We compared the in-vitro antimycobacterial activities of rifabutin and KRM-1648, two rifamycin derivatives, with that of rifampicin against 163 strains of Mycobacterium tuberculosis. We also evaluated the correlation between the level of resistance to rifampicin, rifabutin and KRM-1648 and genetic alterations in the rpoB gene. All 82 strains susceptible to rifampicin or resistant to rifampicin with MICs < or = 16 mg/L were susceptible to rifabutin and KRM-1648 with MICs < or = 1 mg/L. Seventy-six of 81 strains resistant to rifampicin with MICs > or = 32 mg/L were resistant to both rifabutin and KRM-1648, but with lower MICs than those of rifampicin. KRM-1648 showed more potent antimycobacterial activity than rifabutin against organisms with low MICs (< or = 1 mg/L), while rifabutin was more active than KRM-1648 against organisms with high MICs (> or = 2 mg/L). A total of 96 genetic alterations around the 69 bp core region of the rpoB gene were detected in 92 strains. Alterations at codons 515, 521 and 533 in the rpoB gene did not influence the susceptibility to rifampicin, rifabutin and KRM-1648. Point mutations at codons 516 and 529, deletion at codon 518 and insertion at codon 514 influenced the susceptibility to rifampicin but not that to rifabutin or KRM-1648. With the exception of one strain, all alterations at codon 513 and 531 correlated with resistance to the three test drugs. The resistant phenotype of strains with an alteration at codon 526 depended on the type of amino acid substitution. Our results suggest that analysis of genetic alterations in the rpoB gene might be useful not only for predicting rifampicin susceptibility, but also for deciding when to use rifabutin for treating tuberculosis. Further studies may be required to determine the usefulness of KRM-1648.

Five 14C-labelled macrolide antibiotics (erythromycin, josamycin, clarithromycin (TE-031), rokitamycin and roxithromycin) were studied for their transport into human polymorphonuclear leucocytes. Intracellular/extracellular concentration ratios (transport ratios) of these macrolides were quite high: erythromycin, 6.6; josamycin, 15.5; clarithromycin, 16.4; rokitamycin, 30.5; and roxithromycin, 21.9. When polymorphonuclear leucocytes were pre-treated with formaldehyde or incubated at 4 degrees C, or at low pH, transport ratios were reduced. When extracellular macrolide was removed, intracellular macrolide concentrations became as low as 30% of the pre-wash concentrations in 5 min. KF lowered the transport ratios of josamycin and rokitamycin in particular and NaCN reduced the transport ratios of erythromycin and josamycin strikingly. Ouabain slightly lowered transport ratios of all the antibiotics tested except roxithromycin, and 2, 4-dinitrophenol decreased the transport ratio of clarithromycin markedly. The addition of various amino acids or hexose did not inhibit the transfer. Adenosine, however, inhibited the transfer of these antibiotics except erythromycin and lowered transport ratios by 83 to 92%. Puromycin reduced transport ratios of the same antibiotics by 59 to 95%. With polymorphonuclear leucocytes that had phagocytosed Legionella pneumophila serogroup 1, transport ratios of all five drugs tended to decrease. However, when Staphylococcus aureus ATCC 25923 or opsonized zymosan was phagocytosed, transport ratios for macrolides, except for roxithromycin, increased.

High-performance liquid chromatography was used to determine the penetration of 19 antimicrobial agents into human polymorphonuclear leukocytes. The ratios of the intracellular concentration to the extraceHlular concentration of ampicillin, piperacillin, cefazolin, ceftizoxime, cefpimizole, and ceftazidime were all less than 0.6. Lincomycin showed a high intracellular-to-extraceliular ratio (3.0), while clindamycin achieved a ratio of 15.5, which was the highest ratio of all of the 19 tested antibiotics. Ratios for rifampin, isoniazid, chloramphenicol, and trimethoprim were 8.2, 1.1, 9.6, and 6.1, respectively. Six quinolone-class antimicrobial agents had ratios from 2.2 to 8.2. Flucytosine showed a penetration ratio of 4.6. Clindamycin uptake was temperature dependent and occurred best with live polymorphonuclear leukocytes; sodium fluoride, adenosine, and puromycin were inhibitory. The results obtained in this study correlate well with the results of other studies involving radioisotopic methods. This indicates that high-performance liquid chromatography is a useful method for determining the intracellular penetration of antimicrobial agents.

We analyzed the relationship between rifampin MICs and rpoB mutations of 40 clinical isolates of Mycobacterium tuberculosis. A point mutation in either codon 516, 526, or 531 was found in 13 strains requiring MICs of > or = 64 micrograms/ml, while 21 strains requiring MICs of < or = 1 microgram/ml showed no alteration in these codons. However, 3 of these 21 strains contained a point mutation in either codon 515 or 533. Of the other six strains requiring MICs between 2 and 32 micrograms/ml, three contained a point mutation in codon 516 or 526, while no alteration was detected in the other three. Our results indicate that the sequencing analysis of a 69-bp fragment in the rpoB gene is useful in predicting rifampin-resistant phenotypes.

The antimicrobial activity of ofloxacin was tested against 15 standard strains and 37 clinical and environmental strains of Legionella pneumophila by agar dilution susceptibility studies with a new growth medium. The ofloxacin MICs were inoculum dependent and ranged from 0.03 to 0.125 ,ug/ml. The antibacterial activities of other agents tested relative to ofloxacin were rifampin > ofioxacin > josamycin > pipemidic acid. Ofloxacin, at concentrations equal to or greater than 0.05 ,ug/ml, inhibited the growth of L. pneumophila grown in human monocytes. The therapeutic efficacy of ofloxacin in experimental guinea pig L. pneumophila pneumonia was greater than that observed with erythromycin or josamycin therapy; it was less effective than was rifampin. Ofloxacin was very active against intracellular L. pneumophila in these experiments and should be studied in the therapy of human Legionnaires disease.

In a newly established murine model of chronic Pseudomonas aeruginosa respiratory infection mimicking diffuse panbronchiolitis (DPB), we investigated the effect of oral administration of clarithromycin on lymphocyte accumulation in the lung. Infection was produced by placement of a plastic tube precoated with P. aeruginosa in the bronchus. The number of bacteria on the tube was 6.25 +/- 0.22 log10 colony-forming units (cfu)/ml. Viable bacteria were constantly isolated at 10(5) to 10(6) cfu/specimen from the lungs for more than 1 yr. The histopathologic features resembled those of DPB consisting of massive accumulation of lymphocytes in the lung. The total number of pulmonary lymphocytes started to increase on Day 7, reaching a peak level within 12 d of intratracheal challenge. The number remained steady at that level for up to 120 d. There was also a steady fall in the CD4+/CD8+ ratio in the lungs, commencing on Day 7 and persisting to Day 120. A 10-d course of oral clarithromycin (10 mg/kg/d) from Day 7 resulted in a reduction of lymphocyte numbers to baseline level, although the dose did not influence the number of bacteria in the lungs. Treatment also increased the CD4+/CD8+ ratio to the baseline level from Day 7 to 17. Our results were similar to those detected in bronchoalveolar lavage fluid of patients with DPB and suggest that the therapeutic benefits of clarithromycin are due to its anti-inflammatory properties rather than antimicrobial effect.

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The inhibitory effects of azithromycin (AZM), a new 15-membered macrolide antibiotic, on the production of exotoxin A, total protease, elastase, and phospholipase C by Pseudomonas aeruginosa were determined, and the virulence-suppressing effects of AZM were compared with those of erythromycin (EM), roxithromycin (RXM), and rokitamycin (RKM). The elfect of exposure ofP. aeruginosa PA103 or B16 in cultures to sub-MICs of these macrolide antibiotics on the production of exoenzymes was determined. AZM suppressed the in vitro production of extracellular and intracellular exotoxin A by P. aeruginosa PA103 more than did EM, even at a concentration of only 2 ,ug/ml. At concentrations of between 4 and 32 ,ug/ml, AZM also inhibited total protease, elastase, and phospholipase C production by P. aeruginosa B16 more than did EM, RXM, and RKM. AZM was effective in suppressing exotoxin A and total protease production through 24 h of incubation in the presence of drug at sub-MICs, but it had no significant effect on either the growth of P. aeruginosa or its total protein production. Moreover, at a concentration of 4 ,ug/ml, AZM suppressed exoenzyme production by other strains of P. aeruginosa more than did EM. These findings indicate that AZM, EM, RXM, and RKM each has an inhibitory effect on exoenzyme production separate from the antimicrobial effect and that, of these macrolides, AZM has the strongest virulence-suppressing effect.Pseudomonas aeruginosa is an opportunistic pathogen which frequently causes severe septicemia in immunocompromised hosts (4, 5). It is also the principal pulmonary pathogen in patients with cystic fibrosis (24, 33) and diffuse panbronchiolitis (16). P. aeruginosa exoenzymes, including exotoxin A, alkaline protease, elastase, and phospholipase C, are known to be virulence factors of importance in the various types of P. aeruginosa infections (26). The findings of our previous studies have suggested that P. aeruginosa isolates from blood cultures produce large quantities of exotoxin A and total protease (10) and that the production of exotoxin A plays an important role in the pathogenesis of endogenous P. aeruginosa septicemia in mice (13).It has recently been reported that treatment with erythromycin (EM) improves the clinical symptoms and prognosis of patients with chronic pulmonary infections (18). Kita et al. (18) have shown that erythromycin stearate (EMS) suppresses the production of protease and leukocidin by P. aeruginosa without affecting cell growth and speculated that EMS may affect the virulence factors of this organism (18) and host defense mechanisms (7, 9, 17). More recently, we have shown that erythromycin lactobionate (EML) suppresses the in vitro pro-