Intracellular Legionella Pneumophila Research Articles

Effects of Oakmoss Components on Extra- and Intracellular Legionella pneumophila and Its Host Acanthamoeba castellanii.

Acanthamoeba castellanii is a ubiquitous organism found in environmental water. The amoeba is pathogenic to toward humans and is also a reservoir of bacteria of the genus Legionella, a causative agent of legionellosis. Oakmoss, a source of natural fragrance ingredients, and its components are antibacterial agents that are specifically active against the genus Legionella. In the present study, oakmoss and its components were investigated for their inhibitory effects on total (extra- and intracellular) Legionella pneumophila within A. castellanii and on L. pneumophila within A. castellanii. Among the oakmoss components, 3-hydroxy-5-methylphenyl 2,4-dihydroxy-6-methylbenzoate (1), 3-methoxy-5-methylphenyl 2,4-dihydroxy-6-methylbenzoate (2), and 8-(2,4-dihydroxy-6-(2-oxoheptyl)phenoxy)-6-hydroxy-3-pentyl-1H-isochromen-1-one (8) reduced the number of total bacteria (extra- and intracellular) in a test culture and also exhibited high amoebicidal activity against L. pneumophila within A. castellanii at concentrations lower than their IC50 values for A. castellanii. In contrast, 6,8-dihydroxy-3-pentyl-1H-isochromen-1-one (5) reduced the total number of L. pneumophila and, also that of total bacteria after 24 h of treatment (P < 0.05), whereas the compound did not exhibit amoebicidal activity against L. pneumophila within A. castellanii at concentrations lower than its IC50 value against A. castellanii. Thus, it is suggested that these oakmoss components could be good candidates for disinfectants to protect from Legionella infection.

DNA Delivery and Genomic Integration into Mammalian Target Cells through Type IV A and B Secretion Systems of Human Pathogens.

We explore the potential of bacterial secretion systems as tools for genomic modification of human cells. We previously showed that foreign DNA can be introduced into human cells through the Type IV A secretion system of the human pathogen Bartonella henselae. Moreover, the DNA is delivered covalently attached to the conjugative relaxase TrwC, which promotes its integration into the recipient genome. In this work, we report that this tool can be adapted to other target cells by using different relaxases and secretion systems. The promiscuous relaxase MobA from plasmid RSF1010 can be used to deliver DNA into human cells with higher efficiency than TrwC. MobA also promotes DNA integration, albeit at lower rates than TrwC. Notably, we report that DNA transfer to human cells can also take place through the Type IV secretion system of two intracellular human pathogens, Legionella pneumophila and Coxiella burnetii, which code for a distantly related Dot/Icm Type IV B secretion system. This suggests that DNA transfer could be an intrinsic ability of this family of secretion systems, expanding the range of target human cells. Further analysis of the DNA transfer process showed that recruitment of MobA by Dot/Icm was dependent on the IcmSW chaperone, which may explain the higher DNA transfer rates obtained. Finally, we observed that the presence of MobA negatively affected the intracellular replication of C. burnetii, suggesting an interference with Dot/Icm translocation of virulence factors.

Activity of macrolides and fluoroquinolones against intracellular Legionella pneumophila

To evaluate the activity of macrolides and fluoroquinolones against Legionella pneumophila by intracellular susceptibility testing. Minimum inhibitory concentration (MIC) was determined by standard agar dilution test according to the CLSI. For intracellular assays, legionella pneumonia was used to infect human monocytic cell line THP-1. Erythromycin, azithromycin, levofloxacin and moxifloxacin at 1 × MIC, 4 × MIC, 8 × MIC were added following phagocytosis. Number of viable bacteria was enumerated at 24 h on BCYE (buffered charcoal yeast extract) agar in duplicates using standard plate count method. The result was expressed as percentage inhibition. Mann-Whitney U test was used to determine the significant differences in mean percentage inhibition between agents. Percentage inhibition at 24 h were as follows: Erythromycin 1 × MIC (50.18 ± 27.29)%, 4 × MIC (79.48 ± 20.08)%, 8 × MIC (91.46 ± 8.70)%; Azithromycin 1 × MIC (66.77 ± 26.18)%, 4 × MIC (91.73 ± 8.72)%, 8 × MIC (97.10 ± 3.37)%; Levofloxacin 1 × MIC (99.84 ± 0.25)%, 4 × MIC (99.99 ± 0.02)%, 8 × MIC (99.99 ± 0.01)%; Moxifloxacin 1 × MIC (99.90 ± 0.10)%, 4 × MIC (99.99 ± 0.03)%, 8 × MIC (99.99 ± 0.03)%. The fluoroquinolones showed greater inhibitory activity than macrolides against legionella pneumophila(u = 1.0, 2.0, 5.0, P < 0.05). Levofloxacin and moxifloxacin had the same intracellular activity against legionella pneumophila (u = 190, 183, 217, P > 0.05). Azithromycin was more effective than erythromycin in inhibiting intracellular legionella pneumophila (u = 132, 125, 128, P < 0.05). The fluoroquinolones were more active than macrolides against legionella pneumophila. The intracellular activity of levofloxacin against legionella pneumophila appeared to be similar to moxifloxacin. Azithromycin was demonstrated to have superior activity against legionella pneumophila compared with erythromycin.

Combating pulmonary Coxiella burnetii infection: The role of innate immunity. (37.54)

Abstract The innate immune system provides first line host defense. However, within the immune privileged site of the lung, immune response must be closely regulated. As a result, tissue resident alveolar macrophages (AM) are typically immunosuppressed. The intracellular bacteria Coxiella burnetii has been observed within AM during infection and little is known about the role of AM in C. burnetii infection. Therefore, the AM deserves focused study. We hypothesized that, due to the immunosuppressed state of resident AM, these macrophages are particularly susceptible to C. burnetii infection and provide a required niche for infection. Dye labeling techniques were employed to discriminate AM and C. burnetii, allowing for the determination of resident and infected cell populations. These techniques were corroborated with antibody labeling and the various populations were analyzed. We report here that resident AM are infected, even at low infective doses. Interestingly, a heterogeneous population of infected cells is also identifiable. This population is comprised of resident CD11c+ AM expressing CD11b and recruited CD11b+ cells expressing CD11c. These findings suggest CD11c expression serves as a marker of cells susceptible to C. burnetii infection. Identification of this susceptible population may aid vaccine development and prove relevant for treatment of other intracellular bacteria, such as, Mycobacterium tuberculosis and Legionella pneumophila.

Distinct roles of ppGpp and DksA in Legionella pneumophila differentiation

To transit between hosts, intracellular Legionella pneumophila transform into a motile, infectious, transmissive state. Here we exploit the pathogen's life cycle to examine how guanosine tetraphosphate (ppGpp) and DksA cooperate to govern bacterial differentiation. Transcriptional profiling revealed that during transmission alarmone accumulation increases the mRNA for flagellar and Type IV-secretion components, secreted host effectors and regulators, and decreases transcripts for translation, membrane modification and ATP synthesis machinery. DksA is critical for differentiation, since mutants are defective for stationary phase survival, flagellar gene activation, lysosome avoidance and macrophage cytotoxicity. The roles of ppGpp and DksA depend on the context. For macrophage transmission, ppGpp is essential, whereas DksA is dispensable, indicating that ppGpp can act autonomously. In broth, DksA promotes differentiation when ppGpp levels increase, or during fatty acid stress, as judged by flaA expression and evasion of degradation by macrophages. For flagella morphogenesis, DksA is required for basal fliA (sigma(28)) promoter activity. When alarmone levels increase, DksA cooperates with ppGpp to generate a pulse of Class II rod RNA or to amplify the Class III sigma factor and Class IV flagellin RNAs. Thus, DksA responds to the level of ppGpp and other stress signals to co-ordinate L. pneumophila differentiation.

Type IV secretion systems: tools of bacterial horizontal gene transfer and virulence

Type IV secretion systems (T4SSs) are multisubunit cell-envelope-spanning structures, ancestrally related to bacterial conjugation machines, which transfer proteins and nucleoprotein complexes across membranes. T4SSs mediate horizontal gene transfer, thus contributing to genome plasticity and the evolution of pathogens through dissemination of antibiotic resistance and virulence genes. Moreover, T4SSs are also used for the delivery of bacterial effector proteins across the bacterial membrane and the plasmatic membrane of eukaryotic host cell, thus contributing directly to pathogenicity. T4SSs are usually encoded by multiple genes organized into a single functional unit. Based on a number of features, the organization of genetic determinants, shared homologies and evolutionary relationships, T4SSs have been divided into several groups. Type F and P (type IVA) T4SSs resembling the archetypal VirB/VirD4 system of Agrobacterium tumefaciens are considered to be the paradigm of type IV secretion, while type I (type IVB) T4SSs are found in intracellular bacterial pathogens, Legionella pneumophila and Coxiella burnetii. Several novel T4SSs have been identified recently and their functions await investigation. The most recently described GI type T4SSs play a key role in the horizontal transfer of a wide variety of genomic islands derived from a broad spectrum of bacterial strains.

Antibacterial activities of gemifloxacin, levofloxacin, gatifloxacin, moxifloxacin and erythromycin against intracellular Legionella pneumophila and Legionella micdadei in human monocytes

The antibacterial activity of a new fluoroquinolone, gemifloxacin, was tested against intracellular Legionella pneumophila and Legionella micdadei and was compared with the activities of levofloxacin, gatifloxacin, moxifloxacin and erythromycin. For intracellular assays, bacteria were used to infect human monocyte-derived macrophages prepared from heparinized blood of healthy volunteers. Antibiotics were added following phagocytosis. Numbers of viable bacteria were determined at 0, 24, 48, 72 and 96 h. The intracellular antibacterial activity of gemifloxacin was concentration- and time-dependent. All of the quinolones had similar activities against L. pneumophila and L. micdadei at 10 x MIC, but there were minor differences: at 24 h moxifloxacin was significantly more active than the other quinolones against L. pneumophila, while gemifloxacin was more active against L. micdadei (P < 0.01). All of the quinolones were markedly more active than erythromycin (P < 0.01). The antibacterial effect of gemifloxacin against L. pneumophila following drug removal at 24 h persisted for 72 h at 20 x MIC but not at 10 x MIC, while for L. micdadei the antibacterial effect persisted for 24 h at 10 x MIC. All of the quinolones had similar activities against intracellular L. pneumophila and L. micdadei and were markedly more effective than erythromycin.

Pharmacodynamic studies of moxifloxacin and erythromycin against intracellular Legionella pneumophila in an in vitro kinetic model

Newer quinolones are highly active against Legionella pneumophila. Since this pathogen is intracellular, standard in vitro susceptibility tests may not accurately predict clinical efficacy. Few models for studies of intracellular Legionella have been described. In this study, we determined the pharmacodynamic activity of moxifloxacin against intracellular L. pneumophila in comparison with erythromycin. A kinetic model for intracellular studies was constructed in which human pharmacokinetics could be simulated. The model consisted of a glass chamber with two exits and a metal rack fitting cell culture inserts. The inserts had a bottom membrane where cells could be cultured while nutrients and antibiotics passed through. The inserts were prepared with a monolayer of HEp-2 cells, which were exposed to a culture of L. pneumophila. At regular intervals cells were harvested and lysed, viable intracellular bacteria counted and compared with untreated controls. The MICs were 0.0156 mg/L for moxifloxacin and 0.5 mg/L for erythromycin. The human pharmacokinetics were simulated in the model with a mean initial antibiotic concentration of 2.4 mg/L for moxifloxacin and 8.4 mg/L for erythromycin. The mean half-life was 9 h for moxifloxacin and 3.4 h for erythromycin. At 12 h, a 2 log(10) reduction in bacterial counts was seen in cells treated with moxifloxacin and no regrowth was detected at 24 h. Cells treated with erythromycin showed no reduction in intracellular L. pneumophilia at 12 h or 24 h. In experiments using static concentrations of 9 mg/L of erythromycin, similar results were obtained. In this model, moxifloxacin exerts a significantly better antibacterial effect against intracellular L. pneumophila compared with erythromycin.

In Vitro and Intracellular Activities of LBM415 (NVP PDF-713) against Legionella pneumophila

LBM415 activity against extracellular and intracellular Legionella pneumophila was studied. The LBM415 MIC50 for 20 Legionella sp. strains was 4 microg/ml, versus 0.06, 0.25, and <or=0.03 microg/ml for azithromycin, erythromycin, and levofloxacin, respectively. LBM415 (0.5 and 16 microg/ml) reversibly prevented intracellular growth of two L. pneumophila strains and was less active than erythromycin.

BMS-284756 (T-3811ME) a new fluoroquinolone: in vitro activity against Legionella, efficacy in a guinea pig model of L. pneumophila pneumonia and pharmacokinetics in guinea pigs.

The activity of BMS-284756 was studied against extracellular Legionella spp. and intracellular Legionella pneumophila, and for the treatment of guinea pigs with L. pneumophila pneumonia. The BMS-284756 MIC(50) of 22 different Legionella spp. strains was 0.008 mg/L, compared with 0.016 and 0.125 mg/L for levofloxacin and azithromycin, respectively. BMS-284756 (1 mg/L) reduced the intracellular concentrations of two L. pneumophila strains grown in guinea pig alveolar macrophages by c. 1.5 log(10 )cfu/mL, and was more active than erythromycin, but less active than azithromycin or levofloxacin at the same drug concentrations. Efficacy studies of BMS-284756, levofloxacin and azithromycin were performed in guinea pigs with L. pneumophila pneumonia. In infected guinea pigs given BMS-284756 10 mg/kg ip, mean peak plasma levels were 1.8 mg/L at 0.5 h and 0.7 mg/L at 1 h post-dose. The elimination half-life in plasma was 0.5 h, and the AUC(0-24 )was 1.7 mg*h/L, about 2% of the AUC(0-24 )for a single 400 mg oral dose in man. Sixteen of 18 L. pneumophila-infected guinea pigs treated with BMS-284756 10 mg/kg ip once daily for 5 days survived for 7 days post-antimicrobial therapy, as did 11 of 12 guinea pigs treated with azithromycin 15 mg/kg ip once daily for 2 days. All 12 animals that were treated with levofloxacin 10 mg/kg ip once daily for 5 days survived. None of 12 control animals treated with saline survived. Animals treated with BMS-284756 had significantly higher residual lung counts of L. pneumophila at the end of therapy than did animals treated with levofloxacin or azithromycin, which may be attributable to the very low drug concentrations that were obtained. BMS-284756 was more active than erythromycin against L. pneumophila in infected macrophages, and effectively treated animals with experimental L. pneumophila pneumonia. These data support further studies of BMS-284756 for the treatment of Legionnaires' disease.

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.

The activity of ABT-773 was studied against extracellular and intracellular Legionella pneumophila and for the treatment of guinea pigs with L. pneumophila pneumonia. The ABT-773 MIC at which 50% of isolates are inhibited (MIC(50)) for 20 different Legionella sp. strains was 0.016 microg/ml, whereas the MIC(50)s of clarithromycin and erythromycin were 0.032 and 0.125 microg/ml, respectively. ABT-773 (1 microg/ml) was bactericidal for two L. pneumophila strains grown in guinea pig alveolar macrophages. In contrast, erythromycin and clarithromycin had easily reversible static activity only. Therapy studies of ABT-773 and erythromycin were performed with guinea pigs with L. pneumophila pneumonia. When ABT-773 was given to infected guinea pigs by the intraperitoneal route (10 mg/kg of body weight), mean peak levels in plasma were 0.49 microg/ml at 0.5 h and 0.30 microg/ml at 1 h postinjection. The terminal half-life phase of elimination from plasma was 0.55 h, and the area under the concentration-time curve from 0 to 24 h (AUC(0-24)) was 0.65 microg. h/ml. For the same drug dose, mean levels in the lung were 15.9 and 13.2 microg/g at 0.5 and 1 h, respectively, with a half-life of 0.68 h and an AUC(0-24) of 37.0 microg. h/ml. Ten of 15 L. pneumophila-infected guinea pigs treated with ABT-773 (15 mg/kg/dose given intraperitoneally once daily) for 5 days survived for 9 days post-antimicrobial therapy, as did 14 of 15 guinea pigs treated with erythromycin (30 mg/kg given intraperitoneally twice daily) for 5 days. All of the ABT-773-treated animals that died appeared to do so because of drug-induced peritonitis rather than overwhelming pneumonia. None of 12 animals treated with saline survived. ABT-773 is as effective as erythromycin against L. pneumophila in infected macrophages and in a guinea pig model of Legionnaires' disease. These data support studies of the clinical effectiveness of ABT-773 for the treatment of Legionnaires' disease.

Efficacy of moxifloxacin, trovafloxacin, clinafloxacin and levofloxacin against intracellular Legionella pneumophila.

This study compares the in vitro activity of novel fluoroquinolones against clinical Legionella pneumophila isolates. We determined both the MICs and intracellular efficacy of moxifloxacin, trovafloxacin, clinafloxacin and levofloxacin in the Mono Mac 6 infection model. Six legionella strains were tested and all proved highly susceptible to the fluoroquinolones. Clinafloxacin and trovafloxacin were found to have the lowest MICs (0.004 mg/L) followed by levofloxacin and moxifloxacin (0.015-0.03 mg/L). In the Mono Mac 6 infection model, inhibition of L. pneumophila was achieved at concentrations four times below the respective in vitro MICs. Concentrations as low as 0.015 mg/L of moxifloxacin and levofloxacin, 0.004 mg/L of trovafloxacin and 0.002 mg/L of clinafloxacin markedly decreased viable intracellular bacterial counts. These data strongly support further clinical evaluation of new fluoroquinolones for empirical treatment of lower respiratory tract infection including atypical pneumonia caused by L. pneumophila.

Microbiological profile of telithromycin, the first ketolide antimicrobial

Telithromycin, the first of the ketolide antimicrobials, has been specifically designed to provide potent activity against common and atypical/intracellular or cell-associated respiratory pathogens, including those that are resistant to beta-lactams and/or macrolide-lincosamide-streptograminB (MLS(B)) antimicrobials. Against gram-positive cocci, telithromycin possesses more potent activity in vitro and in vivo than the macrolides clarithromycin and azithromycin. It retains its activity against erm-(MLS(B)) or mef-mediated macrolide-resistant Streptococcus pneumoniae and Streptococcus pyogenes and against Staphylococcus aureus resistant to macrolides through inducible MLS(B) mechanisms. Telithromycin also possesses high activity against the Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis, regardless of beta-lactamase production. In vitro, it shows similar activity to azithromycin against H. influenzae, while in vivo its activity against H. influenzae is higher than that of azithromycin. Telithromycin's spectrum of activity also extends to the atypical, intracellular and cell-associated pathogens Legionella pneumophila, Mycoplasma pneumoniae and Chlamydia pneumoniae. In vitro, telithromycin does not induce MLS(B) resistance and it shows low potential to select for resistance or cross-resistance to other antimicrobials. These characteristics indicate that telithromycin will have an important clinical role in the empirical treatment of community-acquired respiratory tract infections.

The effect of azithromycin on intracellular Legionella pneumophila in the Mono Mac 6 cell line at serum concentrations attainable in vivo.

We investigated the antimicrobial efficacy of clinically meaningful, low concentrations of azithromycin against intracellular growth of two clinical isolates of Legionella pneumophila. The mature monocytic cell line Mono Mac 6 was used as a model to investigate the effects of antimicrobial agents on L. pneumophila. Extracellular susceptibility was determined by microdilution susceptibility testing in BYEalpha broth after 48 h of incubation. Mono Mac 6 cells infected with L. pneumophila were incubated with various concentrations of azithromycin. After 2 days of incubation, intracellular bacteria were released from the phagocytes and plated on to BCYEalpha agar. Addition of the intracellular-acting antibiotics azithromycin or ciprofloxacin at their MICs (0.5 and 0. 015 mg/L, respectively) resulted in a significant decrease in cfu, of up to approximately 1 log(10) after 48 h of incubation. In contrast, incubation of intraphagocytic L. pneumophila in the presence of antibiotics without intracellular activity (ceftizoxime, imipenem or amoxycillin-clavulanic acid) did not have any effect. Azithromycin inhibited intracellular replication at concentrations as low as 0.125 mg/L, approximately one-quarter of the extracellular MIC. The Mono Mac 6 cell line is a useful infection model for investigating the intracellular activity of antimicrobial agents in vitro. In accordance with clinical data and animal experiments, azithromycin and ciprofloxacin inhibited the intraphagocytic replication of L. pneumophila. In particular, azithromycin killed ingested legionellae in vitro at concentrations below the peak serum concentrations and below the MIC.

In vitro activity of SCH 27899 (Ziracin) against Legionella species

The activities of Sch 27899 (Ziracin), erythromycin, and ofloxacin against 102 Legionella spp. isolates were measured by a microbroth dilution method. The MICs that inhibited 90% of strains tested were 0.25, 0.5, and 0.06 μg/mL for Sch 27899, erythromycin, and ofloxacin, respectively. The activity of Sch 27899 against intracellular Legionella pneumophila could not be determined because of complete inactivation of the drug by tissue culture medium components.

Signal transduction in the protozoan host Hartmannella vermiformis upon attachment and invasion by Legionella micdadei.

The intracellular pathogens Legionella micdadei and Legionella pneumophila are the two most common Legionella species that cause Legionnaires' disease. Intracellular replication within pulmonary cells is the hallmark of Legionnaires' disease. In the environment, legionellae are parasites of protozoans, and intracellular bacterial replication within protozoans plays a major role in the transmission of Legionnaires' disease. In this study, we characterized the initial host signal transduction mechanisms involved during attachment to and invasion of the protozoan host Hartmannella vermiformis by L. micdadei. Bacterial attachment prior to invasion of H. vermiformis by L. micdadei is associated with tyrosine dephosphorylation of multiple host cell proteins, including a 170-kDa protein. We have previously shown that this 170-kDa protein is the galactose N-acetylgalactosamine (Gal/GalNAc)-inhibitable lectin receptor that mediates attachment to and invasion of H. vermiformis by L. pneumophila. Subsequent bacterial entry targets L. micdadei into a phagosome that is not surrounded by the rough endoplasmic reticulum (RER). In contrast, uptake of L. pneumophila mediated by attachment to the Gal/GalNAc lectin is followed by targeting of the bacterium into an RER-surrounded phagosome. These results indicate that despite similarities in the L. micdadei and L. pneumophila attachment-mediated signal transduction mechanisms in H. vermiformis, the two bacterial species are targeted into morphologically distinct phagosomes in their natural protozoan host.

Simplified quantitative assay system for measuring activities of drugs against intracellular Legionella pneumophila.

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-alpha agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-alpha broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples.

In vitro activity of macrolides against intracellular Legionella pneumophila

The antibacterial effects of clarithromycin, azithromycin, and erythromycin were determined against five strains of Legionella pneumophila including L. pneumophila ATCC 33823 and four clinical isolates. Extracellular minimum inhibitory concentrations (MICs) and MBCs were determined by a microdilution method. Clarithromycin was the most active drug (MIC ⩽ 0.015–0.06), followed by azithromycin (MIC 0.03–0.12) and erythromycin (MIC 0.06–0.25). The antibacterial effect of these macrolides was then determined against L. pneumophila grown intracellularly in MRC-5 human fetal lung fibroblast cells. At two and eight times the extracellular MBC, erythromycin, azithromycin, and clarithromycin were equally effective in inhibiting growth of these five strains of intracellular L. pneumophila.

Cloning and nucleotide sequences of iron and copper-zinc superoxide dismutase genes of Legionella pneumophila and their distribution among Legionella species.

A facultative intracellular parasite Legionella pneumophila has two kinds of superoxide dismutase (SOD), iron-containing superoxide dismutase (Fe-SOD) and copper,zinc-containing one (Cu,Zn-SOD). We cloned both SOD genes of L. pneumophila and determined their DNA sequences. The Fe-SOD gene (sodB), isolated by functional complementation of a SOD-deficient Escherichia coli strain, encoded a protein of 192 amino acids conserving the Fe-SOD-specific amino acid residues. A clone containing entire Cu,Zn-SOD gene (sodC) was constructed by connecting two contiguous DNA fragments; one with a lower part of the gene was obtained by colony hybridization with a probe acquired by polymerase chain reaction (PCR) with degenerate oligonucleotide primers corresponding to conserved regions of known Cu,Zn-SOD genes and the other with an upper part of the gene was by IPCR (inverted PCR). The sodC gene encoded a protein of 162 amino acids, of which the first 20 amino acids inferred a signal peptide similar to other bacterial Cu,Zn-SODs reported previously. Both clones expressed their SOD activities in E. coli K-12 through their own plausible promoters. We examined for SOD genes on chromosomes of several Legionella species. All chromosomes were hybridized with Fe-SOD gene of L. pneumophila, but Cu,Zn-SOD gene did not hybridize to the chromosomes of other than L. pneumophila strains.

Combined therapy with co-amoxiclav and erythromycin against intracellular Legionella pneumophila in cell culture and in an experimental respiratory infection in neutropenic rats.

A combination of co-amoxiclav and erythromycin has been shown in these studies to be more effective than either agent alone against intracellular Legionella pneumophila in MRC-5 cells and following oral administration in an experimental respiratory infection in neutropenic rats,