Development and persistence of multi-resistance to antibiotics in bacteria; an analysis and a new approach to this urgent problem.

The intestinal microflora may have more influence on infectious diseases, than the mere control of growth of opportunistic micro-organisms by colonisation resistance (CR) and unspecific stimulation of the immune system. In compromised patients the CR may become decreased for several reasons but mostly because antibiotics reach the intestine during treatment. The consequence of a CR-decrease is that antibiotic-resistant opportunistic micro-organisms may increase in numbers in the gut. In this context, it is hypothesised that if the CR could be maintained at a normal level, the risk for maintenance and spread of resistant strains could be mitigated. Such maintenance requires absence of active antibiotic substance in the gut. This might be brought by the inactivation of antimicrobial agents by intestinal contents. Intra-intestinal inactivation has been described to occur along two possible routes: (1) inactivation by chemical binding or absorption and (2) by enzymatic destruction. Secondly, the composition of the intestinal microflora should be maintained at a normal level in case of other reasons for CR-decrease than antibiotic activity. Comprehensive study of the composition of normal microflora and the strains of species which play a role in CR with techniques which have become available during last decade, is recommended as well as the application of certain pre- and probiotics. It is concluded that antibiotic inactivation may be an ancient strategy of nature which should become incorporated in antibiotic treatment. Antibiotic use and development of resistance may have occurred when ecosystems formed several billions of years ago. Protection against antibiotics produced by newcomers into the ecosystem may have developed as it was necessary to maintain locally available nutrients for the inhabitants of the ecosystem. Should this hypothesis be correct, it is plausible that antimicrobial inactivation by antibiotic inactivating molecules is ubiquitous. In the ecosystem of the digestive tract, molecules involved in inactivation may predominantly be formed by microorganisms.

Influence of breastmilk on the development of resistance to intestinal colonization in infants born at the Atma Jaya Hospital, Jakarta.

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 < 1, 2, 4 and 6 months, the mean concentration of Enterobacteriaceae was somewhat lower in the breastfed group than in the formula-fed group (only significant at 6 months). This means that the intestinal CR of the breastfed group may have been slightly higher than that in the formula-fed group. In both study groups, the CR was lower in the second and fourth month than soon after birth and at 6 months. For epidemiological reasons, comparison was performed of the Enterobacteriaceae biotypes found in samples from mother and child. The data show that, in the first sampling period, regardless of the theoretical possibility of a 'more intense (skin) contact' during breastfeeding (which might promote transfer of also microorganisms), the breastfed infants had a significantly lower percentage of identical Enterobacteriaceae biotypes than did the formula-fed group. This could possibly be ascribed to a higher CR in the breastfed group. Determination of the concentration of Enterococcus species was found applicable to reproducibly measure the CR in the newborns at 6 months and in the mother-group.

Escherichia coli as a probiotic?

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.

Problems and priorities for controlling opportunistic pathogens with new antimicrobial strategies; an overview of current literature.

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.

In vivo IgA coating of anaerobic bacteria in human faeces.

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.

Micromorphometrical analysis of rodent related (SPF) and unrelated (human) gut microbial flora in germfree mice by digital image processing.

Digital image processing (DIP) of bacterial smears is a new method of analysing the composition of the gut microbial flora. This method provides the opportunity to compare and evaluate differences in the complex highly concentrated anaerobic fraction of gut microbial flora, based on micromorphological differences. There is ample evidence that this fraction can be characterized as related or unrelated to the host organism by its immunogenicity. In this study germfree ND2 mice were associated with either related (rodent) SPF microflora (SPF-MF) or unrelated human MF (HUM-MF). DIP analysis was performed on original SPF-MF and HUM-MF and on the faeces of ex-germfree mice 4 weeks after association. The micromorphological pattern of highly concentrated anaerobic bacteria in faeces of HUM-MF associated ex-germfree mice was significantly different from SPF-MF associated counterparts with regard to the scores for elongation (P < 0.01) and morphological variety (P < 0.05). Moreover, gross morphological variability was present between individual HUM-MF associated mice but not between individual SPF-MF associated animals. No differences were found between original SPF and HUM-MF. The data are discussed with regard to differences in the presence of (non-)immunogenic bacteria and the ability for related and unrelated flora to colonize the murine gut. This study provides evidence that murine host specificity of microbial flora may not only be reflected in the number of non-immunogenic bacteria but also in the micromorphological pattern of highly concentrated anaerobic bacteria in faeces measured by DIP analysis.

Changes in symptoms, peak expiratory flow, and sputum flora during treatment with antibiotics of exacerbations in patients with chronic obstructive pulmonary disease in general practice.

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.

Host-microflora interaction in systemic lupus erythematosus (SLE): circulating antibodies to the indigenous bacteria of the intestinal tract.

Experimental 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 and the antibody-binding capacity of the intestinal bacteria of ten healthy human volunteers.

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.

Computer processing of microscopic images of bacteria: morphometry and fluorimetry.

Several techniques that use computer analysis of microscopic images have been developed to study the complicated microbial flora in the human intestine, including measuring the shape and fluorescence intensity of bacteria. These techniques allow rapid assessment of changes in the intestinal flora and could apply equally to other complex microbial ecosystems.

Direct flow cytometry of anaerobic bacteria in human feces.

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 microns2. 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.

The role of systemic antibodies against intestinal Escherichia coli in the prevention of bacterial translocation of Escherichia coli in a burn model in mice.

Bacterial translocation (BT) from the gastrointestinal (GI) tract has been proposed to play a role in the pathogenesis of septic complications in severely burned patients. It is well known that severely ill patients such as thermally injured patients may acquire new potential pathogenic microorganisms in the GI tract. Because these patients have no antibodies directed against these acquired microorganisms, BT may be facilitated in these patients. To investigate this hypothesis in a burn model, a study was performed in which two groups of C3H-HeN mice underwent a different period of intestinal overgrowth by a single neomycin-resistant (NR) Escherichia coli strain after oral neomycin-bacitracin treatment. Group I underwent a short period (5 days) and group II experienced a long period (44 days) of intestinal overgrowth before a thermal injury was executed. Two days postburn, plasma antibody titers of IgA, IgG, and IgM isotype against NR E. coli were measured by indirect immunofluorescence (IIF) and BT to various organs was determined by culturing. Although there were no significant differences of BT to organs between the groups, the IgG antibody titer against the NR E. coli strain was significantly increased in group II. Antibody titers of IgA and IgM were not significantly different between the groups. Titers of plasma antibodies of IgG isotype against the intestinal NR E. coli did not correlate with BT. We conclude that increased IgG titers against the NR E. coli used are the result of a longer intestinal overgrowth period and are not associated with prevented or decreased BT.

Host-microflora interaction in systemic lupus erythematosus (SLE): colonization resistance of the indigenous bacteria of the intestinal tract.

Experimental data suggest a role for the microflora in Systemic Lupus Erythematosus (SLE). Anti-ds-DNA antibodies may be pathogenic in SLE by forming immune complexes with DNA. Foreign bacteria in the intestines could constitute the stimulus for anti-ds-DNA antibody production in SLE. Colonization Resistance (CR) is the defence capacity of the indigenous microflora against colonization of the intestines by foreign bacteria. A low CR implies increase of translocation of bacteria and a higher chance of subsequent, possibly DNA-cross-reacting antibacterial antibody production. We measured CR by a comprehensive biotyping technique in healthy individuals and patients with inactive and active SLE. CR tended to be lower in active SLE patients than in healthy individuals (P = 0.09, Wilcoxon one sided, with correction for ties). This could indicate that in SLE more and different bacteria translocate across the gut wall due to a lower CR. Some of these may serve as polyclonal B cell activators or as antigens cross-reacting with DNA.

Comparison of imipenem versus cefuroxime plus tobramycin as empirical therapy for febrile granulocytopenic patients and efficacy of vancomycin and aztreonam in case of failure.

143 aplastic episodes with fever in 91 haematological patients with granulocytopenia were treated empirically in a randomized prospective study using either imipenem (Imi) or a combination of tobramycin and cefuroxime (T/C). Response after 72 h was significantly better in patients receiving Imi (44/75 vs 27/68, p < 0.05). This was seen especially in patients with bacteriologically proven infections where the isolated staphylococci and streptococci were more susceptible to Imi. In both groups, patients who failed to respond to the initial antibiotic therapy were given vancomycin and aztreonam (V/A). The response rate after another 72 h, measured using the same criteria as after the first 72 h, did not differ statistically between the groups. One patient in each study group died from the bacterial infection, both from Gram-positive bacteraemia. Duration of fever was significantly shorter in the Imi group (4 days vs 7 days, p < 0.04). Serum peak and trough concentrations of the antibiotics were comparable. Both regimens were well tolerated. Our results show that monotherapy with imipenem is superior to the combination of tobramycin and cefuroxime during the first 72 h of therapy and can be safely administered to neutropenic patients with predominantly Gram-positive infections. A combination of vancomycin and aztreonam, given when initial imipenem treatment has failed, was effective in only a few patients. Adjuvant glycopeptide therapy from the outset in the treatment of febrile granulocytopenic patients did not seem worthwhile.

Oropharyngeal flora in asthma and in chronic obstructive pulmonary disease. Indigenous oropharyngeal microorganisms in outpatients with asthma or chronic obstructive pulmonary disease.

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 in a stable phase of disease throughout the study. The distribution of low (< or = 10(4)/ml) or high (> or = 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 (< or = 10(4)/ml) or high (> or = 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 < 0.005 and p < 0.0005, respectively), but the prevalence of Enterobacteriaceae species was significantly higher (p < 0.0005). No correlation was found between the concentration of viridans streptococci and the prevalence of gram-negative microorganisms. These findings suggest that viridans streptococci are probably not responsible for growth control of gram-negative microorganisms in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of human faecal flora by means of an improved fluoro-morphometrical method.

The 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.

Healthy individuals possess circulating antibodies against their indigenous faecal microflora as well as against allogenous faecal microflora: an immunomorphometrical study.

Healthy 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.

Modulation of the intestinal microflora by the non-absorbable antibiotic vancomycin leads to a reduction of the tumor load in liver and spleen in a leukemic rat model.

Based on previous studies where it was shown that non-absorbable antibiotics can influence the normal hematopoiesis via changes in factors related to the intestinal microflora, the influence of vancomycin on the progression of acute myeloid leukemia was investigated in the BNML rat model. Oral vancomycin, which selectively reduces Gram-positive bacteria in the gut, leads to diminution of the leukemic load in liver and spleen by 30-60%. This 'antileukemic effect' is not dependent on Gram-negative bacteria as source for endotoxin. The presumed mechanism is a decrease of the leukemic growth fraction caused by alterations in the absorption of substances from intestinal Gram-positive bacteria.

The in vitro inactivation of thirteen beta-lactam antibiotics by other mechanisms than adsorption to faecal substance.

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.