Activities of sitafloxacin (DU-6859a), either singly or in combination with rifampin, against Mycobacterium ulcerans infection in mice.

Efficacy of a new fluoroquinolone, sitafloxacin (DU-6859a), against Mycobacterium ulcerans was evaluated in vivo using the mouse footpad system. The growth of M. ulcerans in mouse footpads was completely inhibited when mice were fed with sitafloxacin at a dose of 25 mg/kg body weight per day; on the other hand similar effects were observed with ofloxacin at a dose of 100 mg/kg body weight per day. In the presence of rifampin, the above dose of sitafloxacin could be reduced by 75% to achieve total inhibition, while, under similar circumstances, the dose of ofloxacin could be reduced by only 50%. Either used singly or in combination with rifampin, the effects of sitafloxacin were bactericidal. The results suggest that sitafloxacin should be evaluated as a chemotherapeutic agent against M. ulcerans infection.

In vivo susceptibility of Mycobacterium ulcerans to KRM-1648, a new benzoxazinorifamycin, in comparison with rifampicin. Anti-mycobacterial activity of KRM-1648.

The antibacterial effects of a new benzoxazinorifamycin, KRM-1648 (3'-hydroxy-5'-(4-isobutyl-1-piperazinyl, CAS 129791-92-0), against Mycobacterium ulcerans were evaluated in vivo in mouse foot pads, and the results were compared against those obtained with rifampicin (rifampin, CAS 13292-46-1). When mice were fed with the drugs from the day of footpad inoculations, KRM-1648, at concentrations of 0.001% and higher, mixed in mouse food, was effective in inhibiting the growth of M. ulcerans in the foot pads, and the effects were bactericidal. Effects of KRM-1648 at 0.0005% were bacteriostatic. Similar results were obtained with rifampicin, but only at concentrations of 0.008% and above. In established infection, i.e., when M. ulcerans were growing actively in footpads, bactericidal effects were observed with KRM-1648 at concentrations of 0.002% and above; to obtain similar results with rifampicin, the minimum dose was 0.032%. Thus, the results suggest the superiority of KRM-1648 over rifampicin in the treatment of M. ulcerans infection. The possibility of using KRM-1648 in combination with other antimycobacterial agents is discussed.

Acid tolerance of Escherichia coli O157:H7 and its survival in apple juice.

Outbreaks of diarrhoea and haemolytic uraemic syndrome have been associated with the consumption of apple cider and apple juice. The organism implicated in these outbreaks has been Escherichia coli O157:H7, indicating the resistance of the serotype to acidic pH. On comparing the growth of this serotype with a control strain of E. coli, it was found that strain O157:H7 grew well in trypticase soy broth at pH levels ranging from 2.0 to 9.0, while control strains failed to grow at pH levels below 4.0 and above 9.0. The growth of both strains were inhibited by adding 0.05% of either benzoic acid or sorbic acid. Similarly, O157:H7 grew well in both natural (unpasteurized) as well as in pasteurized apple juice and the growth was inhibited by adding 0.1% of either benzoic acid or sorbic acid. Control strains of E. coli failed to grow in either types of apple juice. The possible sources of contamination of natural apple juice with O157:H7 serotype are discussed.

In vitro activity of KRM-1648, either singly or in combination with ofloxacin, against Mycobacterium ulcerans.

The antimicrobial effect of a benzoxazinorifamycin, KRM-1648, either alone or in combination with ofloxacin, was evaluated in vitro against two type strains and six clinical isolates of Mycobacterium ulcerans. Growth of M. ulcerans was measured by plate counts and the BACTEC radiometric method. The minimal inhibitory concentration as well as minimal bactericidal concentration of KRM-1648 against M. ulcerans was between 0.012 and 0.025 mg/l, while corresponding values for rifampicin and rifabutin were in the range of 0.1-0.8 mg/l and 0.1-0.4 mg/l respectively. When combined with ofloxacin, KRM-1648 exhibited strong synergistic activity while only additive effects were observed with the combination of rifampicin (or rifabutin) and ofloxacin. These results suggest that KRM-1648 has a great potential in the treatment of M. ulcerans infection.

Antimicrobial activities of dihydrofolate reductase inhibitors, used singly or in combination with dapsone, against Mycobacterium ulcerans.

Development of new treatments against Mycobacterium ulcerans infection has become crucial because of its wide-scale prevalence throughout the world. The effects of dihydrofolate reductase inhibitors, used either singly or in combination with dapsone against M. ulcerans were evaluated in vitro. When used singly, epiroprim was the most potent, with MICs between 0.5 and 1.0 mg/L, while trimethoprim was totally ineffective. The MICs of K-130 and brodimoprim ranged from 1.0-2.0 mg/L for the former to 2.0-16.0 mg/L for the latter. When combined with dapsone, synergic effects were observed with epiroprim. These results indicate the great potential of epiroprim in treating M. ulcerans infections.

Search for newer antileprosy drugs.

In 1991 World Health Organization proclaimed the goal of global elimination of leprosy as a public health problem by year 2000 by implementing multidrug therapy (MDT). Since then the prevalence rate has declined by 85%. However, during the same period the incidence rate of leprosy has remained constant or even has been increasing. This suggests that it will take a long time for the eradication of leprosy and that without in-vitro cultivation of M. leprae, eradication of leprosy is not likely to be achieved. While in-vitro cultivation is a long-term goal, as an immediate measure, there is an urgent need for the development of newer drugs and newer multidrug therapy regimens. Using the in-vitro system for screening potential antileprosy drugs and also using the mouse foot-pad system we have evaluated several compounds in four classes of drugs--dihydrofolate reductase inhibitors, fluoroquinolones, rifampicin analogues and phenazines--and identified at least two compounds that appear to be more potent than dapsone, rifampicin and clofazimine. Newer combinations of rifampicin analogues and fluoroquinolones have also been identified that seem to be better than the combination of rifampicin and ofloxacin.

ATP content of Mycobacterium tuberculosis grown in vivo and in vitro.

In order to determine the reason for the slow growth of Mycobacterium leprae either in a host or in vitro, the growth characteristics of Mycobacterium tuberculosis were studied. The ATP content of in vitro-grown M. tuberculosis was about 520 pg/10(6) viable organisms. The ATP levels from in vivo-derived organisms obtained from liver and spleen of mice was about 130 pg (in cases of chronic infection) and about 270 pg (in cases of acute infection). When the in vivo-derived organisms were inoculated into culture medium, the growth rates for both types of organisms, acute as well as chronic infection, were the same and the maximum growth was reached during the fifth subculture. Although the maximum ATP content for both types of organism was the same, it was attained during the 4th subculture for organisms obtained during acute infection and during the 6th subculture for those obtained during chronic infection. The comparison between the ATP content of M. leprae and of M. tuberculosis indicates the reason for the slow growth of M. leprae.

In vitro activity of epiroprim, a dihydrofolate reductase inhibitor, singly and in combination with brodimoprim and dapsone, against Mycobacterium leprae.

The antimicrobial effects of a new dihydrofolate reductase inhibitor, epiroprim, alone and in combination with dapsone and brodimoprim against Mycobacterium leprae were evaluated in vitro in cell-free culture system. Two biochemical parameters were used to measure metabolic activity (and growth) of the organism. The minimal inhibitory activity of epiroprim against M. leprae was 10 mg/l and the action was bactericidal. When combined with dapsone, epiroprim exhibited a strong synergism; on the other hand, combination of epiroprim and brodimoprim provided only additive effects. The results suggest that epiroprim can be a component in multidrug therapy regimen in leprosy.

In vitro and in vivo activity of K-130, a dihydrofolate reductase inhibitor, against Mycobacterium leprae.

The antimicrobial effects of a new dihydrofolate reductase inhibitor, K-130 (2,4-diaminodiphenyl sulfone substituted 2,4-diamino-5-benzylpyrimidine), alone and in combination with dapsone (CAS 80-08-0) against both dapsone-sensitive and dapsone-resistant strains of Mycobacterium leprae were evaluated in vitro, in cell-free culture system, and in vivo, in mouse foot pads. The minimal inhibitory concentration of K-130 against dapsone-sensitive as well as dapsone resistant strains of M, leprae was 0.03 microgram/ml, and the activity was bactericidal in both cases. However, when combined with dapsone, K-130 exhibited synergism in case of dapsone-sensitive M. leprae, while in case of dapsone-resistant M. leprae, the effect was merely additive. Similar synergistic effects were also observed in the mouse foot pad system for both types of M. leprae strains.

In vitro activities of phenothiazine-type calmodulin antagonists against Mycobacterium leprae.

Calmodulin-like protein has been established as the primary receptor for calcium in eukaryotic as well as prokaryotic cells. The calmodulin-calcium complex regulates a variety of enzymes including nucleotide phosphodiesterase. Recently, the presence of this protein in Mycobacterium leprae has been demonstrated and the effects of phenothiazine-type calmodulin antagonists on in vitro growth of M. leprae in a cell-free culture system were investigated. Two biochemical parameters were used to measure metabolic activity and growth of the organism. Among the six phenothiazine derivatives tested, trifluoperazine appeared to be the most potent in inhibiting the in vitro growth of M. leprae, with an MIC of 10 micrograms/ml. Chlorpromazine, triflupromazine and thioridazine were less active than trifluoperazine, with an MIC of 20 micrograms/ml each, while the other two, acetopromazine and fluphenazine, were totally ineffective even at 80 micrograms/ml. All four compounds inhibited the uptake of labelled acetate, glycine and thymidine by whole cells of M. leprae. This suggests that these phenothiazine derivatives have multiple sites of action and probably affect the synthesis of lipids, proteins and DNA.

Factors influencing the in vitro growth of Mycobacterium leprae: effect of inoculum.

Factors responsible for the in vitro growth of Mycobacterium leprae in Dhople-Hanks (DH) medium, and also to improve the technique devised earlier, and the source of the M. leprae used as inoculum, were investigated. M. leprae were obtained from armadillos and nude mice, both inoculated earlier with human- or armadillo-derived M. leprae. The growth of M. leprae in DH medium was monitored using two biochemical indicators. Normal growth was obtained when inocula were from livers and spleens of M. leprae-infected armadillos. The M. leprae harvested from the footpads of nude mice failed to multiply in the same medium. Using inocula from livers and spleens of infected armadillos, a gradual decrease in inoculum size resulted in a proportionately slower multiplication of M. leprae. When the DH medium was supplemented with whole M. leprae, or cell-free extracts of M. leprae, from irradiated livers and spleens of infected armadillos, nude mouse-derived M. leprae exhibited growth in the DH medium in accord with that obtained using armadillo-derived M. leprae. Similar results were obtained with cell-free extracts of M. leprae harvested from non-irradiated livers and spleens of infected armadillos, but no growth was obtained when the medium was supplemented with extracts from livers or spleens of normal armadillos. These results indicate the possible existence of a growth factor in armadillo-derived M. leprae.

The activity of rifabutin against Mycobacterium leprae in armadillos.

The activity of rifabutin (LM 427) against Mycobacterium leprae was evaluated in armadillos inoculated earlier with human-derived M. leprae. Rifabutin was administered daily at a dose of 6 mg/kg body weight/day. The effect of rifabutin on M. leprae harvested from armadillos was determined by measuring the intracellular levels of ATP (an indicator of metabolic activity) of M. leprae and also their ability to multiply in the mouse footpads and in vitro in DH medium. Within 2 weeks of initiating the treatment, ATP levels declined to 21% of the original (pre-treatment level) and these M. leprae failed to multiply in the footpads of mice as well as in the in vitro culture system. This suggests that rifabutin was able to kill all M. leprae within 2 weeks. After 8 weeks the treatment was terminated and results showed that M. leprae from the treated armadillos remained non-viable in the mouse footpad system as well as in the in vitro system, indicating bactericidal action of rifabutin. The results suggest that rifabutin can be a substitute for rifampin in the leprosy multi-drug therapy regimen.

Comparative in vitro activities of rifamycin analogues against Mycobacterium leprae.

Comparative activities of various rifamycin analogues against leprosy were studied by evaluating their effects on in vitro growth of Mycobacterium leprae in DH medium as described earlier. Among the seven analogues studied, KRM-1648 was found to be the most potent in inhibiting the growth of rifampicin-sensitive strains of M. leprae, MIC being 0.05 microgram/ml. This was followed by KRM-2312 and T9 (MIC of each being 0.1 microgram/ml) and rifabutin (MIC, 0.2 microgram/ml). Rifampicin, along with KRM-1657 and KRM-1668, were least effective, with MIC for each being 0.4 microgram/ml. The effects of each at their respective MICs were bactericidal. The results were similar for rifampicin-resistant strains of M. leprae, but the MICs were higher than those obtained with rifampicin-sensitive strains of M. leprae. Thus, even though rifampicin has been the first-line drug in the treatment of leprosy, the results in present studies suggest that other rifamycin analogues are available that are more potent than rifampicin against both rifampicin-sensitive as well as rifampicin-restraint strains of M. leprae.

In vitro activity of a new rifamycin derivative against Mycobacterium leprae.

The antimicrobial effects of T9 (3-(4-cinamyl-1-piperazinyl)iminomethyl rifamycin SV) alone and in combination with ofloxacin, against strains of M. leprae were evaluated, using an in vitro cell-free culture system. The minimum inhibitory concentrations (MICs) of T9 against rifampin-sensitive and rifampin-resistant strains of M. leprae were 0.1 microgram/ml and 0.4 microgram/ml, respectively. Furthermore, in common with rifabutin, but not with rifamycin, T9 demonstrated synergy with ofloxacin against both rifampin-sensitive rifampin-resistant strains of M. leprae. The results suggest that T9, in combination with ofloxacin as part of multidrug regimens, warrants further evaluation as treatment for patients with leprosy.

In vitro susceptibility of Mycobacterium leprae to oxygen-mediated damage.

In order to evaluate factors responsible for the failure of Mycobacterium leprae to multiply in cell-free cultures in vitro studies were undertaken to determine the possible poisoning of the organism by hydroxide and superoxide radicals produced in the growth medium. The superoxide dismutase activity was very low, 10% of the levels found in armadillo cells, while measured activity of catalase and glutathione peroxidase was negligible. Susceptibility of M. leprae to hydrogen peroxide was enhanced by potassium iodide but not by lactoperoxidase. The addition of high amounts of catalase completely prevented hydrogen peroxide-mediated killing of M. leprae. Superoxide generated by the action of xanthine oxidase on xanthine was lethal to M. leprae, but superoxide dismutase added to the reaction mixture gave significant protection. Thus superoxide radicals may be a major cause for the sudden termination of growth of M. leprae in primary cultures and also for failure of subcultures.

Role of iron in the pathogenesis of Mycobacterium avium infection in mice.

Mycobacterial infections are of serious concern to HIV-infected patients, and take a heavy toll of such patients. Mycobacterium avium is the most common opportunistic bacterial infection in patients with AIDS. The overload of iron in serum has been implicated in the pathogenicity of a number of bacterial infections. Since iron storage in cells such as macrophages is increased in AIDS, the role of iron as a possible factor in the pathogenesis of M. avium infection was examined. Supplementing iron to normal laboratory chow resulted in accelerated M. avium infection in mice inoculated earlier with the same organism. The bacterial loads in liver, spleen and lungs were approximately 12-fold higher in mice receiving iron supplementation compared with control groups. This is attributed to an increased percentage saturation of iron in the sera of the mice, thus making more iron available for the replication of bacteria. The addition of beef fat to the diet, together with high iron supplementation, further enhanced the infection. Using smaller inocula, mice receiving chow supplemented with high iron and fat developed disseminated M. avium infection faster than control mice. The results provide strong evidence that iron may play a major role in the pathogenesis of M. avium infection.

In vivo activities of novel benzoxazinorifamycins against Mycobacterium leprae.

Among the four newly synthesized benzoxazinorifamycin derivatives, KRM-1648 and KRM-2312 completely inhibited the multiplication of rifampicin-sensitive as well as rifampicin-resistant strains of M. leprae in the foot-pads of mice. Both were found to be more potent than rifampicin and were bactericidal. In combination with ofloxacin, another potent bactericidal drug against M. leprae, both KRM-1648 and KRM-2312 exhibited synergism. Thus, combination of one of these benzoxazinorifamycin derivatives and ofloxacin in multidrug regimens is worth evaluating in clinical trials.

In vitro activity of levofloxacin, singly and in combination with rifamycin analogs, against Mycobacterium leprae.

The in vitro susceptibility of Mycobacterium leprae to levofloxacin was studied by using two biochemical parameters to measure the metabolic activity of the organism. Levofloxacin consistently exhibited twofold greater bactericidal activity than ofloxacin, with the MIC being 0.75 microgram/ml. When combined with one of the three rifamycin analogs, synergism was obtained with KRM-1648 and rifabutin but not with rifampin.

In vitro activities of 2,2'-bipyridyl analogues against Mycobacterium avium and M. tuberculosis.

SETTING: Because of widespread emergence of resistant Mycobacterium tuberculosis and the high incidence of opportunistic infection caused by M. avium (MAC) in AIDS patients, there is an urgent need for new drugs against these organisms. OBJECTIVE: To evaluate the activity of newly synthesized 2,2'-bipyridyl analogues against MAC and M. tuberculosis. DESIGN: Susceptibility of MAC and M. tuberculosis to VUF-8514 and VUF-8842 were determined by both tube dilution method using 7H9 broth and radiometric (BACTEC) method using 14C-palmitic acid. RESULTS AND CONCLUSIONS: The MICs of 8514 against MAC and M. tuberculosis wee 1 microgram/ml and 0.5 microgram/ml respectively, while for 8842 the respective values were 8 micrograms/ml and 2 micrograms/ml. In general, the MBC values for both drugs were two-fold higher than their corresponding MIC values. However, both drugs exhibited high bactercidal activities against both organisms. The MICs of clinical isolates of both organisms were in the same range as reference strains; furthermore, two isolates of M. tuberculosis that showed resistance to rifampicin were found to be susceptible to 8514. Thus, these two bipyridyl analogues show great promise in chemotherapy of tuberculosis and M. avium infection.

The in-vitro activities of novel benzoxazinorifamycins against Mycobacterium leprae.

The activities of four newly synthesized benzoxazinorifamycin derivatives, either alone or in combination with ofloxacin, against strains of Mycobacterium leprae were determined by assessing their effects on two biochemical parameters of metabolic activity which served as surrogate markers for growth in vitro. KRM-1648 and KRM-2312 were the most active agents tested against both a rifampicin-susceptible isolate (MICs of 0.05 and 0.1 mg/L respectively) and a rifampicin-resistant isolate (MICs of 0.2 and 0.3 mg/L respectively); both compounds were more active than either rifampicin or rifabutin. The activities of the two other derivatives, KRM-1657 and KRM-1668, against a rifampicin- susceptible strain (MICs of 0.3 mg/L) were similar to that of rifampicin, while the MIC of each of these agents for the rifampicin-resistant strain was 1.0mg/L. In common with rifabutin, both of the more active derivatives demonstrated synergy with ofloxacin against the rifampicin-susceptible isolates. The results of this study suggest that these compounds, in combination with ofloxacin as part of multidrug regimens, warrant further evaluation as treatment for patients with leprosy.