs) in the human intestine. Essentially, the technique consists of 2 phases: one in which IgA coated bacteria are detected by immunofluorescence and a second in which these bacteria are subcultured in situ and subsequently identified. In this way transient bacteria are differentiated from resident bacteria. These results show that the resident bacteria are coated with IgA. Resident microorganisms are always highly concentrated in the digestive tract. These results strengthen the hypothesis that only the high antigen concentrations achieved by a large number of resident bacteria are capable of IgA induction.

The Gut Associated Lymphoid Tissue [GALT] is considered to play an important role in infection prevention and in excluding the absorption of antigens [immune exclusion]. Secretory immunoglobulin A [S-IgA] on the mucous membranes may be involved in this protective GALT activity by interfering with bacterial adherence. Attachment to mucosal surfaces via fimbriae may be a prerequisite for many bacteria to colonize and to penetrate the digestive tract. Adherence of fimbriaeted bacteria is apparently inhibited by S-IgA, because the latter blocks interaction fimbriae and mucosal surface. The anti-adhesive or coating phenomenon of S-IgA may therefore be a more important measure of the functional characteristics of S-IgA than the simple quantitation of S-IgA in serum and secretions. In the present study we discuss an in vitro test system, the fluorescence immuno-assay [FIA] to determine the coating’s incidence as a functional parameter of GALT activity [GALT-FIA].

Potentially pathogenic bacteria and yeasts meet a number of resistance factors. These colonization resistance (CR) determining factors control the growth density of potentially pathogenic microorganisms in the various parts of the alimentary canal. The CR of the digestive tract is to be seen partially of host origin but to a greater extent due to direct and indirect activity of the (anaerobic) indigenous microflora. This implies that the CR mechanism is vulnerable to antibiotics which affect the indigenous flora during treatment. A decreased CR enhances colonization by microorganisms that are resistant to the antibiotic(s) used for therapy. Increased population densities of potentially pathogenic bacteria (“overgrowth”) may correlate with invasion of the mucosal tissues often followed by translocation to remote lymphatic organs. Overgrowth also may correlate with development or acquisition of resistance to the antibiotic applied. By screening in experimental animals antibiotics have been selected which do not affect the CR. Such antimicrobial drugs have successfully been used to selectively eliminate potentially pathogenic bacteria and yeasts in neutropenic patients.

The enzymatic inactivation of aztreonam by the faecal flora was investigated in 25 healthy human volunteers. Ten volunteers received aztreonam orally and 15 volunteers did not receive antibiotic treatment. Residual aztreonam and its inactivated open ring form were simultaneously detected by an HPLC-assay. Independent of aztreonam treatment, beta-lactamase activity was detected in the faecal flora of 20 volunteers. The inactivation of aztreonam by the faecal supernatants was inhibited by clavulanic acid.

A method to prevent endogenous infections in patients and laboratory animals with an impaired immune capacity is the total decontamination of the gastrointestinal tract. This is performed by oral administration of nonabsorbable antibiotics. One of the disadvantages of total decontamination is the fact that especially after termination of this treatment the decontaminated individual is easily colonized with organisms from the environment. This is the result of the elimination of the anaerobic part of the microflora, which is responsible for the so-called colonization resistance (CR). This CR is the resistance against exogenous microorganisms to colonize the gastrointestinal tract. In the absence of an anaerobic flora, the colonizing microorganisms can reach abnormally high faecal concentrations, thus increasing the risk for infection. In mice, the implantation of an anaerobic, mouse-derived flora after termination of total decontamination resulted in the restoration of a good CR, as could be shown by orally challenging the animals with a strain of Escherichia coli. Therefore, an anaerobic microflora, free of potentially pathogenic microorganisms, which was isolated from a healthy human volunteer was administered to monkeys and patients after a decontamination period in an attempt to restore CR. In the monkeys, this human donor flora (HDF) did not reduce the faecal concentration of microorganisms that had colonized the gastrointestinal tract before the donor microflora had been established, in contrast to the findings in some of the patients. Qualitative analysis of the microflora of patients which were contaminated with the human donor flora showed that the CR is not of the same quality as is found in healthy individuals. This can be the result of the impaired immune capacity of the patients at the time of HDF implantation. However, the results obtained show that implantation of the HDF in monkeys and patients after a decontamination period allows reconventionalization without an undue risk of microbial infection.

Since antimicrobial drugs first became available for the treatment of infections, resistance has developed (1–12). The increase in the number of antibiotics and in the diversity of their spectrum of activity in the last few decades has not solved the problem: the use of new antibiotics has invariably led to the development of resistance after some time. Bacteria of many different species have proven to have an almost endless capacity to develop sublines of strains that are resistant, often to more than one antibiotic.