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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.

Selective decontamination of the digestive tract was developed following the observation that the digestive tract normally has considerable resistance to colonization by newly ingested bacteria. The research that eventually led to selective decontamination was performed because in the late 1960s and early 1970s, the need for prophylaxis against Gram-negative infections in immuno-compromized patients became evident. At that time, the relatively small number of antibiotics available for therapy of serious infections often lead to treatment failure. To introduce the subject of selective decontamination, this paper, therefore, starts with a short historical overview of the kind of infectious agents as well as the antibiotics available in the 1960s; particularly regarding the type and treatment of infections in severely compromised patients. The fact that the possibilities in infectious diseases were limited was the reason for our experimental search for ways of successful prophylactic treatment with minimal risk of development of resistance.

Bacterial translocation (BT) and release of endotoxin from the gut may contribute to septic complications in severely burned patients. In earlier experiments, it was shown in a burn model that BT exceeded the role of wound dissemination when burn wounds were colonized with Pseudomonas aeruginosa. Burn wound colonization with Pseudomonas aeruginosa also enhanced BT. It was concluded that endotoxin from gram-negative micro-organisms colonizing the burn wound appeared to play a role in the increased BT. In this study, the contribution of burn wound colonization with the gram-positive micro-organisms S. aureus and Str. pyogenes to BT and endotoxemia has been examined in Escherichia coli monoassociated mice with a 30% scald. The animals were sacrificed two days post-burn. The peritoneal cavity, the heart, one lung, the liver, the spleen and the cecum were cultured. Endotoxin in plasma was determined by means of the Limulus amoebocyte lysate assay. It appeared that dissemination of E. coli from the gut played a more important role than S. aureus or Str. pyogenes dissemination from the burn wound in this model. In only one of the 15 surviving Str. pyogenes contaminated animals dissemination had taken place from the burn wound. Dissemination of S. aureus was not seen in the S. aureus contaminated group. The incidence of E. coli translocation two days postburn, to the spleen (p

SUMMARYThe colonization resistance (CR) of the digestive tract was determined in 10 healthy volunteers by oral contamination with a neomycin resistantEscherichia coli(NR-E. coli) strain and measurement of the faecal concentration of this strain during 14 days after the contamination. This ‘gold standard’ was compared with another parameter of CR; the determination of the mean number of different biotypes of Enterobacteriaceae isolated from four faecal samples per volunteer. Both measures are significantly correlated (P < 0.01). The NR-E. colistrain could be cultured from faecal samples of 4/10 volunteers as long as 300 days after contamination. Serum antibody titres against endogenousE. colistrains and the NR-E. colistrain used for experimental oral contamination were measured by an indirect immunofluorescence (IIF) assay. The assay was read by a video camera connected to an image processing system. The 95% confidence limits of antibody titres (log2) against endogenousE. colistrains ranged between < 3 and 7.1 for IgA, between < 3 and 8.7 for IgG and between < 3 and 7.4 for IgM. Antibody titres against the NR-E. coli4 strain were within this (normal) range. The serum antibody titres against the NR-E. colistrain increased slowly after oral contamination, especially IgG and IgM. Little increase in IgA titres could be observed. An increase of serum antibody titres did not correlate with the elimination of the oral contaminant from the intestines. Therefore, we conclude that the CR is not IgG nor IgM antibody mediated.

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.

Infection is a common problem in hepatobiliary surgery. Keighly and Lewis et al. reported infected bile in 31-33% of patients after an operation for biliary disease. Such patients had an increased risk of post-operative infections compared to patients with sterile bile1’2. These findings are confirmed by reports from Flemma et al. and Maddocks et al.. They found that bacteria are common in bile under pathological conditions like stones in the common bile duct, the presence of strictures especially partial strictures or cholangitis3’4. Keighly and Blenkhard also reported a strong relation between per-operative bile cultures and cultures from wound abscesses and blood after surgery on the biliary tract in patients with infected bile. In 64% of the wound abscesses and in 90% of the episodes of bacteraemia the same micro-organisms were observed as had previously been identified in the bile5. Micro-organisms frequently found in these cultures are gram-negative bacteria like E coli, Klebsiella spp or Pseudomonas spp. Sometimes gram-positives are found like Streptococcus faecalis. In patients with a biliodigestive anastomosis anaerobes may be found2’5. Intra-abdominal infections after liver resection are common. Incidences varying between 8 and 30% have been reported from different institutions6’7’8. Moreover, in liver transplant programs infection is 9 10 11 still the major cause of morbidity and mortality’ Therefore, the possible mechanisms of infection of the hepatobiliay system should be one of the major interests in hepatobiliary surgery. Bacteria may enter the liver and biliary tract in two ways. Either directly via the transsphincteric route or indirectly by translocation through the intestinal mucosa via the portal blood or the lymphatic system12’13, In both instances the gastrointestinal tract is the source of the invading micro-organisms. Normally, the upper part of the gastro-intestinal tract contains only few bacteria. This is due to the combined influence of acid secretion of the stomach and the inhibitory action of bile and pancreatic juices on many micro-organisms. The majority of the bacteria present are gram-negative cocci and baccilli. Occasionally

SUMMARYThe present study has attempted to determine the colonization resistance (CR) of the digestive tract by biotyping Enterobacteriaceae in four faecal samples per subject of five different animal species as well as man. The results indicate that the degree of bacterial contamination with Enterobacteriaceae from the environment may strongly influence the outcome. Both conventionally living chicken and man, showed a much wider range of the ‘confidence limits of the mean’ of the mean number of biotypes per faecal sample between individual subjects, than was found between subjects maintained under laboratory circumstances. Yet there appeared a statistically significant difference in CR between some of the animal species as a group. Man did not differ from monkeys, however, both differed from the rodents species studied. Monkeys differed also from dogs and the latter from rodents. It is concluded that the CR measured by determining the mean number of biotypes of Enterobacteriaceae can only be used for accurate comparison of the CR between subjects, if the ‘bacteriological environment’ is known; i.e. the sources of contamination.

A study of intestinal colonization resistance (CR) in breastfed versus formula-fed newborns at 4 intervals after birth in Jakarta, Indonesia, is described. To measure the intestinal CR for gram-negative enterobacilli, mean values of Enterobacteriaceae concentrations and mean numbers of Enterobacteriaceae biotypes were determined. The CR values found in this study show, that in all 4 sampling periods, at

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.

An International Study Group on New Antimicrobial Strategies (ISGNAS) has been formed in response to the recognition that development of microbial resistance to antibiotics is becoming a serious, world-wide problem. The group met in 1993 for the first time to discuss the feasibility of developing rational alternatives to the use of antibiotics and prepared, as a result, a comprehensive overview of normal (physiological) mechanisms involved in the control of potentially pathogenic (oppotunistic) microorganisms. One objective of ISGNAS is to understand the conditions which allow opportunistic microbes present among the symbionts to cause an infection. There is a need for more coherent information concerning the habitat, growth requirements and host and pathogen properties which allow opportunistic pathogens to cause life-threatening infections. In particular, information is urgently being sought to understand the complexity of the interactions between the vast number of microbial species, and the interactions between the microbes and their host. Another goal is to inspire and enable basic and clinical research that will lead to the development of new therapies for regulating colonization, translocation and infection by opportunistic micro-organisms in patients during periods of decreased resistance. With a sufficient amount of knowledge of how healthy individuals keep opportunistic micro-organisms under control, it may become feasible for physicians to maintain host resistance and inter-microbial factors involved in the containment of opportunistic microbes. Therapies aimed at boostering natural resistance mechanisms will be of critical importance to individuals whose resistance has been compromised as a result of another clinical condition.

BACKGROUND--Bacterial infections of the lower airways during an exacerbation in patients with asthma or chronic obstructive pulmonary disease (COPD) may be the cause of an exacerbation or the consequence of a viral infection or an increase in airways limitation. To determine whether bacterial infection is an important component in the pathogenesis of an exacerbation, the effects of antimicrobial treatment must be studied. METHODS--Patients with asthma or COPD seen in general practice were studied in a double blind randomised manner to investigate whether the antimicrobial drugs amoxicillin (500 mg three times daily), cotrimoxazole (960 mg twice daily), or a placebo, each when added to a short course of oral corticosteroids, can accelerate recovery from exacerbations. Patients were instructed to contact their own physician early in the morning when complaints of increased shortness of breath, wheezing, or exacerbations of cough with or without sputum production occurred. Treatment effects were evaluated over the next 14 days by studying symptom scores (wheeze, dyspnoea, cough with and without mucus production, and awakening with dyspnoea), peak expiratory flow values (PEF, expressed as % predicted), and sublingual temperature. Bacteriological study of the sputum was made at the onset of an exacerbation and 7, 21 and 35 days afterwards. RESULTS--Of 195 patients enrolled 71 (36%) contacted their physician for symptoms of an exacerbation. Symptoms improved in all three groups, improvements ranging from 0.54 to 0.75 points per day on a four point scale. PEF% predicted showed improvements in the three groups after the exacerbation, ranging from 0.34% to 0.78% predicted per day, finally returning to baseline values. Sublingual temperature did not change. Six of 71 patients consulted their physician because of a relapse between four and 24 days after the start of treatment. In only two of the 50 sputum samples, collected during an exacerbation, and which contained > or = 10(5) bacteria in culture sensitive to the chosen antibiotic given, did any benefit from antimicrobial treatment occur. During the recovery period sputum purulence improved irrespective of antibiotic treatment. CONCLUSIONS--Antibiotics given with a short course of oral prednisolone during an exacerbation do not accelerate recovery as measured by changes in peak flow and symptom scores in ambulatory patients with mild to moderate asthma or COPD when treated by their general practitioners. Moreover, antibiotics do not reduce the number of relapses after treating an exacerbation.

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.

A system to improve the sensitivity of an industrial charge coupled device (CCD) video camera by increasing the exposure time is presented. The system consists of an expansion board for the IBM-PC, and interface software. The gain in sensitivity is shown to be a linear function of exposure time. Exposures of up to 8.5 seconds have been tested and they show that a 256-fold increase in sensitivity is attainable.

Im Zeitraum der letzten 10 Jahre konnten in der antimikrobiellen Behandlung von stark immunsupprimierten Patienten bedeutsame Fortschritte erreicht und eine grose Anzahl von Antibiotika mit einer ein breites Spektrum abdeckenden Aktivitat entwickelt werden. Die Bedeutung der sofortigen empirischen oder kalkulierten Antibiotikatherapie bei Ausbruch fieberhafter Episoden granulozytopenischer Patienten ist seit den 70er Jahren allgemein anerkannt (EORTC 1978 a). Diese haufig kostenwirksame parenterale Behandlung mit Antibiotikakombinationen (Tattersal et al. 1972; Klastersky et al. 1974; Issel et al. 1979; Schimpff 1979; Menichetti et al. 1986) ist bis zum Ende der Knochenmarksuppression fortzufuhren, um die Wahrscheinlichkeit einer Infektion zu vermindern. Hierbei ist allerdings die Moglichkeit der Ausbildung und Selektion von Resistenzphanomenen gegen primar wirksame Antibiotika gegeben, da am Standort potentiell pathogener Bakterien unvermeidlich auch subinhibitorisch wirksame Konzentrationen von Antibiotika auftreten konnen. Im Fall einer erworbenen Resistenz konnen die resistenten Bakterien die durch Antibiotika gehemmte Standortflora uberwuchern und auf andere Patienten der Station ubertragen werden (Schimpff et al. 1972). Dies ist insofern von groser klinischer Bedeutung, da auf einer hamatologischonkologischen Station bei praktisch allen Patienten im Laufe der ersten Wochen einer Remissions-Induktionstherapie Fieber auftritt.

In order to obtain a method that could predict the in vitro inactivation of an antibiotic in the digestive tract, the non-enzymatic inactivation of 13 beta-lactam antibiotics by human faeces was investigated. Benzylpenicillin, amoxicillin, amoxicillin/clavulanate, cloxacillin, piperacillin, temocillin, cefuroxime, cefamandole, cephradine, cefotaxime, ceftazidime, aztreonam and imipenem were mixed in six graded concentrations with faecal suspensions of 30 healthy volunteers. After incubation the remaining antimicrobial activity was measured by means of a serial dilution method. A relationship between the initial antibiotic concentration (Aia) and the remaining antimicrobial activity after incubation (Asd) was derived, namely: Asd = 1/2[(Aia-K-F)+square root ((Aia-K-F)2 + 4 K Aia)]. The parameters K and F represent the intrinsic dissociation constant and the amount of available bindingsites in the faeces, respectively. Asd values were predicted with a reasonable degree of accuracy for all thirteen antibiotics. Dissociation constants differed significantly between the penicillins and the cephalosporins but F- values did not differ between cephalosporins and penicillins.

Bacterial translocation (BT) from the gastrointestinal tract has been proposed to play a role in the pathogenesis of septic complications in severely burned patients. In a burn model the effect of a subtherapeutic dose of polymyxin B-sulfate (PB) at BT was examined in Escherichia coli-monoassociated mice with Pseudomonas aeruginosa-inoculated burn wounds. The BT incidence and number of translocating microorganisms to the spleen (p less than 0.01), liver (p less than 0.01), lung (p less than 0.05) and heart (p less than 0.05) were diminished significantly in the PB-treated versus the untreated group. Endotoxin in plasma was detectable in one of the 16 PB-treated versus 6 of the 17 control mice (p less than 0.05). The relation of Pseudomonas burn wound inoculation, BT, endotoxin and the endotoxin-neutralizing properties of PB will be discussed.

11 volunteers received 1 g ceftriaxone i.m. every 24 h for 5 consecutive days. Volunteers were selected on the basis of the "ceftriaxone inactivating capacity" of their faeces, which should be 250 mg/kg faeces. The effect of treatment on the aerobic oral and faecal flora was studied. In the oral flora a temporary suppression of the viridans streptococci occurred, which normalised already during treatment. More spectacular was the effect on the faecal flora: suppression of aerobic Gram-negative bacilli with overgrowth of yeasts and enterococci. Two weeks after treatment the results of the faecal cultures were almost the same as before treatment in most of the volunteers. One volunteer experienced severe diarrhoea, starting the day after completion of ceftriaxone treatment and lasting 2 days.

In a randomized study comparing cotrimoxazole plus colistin with ciprofloxacin, each in combination with nonabsorbable antimycotics, the incidence of major infections in terms of septicemias and pneumonias as well as of minor infections and episodes of unexplained fever (FUO) was higher in patients treated with ciprofloxacin. In cases of microbiologically documented infections, gram-positive cocci dominated by far. In surveillance cultures of oral washings and of feces, gram-negative enterobacteria were only rarely detected; however, large numbers of cultures were positive for Acinetobacter species. There were four cases of documented Pneumocystis carinii pneumonia in patients not receiving cotrimoxazole. The incidence of documented mycotic infections as well as the detection of fungi in surveillance cultures was similar in both treatment groups. A decrease in the number of adverse events, especially of allergic reactions, could not be achieved by the administration of ciprofloxacin. In conclusion, cotrimoxazole plus colistin in combination with nonabsorbable antimycotics remains the standard regimen for prevention of infection in patients with acute leukemia undergoing aggressive remission induction therapy. A detailed analysis of study II will be prepared for publication.

In this study the colonization pattern was investigated of a mouse-related (Colonization Resistance-) microflora (CRF-MF) and an unrelated human microflora (HUM-MF) in germfree isolated C3H/Rij mice. The composition of the HUM-MF appeared to change whereas the CRF-MF did not. HUM-MF associated animals also mounted a higher serum antibody level against 50–40% of the high concentrated obligate anaerobic bacteria, whereas this fraction was less than 10% in CRF-MF mice. We conclude that the colonization of the gut is determined immunologically by the antigenic composition of the microflora for which tolerance appears to exist.

SUMMARYThe influence of treatment with increasing oral doses of three absorbable antibiotics on the colonization resistance of the digestive tract was investigated in mice. Mice treated with ampicillin or epicillin in any of the applied doses had a strongly decreased colonization resistance as demonstrated by ‘bacterial over-growth’ after contamination with resistant strains ofEscherichia coli. After a treatment period of 2 weeks,Streptococcus faecalisbecame resistant in a number of animals. Oral treatment with cephradine on the other hand had no obvious influence on the endogenous flora of the mice, nor was the colonization resistance decreased.

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.

We describe the bacteriological results of a controlled clinical trial of selective decontamination of the digestive tract as a method of infection prevention in granulocytopenic patients. Selective elimination of Enterobacteriaceae and Pseudomonadaceae species was accomplished by the oral administration of nalidixic acid, co-trimoxazole, or polymyxin. Yeasts were eliminated selectively by amphotericin B or nystatin treatment. The drugs used in this study were chosen because of their capacities to selectively eliminate gram-negative rods and yeast without affecting the anaerobic part of the gut flora which is responsible for colonization resistance. Compared with the control group, the selectively decontaminated patients had significantly fewer (P less than 0.0005) gram-negative rods or yeasts or both in their throat swab cultures and in their feces. This reduction may explain the clinical effectiveness of selective decontamination.

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.

Summary Oral treatment of groups of four mice with different daily dosages of three related antibiotics, amoxycillin, augmentin and bacampicillin, has indicated the influence of the amount of the dose that reaches the intestine in a biologically active form. Augmentin (amoxycillin plus clavulanic acid to protect it against enzymatic hydrolysis) appeared to have a suppressive effect on the indigenous colonization-resistance-associated microflora. Dose-effect curves of amoxycillin alone, showed the same shape but at a lower level. Bacampicillin treatment practically did not have an effect on the faecal flora. Only at doses of bacampicillin of well above 1.5 mg per day, an indication was seen of CR-flora disturbance. At a dose level of 2 mg and more per day, a low concentration of β-aspartylglycine was found in the faeces. A normal undisturbed intestinal flora normally produces in mice sufficient enzyme to degrade completely this dipeptide released by the host organism into the intestinal tract.

SummaryThe clearance of neomycin and kanamycin from the intestines after stopping oral supply has been determined in mice. Both antibiotics, although given in different doses, were excreted in essentially the same way; the clearance being a little faster than logarithmically in both cases. The importance of this observation with regard to isolation and the moment of reconventionalization is discussed.

After at least two weeks of decontamination, rhesus monkeys were submitted to a total body irradiation of 8.5 Gy, followed by a bone marrow graft. In the following weeks, a number of colonizations were found. Twenty-seven colonizations were known to be present on the day of irradiation. Sixteen colonizations were due to microorganisms suppressed before irradiation. Thirty-two colonizations were considered as exogenous. Only a few colonizations in the irradiated animals could not be controlled. Irradiation caused severe diarrhoea in decontaminated animals, leading to a life-threatening loss of water and electrolytes. When this loss was corrected, the monkeys survived for prolonged periods following irradiation. The amount of food consumed, which contained antibiotics, had no effect on the faecal concentration. The addition of solid food particles, however, resulted in a much lower faecal concentration. No significant antibiotic serum levels were found.

SUMMARYOral treatment of mice with various doses of neomycin or polymyxin B was performed in order to determine which dose caused substantial suppression of aerobic gram-negative rods. In addition the effect of the various doses on Streptococcus faecalis and on other factors of the colonization resistance (CR) of the digestive tract were studied. It was found that polymyxin B was effective in suppressing sensitive gram-negative bacteria following daily doses of 3·2 mg/ mouse, and that even extremely high daily doses of 9·7 mg/mouse did not affect the CR. Neomycin was effective in suppressing Enterobacteriaceae species following oral daily doses of 5·4 mg/mouse. With this dose, however, the CR was somewhat decreased which was also evidenced by the increased concentration of beta-aspartyiglycine in the faeces and the increased size (weight) of the caecum in these animals. Suppression of Sir. faecalis was seen from doses of 24 mg/mouse on.

Tobramycin has been given orally to eight human volunteers for four successive days, to investigate its effect on the Gram-negative enterobacilli as well as on the endogenous anaerobic microflora. The effect was investigated in three treatment legs; i.e. in daily doses of 300 mg, in daily doses of 500 mg and thirdly in daily doses of 200 mg in combination with 1000 mg of neomycin. With 300 mg tobramycin daily, seven of eight volunteers had no Gram-negative bacilli in their faecal cultures by about four days after the onset of treatment. Their anaerobic micro-flora was slightly affected during treatment as evidenced by the appearance of low concentrations of beta-aspartylglycine in their stools. The other dose regimens were not significantly more effective in eliminating Gram-negative bacilli from the intestines; the anaerobic flora however, was more severely affected. Because evidence of induction of resistance was noticed it is recommended that if tobramycin is used for selective decontamination of the intestinal tract it should be given in combination with another antimicrobial drug such as polymyxin.

2% amphotericin B in orabase® was applied to the buccal mucous membranes of healthy volunteers. Amounts of 1 g and 0.5 g were used. The experiments were performed during the day time as well as during the sleeping hours at night. At several intervals after application saliva samples were collected and the amphotericin B concentrations were determined. Application of 1 g of orabase® with 2% amphotericin B appears to maintain a sufficiently high concentration for four hours, 0.5 g for three hours. Application of these amounts before going to sleep guarantees a sufficient concentration until awakening the next morning.

Four broad-spectrum antibiotics-azlocillin, mezlocillin, cefuroxime, and moxalactam - were injected subcutaneously into mice twice a day. The animals divided into treatment groups for each antibiotic and then into subgroups, each subgroup receiving a different dose of antibiotic. The effect of treatment on the resistance of the digestive tract to colonization and the effect of treatment on endogenous gram-negative and intestinal streptococcal flora was studied: resistance to colonization decreased during treatment with approximately 0.9 mg of antibiotic per mouse per day. During treatment with this dose or a higher dose, Escherichia coli or a strain of Enterobacter resistant to the antibiotic being used grew to significantly higher numbers per gram of feces than in the control groups. The resistance of the digestive tract to colonization in mice decreases during systemic treatment with all four antibiotics at or above certain dose levels, a result also likely to occur in humans.

Experimentally induced ulcerative disease of the large intestine in guinea pigs was significantly mitigated by selective elimination of the aerobic gram-negative intestinal microflora. Lesions were induced with 2 or 5% degraded carrageenan administered in the drinking water for 30 to 44 days. Craterous cecal or colonic ulcers, crypt abscesses, mucosal distortion, mesenteric lymphadenopathy, marked cecal or colonic lymphoid hyperplasia, and other lesions were seen in conventional guinea pigs treated with degraded carrageenan. Other guinea pigs that were treated with 2% degraded carrageenan, but freed of Enterobacteriaceae species by the administration of trimethoprim with sulfamethoxazole, and subsequently maintained in isolators, exhibited virtually no lesions. Enterobacteriaceae-free animals that were treated with 5% degraded carrageenan had significantly fewer lesions than were observed in the conventional degraded carrageenan-treated animals. Biotyping of bacterial isolates indicated that the family of Enterobacteriaceae species generally, and not a single biotype, was associated with the lesions. The bacteria presumably stimulated an immunological response after penetration of the intestinal mucosal barrier; thus, inflammatory bowel disease in guinea pigs, induced with degraded carrageenan, may provide a useful model for investigating immunological aspects of human inflammatory bowel disease.

Summary The present paper deals with the influence of a 10 days treatment with mezlocillin, piperacillin, cefotaxime, clindamycin or gentamicin on the endogenous intestinal microflora of Balb/c-mice and on the local growth of sarcoma L-1 tumor. Clindamycin and gentamicin demonstrated no influence, whereas cefotaxime and piperacillin caused the eradication of gram-negative resp. gram-positive bacteria but these antibiotics didn't produce a growth inhibition of local L-1 sarcoma tumor. The oral or parenteral application of mezlocilllin (a 3 days treatment was sufficient) eradicated the complete aerobic and anaerobic intestinal microflora. This effect was significantly correlated with an increase of the cecum weight and the inhibition of local tumor growth. Possible mechanisms of these effects are discussed.