Reversal of drug resistance in Mycobacterium leprae by ampicillin/sulbactam.

The multiplication of Mycobacterium leprae in foot pads of experimentally-infected mice was suppressed by intramuscular administration of ampicillin combined with sulbactam or YTR-830H, two potent inhibitors of beta-lactamase in the bacteria. The antibiotic or the inhibitors by themselves were inactive. Ampicillin/sulbactam also inhibited the growth of drug-resistant M. leprae which grew in the presence of rifampin or dapsone. The finding provides a new approach to treat leprosy and to overcome drug resistance of the mycobacteria.     

Effect of lysozyme on mycobacteria

The effect of lysozyme on the growth of several strains of mycobacteria was examined at pH 5.0-7.0 in Dubos medium containing various concentrations of lysozyme (100-2,000 microgram/ml). Mycobacterium smegmatis and M. phlei were susceptible to lysozyme at pH 5.0-7.0. The effect of lysozyme was marked between pH 6.0 and 7.0 and the colony counts were reduced to approximately 0.1-10% after incubation with 100 micrograms of lysozyme per ml for 48 hr. At pH 5.0, 10-40% of the organisms survived treatment with 1,000 micrograms of lysozyme per ml for 48 hr. M. bovis strain BCG, M. tuberculosis, and M. fortuitum appeared to be more resistant to lysozyme than M. smegmatis and M. phlei. M. smegmatis and M. phlei did not contain detectable amounts of poly-L-glutamic acid, although the susceptibility of the mycobacteria to lysozyme did not correlate with the amounts of the polymer in the cell walls. The role of lysozyme in animal infections with so-called saprophytic mycobacteria is discussed.     

Antitubercular activity of trifluoperazine, a calmodulin antagonist

Trifluoperazine, a calmodulin antagonist, completely inhibited the growth of mycobacteria. The minimum inhibitory concentrations in shake cultures in a synthetic medium containing 0.2% Tween 80 were 5 and 8 micrograms/ml, respectively, for the human pathogenic strain Mycobacterium tuberculosis H37Rv and M. tuberculosis resistant to isoniazid. When added to a growing culture of M. tuberculosis H37Rv on the 10th day (mid exponential phase), trifluoperazine 50 micrograms/ml further arrested growth of this organism. It is suggested that trifluoperazine or similar calmodulin antagonists might be useful as antitubercular drugs.     

In Vitro Effect of Rifampin on Mycobacteria

Rifampin inhibited 20 strains of Mycobacterium tuberculosis in concentrations of 0.005 to 0.02 mug/ml in 7H-9 broth with Tween 80 and killed all or nearly all of the inoculum in four to eight times greater concentrations. In the same medium without Tween 80, as well as on 7H-10 agar, about 16 to 64 times these amounts were required to produce the same effect. Rifampin was also active against M. kansasii and some of the nonchromogenic mycobacteria. The incidence of mycobacterial cells resistant to rifampin within the cultures studied was in the range of one to four per 10(8) to 10(9) colony-forming units with concentrations of 4 to 125 mug of rifampin per ml. Only one of the Battey cultures and that of M. fortuitum yielded cells resistant to rifampin at 125 mug/ml but not at 500 mug/ml. The same strains yielded more than double that number of organisms resistant to streptomycin and up to 100 times more organisms resistant to isoniazid. All three drugs stopped the growth or reduced the mycobacterial population in growing cultures after contact for 24 to 48 hr. Complete inhibition of growth was produced by rifampin at 1.0 mug/ml in an average of 6 days and by streptomycin at 5.0 mug/ml in 3 days. After an average contact of 10.7 days with rifampin, five of seven strains resumed growth and all strains began regrowth after exposure to streptomycin for 9.4 days. The marked susceptibility of M. tuberculosis and of atypical mycobacteria to rifampin in vitro and the relatively low incidence of resistant mutants suggests that this agent may have clinical usefulness in the treatment of tuberculosis and some other mycobacterioses.     

Inhibition of microbial growth by ajoene, a sulfur-containing compound derived from garlic.

Ajoene, a garlic-derived sulfur-containing compound that prevents platelet aggregation, exhibited broad-spectrum antimicrobial activity. Growth of gram-positive bacteria, such as Bacillus cereus, Bacillus subtilis, Mycobacterium smegmatis, and Streptomyces griseus, was inhibited at 5 micrograms of ajoene per ml. Staphylococcus aureus and Lactobacillus plantarum also were inhibited below 20 micrograms of ajoene per ml. For gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae, and Xanthomonas maltophilia, MICs were between 100 and 160 micrograms/ml. Ajoene also inhibited yeast growth at concentrations below 20 micrograms/ml. The microbicidal effect of ajoene on growing cells was observed at slightly higher concentrations than the corresponding MICs. B. cereus and Saccharomyces cerevisiae were killed at 30 micrograms of ajoene per ml after 24 h of cultivation when cultivation was started at 10(5) cells per ml. However, the minimal microbicidal concentrations for resting cells were at 10 to 100 times higher concentrations than the corresponding MICs. The disulfide bond in ajoene appears to be necessary for the antimicrobial activity of ajoene, since reduction by cysteine, which reacts with disulfide bonds, abolished its antimicrobial activity.     

An evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs

Sodium ampicillin was administered subcutaneously to 350-550 g male Dunkin Hartley guinea pigs at doses of 6, 8 and 10 mg/kg tid for 5 days. Over a period of 12 days, the lowest ampicillin dose appeared to be tolerated well. However, significant body weight reduction and mortality occurred with the two higher dosage regimens. Cecal cultures of dead animals confirmed the presence of Clostridium difficile, an organism associated with antibiotic-induced enterotoxemia. Assay of serum collected from ampicillin-treated animals revealed ampicillin concentrations of approximately 10 micrograms/ml at 5 minutes post-dosing which fell precipitously to less than 0.2 micrograms/ml at 60 minutes. Determination of biliary ampicillin levels during the 60 minutes after administration of a single 10 mg/kg SQ dose revealed concentrations ranging from 18 micrograms/ml to 90 micrograms/ml. Estimates of total urinary ampicillin content after a single 10 mg/kg SQ dose were less than 500 micrograms/animal at 7.5 minutes, but increased to greater than 2000 micrograms/animal at 60 minutes after dosing. Results of this study indicated that due to its short serum half-life, sodium ampicillin probably has little systemic therapeutic efficacy in guinea pigs. Because high concentrations of ampicillin accumulated in the urine and bile, the antibiotic probably would have therapeutic efficacy for urinary and intestinal infections. However, its associated toxicity at large doses probably precludes its use. In view of the rapid clearance of ampicillin in guinea pigs in comparison to other species, the pharmacokinetics of other antibiotics, especially those reported to be less toxic for guinea pigs, should be considered.     

The use of ampicillin/sulbactam (Unasyn) in treating inflammatory urological diseases

Unasyn is a combination of ampicillin, a bactericidal antibiotic, and sulbactam, an inhibitor of beta-lactamases. It was used in treatment of 36 patients with urogenital infections. The combination was administered intravenously and in the main intramuscularly. The treatment course amounted to 7-10 days. The average daily dose was 6 to 9 g. 22 patients with acute nonocclusive pyelonephritis were treated with the combination and its clinical and bacteriological efficacy was stated in 95 per cent of the cases. An excellent clinical effect of the combination was observed in 6 patients with acute epididymitis. A clinical improvement was also observed in the treatment of the patients with acute prostatitis and chronic renal infections. Unasyn proved to be a highly efficient antibacterial combination with regard to gram-positive flora and colon bacilli as representatives of gram-negative organisms. Satisfactory results were also stated in the treatment of infections caused by Proteus spp. Complete elimination of the pathogen was achieved in 57.7 per cent of the cases. No adverse reactions to Unasyn except pain in the site of the injection were recorded.     

Histidine-rich glycoprotein plus zinc reverses growth inhibition of vascular smooth muscle cells by heparin

Vascular smooth muscle cell (SMC) hyperplasia is known to be an important component in the pathogenesis of arteriosclerosis and restenosis. Although heparin has been well recognized as the representative molecule suppressing SMC growth in vitro, attempts to use heparin as a therapeutic anti-restenosis drug have not favorably influenced the angiographic or clinical outcome after angioplasty in some clinical trials. In this study, we have examined the effect of histidine-rich glycoprotein (HRG), a relatively abundant serum glycoprotein (~100 micrograms/ml in human serum), on the growth inhibition of cultured vascular SMC by heparin. Vascular SMC growth was significantly inhibited by heparin, giving nearly 85% inhibition with 100 micrograms/ml heparin. HRG reversed heparin-induced SMC growth inhibition in a dose dependent manner; 75% restoration of cell growth was observed when 100 micrograms/ml of HRG was co-added with 100 micrograms/ml heparin. Interestingly, micromolar concentrations of the zinc ion (0-10 microM), compatible with concentrations released from activated platelets, were found to enhance the restorative action of HRG. Western blot experiment demonstrated no significant amounts of the HRG moiety in fetal bovine serum, eliminating the possible contribution of contaminant HRG from culture media. These findings indicate that HRG, in combination with the zinc ion, plays a role in modulating the SMC growth response in pathophysiological states and explain the lack of success of heparin as a therapeutic anti-restenosis drug in clinical trials.     

PDGF-stimulated fibroblast proliferation is enhanced synergistically by receptor-recognized alpha 2-macroglobulin.

alpha-Macroglobulins derived from plasma or secreted by macrophages are platelet-derived growth factor (PDGF) binding proteins that compete with cell-surface receptors on fibroblasts for PDGF binding. alpha 2-Macroglobulin (alpha 2M) derived from bovine plasma was tested for its ability to modulate the PDGF-induced proliferation of primary passage rat lung fibroblasts (RLFs) and a human skin fibroblast cell line (CRL 1508). Fibroblasts were grown in 10% fetal bovine serum (FBS) for 24 hr, then washed with serum-free medium before adding serum-free defined medium (SFDM) containing insulin and transferrin. To this medium were added varying concentrations of human plasma-derived AB-PDGF and alpha 2 M, alone or in combination. Receptor-recognized alpha 2M was prepared by treatment with methylamine. Both native alpha 2M and the alpha 2M-methylamine (alpha 2M-MA) were tested for growth promoting activity in the absence or presence of PDGF. After 3 days, a concentration-dependent growth curve of fibroblast proliferation was demonstrated for PDGF alone, with near maximal stimulation reached at 15-20 ng/ml PDGF. alpha 2M and alpha 2M-MA alone had no effect on cell proliferation. However, alpha 2M-MA concentrations above 32 micrograms/ml synergistically enhanced PDGF-stimulated proliferation greater than 100% in the presence of 15 ng/ml PDGF. Native alpha 2M enhanced PDGF-stimulated growth 80-100% above PDGF controls only at low concentrations (32-64 micrograms/ml alpha 2M). High concentrations of native alpha 2M (128-256 micrograms/ml) either had no effect on growth or were inhibitory to PDGF-stimulated growth, depending on the cell type tested. Rat lung fibroblasts were shown to secrete a factor(s) that inhibited the trypsin-binding capacity of native alpha 2M. We further demonstrated that early passage RLFs possess specific cell-surface receptors for [125I]-PDGF and [125I]-alpha 2M-MA, and preincubation of RLFs with alpha 2M-MA increased the specific binding of [125I]-PDGF to the cell surface of these fibroblasts. Considered together, these data support the view that receptor-recognized alpha 2M synergistically enhances the proliferative capacity of PDGF. We postulate that receptor-recognized alpha Ms enhance PDGF-stimulated growth by increasing the local concentration of PDGF at the cell surface, where the PDGF could be released in close proximity to its own receptors.     

Characterization of the effect of aphidicolin on adenovirus DNA replication: evidence in support of a protein primer model of initiation

Adenovirus DNA replication is inhibited by aphidicolin but the inhibition clearly has different parameters than the inhibition of purified DNA polymerase alpha. In adenovirus infected Hela cells, 10 micrograms/ml of aphidicolin reduced viral DNA synthesis by 80%. Cellular DNA synthesis was inhibited by 97% at 0.1 microgram/ml. 10 micrograms/ml of drug had no effect on virus yield or late protein synthesis though higher concentrations of drug (50 micrograms/ml) caused an abrupt cessation of late protein synthesis and 100 micrograms/ml reduced virus yield by 3 logs. Concentrations of the drug from 0.5 microgram/ml to 10 micrograms/ml were found to dramatically slow the rate of DNA chain elongation in vitro but not stop it completely, so that over a long period of time net incorporation was reduced only slightly compared to the control. 50 micrograms/ml or 100 micrograms/ml of drug completely inhibited incorporation in vitro. Initiation of viral DNA replication - covalent attachment of dCMP to the preterminal protein - occurs in vitro. This reaction was found to be insensitive to inhibition by aphidicolin. We thus conclude that aphidicolin exerts its effect on adenovirus DNA chain elongation, but not on the primary initiation event of protein priming.     

Ampicillin inactivation in the caecum of axenic, gnotoxenic and conventional lambs: interaction with resistant or sensitive Escherichia coli

The fate of orally administered ampicillin was studied in axenic lambs, in gnotoxenic lambs given a complex microflora and a mixture of ampicillin resistant and/or sensitive strains of Escherichia coli, and in conventional lambs. In axenic lambs or animals with a sensitive microflora, antibiotic concentrations of 500-1600 micrograms ml-1 were detected in the intestine, and most of the ampicillin passed through the small intestine and entered the large intestine, within 12-15 h of administration. These antibiotic concentrations were sufficient to decrease the numbers of ampicillin-sensitive E. coli from 10(8)-10(9) bacteria ml-1 to about 10(5)-10(6) bacteria ml-1 by 8 h after ampicillin administration. Second and third doses of antibiotic had no further effect on the bacterial count. Administration of ampicillin to animals hosting ampicillin-resistant E. coli resulted in a significant inactivation of the antibiotic in the intestine. As might be expected there was little reduction in the numbers of these organisms. These results are similar to those observed in conventional lambs hosting resistant E. coli as the dominant colibacillary flora.     

The effect of praziquantel on the pseudophyllidean cestode Bothriocephalus acheilognathi in vitro

Adult Bothriocephalus acheilognathi were incubated in solutions containing 0 (control), 0.1, 1.0, 10.0 and 100 micrograms praziquantel per ml (0, 10(2), 10(3), 10(4) and 10(5) micrograms l-1) of 0.9% saline for 5, 15 and 60 min at a temperature of 18 degrees C. The worms contracted immediately upon being placed in the drug. Scanning and transmission electron microscopy revealed considerable tegumental damage particularly in the neck region. Vacuolization and 'bubbling' of the tegument occurred in all of the drug solutions tested. Exposure to drug concentrations of more than 1.0 micrograms ml-1 (10(3) micrograms l-1) praziquantel for 15 min or greater resulted in many of the 'bubbles' bursting and releasing their contents to the exterior. Mature proglottides were distorted and had occasional large swellings resulting in the mass expulsion of eggs. Praziquantel had no ovicidal activity. Exposure to drug concentrations of 100 micrograms (10(5) micrograms l-1) praziquantel per ml saline for 24 h was not lethal to the worms.     

Disinfectant effects of hot water, ultraviolet light, silver ions and chlorine on strains of Legionella and nontuberculous mycobacteria

The disinfectant effects on Legionella and nontuberculous mycobacteria of hot water, ultraviolet light, silver ions and chlorine, were evaluated. The bacterial strains Legionella pneumophila ATCC33152 and Mycobacterium avium ATCC25291 and strains of L. pneumophila and M. avium which had been isolated from a 24 h bath, were examined for their resistance to treatments. All strains were killed within 3 min on exposure to hot water at 70 degrees C and exposure to ultraviolet light at 90 mW.s/cm2. The strains of L. pneumophila tested were killed within 6 h on exposure to a solution of silver ions at 50 micrograms/l. The number of viable cells of strains of M. avium fell from 10(5) CFU/ml to 10(3) CFU/ml after exposure to an aqueous solution of silver ions at 100 micrograms/l for 24 h. Chlorine effectively killed strains of Legionella which were exposed to an aqueous solution of chlorine at 2 mg/l within 3 min, but strains of Mycobacterium survived exposure to chlorine at 4 mg/l for more than 60 min.     

Growth of phenol-mineralizing microorganisms in fresh water.

A method was developed to enumerate the procaryotic and eucaryotic phenol-mineralizing microorganisms present in samples of fresh water. Sixty-five percent or greater mineralization of [U-14C]phenol was considered a positive tube (contained phenol-mineralizing microorganisms) in the most-probable-number technique. Replicate most-probable-number tubes contained no microbial inhibitors, streptomycin and tetracycline, or cyclohexamide and nystatin plus 200 pg to 100 micrograms of phenol per ml. Phenol mineralization rates were obtained by measuring the amount of exogenous phenol that disappeared from solution over time in the presence or absence of the microbial inhibitors. Initially, less than 100 phenol-mineralizing bacteria per ml and 1 phenol-mineralizing fungus per ml were present at both 200 pg and 100 micrograms of phenol per ml. Phenol mineralization rates were 6.3 times greater for the mineralizing bacteria than for the fungi at 200 pg of phenol per ml. Phenol concentrations above 10 micrograms/ml were inhibitory to the microorganisms capable of mineralizing phenol. The phenol mineralizers grew in the water samples in the absence of phenol, indicating that there were sufficient indigenous nutrients in the lake water to support growth. There was no difference in the growth rate of these microorganisms in the presence or absence of 1 ng of phenol per ml, whereas the growth rate was more rapid at 1 microgram of phenol per ml than in its absence. There was a correlation between microbial growth and the amount of phenol mineralized at 1 microgram but not at 1 ng of phenol per ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of theophylline on chromosomal breakage and sister-chromatid exchange

Frequencies of both sister-chromatid exchange (SCE) and chromosomal breakage (CB) were studied in the lymphocytes of normal individuals (10 and 7 individuals respectively). The cells were exposed in vitro to 3 different concentrations of theophylline (1, 10 and 100 micrograms/ml). A significant concentration effect of the drug was demonstrated for both SCEs and CB. Utilizing a Dunnett's test for individual comparisons, the 10 and 100 micrograms/ml concentrations both demonstrated a significant elevation of SCEs and CB compared to the untreated control cultures. This study suggests that in vitro concentrations of theophylline equal to or greater than 10 micrograms/ml, corresponding to serum levels attained during therapy, increase the frequency of SCEs and chromosome breakage in human lymphocytes.     

Growth of phenol-mineralizing microorganisms in fresh water

A method was developed to enumerate the procaryotic and eucaryotic phenol-mineralizing microorganisms present in samples of fresh water. Sixty-five percent or greater mineralization of [U-14C]phenol was considered a positive tube (contained phenol-mineralizing microorganisms) in the most-probable-number technique. Replicate most-probable-number tubes contained no microbial inhibitors, streptomycin and tetracycline, or cyclohexamide and nystatin plus 200 pg to 100 micrograms of phenol per ml. Phenol mineralization rates were obtained by measuring the amount of exogenous phenol that disappeared from solution over time in the presence or absence of the microbial inhibitors. Initially, less than 100 phenol-mineralizing bacteria per ml and 1 phenol-mineralizing fungus per ml were present at both 200 pg and 100 micrograms of phenol per ml. Phenol mineralization rates were 6.3 times greater for the mineralizing bacteria than for the fungi at 200 pg of phenol per ml. Phenol concentrations above 10 micrograms/ml were inhibitory to the microorganisms capable of mineralizing phenol. The phenol mineralizers grew in the water samples in the absence of phenol, indicating that there were sufficient indigenous nutrients in the lake water to support growth. There was no difference in the growth rate of these microorganisms in the presence or absence of 1 ng of phenol per ml, whereas the growth rate was more rapid at 1 microgram of phenol per ml than in its absence. There was a correlation between microbial growth and the amount of phenol mineralized at 1 microgram but not at 1 ng of phenol per ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maximum growth rate of Mycobacterium avium in continuous culture or chronically infected BALB/c mice

Mycobacterium avium is a human pathogen which may cause either chronic or disseminated disease and the organism exhibits a slow rate of growth. This study provides information on the growth rate of the organism in chronically infected mice and its maximal growth rate in vitro. M. avium was grown in continuous culture, limited for nitrogen with 0.5 mM ammonium chloride and dilution rates that ranged from 0.054 to 0.153 h-1. The steady-state concentration of ammonia nitrogen and M. avium cells for each dilution rate were determined. The bacterial saturation constant for growth-limiting ammonia was 0.29 mM (4 micrograms nitrogen/ml) and, from this, the maximal growth rate for M. avium was estimated to be 0.206 h-1 or a doubling time of 3.4 h. BALB/c mice were infected intravenously with 3 x 10(6) colony-forming units and a chronic infection resulted, typical of virulent M. avium strains. During a period of 3 months, the number of mycobacteria remained constant in the lungs, but increased 30-fold and 8,900-fold, respectively, in the spleen and mesenteric lymph nodes. The latter increase appeared to be due to proliferation in situ. The generation time of M. avium in the mesenteric lymph nodes was estimated to be 7 days.     

Enhancement of mycobacterial growth in middlebrook 7H12 medium by polyoxyethylene stearate

Studies were carried out to enhance mycobacterial growth in 7H12 (BACTEC 12A) radiometric medium. Out of 10 different additives investigated, addition of 100 mcg polyoxy-ethylene stearate (POES)/ml enhanced growth of many mycobacteria, especially ofMycobacterium tuberculosis andM. bovis (TB), with no adverse effects.Evaluation of the growth-enhancing effect of POES in 7H12 medium during primary isolation of 117 mycobacterial cultures from clinical specimens indicated significant time saving due to the rapid growth of mycobacteria, especially of TB. The growth index (GI) in 7H12 medium increased much more rapidly in the POES-containing medium, with smears for acid-fast bacilli available sooner owing to higher biomass. This enhanced growth also allowed earlier differentiation of TB from other mycobacteria by the BACTEC NAP test and earlier initiation of indirect drug susceptibility tests, with results available approximately 2–3 days sooner.     

Experimental criteria for interpretation of bacterial sensitivity to dioxidine determined by diffusion from the disks

Antibacterial activity of dioxidine against aerobic and facultative anaerobic organisms under conditions of anaerobiosis i. e. conditions really observed for example in abscess cavities or necrotic tissues is 30 to 100 times as high as that under aerobic conditions. There is a relationship between sensitivity of bacteria to dioxidine under aerobic and anaerobic conditions which is expressed by the regression equation. Therefore, comparison of the MICs determined under anaerobic conditions with the growth inhibition zones formed by disks with the drug under aerobic conditions is possible. The MIC of dioxidine was determined under anaerobic conditions for 179 clinical strains of aerobic and facultative anaerobic bacteria and the growth inhibition zones of the same bacteria under aerobic conditions were evaluated with the use of disks containing 100, 75, 50, 25, 20, and 15 micrograms of the drug. The border line. MIC differentiating between resistant and sensitive strains was chosen to be equal to 4 micrograms/ml. Differentiation of the strains into sensitive and resistant ones by the values of the growth inhibition zones was performed with the method of error minimization described by C. Metzler and R. De Haan in 1974. Disks containing 25 micrograms of the drug allowed one to differentiate the strains under aerobic conditions into sensitive and resistant ones: the growth inhibition zones greater than 11 mm corresponded to the sensitive strains (the MIC smaller than 4 micrograms/ml) and the growth inhibition zones smaller than 11 mm corresponded to the resistant strains (the MIC greater than 4 micrograms/ml).     

Protection of Chinese hamster ovary cells from hyperthermic killing by cycloheximide or puromycin

Cycloheximide (CHM) and puromycin (PUR) were used at various concentrations up to maxima of 10 micrograms/ml and 100 micrograms/ml, respectively, which inhibited protein synthesis by 95% without any cytotoxicity. The drugs were added to the cells for a maximum period of 7 h, with various combinations for treatment before, during, and after heating. Maximum protection, i.e., a 10,000-fold increase in survival from 5 X 10(-6) to 5 X 10(-2) after 4 h at 43 degrees C, required both 1-2 h of treatment before heating and 1-2 h of treatment during heating. For treatments at 45.5 degrees C, the protection was less, i.e., a 100-fold increase in survival from 10(-5) to 10(-3). Little or no protection was observed if after treatment, the drug was removed before heating, or if the drug was added at the start of heating and left on for 5 min to 3 h after heating. For both drugs, the amount of protection increased as inhibition of protein synthesis increased. However, the amount of protection from the drugs was the same only at about 95% inhibition; at 60-85% inhibition, CHM afforded more protection than PUR. Therefore, the modes of action of the drugs might be common at high drug concentrations, but different when intermediate concentrations are used.