Minimum Growth Inhibitory Concentration Research Articles

Radiochemical assay to measure the biofilm produced by coagulase-negative staphylococci on solid surfaces and its use to quantitate the effects of various antibacterial compounds on the formation of the biofilm.

A firmly adherent mass of slime plus organisms (biofilm) accumulates on the sides of culture tubes when some strains of coagulase-negative staphylococci are grown in a chemically-defined medium containing [14C]glucose. This mass was washed (to remove labelled medium) and then counted after adding scintillation fluid. Organisms from the liquid culture were also washed and counted to check that [14C]glucose had been utilised to label the bacteria. Nine strains were examined in this way, and the results were compared with those obtained with four older techniques for recognising slime production or adherent bacteria. The new method is quick, and has advantages of reproducibility and good discrimination between strains; there was a 15-fold difference in counts in the biofilm between slime-producing and non-producing strains respectively. With the new radiolabel assay, the effects of several antibacterial compounds on the build-up of the biofilm were investigated with four slime-producing strains. Tunicamycin, chloramphenicol and 5-fluorouracil, at levels below their minimum growth-inhibitory concentrations, each greatly diminished biofilm formation; several other drugs had less effect.

Ultrastructural alterations induced by ICI 195,739, a bis-triazole derivative with strong antiproliferative action against Trypanosoma (Schizotrypanum) cruzi

The ultrastructural alterations induced in vitro by ICI 195,739, a recently developed bis-triazole derivative with potent antiproliferative effects on Trypanosoma (Schizotrypanum) cruzi, are reported. On epimastigotes, the triazole at its minimum growth-inhibitory concentration (0.1 microM) produced immediately (within 24 h) gross alterations in the organization of chromatin and the appearance of large electron-dense granules; at this time, many cells were binucleated, indicating a blockade in cytokinesis. At later times (120 h), mitochondrial swelling, a characteristic effect reported previously for the dioxolane-imidazole ketoconazole when the performed ergosterol pool is depleted, was the predominant effect and led to cell lysis. In amastigotes proliferating in Vero cells, the drug at 10 nM produced mitochondrial swelling, autophagic vacuoles, and massive alterations of the plasma membrane, leading to complete parasite destruction after 96 h of incubation of the infected monolayers with the drug. The results support previous conclusions that ICI 195,739 has a dual mechanism of action against T. cruzi, involving blockade of ergosterol biosynthesis and a direct effect on cell division which cannot be reversed by addition of exogenous ergosterol.

State of penicillin-binding proteins and requirements for their bactericidal interaction with β-lactam antibiotics in Serratia marcescens highly resistant to extended-spectrum β-lactams

The quantities of penicillin-binding proteins (PBPs), and sensitivity to extended-spectrum beta-lactams, were measured in isogenic strains of Serratia marcescens with high (HR) and low (LR) resistance to extended-spectrum beta-lactam antibiotics and with constitutively overproduced chromosomal beta-lactamase in the periplasm. The binding of structurally different beta-lactams to PBPs in growing resistant bacteria was determined quantitatively. In S. marcescens HR, the amounts of PBPs 3 and 6 were, respectively, 1.5 and 2 times those in strain LR and in sensitive reference strains. Sensitivities of the essential PBPs in S. marcescens LR and HR to the tested beta-lactams were identical. Only a single target, PBP 3, was highly sensitive to cefotaxime, ceftazidime and aztreonam. In contrast, three PBPs (2, 1A and 3) were highly sensitive to imipenem. In growing S. marcescens HR and LR, all antibiotics, even at fractions of their minimal growth inhibitory concentrations (MICs), bound extensively to those PBPs which were highly sensitive to them. Thus, overproduced beta-lactamase did not prevent PBP-beta-lactam interaction. Only at or above their (high) MICs did cefotaxime, ceftazidime and aztreonam bind to multiple targets. Growth inhibition of the otherwise highly resistant S. marcescens HR at the lower MIC of imipenem was correlated with the binding of this antibiotic to multiple, highly sensitive targets in the bacteria. Killing of the bacteria by inactivation of multiple targets was suggested. This assumption was supported by the synergistic killing of HR bacteria by combinations of the PBP-2-specific mecillinam with PBP-3-specific beta-lactams.

Growth inhibitory and biocidal activity of some isothiazolone biocides

Similar patterns of growth inhibition were observed for the three biocides, benzisothiazol-3-one (BIT), 5-chloro-N-methylisothiazol-3-one (CMIT) and N-methylisothiazol-3-one (MIT) against Escherichia coli ATCC 8739 and Schizosaccharomyces pombe NCYC 1354. After periods of induced stasis, proportional to biocide concentration, growth proceeded at an inhibited rate. Extrapolation of the static periods and inhibited growth rates against biocide concentration gave minimum growth inhibitory concentration estimates of 0.1-0.5 micrograms/ml for CMIT, 15-20 micrograms/ml for BIT and 40-250 micrograms/ml for MIT. Patterns of growth inhibition by CMIT and induced morphological changes in inhibited cultures suggested this compound to also inhibit initiation of DNA replication. Growth inhibitory activity was rapidly quenched by the addition of thiol-containing materials such as glutathione and cysteine. The activity of CMIT was additionally quenched by the presence of the non-thiol amino acids valine and/or histidine. These results suggest that the chlorinated isothiazolones can react with amines as well as with essential thiol groups.

β-Lactamase-Producing Mutants of Streptococcus cremoris

Penicillin-resistant mutants were isolated for six strains of Streptococcus cremoris used in commercial Cheddar cheese manufacture after treatment with N-methyl-N-nitro-N-nitrosoguanidine. The resistant mutants had an elevated minimal growth inhibitory concentration, 2.5μg (4.13 units)/ml, for penicillin G and other β-lactam antibiotics as compared with the penicillin-susceptible parent strains, which were each sensitive to .05μg (.08 units)/ml. Penicillin resistance was due to the production of β-lactamase. Plasmid DNA was not demonstrated in partially purified lysates of four mutants. Mutants had normal cellular morphology but altered phage sensitivity patterns. All except one strain were able to support complete phage adsorption. Resistance was retained after 20 passages in absence of penicillin.

Progress of O-acetylation and cross-linking of peptidoglycan in Neisseria gonorrhoeae grown in the presence of penicillin.

The synthesis, cross-linking and O-acetylation of gonococcal peptidoglycan in growing cells were studied by following incorporation of radioactive glucosamine and separation of the SDS-insoluble peptidoglycan into uncross-linked (monomer) and cross-linked (dimer and oligomer) fragments. Cultures to which penicillin or piperacillin at concentrations near the minimum growth inhibitory concentration (MIC) had been added 20 min before the radioactive label were compared with controls. The beta-lactams affected the early stage of cross-linking (up to 3 min) but had no effect thereafter. The deficit of cross-linking, however, did not recover. The O-acetylation, particularly of the monomer fraction, was decreased by beta-lactams even at concentrations that had no effect on culture optical density, viable counts or overall peptidoglycan synthesis. These effects on O-acetylation occurred mainly after the first 3 min of incorporation, rather than before. O-Acetylation of the oligomer fraction was also followed. Here penicillin led to increased levels of O-acetylation during the early stages of incorporation but the final value was never exceeded; indeed at higher drug concentrations the later stages of O-acetylation of oligomers were inhibited (e.g. almost completely at 2.5 X MIC).

In vivo target of benzylpenicillin in Gaffkya homari.

It has been established that the DD-carboxypeptidase is the primary in vitro target of benzylpenicillin in Gaffkya homari (W. P. Hammes, Eur. J. Biochem. 70:107-113, 1976). To determine whether this enzyme is also the primary target of benzylpenicillin in vivo, we compared the effects of this beta-lactam, cefmenoxime, cephalothin, and cefoxitin on growth with their acylation of penicillin-binding protein (PBP) 9, the DD-carboxypeptidase. Results of three types of experiments with membrane-walls indicated that PBP 9 is this enzyme and that it is the primary in vitro target of these beta-lactams in the synthesis of sodium dodecyl sulfate (SDS)-insoluble peptidoglycan. First, the acylation of PBP 9 by these beta-lactams paralleled the inhibition of DD-carboxypeptidase and the inhibition of SDS-insoluble peptidoglycan synthesis. Second, the rate of benzylpenicillin release from PBP 9 correlated with the recovery of DD-carboxypeptidase. Third, DD-carboxypeptidase activity was detected in a protein with the same apparent molecular weight as PBP 9 after elution from an SDS-polyacrylamide gel. When intact cells were treated with benzylpenicillin, the minimum growth inhibitory concentration (MGIC) correlated with the concentration of [35S]benzylpenicillin required to acylate PBPs 6 and 9 by 50%. When intact cells were treated with cefmenoxime, cephalothin, or cefoxitin, the MGICs correlated with the concentration of unlabeled beta-lactam required to reduce the subsequent binding of [35S]benzylpenicillin by 50% (ED50) for PBP 6. In contrast, the MGICs of these beta-lactams did not correlate with the ED50s for PBP 9. PBP 9 was not acylated by cefmenoxime or cephalothin at their MGICs, whereas this PBP was fully acylated by cefoxitin at one-tenth of its MGIC. It is suggested that PBP 6 may be a primary target of growth inhibition by benzylpenicillin, cefmenoxime, cephalothin, and cefoxitin; PBP 9, the DD-carboxypeptidase, is dispensable for growth under laboratory conditions; and PBP 9 does not appear to be a primary in vivo target of these beta-lactams, even though this PBP is their primary target in vitro.

Involvement of the relA gene in the autolysis of Escherichia coli induced by inhibitors of peptidoglycan biosynthesis.

It is generally assumed that inhibitors of peptidoglycan biosynthesis do not kill nongrowing bacteria. An exceptional case is reported here. The addition of chloramphenicol to amino acid-deprived cultures of relA+ strains of Escherichia coli which were treated with beta-lactam antibiotics, D-cycloserine, or moenomycin resulted in lysis. This phenomenon is termed chloramphenicol-dependent lysis. To be effective, chloramphenicol had to be present at its minimum growth-inhibitory concentration (or higher). Analogs of chloramphenicol which did not bind to ribosomes were completely ineffective. Amino acid deprivation was actually not required to demonstrate chloramphenicol-dependent lysis, and cultures treated with growth-inhibitory levels of chloramphenicol alone were lysed when challenged with inhibitors of peptidoglycan synthesis. Peptidoglycan synthesis has been shown previously to be under stringent (relA+) control, and chloramphenicol is known to be an antagonist of stringent control. Thus, it is proposed that the mechanism of chloramphenicol-dependent lysis is based on the ability of chloramphenicol to relax peptidoglycan synthesis in nongrowing relA+ bacteria. This is also consistent with the observation that treatment of amino acid-deprived relA mutants with inhibitors of peptidoglycan synthesis resulted in lysis, i.e., without the mediation of chloramphenicol.

Inhibition of microbial growth at different pH levels by benzoic and propionic acids and esters of p-hydroxybenzoic acid

The minimum growth inhibitory concentration of benzoic and propionic acids and esters of p-hydroxybenzoic acid (parabens) has been determined for Bacillus subtilis, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. For benzoic and propionic acid, the inhibition was shown to be due to both undissociated and dissociated acid, and the effect of each has been calculated in accordance with a proposed mathematical model which has earlier been applied to sorbic acid. Parabens showed some pH dependence for the action on bacteria, but not on yeast. This pH dependence was unrelated to dissociation.

Physiological activities of 3,3',4,5'-tetrahydroxystilbene isolated from the heartwood of Cassia garrettiana CRAIB.

3, 3', 4, 5'-Tetrahydroxystilbene (1) isolated from the heartwood of Cassia garrettiana CRAIB. showed a broad spectrum of physiological activities, including antifungal, phytogrowthinhibitory and ichthyotoxic activities. Firstly. 1 showed antifungal activity ; the minimal growthinhibitory concentration (MIC) was 25 μg/ml against Penicillium citrinum and Rhizopus nigricans. Secondly, 1 showed rather strong inhibitory activity on the growth of five plant species at a concentration of 500ppm. Thirdly. 1 had ichthyotoxic activity. The median tolerance limit (TLm) at 48h was 26.5ppm in Oryzias latipes TEMMINCK et SCHLEGEL and 31.5 ppm in Carassius auratus LINNE. Studies were made on the activities of derivatives of 1, i.e., 3, 3', 4, 5'-tetrahydroxystilbenetetraacetate (2), 3, 3', 4, 5'-tetrahydroxystilbene-tetramethyl ether (3), and 3, 3', 4, 5'-tetrahydroxybibenzyl (4). The activities of these derivatives, however, were lower than those of 1, except for the phytogrowth-inhibitory and ichthyotoxic activities of 4. It is clear that for antifungal activity, both the hydroxyl groups attached to the benzene rings and the trans-olefin structure are necessary, while for the phytogrowth-inhibitory and ichthyotoxic activities, only the hydroxyl groups attached to the benzene rings are necessary.

Penicillin-resistant and penicillin-tolerant mutants of group A Streptococci.

Penicillin-resistant and penicillin-tolerant mutants have been isolated from group A streptococci mutagenized by ethyl methane sulfonate. The resistant mutants had an elevated minimal growth inhibitory concentration for benzylpenicillin (minimal inhibitory concentration, 0.2 microgram/ml, as compared with the minimal inhibitory concentration of 0.006 microgram/ml in the penicillin-susceptible parent strain); they also had an abnormal cellular morphology and showed altered penicillin-binding proteins. Penicillin-tolerant mutants were killed more slowly than were the parental cells during treatment with penicillin; they had virtually unchanged minimal inhibitory concentration values for penicillin and normal cellular morphology and penicillin-binding proteins.

In vitro studies of aculeacin A, a new antifungal antibiotic.

Aculeacin A is a cyclopeptide-containing long-chain fatty acid, representing a new class of antibiotics. It has a relatively narrow antifungal spectrum in vitro and is highly active against some groups of yeasts. Of 31 strains of Candida ad Torulopsis tested, the majority were susceptible to aculeacin A at 0.31 micrograms per ml or less. On the other hand, the antibiotic was scarcely active or inactive against other yeasts, such as Cryptococcus neoformans, and all filamentous and dimorphic fungi tested. A distinct inoculum effect has been observed in vitro with a number of strains of C. albicans; minimum growth-inhibitory concentrations (MIC) have tended to increase with increased incubation time. MIC values were also increased in the presence of serum. Aculeacin A is fungicidal for growing cells of C. albicans. It was most lethal against sensitive yeasts at 0.08 to 0.31 micrograms per ml, and increases in the concentrations of the drug above this range reduced, rather than increased, its lethal effect.

Autolytic Activity and an Autolysis-Deficient Mutant of Clostridium acetobutylicum

The optimum conditions for autolysis and autoplast formation in Clostridium acetobutylicum P262 have been defined. Autolysis was optimal at pH 6.3 in 0.04 M sodium phosphate buffer, and the bacterium produced latent and active forms of an autolytic enzyme. The ability of cells to autolyze decreased sharply when cultures entered the stationary phase. Autoplasts were induced by 0.25 to 0.5 M sucrose and were stable in media containing sucrose, CaCl(2), and MgCl(2). A pleiotropic autolysis-deficient mutant (lyt-1) was isolated. The mutant produced less autolysin than did the parent P262 strain, and it had an altered cell wall which was more resistant to both its own and P262 autolysins. The mutant formed long chains of cells, and lysozyme was required for the production of autoplasts. Growth of the P262 strain or the lyt-1 mutant was inhibited by the same concentrations of penicillin, ampicillin, and vancomycin. The lyt-1 mutant strain treated with the minimum growth-inhibitory concentration of penicillin autolyzed upon the addition of wild-type autolysin to the autolysis buffer at the same rate as did the untreated P262 strain. Chloramphenicol did not protect the penicillin-treated lyt-1 cells against autolysis enhanced by exogenous wild-type autolysin.

The role of penicillin-proteins in the action of cephalosporins against Escherichia coli and Salmonella typhimurium.

The binding of two radioactively-labelled cephalosporins, LY97962 and LY121998, to the penicillin-binding proteins of Escherichia coli and Salmonella typhimurium has been studied in detail. The concentrations of the cephalosporins that resulted in 50% saturation of the penicillin-binding proteins were measured in growing cells as well as in preparations of disrupted cell envelopes. This technique has allowed the crypticity of an organism towards a particular β-lactam to be measured directly, i.e. the extent to which a penicillin-binding protein is accessible in intact cells. The cephalosporin LY97962 appeared to have no difficulty in penetrating the outer layers of the cell whereas access of cephalosporin LY121998 to the penicillin-binding proteins appeared to be somewhat restricted. At concentrations below the minimum growth inhibitory concentration the two cephalosporins inhibited cell division and caused the cells to grow as long filaments. When growing cells were incubated with the radioactive β-lactam antibiotics at these concentrations, the β-lactams bound almost exclusively to penicillin-binding protein 3. The degree of binding of the cephalosporins to protein 1 A in cells growing in the presence of the minimum growth inhibitory concentrations of antibiotics varied with the duration of the incubation. After exposure for 5 min, very little binding to protein 1 A was apparent, whereas substantial binding was observed after 60–180 min. It was only at the end of this period of growth in the presence of the cephalosporins that lysis of the cells became apparent. This provided evidence that the binding of β-lactams to protein 1 A of gramnegative organisms is an important step in the bactericidal action of certain cephalosporins. Protein 1 B was excluded as a target for the lethal action of these cephalosporins because binding to this protein was observed only at concentrations greater than those needed to inhibit growth of the cells. Microscopical examination of the growth of S. typhimurium in the presence of cefotaxime at sub-lytic concentrations showed that cells grew as filaments for at least six generations without lysis. Observations of the division of the filament into individual cells after removal of the antibiotic showed that the entire length of the filament was viable. It is concluded that inhibition of cell division as a result of the binding of β-lactams to protein 3 of gram-negative organisms is not in itself a lethal event for the cells, but requires concomitant binding of antibiotic to protein 1 A before lysis is initiated.

Effects of imazalil on a wild-type and fungicide-resistant strain of Aspergillus nidulans

The effects of different incubation conditions on toxicity and uptake of imazalil by mycelium of a wild-type (003) and fungicide-resistant (R264) strain of Aspergillus nidulans were determined. In agar plate and liquid shake culture, imazalil was 200 and 40 times less toxic to the R264 strain, respectively, than to the wild-type strain. Inhibition of C-4 desmethyl sterol (ergosterol) biosynthesis occurred rapidly in mycelium of both strains at minimum growth inhibitory concentrations. Imazalil was neither detoxified nor converted into a toxic compound by mycelial suspension. Increased uptake of imazalil by the two strains occurred as concentrations of the fungicide were increased. However, the percentage uptake of imazalil by the wild-type strain was highest at the lowest concentration. These results suggest that binding in the wild-type strain involved a small number of high-affinity sites which became saturated as fungicide concentrations increased; and that at higher concentrations considerable nonspecific binding occurred in both strains. Uptake of imazalil during the initial 10 min of incubation was considerably lower in resistant than in the wild-type strain. However, upon prolonged incubation, both strains took up near equal amounts of fungicide. Uptake of fungicide by both strains was not inhibited by incubation at low temperature but was stimulated by respiratory inhibitors. These data support, in part, the hypothesis that resistance to imazalil, as reported previously for fenarimol (M. A. de Waard and J. G. M. van Nistelrooy, Pestic. Biochem. Physiol. 13, 255 (1980)), is based on reduced uptake of fungicide by mycelium of the resistant mutant.

Effects of amphotericin B, nystatin, and other polyene antibiotics on chitin synthase.

The effects of amphotericin B (AmB), nystatin, filipin, and pimaricin were tested chitin synthase (EC 2.4.1.16) (chitosomes from yeast cells of Mucor rouxii). AmB and nystatin inhibited the enzyme at concentrations greater than or equal to 10 micrograms/ml, filipin was weakly inhibitory, and pimaricin had no effect. The inhibition of chitin synthase by AmB appears to be noncompetitive, with a Ki value of about 0.13 mM. the effect of nystatin was more complex and included a sharp stimulation of chitin synthase activity at approximately 50 micrograms/ml. Our findings suggest the existence of binding sites (sterols?) on the chitosome that are selective for certain polyenes and that play a role in the operation of chitin synthase. Because the minimal growth inhibitory concentrations of AmB or nystatin are lower than the concentrations that inhibit chitin synthase in vitro, the possibility of chitosomal chitin synthase being a primary target for the antifungal action of these polyenes seems unlikely.

Triggering of autolytic cell wall degradation in Escherichia coli by beta-lactam antibiotics.

A biochemical method was developed to quantitatively compare the effectiveness of beta-lactams in triggering murein degradation (autolysin activity) in Escherichia coli. Bacteria prelabeled in their cell walls with radioactive diaminopimelic acid in growth medium were exposed for 10 min to the antibiotics at the appropriate minimal growth inhibitory concentrations and at multiples of these values, and the rate of cell wall degradation was followed during subsequent penicillin-binding protein (PBP)-1 were the most effective triggers of autolytic wall degradation; beta-lactams selective for PBP-2 were the poorest; and antibiotics preferentially binding to PBP-3 showed intermediate activities. The relative effectiveness of beta-lactams in autolysin triggering was found to parallel the effectiveness of the same drugs in causing rapid loss of viability, culture lysis, and spheroplast formation. Autolysin triggering was suppressed by inhibitors of protein and ribonucleic acid biosynthesis but not by inhibitors of deoxyribonucleic acid synthesis. The beta-lactam-induced cell wall degradation did not seem to involve a direct stimulation of enzyme activity or synthesis of new enzyme molecules, and murein sacculi isolated from cells that had been preexposed to a triggering dose of beta-lactam treatment exhibited the same sensitivity to crude, homologous autolysins as sacculi prepared from untreated control bacteria. On the basis of these observations, mechanisms are considered for the triggering of E. coli autolysins and for the role of autolytic activity in bacterial spheroplast formation, lysis, and death.

Rapamycin (AY-22,989), a new antifungal antibiotic. IV. Mechanism of action.

Rapamycin, an antifungal antibiotic produced by Streptomyces hygroscopicus showed a strong candicidal activity, which could not be reversed by sterols. It has no effect on efflux of K+, Pi ir U.V. absorbing materials and cell permeability of Candida albicans. Thus, in its action it differs from the polyenes. Mechanism of action of rapamycin appears to be different from many known antifungal agents. In C. albicans, rapamycin at the minimum growth inhibitory concentration inhibited: 1) phosphate incorporation into nucleic acids, 2) acetate incorporation into lipids and 3) substrate respiration of amino acids. The effect on amino acid metabolism was expressed as inhibition of oxidative deamination. At low concentrations rapamycin caused degradation of P32-labeled intracellular macromolecules. Inhibition of threonine incorporation into cell wall and leucine incorporation into cellular protein was observed at relatively higher concentrations of rapamycin. The antibiotic showed no effect on cell-free protein synthesizing systems of Escherichia coli, rat liver and C. albicans and in the mitochondrial enzyme systems. Whether the lethal action of rapamycin on C. albicans is primarily due to one of the above effects or is the result of combined effect on some of these biosynthetic parameters remains to be established.

Effect of two imidazole antimycotics, clotrimazole and miconazole on amino acid transport inCandida albicans

Effect of the two imidazole antimycotics, clotrimazole and miconazole, on the uptake of leucine, lysine and several other amino acids by starved cells of Candida albicans was studied. Both imidazoles at concentrations around the minimum growth-inhibitory concentration substantially decreased the rate of uptake and the level in the pool of these amino acids when the cells were incubated in the absence of glucose. Inhibition by the 2 imidazoles, particularly clotrimazole, of uptake of leucine or lysine was markedly antagonized by exogenous glucose. However, such a protective effect of glucose was completely nullified when blasticidin S, a preferential inhibitor of protein synthesis, was added to the cells along with glucose. Under all of these experimental conditions, the 2 imidazoles inhibited the accumulation of leucine in the intracellular pool more profoundly than the corresponding overall incorporation of this amino acid into proteins. The imidazoles caused an efflux of previously accumulated leucine a...

Bladder Instillations with Trimethoprim-Sulfamethoxazole in the Treatment of Urinary Infection

205 paraplegic inpatients with urinary infections were treated with trimethoprim-sulfamethoxazole (TMP-SMZ) bladder instillations (160 mg TMP 800 mg SMZ daily for 2 consecutive weeks). 89.3% out of 84 patients with recent vertebral trauma and no history of previous urinary infection (group A), and 68.6% out of 121 patients with paraplegia since more than 2 years, severe chronic infection and various pathological conditions of the urinary tract (group B) were found to be sterile at the end of the trial. The intravesical concentrations of 3.2 mg/ml TMP and 16 mg/ml SMZ which are reached immediately after instillation are 50--100 times superior to the minimal growth-inhibitory concentrations for most sensitive germs as determined with agar dilution tests. Several strains of Klebsiella and Proteus which had previously proved resistant to oral treatment were eliminated with intravesical TMP-SMZ. The incidence of reinfection, evaluated 1 month after interruption of treatment, was particularly high (62.7%) in patients with known involvement of the upper tract of intravesical obstruction with postmicturition residuals above 75 ml. On the other hand, 28% only among the patients with recent spinal injury and integrity of the upper urinary tract became reinfected after the same interval. The absence of local intolerance could be confirmed in all 21 cystoscopically examined patients between the 6th and 11th day of treatment. Hematuria was never encountered. Systemic side effects were noted in 7.8% Serum concentrations of both drug components are being investigated; results will be published elsewhere.