Choline Supplementation Sensitizes Legionella dumoffii to Galleria mellonella Apolipophorin III.

The growth of Legionella dumoffii can be inhibited by Galleria mellonella apolipophorin III (apoLp-III) which is an insect homologue of human apolipoprotein E., and choline-cultured L. dumoffii cells are considerably more susceptible to apoLp-III than bacteria grown without choline supplementation. In the present study, the interactions of apoLp-III with intact L. dumoffii cells cultured without and with exogenous choline were analyzed to explain the basis of this difference. Fluorescently labeled apoLp-III (FITC-apoLp-III) bound more efficiently to choline-grown L. dumoffii, as revealed by laser scanning confocal microscopy. The cell envelope of these bacteria was penetrated more deeply by FITC-apoLp-III, as demonstrated by fluorescence lifetime imaging microscopy analyses. The increased susceptibility of the choline-cultured L. dumoffii to apoLp-III was also accompanied by alterations in the cell surface topography and nanomechanical properties. A detailed analysis of the interaction of apoLp-III with components of the L. dumoffii cells was carried out using both purified lipopolysaccharide (LPS) and liposomes composed of L. dumoffii phospholipids and LPS. A single micelle of L. dumoffii LPS was formed from 12 to 29 monomeric LPS molecules and one L. dumoffii LPS micelle bound two molecules of apoLp-III. ApoLp-III exhibited the strongest interactions with liposomes with incorporated LPS formed of phospholipids isolated from bacteria cultured on exogenous choline. These results indicated that the differences in the phospholipid content in the cell membrane, especially PC, and LPS affected the interactions of apoLp-III with bacterial cells and suggested that these differences contributed to the increased susceptibility of the choline-cultured L. dumoffii to G. mellonella apoLp-III.

Omadacycline: development of a novel aminomethylcycline antibiotic for treating drug-resistant bacterial infections.

Omadacycline is a first-in-class aminomethylcycline antibiotic that circumvents common tetracycline resistance mechanisms. In vitro omadacycline has potent activity against Gram-positive aerobic bacteria including methicillin-resistant Staphylococcus aureus, penicillin-resistant and multidrug-resistant Streptococcus pneumoniae, and vancomycin-resistant Enterococcus spp. It is also active against common Gram-negative aerobes, some anaerobes and atypical bacteria including Legionella spp. and Chlamydia spp. Ongoing Phase III clinical trials with omadacycline are investigating once daily doses of 100 mg intravenously followed by once-daily doses of 300 mg orally for the treatment of acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia. This paper provides an overview of the microbiology, nonclinical evaluations, clinical pharmacology and initial clinical experience with omadacycline.

The lipid composition of Legionella dumoffii membrane modulates the interaction with Galleria mellonella apolipophorin III.

Apolipophorin III (apoLp-III), an insect homologue of human apolipoprotein E (apoE), is a widely used model protein in studies on protein-lipid interactions, and anti-Legionella activity of Galleria mellonella apoLp-III has been documented. Interestingly, exogenous choline-cultured Legionella dumoffii cells are considerably more susceptible to apoLp-III than non-supplemented bacteria. In order to explain these differences, we performed, for the first time, a detailed analysis of L. dumoffii lipids and a comparative lipidomic analysis of membranes of bacteria grown without and in the presence of exogenous choline. (31)P NMR analysis of L. dumoffii phospholipids (PLs) revealed a considerable increase in the phosphatidylcholine (PC) content in bacteria cultured on choline medium and a decrease in the phosphatidylethanolamine (PE) content in approximately the same range. The interactions of G. mellonella apoLp-III with lipid bilayer membranes prepared from PLs extracted from non- and choline-supplemented L. dumoffii cells were examined in detail by means of attenuated total reflection- and linear dichroism-Fourier transform infrared spectroscopy. Furthermore, the kinetics of apoLp-III binding to liposomes formed from L. dumoffii PLs was analysed by fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy using fluorescently labelled G. mellonella apoLp-III. Our results indicated enhanced binding of apoLp-III to and deeper penetration into lipid membranes formed from PLs extracted from the choline-supplemented bacteria, i.e. characterized by an increased PC/PE ratio. This could explain, at least in part, the higher susceptibility of choline-cultured L. dumoffii to G. mellonella apoLp-III.

Controlling Legionella and Pseudomonas aeruginosa re-growth in therapeutic spas: implementation of physical disinfection treatments, including UV/ultrafiltration, in a respiratory hydrotherapy system.

The study aimed to assess the efficacy of an integrated water safety plan (WSP) in controlling Legionella re-growth in a respiratory hydrotherapy system located in a spa centre, supplied with sulphurous water, which was initially colonized by Legionella pneumophila. Heterotrophic plate counts, Pseudomonas aeruginosa, Legionella spp. were detected in water samples taken 6-monthly from the hydrotherapy equipment (main circuit, entry to benches, final outlets). On the basis of the results obtained by the continuous monitoring and the changes in conditions, the original WSP, including physical treatments of water and waterlines, environmental surveillance and microbiological monitoring, was integrated introducing a UV/ultrafiltration system. The integrated treatment applied to the sulphurous water (microfiltration/UV irradiation/ultrafiltration), waterlines (superheated stream) and distal outlets (descaling/disinfection of nebulizers and nasal irrigators), ensured the removal of Legionella spp. and P. aeruginosa and a satisfactory microbiological quality over time. The environmental surveillance was successful in evaluating the hazard and identifying the most suitable preventive strategies to avoid Legionella re-growth. Ultrafiltration is a technology to take into account in the control of microbial contamination of therapeutic spas, since it does not modify the chemical composition of the water, thus allowing it to retain its therapeutic properties.

Reduction of Legionella spp. in water and in soil by a citrus plant extract vapor.

Legionnaires' disease is a severe form of pneumonia caused by Legionella spp., organisms often isolated from environmental sources, including soil and water. Legionella spp. are capable of replicating intracellularly within free-living protozoa, and once this has occurred, Legionella is particularly resistant to disinfectants. Citrus essential oil (EO) vapors are effective antimicrobials against a range of microorganisms, with reductions of 5 log cells ml(-1) on a variety of surfaces. The aim of this investigation was to assess the efficacy of a citrus EO vapor against Legionella spp. in water and in soil systems. Reductions of viable cells of Legionella pneumophila, Legionella longbeachae, Legionella bozemanii, and an intra-amoebal culture of Legionella pneumophila (water system only) were assessed in soil and in water after exposure to a citrus EO vapor at concentrations ranging from 3.75 mg/liter air to 15g/liter air. Antimicrobial efficacy via different delivery systems (passive and active sintering of the vapor) was determined in water, and gas chromatography-mass spectrometry (GC-MS) analysis of the antimicrobial components (linalool, citral, and ß-pinene) was conducted. There was up to a 5-log cells ml(-1) reduction in Legionella spp. in soil after exposure to the citrus EO vapors (15 mg/liter air). The most susceptible strain in water was L. pneumophila, with a 4-log cells ml(-1) reduction after 24 h via sintering (15 g/liter air). Sintering the vapor through water increased the presence of the antimicrobial components, with a 61% increase of linalool. Therefore, the appropriate method of delivery of an antimicrobial citrus EO vapor may go some way in controlling Legionella spp. from environmental sources.

Anti-Legionella activity of essential oil of Satureja cuneifolia.

The essential oil of Satureja cuneifolia Ten. was characterized by a high concentration of the phenolic compounds carvacrol (21.3%) and thymol (9.2%). The in vitro activity of the essential oil against Legionela pneumophila serogroups (SG) I and 2-15 and Legionella spp. from different sources, using microdilution, showed that L. pneumofila is sensitive to the oil, with MICs ranging from 0.12 to 0.5%, v/v, and a MBC at 0.5 to 1%, v/v. The essential oil of S. cuneifolia was effective in the reduction of Legionellosis infections.

In vitro and in vivo activity of olamufloxacin (HSR-903) against Legionella spp.

The activity of the fluoroquinolone olamufloxacin (HSR-903) against Legionella spp. was studied in vitro and in vivo. The olamufloxacin MIC at which 50% of isolates are inhibited (MIC50) for 81 different Legionella spp. strains (59 type strains and 22 clinical isolates) was 0.008 mg/L, which was identical to sparfloxacin, whereas the MIC50s for erythromycin, levofloxacin and ciprofloxacin were 0.25, 0.032 and 0.032 mg/L, respectively. Olamufloxacin and sparfloxacin (at 0.008 mg/L) inhibited intracellular growth and subsequent cytotoxicity of L. pneumophila 80-045 in J774.1 macrophages, whereas levofloxacin and ciprofloxacin did not, at the same concentration. When olamufloxacin was given to the infected guinea pigs orally (5 mg/kg of body weight), peak levels in the lung were 3.02 mg/kg at 2 h post-administration, with a half-life of 3.41 h and an AUC0-12 of 12.31 mg.h/kg. The 2 day post-infection bacterial burden of the lung in the animals treated with olamufloxacin (5 and 1.25 mg/kg given orally twice a day) was much lower than in those treated with levofloxacin (same dose as olamufloxacin) or erythromycin (10 mg/kg given orally twice a day). When treated with olamufloxacin (5 mg/kg given orally twice a day) for 7 days, 11 of 12 L. pneumophila-infected guinea pigs survived for 14 days post-infection, as did all 12 guinea pigs treated with levofloxacin (5 mg/kg given orally twice a day) for 7 days. In contrast, only two of 12 animals treated with erythromycin survived and 10 of 11 died in the physiological saline group. Olamufloxacin was as effective as levofloxacin in a guinea pig model of Legionnaires' disease. These data warrant further study of whether olamufloxacin is an option for the treatment of Legionella infections.

In vitro antimycobacterial and antilegionella activity of licochalcone A from Chinese licorice roots.

Licochalcone A, extracted and purified from Chinese licorice roots, showed in vitro inhibitory effect on human pathogenic Mycobacteria species and Legionella species. M. tuberculosis, M. bovis and BCG were inhibited by < 20 mg/l licochalcone A, whereas all non- M. tuberculosis complex species were resistant to > 20 mg/l Legionella pneumophila (serogroups 1 - 7) and L. bozemanii, L. dumoffii, L. feelei, L. longbeacheae and L. wadsworthii were inhibited by licochalcone A 1 - 4 mg/l, whereas L. gormanii and L. micdadei were inhibited by licochalcone A 500 - 1000 mg/l. These data indicate that licochalcone A might be of interest as a new class of antibacterial drug in the treatment of severe lung-infections.

Activity of trovafloxacin (CP-99,219) against Legionella isolates: in vitro activity, intracellular accumulation and killing in macrophages, and pharmacokinetics and treatment of guinea pigs with L. pneumophila pneumonia.

The activity of trovafloxacin against 22 clinical Legionella isolates was determined by broth microdilution susceptibility testing. The trovafloxacin concentration required to inhibit 90% of strains tested was < or = 0.004 micrograms/ml, in contrast to 0.032 micrograms/ml for ofloxacin. In guinea pig alveolar macrophages, trovafloxacin achieved intracellular levels up to 28-fold over the extracellular concentration, which was similar to the levels obtained with erythromycin. Trovafloxacin (0.25 micrograms/ml) reduced bacterial counts of two L. pneumophila strains grown in guinea pig alveolar macrophages by > 2 log10 CFU/ml, without regrowth, under drug-free conditions over a 3-day period; trovafloxacin was significantly more active than ofloxacin or erythromycin (0.25 to 1 microgram/ml) in this assay. Single-dose (10 mg of prodrug CP-116,517-27 per kg of body weight given intraperitoneally [i.p.], equivalent to 7.5 mg of trovafloxacin per kg) pharmacokinetic studies performed in guinea pigs with L. pneumophila pneumonia revealed peak serum and lung trovafloxacin levels to be 3.8 micrograms/ml and 5.0 micrograms/g, respectively, at 0.5 h and 4.2 micrograms/ml and 2.9 micrograms/g, respectively, at 1 h. Administration of a lower prodrug dose (1.4 mg of trovafloxacin equivalent per kg i.p.) gave levels in lung and serum of 0.4 microgram/g and 0.4 microgram/ml, respectively, 1 h after drug administration. The terminal half-lives of elimination from serum and lung were 0.8 and 1.1 h, respectively. All 15 infected guinea pigs treated for 5 days with CP-116,517-27 once daily (10 mg/kg/day i.p., equivalent to 7.5 mg of trovafloxacin per kg/day) survived for 10 days after antimicrobial therapy, as did all 15 guinea pigs treated with ofloxacin once daily (10 mg/kg/day i.p.) for 5 days. None of 13 animals treated with saline survived. In a second experiment with animals, trovafloxacin (1.4 mg/kg/day i.p. for 5 days) protected all 16 guinea pigs from death, whereas all 15 animals treated with saline died. Trovafloxacin is an effective antimicrobial agent against Legionella in vitro and in vivo, with the ability to concentrate in macrophages and kill intracellular organisms.

[Bactericidal effect of chlorine on strains of Legionella species].

We have previously reported that the hot water of 17 (42.5%) out of 40 thermal baths were contaminated with legionellae. Our recent investigation revealed that legionellae inhabited 39 (66.1%) of the 59 thermal bath water, and their viable counts were at the level of 10(4) CFU/100 ml in 5 baths and 10(5) CFU/100 ml in another 5. Accordingly, the bactericidal effects of free chlorine on 102 strains of 22 Legionella species were tested, in order to find a method of controlling legionellae in thermal bath water. The test strains were the type strains of 22 species, 35 strains of 4 species from patients with Legionella pneumonia in Japan, and 45 strains of 4 species from thermal bath water. Viable cells of all 102 strains suspended at the concentration of 10(5) CFU/100 ml in sodium hypochloride solution with 0.4 mg/l free chlorine became undetectable within 15 min.

Rifampin resistance of Legionella pneumophila is not increased during therapy for experimental Legionnaires disease: study of rifampin resistance using a guinea pig model of Legionnaires disease.

Isolates of Legionella pneumophila serogroup 1, obtained from guinea pigs with experimentally induced Legionnaires disease, were tested for rifampin resistance. Thirteen isolates were from animals treated with rifampin alone, four isolates were from animals treated with saline, and three isolates each were from animals treated with erythromycin or erythromycin plus rifampin; all of these isolates were derived from the same parent strain, F889. Most of the isolates were obtained from rifampin-treated animals that survived infection but had persistence of bacteria in their lungs at necropsy. No differences in rifampin agar dilution MICs were detected for the 23 isolates and parent strain that were tested. None of the 13 isolates from animals treated with rifampin alone had a high number of resistant organisms detected by using a rifampin gradient plate assay. Thirteen isolates plus the parent strain were tested by using a quantitative method of determining resistance frequency. Considerable heterogeneity among isolates was observed, but there was no evidence of increased resistance for any treatment group. The range of rifampin resistance frequencies was 10(-7) to 10(-8). No evidence for rifampin-induced resistance of L. pneumophila was found in this study.

Pathogenesis and chemotherapy of experimental Legionella pneumophila infection in the chick embryo.

The pathogenicity of Legionella pneumophila, serogroup 1, strain Nottingham N7, was assessed in terms of LD50 data and the ability of the organism to induce pathological lesions in the fertile hen's egg. Histopathological examination of embryo organs after inoculation with 1, 10, 100 and 1000 times the yolk sac LD50 revealed a disseminated infection. Systemic spread of the organism resulted in widespread necrosis and evidence of consolidation together with generation of copious amounts of oedema fluid. These were particularly severe in the liver, heart, spleen and kidney. The infection elicited a massive inflammatory response typified by infiltration with polymorphonuclear leucocytes and lymphocytes. Selected antimicrobial chemotherapeutic agents were investigated in protection studies for their capacity to ameliorate or control disease processes in this test system. Of those examined ciprofloxacin was most effective in reducing the incidence of lesions in these tissues and for prolonging embryo viability. Rifampicin, and to a lesser degree, erythromycin and doxycycline, also showed antimicrobial activity in these in vivo trials. These results indicate that the fertile hen's egg may be a useful alternative to other animal systems for the in vivo testing of clinically putative antimicrobial agents in the treatment of Legionnaires' disease.

Characterization of the selective inhibition of growth of virulent Legionella pneumophila by supplemented Mueller-Hinton medium.

The phenotypic difference between virulent and avirulent Legionella pneumophila with regard to growth on supplemented Mueller-Hinton (SMH) agar was investigated. Defined populations of virulent and avirulent L. pneumophila were inoculated onto hybrid growth media containing the combination of SMH agar components with potassium phosphate-buffered charcoal-yeast extract agar. The casein acid hydrolysate component of SMH agar was found to be inhibitory to the growth of virulent but not avirulent cells. The selectively inhibitory component of the casein acid hydrolysate was identified as NaCl.

[Treatment of severe respiratory infection. Legionella pneumonia].

This paper discusses the evaluation of new macrolides and new quinolones for the treatment of Legionnaires' disease in in vitro susceptibility test, penetration to polynuclear leukocytes and treatment in an animal model. Some kinds of biological response modifier (BRMs) such as granulocyte colony stimulating factor (GCSF), monocyte CSF (M-CSF), GM-CSF, recombinant interleukin-1 (IL-1) and IL-2 were also evaluated. The antimicrobial activity (MIC) of new macrolides to Legionella pneumophila (45 strains) showed the highest activity in TE-031 (Taisho Pharmaceutical Co., Tokyo, Japan) and then rokitamycin (RKM), RU-28965 (Roussel, France), erythromycin (EM), josamycin (JM) in decreasing order of activity. According to the results of the data of cell-penetration and survival rate after treatment of guinea pigs with experimental Legionella pneumonia, the new macrolides such as TE-031, RKM and RU-28965 are expected to be more effective in the treatment of Legionnaires' disease than EM. New quinolones such as ciprofloxatin (CPFX), NY-198 (Hokuriku Pharmaceutical Co., Japan) or T-3262 (Toyama Kagaku Pharmaceutical Co., Toyama Japan) were compared to ofloxacin (OFLX), enoxacin (ENX) or rifampin (RFP) for evaluation. These drugs showed excellent activity against L. pneumophila and good penetration to polynuclear leukocytes. Regarding the treatment of guinea pig with legionella pneumonia, OFLX was the most effective, and NY-198 or T-3262 were more effective than EM treatment. The highest survival rate was obtained with IL-2 in infected guinea pigs. We also observed the efficacy of combined use of IL-2 and HR-8 10 (Horchst FRG) which is a newly developed cephem antibiotic.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of ciprofloxacin and rifampicin in experimental Legionella pneumophila pneumonia.

We evaluated intraperitoneal ciprofloxacin and rifampicin alone and as combination therapy in experimentally induced Legionella pneumophila pneumonia in guinea pigs. Intraperitoneal treatment began 48 h after intratracheal inoculation of 3 X 10(6) L. pneumophila and consisted of sterile saline (0.3 ml bid), ciprofloxacin (30 mg/kg bid), rifampicin (10 mg/kg/bid), or ciprofloxacin plus rifampicin (same doses). Animals were treated for five days and survivors killed after 11 days. Quantitative lung cultures were done post mortem. Respective mean and median days of animal survival were increased by treatment with ciprofloxacin plus rifampicin (8.4 and 9.5 days), ciprofloxacin (8.2 and 7.5 days), or rifampicin (8.3 and 7.5 days), compared with controls (5.5 and 5.0 days). Compared with control animals (log rank test) survival was improved by treatment with ciprofloxacin plus rifampicin (P less than or equal to 0.047) ciprofloxacin (P less than or equal to 0.047) or rifampicin (P less than or equal to 0.047). Quantitative lung cultures (cfu/g) were also decreased by treatment with ciprofloxacin plus rifampicin (2.0 X 10(4)), ciprofloxacin (5.4 X 10(4)), or rifampicin (1.7 X 10(4)) compared with controls (3.2 X 10(8)). No differences in survival, quantitative lung cultures, or animal weights were noted between treatment groups. This study demonstrates that ciprofloxacin is as effective as rifampicin in the treatment of experimentally induced L. pneumophila pneumonia and that the combination of ciprofloxacin plus rifampicin has no advantages over single agent therapy in this model.

Activity of macrolides against organisms responsible for respiratory infection with emphasis on Mycoplasma and Legionella.

The activity of roxithromycin against Legionella pneumophila in vitro was approximately the same as that of rokitamycin and superior to those of erythromycin and josamycin. In experimental pneumonia due to L. pneumophila none of the animals in the roxithromycin and rifampicin groups died by day 10 of the infection. The MIC ranges of roxithromycin, erythromycin and rokitamycin for Mycoplasma pneumoniae were 0.008-0.063, 0.004-0.008 and 0.016-greater than 125, respectively. In experimental pneumonia caused by M. pneumoniae, roxithromycin showed good activity similar to that of erythromycin.

The antimicrobial activity of ciprofloxacin against Legionella species and the treatment of experimental Legionella pneumonia in guinea pigs.

The antimicrobial activity of ciprofloxacin was tested against 15 standard reference strains, and 37 clinical and environmental strains of Legionella pneumophila by an agar dilution method, using a new growth medium (B-SYE agar) which we devised. The minimal inhibitory concentrations of ciprofloxacin were found to be inoculum dependent, and ranged from 0.02 to 0.06 mg/l at 10(4) cfu inoculum and 0.02 by 0.125 mg/l at 10(6) cfu inoculum. The most potent antibacterial activity was shown by rifampicin, followed by ofloxacin, ciprofloxacin, enoxacin, norfloxacin, erythromycin and pipemidic acid in that order. The therapeutic efficacy of ciprofloxacin in experimental guinea pig pneumonia due to L. pneumophila was fairly good with a survival rate of 80%. From other data of ours, its effectiveness in experimental pneumonia was equal to or greater than that of erythromycin. Further studies would be appropriate to investigate the possibility of using ciprofloxacin for the treatment of human L. pneumophila infection.