Legionella prevalence and risk of legionellosis in Japanese households.

This study determined the occurrence of legionellae in private houses for which there were no available data on aquatic environments other than the water supply system. From June 2013 to November 2014, we collected 138 water and 90 swab samples from aquatic environments in 19 houses. Legionella DNA was detected via a loop-mediated isothermal amplification assay in 66 (47·8%) water and 17 (18·9%) swab samples. High Legionella DNA detection rates were observed in water samples from washing machines and aquariums. Legionella spp. was isolated from 9 (6·5%) water and 3 (3·3%) swab samples. Legionella pneumophila SG 1 was detected from the outlet water of a bathtub spout and a bath sponge. Use of amoebic co-culture effectively increased legionellae and Legionella DNA detection rates from all sample types. A logistic regression analysis revealed that the heterotrophic plate count was significantly related to Legionella contamination. Our findings indicate that there is a risk of legionellosis from exposure to Legionella spp. in a variety of aquatic environments in residential houses. Control measures for legionellae in houses should include frequent cleaning and disinfecting to reduce heterotrophic bacteria in water and, where possible, preventing aerosolization from aquatic environments.

Is driving a car a risk for Legionnaires' disease?

Legionnaires' disease (LD) is a major cause of severe community-acquired pneumonia but the source and mode of transmission are not always apparent, especially in sporadic cases. We hypothesized that LD can be acquired from the air-conditioning systems of motor cars. Swabs were taken from the evaporator compartments of the air-conditioning system of scrapped cars. Healthy subjects who were mainly employees of regional transportation companies were tested for antibody to Legionella pneumophila serogroups 1-6; they also completed a questionnaire. Legionella species were detected in 11/22 scrapped cars by the loop-mediated isothermal amplification method. The prevalence of microplate agglutination titres > or =1:32 was significantly higher in subjects who sometimes used car air-conditioning systems. Although we did not prove a direct link between Legionella spp. in the car evaporator and LD, our findings point to a potential risk of car air-conditioning systems in LD, which needs further investigation.

Outbreak of Legionnaires' disease on a cruise ship linked to spa-bath filter stones contaminated with Legionella pneumophila serogroup 5.

In January 2003, two cases of Legionnaires' disease associated with a ship's cruise were registered in the database of National Epidemiological Surveillance of Infectious Diseases. A 70-year-old male heavy smoker with mild emphysema contracted the disease during a cruise. Legionella pneumophila serogroup (sg) 5 was isolated from the patient's sputum and the ship's indoor spa. The isolate from the spa matched the patient's isolate by genotyping performed by pulsed-field gel electrophoresis (PFGE). The second case was in a 73-year-old female. During epidemiological investigation, a third case of Legionnaire's disease in a 71-year-old male was subsequently diagnosed among passengers on the same ship on the following cruise. Environmental investigation revealed that porous natural stones (Maifanshi) in the filters of the spas had harboured L. pneumophila, a phenomenon which has not been reported except in Japan. This is the first documented evidence of L. pneumophila sg 5 infection on a ship and of porous stones as a source of Legionella infection.

Relative contributions of myeloperoxidase and NADPH-oxidase to the early host defense against pulmonary infections with Candida albicans and Aspergillus fumigatus.

Generation of oxidative products by phagocytic cells is known to be an important host defense mechanism directed toward killing of invading microorganisms. The importance of two major oxidant-producing enzymes, myeloperoxidase (MPO) and NADPH-oxidase, in in vivo fungicidal action was directly compared in genetically engineered mice. Both MPO-deficient (MPO-/-) and NADPH-oxidase-deficient (X-linked chronic granulomatous disease [X-CGD]) mice showed increased susceptibility to pulmonary infections with Candida albicans and Aspergillus fumigatus compared with normal mice, and the X-CGD mice exhibited shorter survivals than MPO-/- mice. This increased mortality of X-CGD mice was associated with a 10- to 100-fold increased outgrowth of the fungi in their organs during the first 6 days. These results suggest that superoxide (O2-) produced by NADPH-oxidase is more important than hypochlorous acid (HOCl) produced by MPO, although both oxidative products obviously contribute to the host defense against pulmonary infection with those fungi. We also observed that MPO-/-/X-CGD double knockout mice showed comparable levels of susceptibility to the X-CGD mice against C. albicans and A. funigatus, indicating that MPO is unable to play a role in host defense in the absence of NADPH-oxidase. This strongly suggests that hydrogen peroxide, the precursor of HOCl, is solely derived from O2- produced by NADPH-oxidase.

Differential host susceptibility to pulmonary infections with bacteria and fungi in mice deficient in myeloperoxidase.

Myeloperoxidase (MPO), which is located within neutrophils capable of producing hypochlorous acid, is active in vitro against bacteria and fungi. However, MPO-deficient persons are usually healthy. To define the in vivo contribution of MPO to early host defense against pulmonary infections, MPO-deficient and control mice were intranasally infected with various fungi and bacteria, and the number of residual microorganisms in lungs was compared 48 h later. MPO-deficient mice showed severely reduced cytotoxicity to Candida albicans, Candida tropicalis, Trichosporon asahii, and Pseudomonas aeruginosa. However, the mutant mice showed a slight but significantly delayed clearance of Aspergillus fumigatus and Klebsiella pneumoniae and had comparable levels of resistance to the wild type against Candida glabrata, Cryptococcus neoformans, Staphylococcus aureus, and Streptococcus pneumoniae. These results suggest that the MPO-dependent oxidative system is important for host defense against fungi and bacteria, although the effect varies by pathogen species.

Modulation of chemotaxis, O(2)(-) production and myeloperoxidase release from human polymorphonuclear leukocytes by the ornithine-containing lipid and the serineglycine-containing lipid of Flavobacterium.

The ornithine-containing lipid (OL) and the serineglycine-containing lipid (SGL) of Flavobacterium activated and modulated the functions of human polymorphonuclear leukocytes (PMNs). The OL and the SGL strongly activated fMet-Leu-Phe- and interleukin-8-induced chemotaxis of PMNs at the concentration of 0.1 microg ml(-1), and a synthetic OL also activated the function of PMNs. Further, the OL strongly activated O(2)(-) production from PMNs. Although the OL and the SGL slightly modulated myeloperoxidase release from PMNs, inhibition effects of their component fatty acid analogues were observed. O(2)(-) production-inducing activity is a common biological activity between the OL and bacterial lipopolysaccharides, but OL and SGL, unlike lipopolysaccharide, are potent activators of PMN chemotaxis.

Yersinia pseudotuberculosis blocks the phagosomal acidification of B10.A mouse macrophages through the inhibition of vacuolar H(+)-ATPase activity.

Yersinia pseudotuberculosis survived and multiplied in the phagosomes of B10.A mouse peritoneal macrophages. As one of the possible mechanisms for the bacteria's survival in the phagosomes, we demonstrated that live Y. pseudotuberculosis inhibited the phagosomal acidification; pH within phagosomes containing the live Y. pseudotuberculosis remained at about 6.0, whereas pH within phagosomes containing the dead Y. pseudotuberculosis fell to about 4. 5. This ability to inhibit intraphagosomal acidification was also shared by mutants lacking the 42 Md virulence plasmid, indicating that it is chromosomally encoded. The phagosomes containing dead bacteria raised the pH to 6.2 after the treatment of their macrophages with an inhibitor (bafilomycin A1) specific for V-ATPase. Although the amount of V-ATPase in the A and B subunits on the phagosomes was not significantly different between the live and dead bacteria infection, the phagosomes containing live bacteria had a 10-fold smaller V-ATPase activity than those containing the dead bacteria. These results indicated that the inhibition of phagosomal acidification by Y. pseudotuberculosis infection was due to the attenuation of V-ATPase activity, and not due to the exclusion of V-ATPase subunits from the phagosome membrane as found in Mycobacterium avium.

Identification of a novel periplasmic catalase-peroxidase KatA of Legionella pneumophila.

A gene katA that encodes a novel catalase-peroxidase was cloned from the chromosome of Legionella pneumophila. The nucleotide sequence revealed that KatA was highly homologous to members of the bacterial bifunctional catalase-peroxidase family. In addition, KatA has a N-terminal signal sequence and was considered to be present in the periplasm of the bacterium.

Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase.

Myeloperoxidase (MPO) catalyzes the reaction of hydrogen peroxide with chloride ion to produce hypochlorous acid (HOCl), which is used for microbial killing by phagocytic cells. Despite the important role of MPO in host defense, however, MPO deficiency is relatively common in humans, and most of these individuals are in good health. To define the in vivo role of MPO, we have generated by gene targeting mice having no MPO activity in their neutrophils and monocytes. The mice without MPO developed normally, were fertile, and showed normal clearance of intraperitoneal Staphylococcus aureus. However, they showed increased susceptibility to pneumonia and death following intratracheal infection with Candida albicans. Furthermore, the lack of MPO significantly enhanced the dissemination of intraperitoneally injected C. albicans into various organs during the first 7 days. Thus, MPO is important for early host defense against fungal infection, and the inability to generate HOCl cannot be compensated for by other oxygen-dependent systems in vivo in mice. The mutant mice serve as a model for studying pulmonary and systemic candidiasis.

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.

Difference in Legionella pneumophila growth permissiveness between J774.1 murine macrophage-like JA-4 cells and lipopolysaccharide (LPS)-resistant mutant cells, LPS1916, after stimulation with LPS.

To elucidate the role of the oxidative burst in macrophage resistance to Legionella infection, we examined a murine macrophage-like cell line, J774.1, for permissiveness to Legionella growth, using a mutant that has a selective defect in the oxidative burst after lipopolysaccharide (LPS) stimulation. Legionella pneumophila serogroup 1 was infected into J774.1 monolayers, and then the extent of bacterial growth was estimated by a CFU assay. Both the parental cell line, JA-4, and the LPS-resistant mutant, LPS1916, were permissive for Legionella growth but became nonpermissive after pretreatment with gamma interferon. However, pretreatment of LPS1916 cells with LPS failed to inhibit bacterial growth, although LPS-treated JA-4 cells exhibited inhibited multiplication of the bacteria. The bacterial growth inhibition in JA-4 and mutant LPS1916 cells was correlated with the extent of the oxidative burst in the cells, as judged by cytochrome c reduction but not nitrite production. Neither transferrin receptor expression nor the iron content in JA-4 and LPS1916 cells, with or without LPS treatment, was correlated with suppression of Legionella growth. These results suggest that the restriction of Legionella growth in J774.1 cells is due to a bactericidal effect of the oxidative burst rather than reduction of the iron supply to the intracellular bacteria and that the effectors are reactive oxygen intermediates and not reactive nitrogen intermediates.

Mechanism in 1,25(OH)2D3-induced suppression of helper/suppressor function of CD4/CD8 cells to immunoglobulin production in B cells.

On the basis of previous data that 1,25(OH)2D3 suppressed both helper and suppressor activities of CD4 and CD8 cells in the pokeweek mitogen-stimulated culture, we examined the further effect of 1,25(OH)2D3 on both cells to define how 1,25(OH)2D3 is involved in the deterioration of their functions. 1,25(OH)2D3 suppressed the pokeweed mitogen and phytohemagglutinin-induced DNA synthesis of CD4 and CD8 cells. The suppression by 1,25(OH)2D3 of DNA synthesis was caused by a time lag in reaching maximal response. 1,25(OH)2D3 also suppressed interleukin-2 production of CD4 and CD8 cells. 1,25(OH)2D3 did not, however, affect their interleukin-2 receptor expression detected within 24 hr after phytohemagglutinin stimulation. In addition, 1,25(OH)2D3 failed to suppress DNA synthesis of CD4 and CD8 cells when cultured with a large amount of interleukin-2. Suppression by 1,25(OH)2D3 of proliferation and interleukin-2 production in CD4 and CD8 cells would bring about the decrease of their helper or suppressor functions by inhibiting their expansion or maturation.

The effect of 1,25-dihydroxyvitamin D3 on in vitro immunoglobulin production in human B cells.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently suppressed immunoglobulin (Ig) production of human B cells, as evaluated by IgG-plaque-forming cells (IgG-PFC) in the culture of pokeweed mitogen (PWM)-activated B cells. Similar suppressive effect of 1,25(OH)2D3 on Ig production of B cells was observed in the Staphylococcus aureus Cowan I(SAC)-induced Ig-producing system. The mean percentage of inhibitions at a concentration of 10(-9) M were 60.0 +/- 8.2% (mean +/- SE, n = 6) and 65.1 +/- 4.7% (n = 10) in PWM- and SAC-stimulated cultures, respectively. The suppression was strongly exhibited only when 1,25(OH)2D3 was added at the start of the 6-day culture, accompanied by a decrease in DNA synthesis of B cells in both culture systems. On the other hand, the addition of 1,25(OH)2D3 on day 4, when DNA synthesis reached at plateau and IgG-PFC began to be detectable, had no noticeable affect on either the number of PFC or DNA synthesis of B cells. Furthermore, 1,25(OH)2D3 suppressed Ig production even when B cells were exposed to the agent for 4 hr after the activation with PWM or SAC, but not before the activation. These results indicate that 1,25(OH)2D3 inhibits B cell proliferation before differentiation to Ig-secreting cells, consequently reducing Ig production; and that its action appears to be mediated by the cytosol receptors expressed on activated B cells. Thus, the agent may serve as an immunoregulating hormone in vivo, as well as in vitro.

Effect of 1,25-dihydroxyvitamin D3 on the immunocompetent cells.

This paper deals with the immunosuppressive effects of 1,25-dihydroxy vitamin D3 (1,25(OH)2, D3), a metabolic product which acts in vivo as the active form vitamin D3. Dose and time of exposure-dependent suppressive effects were demonstrated in vitro on PHA blastogenesis of mononuclear cells, IL-2 production of T cells and IL-1 production of macrophages when these human immunocytes were treated with 1,25(OH)2D3. Addition of exogenous IL-1 and IL-2 partially abrogated the suppressive effects of 1,25(OH)2 D3 on IL-2 production and PHA blastogenesis, respectively. Thus, it is suggested that inhibition of PHA blastogenesis results from the reduced production of such growth factors in this IL-1 -IL-2 cascade system. PWM-induced immunoglobulin (Ig) synthesis of B cells was also markedly inhibited by 1,25(OH)2 D3, which was caused not only by direct suppression toward B cells but also by indirect suppression due to reduced helper factor from T cells when these cells were treated with 1,25(OH)2 D3. It is proposed that 1,25(OH)2D3 may act as one of the immunoregulatory factors in vivo, in addition to its well-known action in calcium metabolism.

The role of monocytes in the suppression of PHA-induced proliferation and IL 2 production of human mononuclear cells by 1,25-dihydroxyvitamin D3.

1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibited phytohemagglutinin (PHA)-induced proliferation of human blood mononuclear cells (MNC) at concentrations of 10(-11) M or more. Interleukin 2 (IL 2) production of T cells activated with PHA was also inhibited by 1,25(OH)2D3. Furthermore, 1,25(OH)2D3 suppressed interleukin 1 (IL 1) production of monocytes (Mo), and the agent-treated Mo were unable to promote IL 2 production of non-adherent cells (NAC). Thus, the reduction of proliferative response of MNC to PHA by 1,25(OH)2D3 appeared to have resulted from the inhibitory effects of the agent on both IL 2 and IL 1 production. From these data, 1,25(OH)2D3 appears to play an important role in the immunoregulatory system.

Ontogeny of 'macrophage' function. III. Manifestation of high accessory cell activity for primary antibody response by Ia+ functional cells in newborn mouse spleen in collaboration with Ia- macrophages.

The ontogenesis of the responsiveness of murine whole spleen cells in the in vitro primary antibody response paralleled not only the development of competent lymphoid cells but also that of the accessory cell (A-cell) activity of spleen adherent cells (SAC). The Ia+ cell content of SAC (and also peritoneal exudate cells) was very low until 2 weeks of age. The phagocytic activity of macrophages in SAC was higher in newborns than in adults, though no significant difference was observed between Ia- and Ia+ macrophages in phagocytic activity. We attempted to reveal a high A-cell activity using cells in newborn spleen by means of our experimental strategy documented previously in adult mice (Inaba, Nakano & Muramatsu, 1981): though neither Ia- macrophage population (Ia- SAC) nor a temporarily adherent spleen cell population containing few phagocytic macrophages (crude non-macrophage cell fraction, CF) serves as an autonomous A-cell source, the collaboration of Ia+ non-macrophage cells in CF with Ia- SAC causes the manifestation of A-cell activity. Adult CF collaborated as well with newborn SAC as with adult Ia- SAC, indicating that newborn Ia- macrophages are functionally comparable with adult Ia- macrophages in the ability to collaborate with Ia+ non-macrophage cells. On the other hand, a high A-cell activity was generated by the combination of adult Ia- SAC with a large number of newborn CF cells, indicating that there exist competent Ia+ cells in newborn spleen, though much fewer than in adult spleen.