WIN 57273 is bactericidal for Legionella pneumophila grown in alveolar macrophages

Abstract

The in vitro antimicrobial activity of WIN 57273, a new quinolone antimicrobial agent, was determined for 21 LegioneUa strains, using broth macrodilution and agar dilution testing methods; ciprofloxacin and erythromycin were tested as well. Three different buffered yeast extract media were used for the agar dilution studies, two of which were made with starch rather than charcoal. Broth macrodilution susceptibility testing was performed with buffered yeast extract broth and two Legionella pneumophila strains. Antimicrobial inhibition of L. pneumophila growth in guinea pig alveolar macrophages was also studied, using a method able to detect bacterial killing. The MICs for 90% of the 21 strains of Legionella spp. grown on buffered charcoal yeast extract medium were 0.125 ,g/ml for WIN 57273, 0.25 ,Ig/ml for ciprofloxacin, and 1.0 ,ug/ml for erythromycin. These MICs were falsely high, because of inhibition of drug activity by the medium used. Use of less drug-antagonistic, starch-containing media did not support good growth of the test strains. The broth macrodilution MICs for two strains of L. pneumophila serogroup 1 were s0.03 ,ug/ml for WIN 57273 and ciprofloxacin and 0.125 ,ug/mI for erythromycin. WIN 57273, ciprofloxacin, and erythromycin all inhibited growth of L. pneumophila in guinea pig alveolar macrophages at concentrations of 1 ,ug/ml, but only WIN 57273 prevented regrowth or killed L. pneumophila after removal of extracellular antimicrobial agent.Erythromycin is considered the treatment of choice for Legionnaires disease on the basis of retrospective studies. However, a major limitation of erythromycin therapy is that a long treatment course is required to prevent disease relapse (14). The search for alternatives to erythromycin therapy is compromised by the poor correlation of results of classical in vitro antimicrobial susceptibility testing with clinical experience (7,11,14). This discrepancy is likely due to antimicrobial antagonism by components of the media and to the protected intracellular site in which legioneilae exist.Some investigators have claimed the superiority of one medium over another for the purposes of susceptibility testing, mainly in terms of limited inhibition of antimicrobial agents, but few comparative studies have been performed (2,8,13,20).Antimicrobial treatment of an animal model of Legionnaires disease has demonstrated good correlation with clinical observations, but is too expensive for screening purposes (3, 7, 10, 18-20). To address this, several investigators have validated the use of monocyte or macrophage growth inhibition tests to predict the effectiveness of antimicrobial agents in animal models (1,9,11,(20)(21)(22). However, reversible inhibition of bacterial growth has been detected in these cellular systems (12, 21).We performed susceptibility testing of legionellae with erythromycin, ciprofloxacin, and WIN 57273, a new quinolone antimicrobial agent, to determine whether use of different media influenced susceptibility results and inhibition of antimicrobial agen...

Pore Formation Triggered byLegionellaspp. Is an Nlrc4 Inflammasome-Dependent Host Cell Response That Precedes Pyroptosis

“…GFP expression was induced with 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG). F2111, a clinical isolate of L. pneumophila was previously described (18). The Legionella species…”

In Vitro Activity of ABT-773 against Legionella pneumophila , Its Pharmacokinetics in Guinea Pigs, and Its Use to Treat Guinea Pigs with L. pneumophila Pneumonia

“…We have previously used these 20 strains in other studies of antimicrobial activity against Legionella spp. (11,13,15,17). Included among these strains were L. pneumophila strains F889 and F2111, which have been extensively studied in a cell model of L. pneumophila infection.…”

“…Guinea pig pulmonary alveolar macrophages were harvested and purified as described previously (11). The final concentration of macrophages was approximately 10 5 cells per well.…”

“…Approximately 10 4 bacteria were added to each well. The bacteria were incubated with macrophages for 1 h in a shaking incubator and then for 1 day in stationary culture, as described previously (11). One set of replicate wells was washed (500 l) three times with tissue culture medium and was then sonicated at low energy to release the intracellular bacteria, which were quantified with BCYE␣ agar.…”

MyD88-Dependent Responses Involving Toll-Like Receptor 2 Are Important for Protection and Clearance ofLegionella pneumophilain a Mouse Model of Legionnaires' Disease

“…L. pneumophila serogroup 1 strain JR32 (38), serogroup 1 clinical isolate F2111 (11), and an isogenic dotA mutant of strain F2111 obtained by sodium selection were used in this study. L. pneumophila strains were cultured on charcoal-yeast extract (CYE) agar (12) For enzymelinked immunosorbent assay (ELISA) studies and in vivo growth assays, bacteria were grown to an optical density at 600 nm of 1 in AYE broth.…”