Michio Koide

Isolation of Legionella longbeachae Serogroup 1 from Potting Soils in Japan

resistance cannot be ignored. In contrast, therapy with a 1-g dose of azithromycin would be expected to provide intracellular levels of drug that exceed the MIC for Shigella for as long as 14 days [7]. The data presented here expand the experience reported from Bangladesh, where 5 days of azithromycin (total dose, 1.5 g) was as effective against shigellosis as 5 days of ciprofloxacin [5]. Although the use of azithromycin in the treatment of other diarrheal diseases is still under evaluation, a single 1-g dose of azithromycin is effective and welltolerated treatment of epidemic dysentery.

Legionella pneumophila infection induces programmed cell death, caspase activation, and release of high-mobility group box 1 protein in A549 alveolar epithelial cells: inhibition by methyl prednisolone

BackgroundLegionella pneumophila pneumonia often exacerbates acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Apoptosis of alveolar epithelial cells is considered to play an important role in the pathogenesis of ALI and ARDS. In this study, we investigated the precise mechanism by which A549 alveolar epithelial cells induced by L. pneumophila undergo apoptosis. We also studied the effect of methyl prednisolone on apoptosis in these cells.MethodsNuclear deoxyribonucleic acid (DNA) fragmentation and caspase activation in L. pneumophila-infected A549 alveolar epithelial cells were assessed using the terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP)-biotin nick end labeling method (TUNEL method) and colorimetric caspase activity assays. The virulent L. pneumophila strain AA100jm and the avirulent dotO mutant were used and compared in this study. In addition, we investigated whether methyl prednisolone has any influence on nuclear DNA fragmentation and caspase activation in A549 alveolar epithelial cells infected with L. pneumophila.ResultsThe virulent strain of L. pneumophila grew within A549 alveolar epithelial cells and induced subsequent cell death in a dose-dependent manner. The avirulent strain dotO mutant showed no such effect. The virulent strains of L. pneumophila induced DNA fragmentation (shown by TUNEL staining) and activation of caspases 3, 8, 9, and 1 in A549 cells, while the avirulent strain did not. High-mobility group box 1 (HMGB1) protein was released from A549 cells infected with virulent Legionella. Methyl prednisolone (53.4 μM) did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells.ConclusionInfection of A549 alveolar epithelial cells with L. pneumophila caused programmed cell death, activation of various caspases, and release of HMGB1. The dot/icm system, a major virulence factor of L. pneumophila, is involved in the effects we measured in alveolar epithelial cells. Methyl prednisolone may modulate the interaction of Legionella and these cells.

Detection of Legionella Species in Clinical Samples: Comparison of Polymerase Chain Reaction and Urinary Antigen Detection Kits

Background:Recently, two excellent methods have been used for the diagnosis of Legionnaires’ disease: urinary antigen detection and PCR. The purpose of the present study is to analyze and evaluate the sensitivity and specificity of three different urinary antigen detection kits as well as PCR.Materials and Methods:A total of 148 samples were collected from 33 patients between 1993 and 2004. These consisted of 73 urine samples obtained from 33 patients, 57 serum samples provided by 29 patients, and 18 respiratory tract specimens from 13 patients. Three commercially available kits were used to detect urinary antigen. For the 5S PCR reaction, primers L5SL2 and L5SR84 were used.Results:Positive results were shown in all patients’ urine (representing 79.5% of total samples) using the Binax EIA kit, in 93.9% patients (representing 75.3% samples) using the Binax NOW immunochromatographic kit, and in 90.9% (representing 72.6% samples) using the Biotest EIA kit. Urine samples from 12.1% patients (representing 6.8% of total samples), serum samples from 41.4% patients (representing 35.1% of total samples), and respiratory samples from 84.6% patients (representing 88.9% of total samples) showed positive results with PCR.Conclusion:In testing urine of legionellosis patients, it was suggested that three kits were all valuable tools for diagnosis of legionellosis. Since over one-third of patients’ serum samples and most respiratory specimens showed positive results with PCR, the addition of PCR for testing of these samples might be useful, particularly in cases of culture negative and serum antibody negative patients.

Comparison of Polymerase Chain Reaction and Two Urinary Antigen Detection Kits for Detecting Legionella in Clinical Samples

In the study presented here, two urinary antigen tests (Legionella Urinary Antigen EIA; Binax, Portland, ME, USA and Biotest Legionella Urin Antigen EIA; Biotest, Dreieich, Germany) and one PCR with a 5S-primer were used to detect Legionella in the clinical samples of six patients diagnosed with legionellosis between 1997 and 1999. Initially, clinical specimens that could be collected in a series were obtained from each patient, resulting in a total of 68 samples (34 urine, 24 serum, 7 sputum, and 3 respiratory). These samples were tested with culture, serum antibody and urinary antigen tests, then stored at – 80�C until further testing in the present study. Upon retesting with the two urinary antigen tests and the 5Sprimer PCR system, Legionella infections were identified using culture and urinary antigen tests in three cases, with culture alone in one case, and with urinary antigen tests alone (both Binax and Biotest) in two cases. The 5Sprimer PCR system detected 26 of 34 Legionella spp. tested. Patient 1 had undergone a cone resection for earlystage uterine cervical carcinoma 1 year prior to presentation. A diagnosis of Pneumocystis carinii pneumonia and pulmonary infiltration of adult T-cell leukemia cells was made. Legionella pneumophila serogroup 1 was later isolated from bronchial lavage fluid and, over time, the serum indirect immunofluorescent antibody (IFA) titer against Legionella pneumophila serogroup 1 increased from 1:128 to 1:512. The IFA test was performed as described previously [1]. The patient was treated with antibiotics (erythromycin, sparfloxacin, and trimethoprim-sulfamethoxazole) and antineoplastic agents (vincristine, cyclophosphamide and adriamycin) and was discharged 5 5 months after admission, with ATL in

Induction of apoptosis of human macrophages in vitro by Legionella longbeachae through activation of the caspase pathway

The cytotoxicity of the facultative intracellular bacterium, Legionella longbeachae, an important cause of legionellosis, was characterised. Apoptosis was induced in HL-60 cells, a human macrophage-like cell line, during the early stages of infection and induction of apoptosis correlated with cytotoxicity. Apoptosis was confirmed by agarose gel electrophoresis of fragmented DNA, surface exposure of phosphatidylserine and propidium iodide labelling of host cell nuclei. The involvement of macrophage infectivity potentiator (Mip) protein, a known virulence factor of L. longbeachae, was also examined. A mip mutant of L. longbeachae induced apoptosis of HL-60 cells but failed to multiply intracellularly, suggesting that intracellular replication of L. longbeachae is not essential for the induction of apoptosis of HL-60 cells. Furthermore, induction of apoptosis of L. longbeachae-infected macrophages was mediated by activation of the caspase pathway but might be independent of tumour necrosis factor-alpha- and Fas-mediated signal transduction pathways.

Hepatocyte growth factor levels in Legionella pneumonia: a retrospective study.

BackgroundHepatocyte growth factor (HGF) is known to be involved in the resolution of pulmonary inflammation and repair of acute lung injury. Legionella pneumonia is sometimes complicated by acute lung injury. Our study aimed to determine the role of serum HGF levels in Legionella pneumonia.MethodsSera from patients with Legionella pneumonia (42 cases), other bacterial pneumonia (33 cases), pulmonary tuberculosis (19 cases), and normal controls (29 cases) were collected. The serum HGF levels for each serum sample were determined by sandwich ELISA. Clinical and laboratory data were collected by reviewing the medical charts.ResultsSerum HGF levels were higher in patients with Legionella pneumonia than in those with other bacterial pneumonia, pulmonary tuberculosis, and controls. The HGF levels were compared with white blood cell counts, C-reactive protein, Alanine amino- transferase, and lactate dehydrogenase (LDH). The HGF levels were correlated to serum LDH levels. Moreover, serum HGF levels were significantly higher in non-survivors than in survivors.ConclusionsHGF levels increased in severer pneumonia caused by Legionella, suggesting that HGF might play a significant role in the Legionella pneumonia.

Legionella pneumonia caused by Legionella pneumophila serogroup 2: second case report in Japan

A 56-year-old man with a 3-day history of a chilly sensation and general fatigue presented to a hospital in his neighborhood. He was diagnosed as having pneumonia and immediately treated with intravenous ceftriaxone sodium, but his respiratory condition deteriorated and he developed symptoms of restlessness. Although Legionella urinary antigen detection tests were negative, his clinical course suggested Legionella pneumonia. After his treatment was changed to intravenous ciprofloxacin and oral clarithromycin, his general condition gradually improved. Later, Legionella pneumophila serogroup 2 was isolated from a bronchoalveolar lavage specimen. This was considered to be the causative organism. In our literature search, this was only the second case of Legionella pneumonia caused by Legionella pneumophila serogroup 2 in Japan.

Identification of Legionella pneumophila serogroups and other Legionella species by mip gene sequencing

The virulence factor known as the macrophage infectivity potentiator (mip) is responsible for the intracellular survival of Legionella species. In this study, we investigated the potential of the mip gene sequence to differentiate isolates of different species of Legionella and different serogroups of Legionella pneumophila. We used 35 clinical L. pneumophila isolates and one clinical isolate each of Legionella micdadei, Legionella longbeachae, and Legionella dumoffii (collected from hospitals all over Japan between 1980 and 2007). We used 19 environmental Legionella anisa isolates (collected in the Okinawa, Nara, Osaka, and Hyogo prefectures between 1987 and 2007) and two Legionella type strains. We extracted bacterial genomic DNA and amplified out the mip gene by PCR. PCR products were purified by agarose gel electrophoresis and the mip gene was then sequenced. The L. pneumophila isolates could be divided into two groups: one group was very similar to the type strain and was composed of serogroup (SG) 1 isolates only; the second group had more sequence variations and was composed of SG1 isolates as well as SG2, SG3, SG5, and SG10 isolates. Phylogenetic analysis displayed one cluster for L. anisa isolates, while other Legionella species were present at discrete levels. Our findings show that mip gene sequencing is an effective technique for differentiating L. pneumophila strains from other Legionella species.

Diagnosis of Legionella infection by reverse transcription of 5S-ribosomal RNA and polymerase chain reaction

We investigated a diagnostic method for legionellosis that uses detection ofLegionella 5S ribosomal RNA by reverse transcription-polymerase chain reaction (RT-PCR). Primer L5SR105, located between base pairs 105 and 119 of theLegionella 5S ribosomal RNA, was used for reverse transcription. Primer L5SL2, located between 2 and 21 bp of theLegionella 5S ribosomal RNA, and primer L5SR84, located between 84 and 103 bp, were used for the polymerase chain reaction (PCR). We found that 8-methoxypsoralen at a concentration of 25μg/mL followed by 20 minutes of ultraviolet irradiation (366 nm) was adequate for elimination of DNA contamination when using Sorenson-transparent microtubes. The specificity of the RT-PCR system was determined by using 27Legionella species (42 strains) and 9 non-Legionella strains. Of these, 23 species (38 strains) showed positive results. Subsequently, the elimination step for PCR mixtures was followed by RT-PCR of clinical samples. We examined 6 samples from 2 patients with positive cultures with this RT-PCR method, and showed results 102 to 103 times more sensitive than themip-primer-PCR method. Furthermore, 2 of the patient samples negative by culture andmip-primer-PCR assay showed a positive reaction with this RT-PCR method. Our results suggest that this RT-PCR technique may be a useful diagnostic tool for legionellosis.

In Vitro and In Vivo Evaluation of the Antimicrobial Activity of Azithromycin against Legionella Species

The antimicrobial activity of azithromycin (AZM) was evaluated againstLegionella species using an experimental model of legionellosis. The minimum inhibitory concentration90s (MIC90s) of AZM against 35 standard strains (0.25 mg/L) and 22 Japanese clinical isolates ofLegionella pneumophila (0.063 mg/L) were lower than those of erythromycin (EM) and almost equal to those of clarithromycin and roxythromycin. Using14C-labeled antibiotic, AZM and EM were observed concentrated inside human polymorphonuclear leukocytes (PMN) with intracellular/extracellular concentration ratios of AZM and EM at 120 minutes after dosing of 27.3 and 22.2, respectively. AZM inhibited the growth ofL. pneumophila (80-045 strain) from cultured guinea pig alveolar macrophages, even when the extracellular AZM was washed out on day 2 of culture. However, the addition of identical concentrations of EM failed to produce a similar inhibition. Pharmacokinetic studies of AZM tissue distribution in guinea pigs infected withL. pneumophila 80-045 revealed high drug concentrations in the lung and liver and a longer half-life in these organs compared with those in plasma. Treatment of guinea pigs with experimentally-induced legionellosis with oral AZM (10 mg/kg/day for 2 days) was more effective than EM treatment (10 mg/kg/day for 4 days) or a placebo, and resulted in a significant improvement in the survival rate. Our results suggest that AZM is a promising new drug for the treatment of legionellosis using a short-term dosing regimen.