Legionella pneumophila contamination of hospital dishwashers.

In a tertiary hospital, Legionella spp were isolated from taps and from ward dishwashers connected to contaminated tap piping. Our investigation revealed favorable conditions for growth of Legionella, and showed that Legionella pneumophila SG6 isolates from the taps and dishwashers were all genetically identical by repetitive-element polymerase chain reaction. These results suggest that contaminated dishwashers might be a potential reservoir for the spread of Legionella in health care facilities.

Clinical evaluation of high mobility group box 1 protein in Legionella pneumophila pneumonia.

High mobility group box 1 (HMGB-1) protein is involved in acute lung injury due to various etiologies. We evaluated HMGB-1 levels in sera and bronchoalveolar fluids in patients with pneumonia caused by Legionella pneumophila. Levels of HMGB-1 in the sera of patients with L. pneumophila pneumonia (32 cases) and control subjects (24 cases) were determined. Serum HMGB-1 levels in Legionella pneumonia were similar to those of the control subjects. No significant correlation between HMGB-1 levels and other biomarkers and the outcome of cases was observed. In contrast, HMGB-1 levels, as well as interferon-γ, in bronchoalveolar (BA) fluids from severe L. pneumophila pneumonia (7 cases) were significantly higher than those in the sera of identical patients. HMGB-1 levels in BA fluids were relatively higher in pneumonia cases with ALI than those without ALI. Our findings suggest that intra-pulmonary HMGB-1 may be involved in the pathophysiology of pneumonia caused by L. pneumophila.

Role of Brevundimonas vesicularis in supporting the growth of Legionella in nutrient-poor environments.

In 1986, we encountered the first case of Legionella micdadei pneumonia in Japan. In the follow-up study to determine the infection route of L. micdadei, we isolated Brevundimonas vesicularis from the shower hose of the patient�s home. This motivated us to explore the symbiosis between B. vesicularis and Legionella in this study. B. vesicularis type strain, B. vesicularis Kobe strain, Legionella pneumophila serogroup 1 type strain, and L. micdadei Kobe strain were used. B. vesicularis was inoculated into 0.01 M phosphate buffer solution containing artificial sand, and varying concentrations of glucose at 0.1%, 0.01%, and 0.001%. Legionella was added to the cultures after ten days of incubation, and Legionella viable counts were monitored over time. After three days of incubation, Legionella counts increased approximately twofold in flasks containing 0.001% glucose, but Legionella counts decreased in both B. vesicularis inoculated and non-inoculated flasks containing higher concentrations of glucose. The counts were significantly higher in flasks inoculated with B. vesicularis than in non-inoculated flasks throughout the experiments. Under the nutrient-poor conditions, the presence of B. vesicularis was found to aid a further increase in Legionella counts. Further research is necessary to understand the symbiotic conditions most supporting the growth of L. micdadei.

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.

[Comparison of pneumonia severity indices between modified A-DROP system and current A-DROP system predicting outcomes for patients hospitalized with community-acquired pneumonia].

BACKGROUND: The A-DROP is a predicting pneumonia severity index which is adopted in the Japanese Respiratory Society (JRS) guidelines. For community-acquired pneumonia, we made a modified A-DROP, adding two new index items to the current A-DROP. Then, we retrospectively compared the modified A-DROP with the current A-DROP regarding 30-day mortality. METHODS AND RESULTS: We analyzed consecutive 227 patients hospitalized with community-acquired pneumonia (mean age 79.0 years). The added index items were respiratory rate > or = 30/min and the presence or absence of underlying diseases. There were 16 fatalities (7.0%). In the extremely severe group, the sensitivities of the 30-day death and odds ratios were 19.9% and 9.5 in the current A-DROP, but 75.0% and 14.1 for the modified A-DROP, respectively. In addition, regarding the receiver-operating characteristic (ROC) area under the curve for the 30-day death ratio, the current A-DROP and modified A-DROP were 0.807 and 0.840, respectively. CONCLUSIONS: The modified A-DROP improved the ability to predict outcomes compared with the current A-DROP.

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

BACKGROUND: Hepatocyte 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. METHODS: Sera 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. RESULTS: Serum 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. CONCLUSIONS: HGF levels increased in severer pneumonia caused by Legionella, suggesting that HGF might play a significant role in the Legionella pneumonia.

Repetitive element-polymerase chain reaction for genotyping of clinical and environmental isolates of Legionella spp.

Genotyping of Legionella strains is important for the epidemiologic survey of Legionnaires' disease infections. In this study, we investigated the potential of repetitive element-polymerase chain reaction (rep-PCR) for differentiating various isolates of Legionella spp. We used 38 Legionella pneumophila isolates (collected in clinics all over Japan between 1980 and 2007), 19 environmental Legionella anisa isolates (collected in Okinawa, Nara, Osaka, and Hyogo prefecture between 1987 and 2007), and 2 Legionella-type strains. We extracted bacterial genomic DNA and applied it to rep-PCR. PCR products were then converted into bands by agarose gel electrophoresis. The L. pneumophila serogroup (SG) 1 displayed very diverse patterns. Different bands were produced for each species of Legionella, and each species was clearly distinct. Phylogenetic analysis displayed 1 cluster of L. anisa isolates, while other Legionella spp. were present at discrete levels. Our findings show that rep-PCR is an effective, rapid, and simple technique for differentiation of L. pneumophila strains as well as Legionella spp.

Lung abscess caused by Legionella species: implication of the immune status of hosts.

Legionella pneumonia typically presents as lobar pneumonia with multiple-lobe involvement, but Legionella lung abscess is rare. To identify the predisposing factors for Legionella abscess, we analyzed 62 of the 79 case reports on Legionella abscess found in literature; 28 (45.2%) were of hospital-acquired infection and 28 (45.2%), community-acquired infection. Seventeen patients (27.4%) died. L. pneumophila serogroup 1 was the most common, but other serogroups of L. pneumophila, L. micdadei, L. bozemanii, L. dumoffii, and L. maceachernii were also isolated from the abscess. Corticosteroids were administered for underlying diseases to 43 (69.4%) patients. Peripheral neutrophil counts were higher in patients with abscess than in those with only pulmonary infiltration. In certain cases, Legionella abscess developed during neutropenia recovery. However, lymphocyte counts were low in most cases. Clinical factors like corticosteroid treatment, which causes impaired cellular immunity and subsequent neutrophil accumulation in the lesion, might function as predisposing factors for Legionella abscess.

Epidemiology of influenza from 2007 to 2008 in naha area, okinawa prefecture: surveillance of rapid antigen test results.

The incidence of influenza in the Naha city area in the southernmost part of Japan was surveyed in 2007 and 2008. Patients who had influenza-like symptoms and visited one of four general hospitals in Naha City, Okinawa, Japan were included in this study. The nasal or throat swab samples were applied to the rapid test for detecting influenza A and B virus antigens. The positive rate of influenza A and/or B virus antigen was 26.2% (8,480/32,380). Most cases (82.9%) were influenza A. In 2007, influenza A cases were detected during the entire year, and an epidemic peak was also noted in July, while no outbreak occurred in the summer of 2008. The surveillance of the rapid influenza virus antigen test seemed to provide reliable epidemiological data. This finding warrants further study in this region, including study of the influences of climate and socio-behavior patterns of the residents in the region on influenza epidemics.

Characteristics of Legionella pneumophila serogroup 2 strains by colony morphology.

The isolation rate of Legionella pneumophila serogroup 2 from clinical samples is low. In 2007, we encountered the second case known to occur in Japan. As the L. pneumophila serogroup 2 type strain, the Kobe strain isolated in 1988, and the Okinawa strain isolated from the present patient could not be differentiated using the usual biochemical and serological tests, we tried to achieve differentiation by observing colony morphology. In Oxoid BCYEalpha medium, colonies of the Kobe strain developed multiple protuberances on the surface, but these did not develop on the other two strains. In Becton-Dickinson BCYEalpha medium, colonies of the Okinawa strain had several outgrowths from the margin, but the type strain and the Kobe strain did not have any outgrowths. The Okinawa strain isolated from the present case showed intermediate characteristics between the type strain and the Kobe strain in the appearance of colony morphology. It may be useful to conduct an investigation of this rare serogroup.

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.

[A-DROP system might result in underestimation of severity of cases with Legionnaires' disease].

Fifteen cases of legionella pneumonia experienced in our department or associated hospital were included in this study. Each case was classified with the A-DROP system of the Japanese Respiratory Society Guidelines, and guidelines from the Infectious Diseases Society of America (IDSA) (1998) and then we compared the severity of the cases of pneumonia. Although 10 cases were classified as intermediate, 3 as severe, and 2 as extremely severe with the A-DROP system, most cases were classified as severe according to the IDSA guidelines. Among 5 fatal cases, three were ranked as intermediate with the A-DROP system. However all the fatal cases were ranked as severe in the IDSA guidelines. This study suggested that the A-DROP system might underestimate the severity of pneumonia in cases with legionella pneumonia.

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.

BACKGROUND: Legionella 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. METHODS: Nuclear 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. RESULTS: The 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 muM) did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells. CONCLUSION: Infection 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 pneumophila serogroup 1 antigen in respiratory samples using an immunochromatographic membrane test.

The immunochromatographic membrane test (ICT) efficacy of Legionella antigen detection (Binax Now Legionella) was evaluated using respiratory samples, including bronchial washings (44 cases) and sputum (128 cases), from suspected Legionella pneumonia patients. The ICT results using respiratory samples agreed well with isolation of L. pneumophila SG1 and ICT using urines.

Clinical features of two cases of Legionnaires' disease with persistence of Legionella urinary antigen excretion.

The advantages of diagnosing legionellosis by urinary antigen detection are widely recognized, and include early detection, rapidity of testing, and ease of specimen collection. However, the persistence of Legionella urinary antigen excretion has been suggested in some selected patients, although the clinical features of these patients have not yet been clearly described. Here, we describe the clinical features of two patients with Legionnaires' disease with persistence of Legionella urinary antigen excretion (117 days and 247 days). One patient had an underlying disease, adult T-cell leukemia, and the other patient had ulcerative colitis and was receiving oral corticosteroids. Unusual clinical and radiological findings as well as a review of the literature are presented.

Mechanism of formation of the orange-colored sputum in pneumonia caused by Legionella pneumophila.

It has been said that the color of sputum from patients with Legionella pneumophila pneumonia is orange. However, why the color is orange has not been clarified. First, orange-colored sputum obtained from a patient with L. pneumophila pneumonia is presented. Then, the formation of an orange-colored pigment in a culture medium (without charcoal) after the growth of L. pneumophila is demonstrated.

Comparative evaluation of Duopath Legionella lateral flow assay against the conventional culture method using Legionella pneumophila and Legionella anisa strains.

Duopath Legionella is a new immunochromatographic assay for the identification of Legionella pneumophila and Legionella spp. As excellent specificity has been previously reported for this kit, we attempted an evaluation of its sensitivity using L. pneumophila serogroup 1 and Legionella anisa strains. Bacterial suspensions of L. pneumophila at concentrations of 1.2 x 10(8) cfu/ml and 1.2 x 10(7) cfu/ml were detected, but those below 1.2 x 10(6) cfu/ml were not recognized by the Duopath kit. After centrifugation and the sediment resuspension, a 2.8 x 10(7) cfu/ml concentration of bacterial suspension showed a positive result, but negative results were obtained below 2.8 x 10(6) cfu/ml. Also, bacterial suspension with concentrations of 3.2 x 10(9) cfu/ml and 1.4 x 10(9) cfu/ml after centrifugation of L.anisa were detected, but those below 3.2 x 10(8) cfu/ml and 1.4 x 10(8) cfu/ml after centrifugation were not recognized by the Duopath kit. Meanwhile, this kit was less sensitive to the L. pneumophila serogroup 1 suspension, and was more sensitive to the L. pneumophila serogroup 2 and L.anisa than the Binax NOW immunochromatographic kit. It was realized that the sensitivity of this kit is too low for determining the presence of Legionella in water samples. Although this kit may have excellent specificity, it has low sensitivity.

Intravenous ciprofloxacin versus erythromycin in the treatment of Legionella pneumonia.

BACKGROUND: Erythromycin (EM) and rifampicin (RFP) have mainly been used to treat patients with Legionella pneumonia. Since intravenous ciprofloxacin (CPFX) became available in Japan from 2000, many reports have been published detailing successful treatment of Legionella pneumonia with CPFX. In this study, we compared the evolution of patients with Legionella pneumonia treated with CPFX to those treated with EM. METHODS: The study included nine patients treated with CPFX and eighteen patients treated with EM. Diagnosis of these patients was made by culture, PCR, urinary antigen assay or a serological method. A comparison was made of the patients' characteristics, severity of pneumonia, efficacy of each agent and the clinical course. RESULTS: No significant differences were observed between the two groups, in regard to age, gender, underlying disease or severity of pneumonia. In addition, the period of time from onset of the disease until appropriate therapy did not differ significantly between the two groups. In the CPFX group, all of the patients were cured and in the EM group 16 out of the 18 patients were cured. Although there were no significant differences, the time to apyrexia, normalization of leukocytosis and a 50% decrease in C-reactive protein (CRP) occurred within a relatively shorter time frame in the CPFX group than in the EM group (3.5 versus 4 days, 4 versus 5.2 days, and 2.9 versus 10.3 days, respectively). And, the duration of antibiotic treatment in the CPFX group was significantly shorter than in the EM group. CONCLUSION: CPFX was as effective as erythromycin in the treatment of Legionella pneumonia. The effects of treatment may appear relatively earlier and the duration of treatment was significantly shorter in patients treated with CPFX therapy than with EM therapy.

Prospective monitoring study: isolating Legionella pneumophila in a hospital water system located in the obstetrics and gynecology ward after eradication of Legionella anisa and reconstruction of shower units.

We previously reported on the sporadic contamination by Legionella anisa of shower units and sink taps at Ryukyu University Hospital. Starting in July 2003, the neonatal area underwent an 8-month reconstruction, and in March 2005, the boiler system was replaced. We therefore examined shower water and tap water for the presence of Legionella just after replacement of the boiler system. In 3 of the 8 water samples collected from the remodeled area, we isolated Legionella pneumophila serogroup 1 and L. anisa. Moreover, L. pneumophila serogroup 1 was isolated in 4 of the 5 water samples gathered from the unreconstructed area of the same floor. Random amplified polymorphic DNA analysis suggested that a single clone of L. pneumophila might exist throughout the floors of the water distribution system. We replaced the shower units at the Legionella-positive site, and began flushing the sink-faucets with water heated to 55N for at least 1 h every morning. As a result, Legionella was not subsequently isolated in water samples. In this prospective study, we identified a central contamination by L. pneumophila serogroup 1 and showed that flushing with hot tap water was effective to counter this situation.

Influence of temperature on growth of Legionella pneumophila biofilm determined by precise temperature gradient incubator.

Bacterial growth is influenced by several different culture conditions. Temperature is one of an essential component which regulates bacterial growth and their morphology. The influence of temperature on the length of bacteria was investigated in broth and on agar in a temperature range from 30.0 degrees C to 47.0 degrees C in 0.5 degrees C steps using a newly developed temperature gradient incubator. The incubator is able to reach a set temperature within 2 h and maintain temperature as accurate as +/-0.1 degrees C of the set temperature. Three Legionella pneumophila serotype 1 strains were incubated for 48 h in BCYE-alpha agar at various temperatures ranging from 30.0 degrees C to 48.0 degrees C and length of bacteria grown at each temperature was microscopically measured. Ability of bacteria to multiply at a given temperature was also determined. L. pneumophila serotype 1 strains ATCC 33152, a clinical isolate Okinawa 02-001 were going to elongate to longer than 100 mum when cultured higher than at 39.5 degrees C and at 41.5 degrees C, respectively. Each strain was unable to multiply when cultured higher than at 44.2 degrees C (ATCC 33152) or at 44.0 degrees C (Okinawa 02-001). Those data would provide insights for establishing regulations in terms of maintaining hot water temperature in a facility where a circulating hot water supply-system is available and contamination with Legionella spp. is likely to happen.