Influence of iron-limited continuous culture on physiology and virulence of Legionella pneumophila.

A virulent strain of Legionella pneumophila serogroup 1, subgroup Pontiac, was grown in continuous culture at a constant growth rate under iron-replete and iron-limited conditions. Iron limitation was achieved by the removal of ferrous sulfate and hemin from the chemically defined medium. Residual contaminating iron, 0.45 microM, was sufficient to support iron-limited growth. Typical iron-replete cultures metabolized 3.3 microM iron. Serine provided the principal source of carbon and energy for both cultures, although iron-replete cultures also depleted a number of other amino acids. There was a 40% decrease in culture biomass under iron-restricted conditions. Iron limitation did not significantly affect carbohydrate metabolism, with the molar growth yield for carbon (Ycarbon) comparable for both cultures. However, under iron-limited conditions a sixfold increase in Yiron correlated with a significant decrease in the iron content of the biomass, as the culture utilized the available iron more efficiently. Highly pleomorphic iron-replete cultures became uniform cultures of short fine rods when adapted to iron-deficient conditions. In addition to the morphological and physiological changes, iron limitation had a critical effect on culture virulence. The virulence of this strain was significantly (P < 0.05) reduced when the culture was subjected to iron-limited conditions. This phenomenon was reversible, with a significant increase in culture virulence upon reversion to iron-replete conditions. When compared in an in vitro macrophage assay, the number of culturable avirulent iron-limited cells located intracellularly after infection was significantly lower than for the virulent replete and control cultures. These results further support the role of environmental parameters in regulating the virulence of L. pneumophila.

Experimental Legionnaires' disease in SCID-Beige mice reconstituted with human leucocytes.

A new small animal model of experimental Legionnaires' disease is described in which the reconstitution of SCID-Beige mice with human peripheral blood leucocytes permits the in-vivo growth of Legionella pneumophila in the lungs of aerosol-challenged mice. Following infection, viable bacterial counts within the lungs of mice increased from 10(5) cfu/lung at the time of inoculation to a maximum of 10(10) cfu/lung by 48 h post-inoculation. Two types of disease were detected in the lungs of infected SCID-Beige mice. An acute exudative bronchiolitis and bronchopneumonia were seen in the most severely affected mice and, in the less severely affected mice, lesions of subacute or chronic disease were seen with thickening of alveolar walls and consolidation of lung tissue. Human cells did not appear to be involved directly in the pathology but were required for the establishment of infection. Immunohistological staining of lung tissue revealed substantial amounts of bacterial antigen distributed in a pattern similar to that seen in human Legionnaires' disease.

Growth temperature reversibly modulates the virulence of Legionella pneumophila.

In chemostat culture, the virulence of two strains of Legionella pneumophila was shown to be significantly (P < 0.05) reduced when the culture temperature was lowered from 37 to 24 degrees C. This modulation was reversed by returning the temperature to 37 degrees C, which resulted in a statistically significant (P < 0.05) increase in virulence.

A comparison of aerosol and intragastric routes of infection with Listeria spp.

Aerosol infection (AI) of Porton outbred mice with Listeria species, exhibiting varying degrees of virulence, was compared with gastric intubation (GI) on the basis of numbers of deaths, 50% lethal dose (LD50) and pattern of listerial infection. The AI route appeared to be more sensitive, efficient and consistent than GI in that it required 10(5) fewer micro-organisms to obtain infection and death then ensued within 4 days, with GI deaths usually occurring on day 7. All the virulent strains tested caused 100% mortality by AI, while virulent and avirulent strains were indistinguishable by GI. Bacterial counts in the livers and spleens of infected mice were consistent with the relative virulence of the infectious agent using AI but not in GI mice. There were higher numbers of micro-organisms and more widespread lesions in the organs of AI mice than in GI. Results indicate that AI is an accurate in vivo indicator of virulence in listeria and using AI, bacterial counts in the liver and spleen could replace LD50 tests, thereby reducing the number of animals required for in vivo virulence testing.

A comparison of the efficacy of azithromycin and clarithromycin in oral therapy of experimental airborne Legionnaires' disease.

The activities of two new macrolides, azithromycin and clarithromycin, were compared in an aerosol-infected guinea-pig model of legionnaires' disease. The results of this study indicate that a low oral dose of azithromycin (3.6 mg/kg) administered once daily gives 100% survival in Legionella pneumophila-infected animals. An eight-fold higher dose of clarithromycin (28.8 mg/kg) given twice-daily was required to achieve the same effect. Similarly, azithromycin was more effective than clarithromycin in preventing pyrexia and in reducing numbers of bacteria and lesions in the lung.

Demonstration of the intracellular production of tissue-destructive protease by Legionella pneumophila multiplying within guinea-pig and human alveolar macrophages.

The major extracellular enzyme of Legionella pneumophila, a metalloprotease, has been proposed as a pathogenic factor in Legionnaires' disease due to its cytotoxic, tissue-destructive, and phagocyte-inhibitory properties. The relevance of these activities depends on the production of the protease during infection, i.e. by L. pneumophila multiplying intracellularly. In this study, L. pneumophila was demonstrated to produce protease in guinea-pig and human alveolar macrophages infected in vitro. After 24 h infection, approximately 0.1 to 0.2 micrograms of protease per 10(6) bacteria was measured by ELISA in culture supernatants and lysates of the infected cells, whereas no protease could be detected immediately after infection. Immunogold labelling using anti-protease antibody showed the enzyme to be located within phagosomes and distributed throughout the macrophages. Recent observations have shown that this protease could modify host defence mechanisms through inhibition of bacterial killing by neutrophils and monocytes. The intracellular production of the enzyme in infected macrophages demonstrated here further supports a role for the protease in the pathogenesis of Legionnaires' disease.

Transposon mutagenesis in Legionella pneumophila. II.--Mutants exhibiting impaired intracellular growth within cultured macrophages and reduced virulence in vivo.

Transposon Tn5 mutants of L. pneumophila were isolated and screened for loss of virulence-associated characteristics. Three mutants were found with normal ability to produce putative pathogenicity determinants and to be endocytosed by guinea pig alveolar macrophages in vitro but with a greatly reduced ability to multiply within them. These mutants showed considerable loss of virulence in an authentic animal model of the pneumonia.

Airborne infection of laying hens with Salmonella enteritidis phage type 4.

Hens were exposed to small-particle aerosols containing different concentrations of Salmonella enteritidis phage type 4. They developed a systemic infection and some birds were still excreting the organism in the faeces when killed 28 days after infection. S enteritidis was present for a similar period in a wide range of alimentary tract issues and in the ovary and oviduct.

Viability and distribution of bacteria after passage through a circle anaesthetic system.

We have assessed in vitro the viability of eight species of micro-organism suspended as aerosols and passed through a soda-lime absorber rebreathing system. As had been predicted, the soda-lime exerted a potent cidal effect on non-sporing organisms, all of which were rendered non-viable. One percent of the spore bearing organism, Bacillus subtilis, was still viable after 30 min contact. Although Bacillus subtilis is an organism of low pathogenicity, spores may be more resistant to the alkaline medium of the soda-lime. The micro-organisms were observed to lodge in all components of the breathing system, with the greatest concentration being recovered from the corrugated tubing. We recommend that disposable components of the circle breathing system should be changed, and the non-disposable parts sterilized or disinfected, daily. Soda-lime cannisters should be sterilized or disinfected when changing the soda-lime.

Efficacy of azithromycin in the treatment of guinea pigs infected with Legionella pneumophila by aerosol.

Azithromycin and erythromycin were compared for efficacy in guinea pigs infected with an aerosol containing Legionella pneumophila. When administered intraperitoneally, azithromycin was very effective in the treatment of experimental Legionnaires' disease. Even at the low dose of 3.6 mg/kg/day it gave 100% survival and eliminated lung infectivity two days following infection. In contrast, erythromycin at a much higher dose (96 mg/kg/day) gave only 83.3% survival and failed to eliminate organisms from the lung six days after infection. The histological findings confirmed the superiority of azithromycin. A single dose of azithromycin given intraperitoneally at 3.6 or 14.4 mg/kg gave survival rates of 83.3 and 100%, respectively. Azithromycin was also found to be superior to erythromycin in eliminating lung infectivity and reducing mortality, when administered orally. However, oral administration of azithromycin was not as effective as intraperitoneal when assessed by lung histopathology, although it was still superior to oral erythromycin treatment.

Bacterial infection (Legionella pneumophila) stimulates fever, metabolic rate and brown adipose tissue activity in the guinea pig.

The objective of this study was to assess whether bacterial infection stimulates oxygen consumption and brown adipose tissue (BAT) activity. Guinea pigs infected with Legionella pneumophila showed marked fever and a significant (33%) increase in resting oxygen consumption (VO2), 24h after infection. At this time, food intake and body weight were normal and the in vitro thermogenic activity of BAT taken from infected animals was elevated by 64% above that of control guinea pigs. VO2 and BAT activity fell to control values by 48h as infected animals became moribund and over this period food intake was markedly reduced.

Effects of inhaled titanium dioxide dust on the lung and on the course of experimental Legionnaires' disease.

Guinea-pigs were exposed for 14 days to an aerosol of titanium dioxide (TiO2) dust to produce macrophage blockade. Groups of the animals were later infected by aerosol with Legionella pneumophila. Histological and ultrastructural studies showed that TiO2 dust alone was inert and non-fibrogenic and even at 6 weeks induced no pathological lesions in the lungs, apart from accumulation of macrophages in interalveolar septa. The macrophage blockade by TiO2 did not alter the animals' susceptibility to Legionnaires' disease nor increase mortality. The blockade was effective in the early stages of the infection and limited multiplication of L. pneumophila in the lungs. Later blood monocytes were recruited into the lungs, where they phagocytosed Legionellae, resulting in lung counts comparable to those of TiO2-free control animals.

The effect of ofloxacin on the intracellular growth of Legionella pneumophila in guinea pig alveolar phagocytes.

Ofloxacin was evaluated as an antibiotic for possible use in the therapy of Legionnaires' disease in relation to its ability to penetrate alveolar phagocytes and inhibit Legionella pneumophila intracellular replication. A comparison with two other antibiotics used in the treatment of Legionnaires' disease, ciprofloxacin and erythromycin, was also made. Ofloxacin was found to be the most effective antibiotic, eliminating viable L. pneumophila from alveolar phagocytes at 0.001 mg/l. This was followed by ciprofloxacin, eliminating intracellular organisms at 0.01 mg/l. Erythromycin was shown to be much less effective, requiring a much higher concentration, of 0.1 mg/l. All three antibiotics had approximately similar MIC values and the considerable differences in intracellular penetration shown by these antibiotics indicate how discrepancies between in-vitro and in-vivo estimates of efficacy can occur.

Effects of polymorphonuclear leucocyte depletion on the pathogenesis of experimental Legionnaires' disease.

Guinea-pigs were depleted of circulating polymorphonuclear leucocytes (PMN) by administration of anti-polymorph serum. Groups of animals were then infected by aerosols containing different doses of Legionella pneumophila and the effects compared with those in intact infected controls. Elimination of PMN lowered the dose of L. pneumophila necessary to establish infection, increased bacterial numbers in the lungs and caused much higher mortality. It did not change the nature or extent of pulmonary lesions. The findings confirm the importance of PMN in defence of the lung against L. pneumophila infection and indicate that PMN and their enzymes are not responsible for the pulmonary lesions, which are probably caused directly by the bacteria.

Experimental infection of monkeys with Leptospira interrogans serovar hardjo.

Grivet monkeys experimentally infected with two different strains of Leptospira interrogans serovar hardjo showed no signs of severe clinical disease. There were no significant macroscopic lesions in any of the tissues examined, but the organisms were demonstrated in various tissues by immunofluorescent technique and were isolated from the blood and urine of two monkeys and the kidney of one. Abraded skin was shown to be a viable route of infection in non-human primates.

The effect of oxygen-dependent antimicrobial systems on strains of Legionella pneumophila of different virulence.

Four strains of Legionella pneumophila of different virulence as identified by ability to produce pneumonia and death in guinea-pigs infected by a fine-particle aerosol were examined for factors which may intracellularly influence virulence. Possible bactericidal mechanisms possessed by alveolar phagocytes were examined. A relationship could be established between resistance to H2O2, catalase activity and virulence amongst the strains. Virulent strains resisted the bactericidal activity generated by the xanthine oxidase system; avirulent strains did not. Incorporation of various specific inhibitors of the xanthine oxidase system indicated that the main bactericidal activities were associated with the production of H2O2 and hydroxyl radicals (.OH). All strains of L. pneumophila were susceptible to the bactericidal activity generated by the myeloperoxidase-H2O2-halide system, confirming earlier observations that polymorphonuclear neutrophil leucocytes (PMNLS) are able to kill both virulent and avirulent strains of L. pneumophila.