Evaluating Fourier-transform infrared spectroscopy with IR Biotyper as a faster and simpler method for investigating the sources of an outbreak of legionellosis.

Fourier-transform infrared (FTIR) spectroscopy using the IR Biotyper and core genome single nucleotide polymorphism (cgSNP) analysis were performed on 12 Legionella isolates associated with an outbreak at a spa house in Kanagawa Prefecture, Japan, and 3 non-outbreak isolates. The discriminative power of FTIR spectroscopy for 48-h incubation conditions of L. pneumophila in this outbreak was lower than cgSNP-based typing but higher than serogroup typing. FTIR spectroscopy could screen outbreak isolates from a group of genetically related isolates and may be useful as an initial typing method in Legionella outbreak investigations.

More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells.

The genus Legionella comprises 65 species, among which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes and Legionella Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens.

Improvements to the Success of Outbreak Investigations of Legionnaires' Disease: 40 Years of Testing and Investigation in New York State.

Since 1978, the New York State Department of Health's public health laboratory, Wadsworth Center (WC), in collaboration with epidemiology and environmental partners, has been committed to providing comprehensive public health testing for Legionella in New York. Statewide, clinical case counts have been increasing over time, with the highest numbers identified in 2017 and 2018 (1,022 and 1,426, respectively). Over the course of more than 40 years, the WC Legionella testing program has continuously implemented improved testing methods. The methods utilized have transitioned from solely culture-based methods for organism recovery to development of a suite of reference testing services, including identification and characterization by PCR and pulsed-field gel electrophoresis (PFGE). In the last decade, whole-genome sequencing (WGS) has further refined the ability to link outbreak strains between clinical specimens and environmental samples. Here, we review Legionnaires' disease outbreak investigations during this time period, including comprehensive testing of both clinical and environmental samples. Between 1978 and 2017, 60 outbreaks involving clinical and environmental isolates with matching PFGE patterns were detected in 49 facilities from the 157 investigations at 146 facilities. However, 97 investigations were not solved due to the lack of clinical or environmental isolates or PFGE matches. We found 69% of patient specimens from New York State (NYS) were outbreak associated, a much higher rate than observed in other published reports. The consistent application of new cutting-edge technologies and environmental regulations has resulted in successful investigations resulting in remediation efforts. IMPORTANCE Legionella, the causative agent of Legionnaires' disease (LD), can cause severe respiratory illness. In 2018, there were nearly 10,000 cases of LD reported in the United States (https://www.cdc.gov/legionella/fastfacts.html; https://wonder.cdc.gov/nndss/static/2018/annual/2018-table2h.html), with actual incidence believed to be much higher. About 10% of patients with LD will die, and as high as 90% of patients diagnosed will be hospitalized. As Legionella is spread predominantly through engineered building water systems, identifying sources of outbreaks by assessing environmental sources is key to preventing further cases LD.

Detection of Legionella species, the influence of precipitation on the amount of Legionella DNA, and bacterial microbiome in aerosols from outdoor sites near asphalt roads in Toyama Prefecture, Japan.

BACKGROUND: Legionellosis is caused by the inhalation of aerosolized water contaminated with Legionella bacteria. In this study, we investigated the prevalence of Legionella species in aerosols collected from outdoor sites near asphalt roads, bathrooms in public bath facilities, and other indoor sites, such as buildings and private homes, using amoebic co-culture, quantitative PCR, and 16S rRNA gene amplicon sequencing. RESULTS: Legionella species were not detected by amoebic co-culture. However, Legionella DNA was detected in 114/151 (75.5%) air samples collected near roads (geometric mean ± standard deviation: 1.80 ± 0.52 log10 copies/m3), which was comparable to the numbers collected from bathrooms [15/21 (71.4%), 1.82 ± 0.50] but higher than those collected from other indoor sites [11/30 (36.7%), 0.88 ± 0.56] (P < 0.05). The amount of Legionella DNA was correlated with the monthly total precipitation (r = 0.56, P < 0.01). It was also directly and inversely correlated with the daily total precipitation for seven days (r = 0.21, P = 0.01) and one day (r = - 0.29, P < 0.01) before the sampling day, respectively. 16S rRNA gene amplicon sequencing revealed that Legionella species were detected in 9/30 samples collected near roads (mean proportion of reads, 0.11%). At the species level, L. pneumophila was detected in 2/30 samples collected near roads (the proportion of reads, 0.09 and 0.11% of the total reads number in each positive sample). The three most abundant bacterial genera in the samples collected near roads were Sphingomonas, Streptococcus, and Methylobacterium (mean proportion of reads; 21.1%, 14.6%, and 1.6%, respectively). In addition, the bacterial diversity in outdoor environment was comparable to that in indoor environment which contains aerosol-generating features and higher than that in indoor environment without the features. CONCLUSIONS: DNA from Legionella species was widely present in aerosols collected from outdoor sites near asphalt roads, especially during the rainy season. Our findings suggest that there may be a risk of exposure to Legionella species not only in bathrooms but also in the areas surrounding asphalt roads. Therefore, the possibility of contracting legionellosis in daily life should be considered.

Legionella septentrionalis sp. nov., isolated from aquatic environments in the northern PR China.

Four strains (km711T, km714, km542 and km524), representing a novel Legionella species, were isolated from aquatic environments in northern PR China. Cells were Gram-stain-negative, rod-shaped, microaerobic, motile and growth depended on l-cysteine. They grew at 25‒42 °C (optimum, 35‒37 °C) and could tolerate up to 1.5 % (w/v) NaCl (optimum, 0.5 %). The major fatty acids (>5 %) of the type strain km711T were C17 : 0 anteiso, C15 : 0 anteiso, iso-C16 : 0 and C16 : 1 ω7c and/or iso-C15 : 0 2OH. The pairwise comparison values were <96.1 % for 16S rRNA gene sequences, 23.3‒28.7 % interspecies variation for mip gene sequences, <93.6 % average nucleotide identity and <72.8 % average amino acid identity between these four strains and related type strains within the genus Legionella. The phylogenetic tree based on the four concatenated genes (16S rRNA, mip, rpoB and rnpB) and protein-concatamer tree based on concatenation of 21 protein markers both revealed that these four strains formed a separate phylogenetic branch cluster within the genus Legionella. The results of phenotypic and genotypic features suggest that these four strains represent a novel species of the genus Legionella, for which the name Legionella septentrionalis sp. nov. is proposed (type strain km711T=KCTC 15655T=NBRC 113219T).

Occurrence of the Legionella species in the respiratory samples of patients with pneumonia symptoms from Ahvaz, Iran; first detection of Legionella cherrii.

BACKGROUND: This study aimed to investigate the occurrence of Legionella species in the respiratory samples of patients with pneumonia symptoms from Ahvaz, Iran by culture and the real-time PCR of 23S-5S rRNA gene spacer region. METHODS AND RESULTS: A total of 123 clinical respiratory samples including 63 pleural aspirates, 57 bronchoalveolar lavage (BAL), and 3 sputum were collected from 65 males and 58 females with pneumonia symptoms. All samples were cultured on the Modified Wadowsky-Yee (MWY) agar. The Legionella species was identified by routine bacteriological tests. The presence of the 16S-23S rRNA spacer region gene was investigated by real-time PCR. The Legionella species were differentiated by sequencing of 16S-23S rRNA gene. A total of 2 (1.6%) BAL specimens were positive for Legionella species by culture method. No Legionella spp. were identified in pleural aspirates and sputum samples by the culture method. Using real-time PCR, 9 (7.3%) samples including 6 BAL, 1 sputum, and 2 pleural aspirates were positive for legionella species. These species were detected in 3 (5.2%) females and 6 males (9.2%). The results of sequencing showed that eight species were L. pneumophila while one was L. cherrii. Also, the 2 isolates that were identified by culture method, were confirmed as L. pneumophila by sequencing. CONCLUSIONS: The results showed that using the real-time PCR has a more efficacy for detecting of Legionella species in respiratory samples. Also, L. pneumophila was the most prevalent species circulating in the southwest region of Iran. So, periodic monitoring programs is recommended to prevent epidemics due to this bacterium.

Isolation and identification of Legionella spp. from hot spring water in Algeria by culture and molecular methods.

AIMS: Due to infectious risk associated with the presence of Legionella in warm water, we determined the prevalence of living Legionella spp. in hot spring water in Algeria. METHODS AND RESULTS: Detection of Legionella by culture was done by using two methods, direct culture on agar plates and co-culture with amoeba. Fifty samples were taken from different hot springs in northern Algeria, including swimming pools, showers and thermal sources. Legionella pneumophila serotypes were predominant, accounting for 60% of positive samples. Direct method allowed the isolation of 13 L. pneumophila only of 50 samples (26%), whereas co-culture using a panel of three free living amoeba allowed the isolation of 119 Legionella species from the same samples (80%) CONCLUSIONS: Amoeba co-culture allowed the isolation of several Legionella sp., while direct culture allowed the isolation of L. pneumophila only. Remarkably, Legionella longbeachae, usually isolated from soil and compost, was isolated for the first time in thermal water in three samples using Vermamoeba vermiformis co-culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Legionella in the water of hot springs in Algeria, which are mainly frequented by individuals at risk of Legionellosis, requires urgent control measures.

Distribution of Legionella species and serogroups in patients with culture-confirmed Legionella pneumonia.

Legionella species are consistently identified as some of the most common causative agents of severe community-acquired pneumonia (CAP) or nosocomial pneumonia. Although the number of reported Legionella infection cases is gradually increasing in Japan, most cases are diagnosed by a urinary antigen test, which identifies only L. pneumophila serogroup 1. Therefore, assessment of pneumonia-causing Legionella species and serogroups would be important. The Japan Society for Chemotherapy Legionella committee has collected the isolates and clinical information on cases of sporadic community-acquired Legionella pneumonia throughout Japan. Between December 2006 and March 2019, totally 140 sporadic cases were identified, in which L. pneumophila was the most frequently isolated species (90.7%) followed by L. bozemanae (3.6%), L. dumofii (3.6%), L. micdadei (1.4%), and L. longbeachae (0.7%). Among 127 isolates of L. pneumophila, 111 isolates were of serogroup 1, two of serogroup 2, four of serogroup 3, one of serogroup 4, one of serogroup 5, seven of serogroup 6, and one was of serogroup 10. We also assessed in vitro activity of antibiotics against these isolates and showed that quinolones and macrolides have potent anti-Legionella activity. Our study showed that pneumonia-causing Legionella species and serogroup distribution was comparable to that reported in former surveillances. L. pneumophila was the most common etiologic agent in patients with community-acquired Legionella pneumonia, and L. pneumophila serogroup 1 was the predominant serogroup.

Taking a Closer Look at the Valid Publication and Authorship of Legionella bozemanae Brenner et al. 1980, Fluoribacter bozemanae Garrity et al. 1980, Legionella pittsburghensis Pasculle et al. 1980, Legionella micdadei Hébert et al. 1980 and Tatlockia micdadei (Hébert et al. 1980) Garrity et al. 1980.

The names Legionella bozemanae Brenner et al. 1980, Fluoribacter bozemanae Garrity et al. 1980, Legionella pittsburghensis Pasculle et al. 1980, Legionella micdadei Hébert et al. 1980 and Tatlockia micdadei (Hébert et al. 1980) Garrity et al. 1980, all appeared in the same issue of the International Journal of Systematic Bacteriology. Fluoribacter bozemanae Garrity et al. 1980 appeared as the name of new taxon at the rank of species and Tatlockia micdadei (Hébert et al. 1980) Garrity et al. 1980 as a new combination, both in the same original article in the International Journal of Systematic Bacteriology. The names Legionella bozemanae Brenner et al. 1980 (originally published as Legionella bozemanii) Legionella pittsburghensis Pasculle et al. 1980 (originally published as Legionella pittsburgensis) and Legionella micdadei Hébert et al. 1980, all appeared initially in effective publications outside of the International Journal of Systematic Bacteriology and were validly published by inclusion in Validation List no 5. While it is evident from the inclusion of the names Legionella bozemanae Brenner et al. 1980, Legionella pittsburghensis Pasculle et al. 1980 and Legionella micdadei Hébert et al. 1980 on Validation List no. 5 that the authors were following the 1975 revision of the International Code of Nomenclature of Bacteria, the wording of Garrity et al. 1980 indicates that they were following the interpretation found in the 1966 revision of the International Code of Nomenclature of Bacteria. Changes to the International Code of Nomenclature of Bacteria between the 1966 and 1975 revisions introduced new criteria for the valid publication of names. In particular, there was a change from all effective publications being accepted as the publication in which valid publication of a name could occur to only one journal being accepted as the publication in which valid publication could occur (the International Journal of Systematic Bacteriology, now the International Journal of Systematic and Evolutionary Microbiology). This change has a direct effect on the order of valid publication of the names Legionella bozemanae Brenner et al. 1980, Fluoribacter bozemanae Garrity et al. 1980, Legionella pittsburghensis Pasculle et al. 1980, Legionella micdadei Hébert et al. 1980 and Tatlockia micdadei (Hébert et al. 1980) Garrity et al. 1980, their authorships, as well as determining which names should be treated as names of new taxa at the rank of species (sp. nov.) vs new combinations (comb. nov.) based on the names of existing taxa. Given the fact that Legionella pittsburghensis Pasculle et al. 1980, Legionella micdadei Hébert et al. 1980 and Tatlockia micdadei (Hébert et al. 1980) Garrity et al. 1980 share the same nomenclatural type, this also has an influence on which epithet has priority and which epithet is illegitimate.

Ten-Year Retrospective Analysis of Legionella Diffusion in Hospital Water Systems and Its Serogroup Seasonal Variation.

INTRODUCTION: Legionella spp. are ubiquitous aquatic organisms found to be associated with community-acquired pneumoniae (CAP) as well as hospital-acquired pneumonia (HAP). Direct inhalation of aerosols from environmental colonisation is typically the source of infection. The aim of this study was to determine the level of colonisation in hospital water supply systems in order to assess the criticality of the water distribution network and strengthen preventive measures. METHODS: From 2009 to 2018, 769 water samples were collected and then analysed according to the standard methods indicated in ISO11731-2:2004 and ISO11731:2017 for Legionella detection. RESULTS: The samples were positive in 37.1% cases (n. 285) and negative in 62.9% cases (n. 484). The threshold of 10,000 CFU/L was exceeded in 15.1% cases and led to decolonisation as indicated by Italian and European ECDC guidelines. In the autumn-winter period SG1 showed a positivity of 41.2% (n. 40) with a decrease in the spring-summer period with 9.6% (n. 18) of positivity. In contrast, SG2-15 showed a positivity of 30.9% (n. 30) in autumn-winter, which tends to increase to 56.9% (n. 112) in spring-summer (p < 0.001). CONCLUSION: Surprisingly, besides showing a seasonal trend already described previously in the literature, the positivity of our sample was not balanced even for serogroups in the two periods. This could be due to genetic differences and ecological niches to be further investigated that could also have links with the greater pathogenicity of SG1. Environmental microbiological surveillance and risk assessment should be performed more frequently and disinfection must be carried out, especially in health facilities where people are more susceptible to infections.

Legionella qingyii sp. nov., isolated from water samples in China.

Three Legionella-like strains, designed km488T, km489 and km521, were isolated from freshwater samples in China. Cells were Gram-stain-negative, rod-shaped and non-spore-forming. Growth was observed on BCYEα agar, but not on BCYEα agar without l-cysteine, chocolate agar with PolyViteX or Columbia blood agar. The major fatty acids (>5 %) of strains km488T, km489 and km521 were C16 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The mip gene sequences (574 nt) showed the isolates were almost identical with more than 99.7 % sequence similarities, and closely matched to L. gormanii ATCC 33297T with 95.4-95.6 % sequence similarities. Phylogenetic analyses based on concatenated gene (16S rRNA, mip, rpoB and rnpB) sequences indicated that the isolates formed a distinct cluster along with L. gormanii within the genus Legionella. Matrix-assisted laser desorption ionization time-of-flight analyses also demonstrated a clear separation between the isolates and other closely and distantly related Legionella species. DNA-DNA hybridization studies demonstrated that the isolates were closely related (92.0 -95.0 % DNA-DNA relatedness) but differentiated from their phylogenetic neighbours (<70 % DNA-DNA relatedness). The whole genome of km488T was sequenced, and showed a G+C content of 37.8 mol%. Based on the findings from this polyphasic taxonomic study, the isolates are considered to represent a single novel species, for which the name Legionella qingyii sp. nov. is proposed. The type strain is km488T (KCTC 15636T=CCTCC AB 2018025T=NRBC 113223T).

Legionella: a reemerging pathogen.

PURPOSE OF REVIEW: The present review summarizes new knowledge about Legionella epidemiology, clinical characteristics, community-associated and hospital-based outbreaks, molecular typing and molecular epidemiology, prevention, and detection in environmental and clinical specimens. RECENT FINDINGS: The incidence of Legionnaire's disease is rising and the mortality rate remains high, particularly for immunocompromised patients. Extracorporeal membrane oxygenation may help support patients with severe respiratory failure. Fluoroquinolones and macrolides appear to be equally efficacious for treating Legionnaires' disease. Whole genome sequencing is an important tool for determining the source for Legionella infections and for understanding routes of transmission and mechanisms by which new pathogenic clones emerge. Real-time quantitative polymerase chain reaction testing of respiratory specimens may improve our ability to diagnose Legionnaire's disease. The frequency of viable but nonculturable organisms is quite high in some water systems but their role in causing clinical disease has not been defined. SUMMARY: Legionellosis remains an important public health threat. To prevent these infections, staff of municipalities and large buildings must implement effective water system management programs that reduce Legionella growth and transmission and all Medicare-certified healthcare facilities must have water management policies. In addition, we need better methods for detecting Legionella in water systems and in clinical specimens to improve prevention strategies and clinical diagnosis.

Diverse Legionella-Like Bacteria Associated with Testate Amoebae of the Genus Arcella (Arcellinida: Amoebozoa).

Diverse species of Legionella and Legionella-like amoebal pathogens (LLAPs) have been identified as intracellular bacteria in many amoeboid protists. There are, however, other amoeboid groups such as testate amoeba for which we know little about their potential to host such bacteria. In this study, we assessed the occurrence and diversity of Legionella spp. in cultures and environmental isolates of freshwater arcellinid testate amoebae species, Arcella hemispherica, Arcella intermedia, and Arcella vulgaris, via 16S rRNA gene sequence analyses and fluorescent in situ hybridization (FISH). Analysis of the 16S rRNA gene sequences indicated that A. hemispherica, A. intermedia, and A. vulgaris host Legionella-like bacteria with 94-98% identity to other Legionella spp. based on NCBI BLAST search. Phylogenetic analysis placed Legionella-like Arcella-associated bacteria (LLAB) in three different clusters within a tree containing all other members of Legionella and LLAPs. The intracellular localization of the Legionella within Arcella hosts was confirmed using FISH with a Legionella-specific probe. This study demonstrates that the host range of Legionella and Legionella-like bacteria in the Amoebozoa extends beyond members of "naked" amoebae species, with members of the testate amoebae potentially serving an ecological role in the dispersal, protection, and replication of Legionella spp. in natural environments.

Detection of Pathogenic and Non-pathogenic Bacteria in Drinking Water and Associated Biofilms on the Crow Reservation, Montana, USA.

Private residences in rural areas with water systems that are not adequately regulated, monitored, and updated could have drinking water that poses a health risk. To investigate water quality on the Crow Reservation in Montana, water and biofilm samples were collected from 57 public buildings and private residences served by either treated municipal or individual groundwater well systems. Bacteriological quality was assessed including detection of fecal coliform bacteria and heterotrophic plate count (HPC) as well as three potentially pathogenic bacterial genera, Mycobacterium, Legionella, and Helicobacter. All three target genera were detected in drinking water systems on the Crow Reservation. Species detected included the opportunistic and frank pathogens Mycobacterium avium, Mycobacterium gordonae, Mycobacterium flavescens, Legionella pneumophila, and Helicobacter pylori. Additionally, there was an association between HPC bacteria and the presence of Mycobacterium and Legionella but not the presence of Helicobacter. This research has shown that groundwater and municipal drinking water systems on the Crow Reservation can harbor potential bacterial pathogens.

Outbreak of Legionnaires' disease associated with cooling towers at a California state prison, 2015.

A large outbreak of Legionnaires' disease occurred at a California state prison in August 2015. We conducted environmental and epidemiological investigations to identify the most likely source of exposure and characterise morbidity. Sixty-four inmates had probable Legionnaires' disease; 14 had laboratory-confirmed legionellosis. Thirteen (17%) inmates were hospitalised; there were no deaths. Ill inmates were more likely to be ⩾65 years old (P < 0.01), have the chronic obstructive pulmonary disease (P < 0.01), diabetes mellitus (P = 0.02), hepatitis C infection (P < 0.01), or end-stage liver disease (P < 0.01). The case-patients were in ten housing units throughout the prison grounds. All either resided in or were near the central clinical building (for appointments or yard time) during their incubation periods. Legionella pneumophila serogroup 1 was cultured from three cooling towers on top of the central medical clinic (range, 880-1200 cfu/ml). An inadequate water management program, dense biofilm within the cooling towers, and high ambient temperatures preceding the outbreak created an ideal environment for Legionella sp. proliferation. All state prisons were directed to develop local operating procedures for maintaining their cooling towers and the state health department added a review of the maintenance plans to their environmental inspection protocol.

Re-evaluation of the etiology and clinical and radiological features of community-acquired lobar pneumonia in adults.

OBJECTIVE: The aims of this study were to elucidate the frequency and etiology of community-acquired lobar pneumonia (CALP) and the clinical and radiological differences between CALP and tuberculous lobar pneumonia (TLP). PATIENTS AND METHODS: We retrospectively reviewed medical records of patients with community-acquired pneumonia (CAP) (n = 1032) and tuberculosis (n = 1101) admitted to our hospital. RESULTS: Sixty-nine (6.7%) patients with CAP and 23 (2.1%) with pulmonary tuberculosis developed CALP. Legionella species were the most common pathogen (27 patients, 39.1%), followed by Streptococcus pneumoniae (19 patients, 27.5%) and Mycoplasma pneumoniae (18 patients, 26.1%). Symptom duration was longer in the patients with TLP than in those with CALP. On chest radiographs, cavitation in the area of lobar pneumonia and nodular shadows were radiological findings predictive of TLP. High-resolution computed tomography showed cavitation in the area of lobar pneumonia, well-defined centrilobular nodules, and tree-in-bud sign to be the radiological findings predictive of TLP by multivariate logistic regression models. CONCLUSION: Common causes of CALP are Legionella species, S. pneumoniae, and M. pneumoniae. TLP should be considered in patients with lobar pneumonia, particularly in patients with long symptom duration, cavitation, and nodular shadows on chest radiographs, and cavitation, well-defined centrilobular nodules, and tree-in-bud sign on CT.

Legionella feeleii: an unusual organism associated with cutaneous infection in an immunocompromised patient.

We report a 23-year-old immunocompromised woman who, following cardiac transplantation, presented with an unusual cutaneous eruption. She developed a widespread pustular rash, systemic symptoms and a high temperature with raised inflammatory markers. The diagnosis was reached when a skin biopsy was cultured onto Legionella agar (buffered charcoal yeast extract) and Legionella feeleii was isolated. The patient was treated with 6 weeks of moxifloxacin and her cutaneous lesions gradually resolved. Cutaneous Legionella infections are uncommon and usually affect immunocompromised patients.

Legionnaires' disease.

Since first identified in early 1977, bacteria of the genus Legionella are recognised as a common cause of community-acquired pneumonia and a rare cause of hospital-acquired pneumonia. Legionella bacteria multisystem manifestations mainly affect susceptible patients as a result of age, underlying debilitating conditions, or immunosuppression. Water is the major natural reservoir for Legionella, and the pathogen is found in many different natural and artificial aquatic environments such as cooling towers or water systems in buildings, including hospitals. The term given to the severe pneumonia and systemic infection caused by Legionella bacteria is Legionnaires' disease. Over time, the prevalence of legionellosis or Legionnaires' disease has risen, which might indicate a greater awareness and reporting of the disease. Advances in microbiology have led to a better understanding of the ecological niches and pathogenesis of the condition. Legionnaires' disease is not always suspected because of its non-specific symptoms, and the diagnostic tests routinely available do not offer the desired sensitivity. However, effective antibiotics are available. Disease notification systems provide the basis for initiating investigations and limiting the scale and recurrence of outbreaks. This report reviews our current understanding of this disease.

Caspase-1 but Not Caspase-11 Is Required for NLRC4-Mediated Pyroptosis and Restriction of Infection by Flagellated Legionella Species in Mouse Macrophages and In Vivo.

Gram-negative bacteria from the Legionella genus are intracellular pathogens that cause a severe form of pneumonia called Legionnaires' disease. The bacteria replicate intracellularly in macrophages, and the restriction of bacterial replication by these cells is critical for host resistance. The activation of the NAIP5/NLRC4 inflammasome, which is readily triggered in response to bacterial flagellin, is essential for the restriction of bacterial replication in murine macrophages. Once activated, this inflammasome induces pore formation and pyroptosis and facilitates the restriction of bacterial replication in macrophages. Because investigations related to the NLRC4-mediated restriction of Legionella replication were performed using mice double deficient for caspase-1 and caspase-11, we assessed the participation of caspase-1 and caspase-11 in the functions of the NLRC4 inflammasome and the restriction of Legionella replication in macrophages and in vivo. By using several species of Legionella and mice singly deficient for caspase-1 or caspase-11, we demonstrated that caspase-1 but not caspase-11 was required for pore formation, pyroptosis, and restriction of Legionella replication in macrophages and in vivo. By generating F1 mice in a mixed 129 × C57BL/6 background deficient (129 × Casp-11(-/-) ) or sufficient (129 × C57BL/6) for caspase-11 expression, we found that caspase-11 was dispensable for the restriction of Legionella pneumophila replication in macrophages and in vivo. Thus, although caspase-11 participates in flagellin-independent noncanonical activation of the NLRP3 inflammasome, it is dispensable for the activities of the NLRC4 inflammasome. In contrast, functional caspase-1 is necessary and sufficient to trigger flagellin/NLRC4-mediated restriction of Legionella spp. infection in macrophages and in vivo.

A gyrB oligonucleotide microarray for the specific detection of pathogenic Legionella and three Legionella pneumophila subsp.

Among the 50 species and 70 serogroups of Legionella identified, Legionella pneumophila, comprising three subsp. (subsp. pneumophila, subsp. fraseri, and subsp. pasculleii), is recognized as the major cause of epidemic legionellosis. Rapid and reliable assays to identify pathogenic Legionella spp., and the three L. pneumophila subsp. in particular, are in great demand. In this study, we analyzed the gyrB genes of eleven Legionella spp. and subsp., comprising L. anisa, L. bozemanii, L. dumoffii, L. feeleii, L. gormanii, L. longbeachae, L. micdadei, L. waltersii, L. pneumophila subsp. pneumophila, L. pneumophila subsp. fraseri, and L. pneumophila subsp. pasculleii. We developed a rapid oligonucleotide microarray detection technique to identify accurately these common pathogenic Legionella spp. and L. pneumophila subsp. To detect multiple Legionella species with high specificity, 31 reproducible probes were designed in the array. Sixty-one strains were analyzed in total, including 37 target pathogens and 24 non-target bacterial species used to validate the microarray. The sensitivity of the detection was 1.0 ng using genomic DNA of three Legionella spp., L. anisa, L. dumoffii, and L. waltersii, or 13 CFU/100 mL using the cultured L. pneumophila subsp. pneumophila. Eight isolated strains were tested using the microarray with 100% accuracy. The data indicated that the technique is an efficient method to diagnose and detect Legionella spp. and subsp. in basic microbiology, clinical diagnosis, epidemiological surveillance, and food safety applications. In addition, a phylogenetic study based on the gyrB gene revealed the genetic relationship among the different Legionella spp. and subsp.