Legionella pneumophila is the cause of Legionnaires' disease, which is a form of potentially fatal pneumonia. To identify genes required for virulence of the bacterium, a library of 1,386 L. pneumophila signature tagged transposon mutants was studied for guinea pig virulence. The mutants were screened in pools of 96 each in a guinea pig model of L. pneumophila pneumonia. Sixteen unique mutant clones were determined to have attenuated virulence after being screened twice in the animal model. All 16 mutants failed to multiply in both lungs and spleens. Four of the sixteen had no apparent defect for intracellular multiplication in macrophages. Partial DNA sequences of the interrupted genes adjacent to the transposon insertions showed that six of them had mutations in five known L. pneumophila virulence genes: dotB, dotF͞icmG, dotO͞icmB, icmX, and proA. Three of the sequenced clones contained mutations in genes without known homology to other published bacterial genes, and seven clones appeared to be homologous to five different known bacterial genes but are still being characterized. With this methodology, we demonstrate the existence of L. pneumophila genes responsible for non-macrophage-related virulence. The discovery of L. pneumophila virulence genes indicates the utility of the signature tagged mutagenesis technique for pulmonary pathogens.

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...

The activities of levofloxacin and ofloxacin against 22 clinical legionella isolates was determined by microbroth dilution susceptibility testing. Growth inhibition of two Legionella pneumophila strains grown in guinea pig alveolar macrophages by levofloxacin, ofloxacin, or erythromycin was also determined. The drug concentrations required to inhibit 90% of strains tested was 0.032 mg/L for levofloxacin or ofloxacin, and was 0.016 mg/L for ciprofloxacin. BYE alpha broth significantly inhibited the activities of all three drugs tested, as judged by the susceptibility of control Escherichia coli strains. Levofloxacin (0.25 mg/L) reduced bacterial counts of two L. pneumophila strains grown in guinea pig alveolar macrophages by 1 log10, but regrowth occurred over a 3 day period; levofloxacin (1 mg/L) reduced bacterial counts by 2-3 log10 cfu/mL. Levofloxacin was significantly more active than erythromycin, and as active as ofloxacin or ciprofloxacin in this assay. Pharmacokinetic and therapy studies of levofloxacin and ofloxacin were performed in guinea pigs with L. pneumophila pneumonia. For the pharmacokinetic study, levofloxacin was given (10 mg/kg) by the intraperitoneal route to infected guinea pigs; mean peak plasma and lung concentrations were 3.4 mg/L and 1.4 micrograms/g, respectively, at 0.5 h and 2.6 mg/L and 0.6 micrograms/g at 1 h. The terminal half-life phase of elimination from plasma and lung was c. 1 h. All 15 infected guinea pigs treated with levofloxacin (10 mg/kg/day given ip once daily) for 5 days survived for 9 days after antimicrobial therapy, as did all 14 guinea pigs treated with the same dose of ofloxacin. None of 13 animals treated with saline survived. Levofloxacin is effective against L. pneumophila in vitro and in a guinea pig model of legionnaire's disease. Levofloxacin should be evaluated as a treatment of human legionnaires' disease.