Legionella feeleii: Ubiquitous Pathogen in the Environment and Causative Agent of Pneumonia.

L. feeleii is one of the most frequent Legionella species isolated from natural pools of the central region of Spain. This study aimed to evaluate its ecology and to identify this Legionella species as a respiratory pathogen. A PCR assay for detecting the L. feeleii mip gene was developed to identify it in clinical and environmental samples. Culture and PCR were performed in environmental samples from four drinking water treatment plants (DWTPs). Free L. feeleii was only detected in raw water samples (3.4%), while L. feeleii as an Acanthamoeba endosymbiont was found in 30.7% of raw water, 11.5% of decanter biofilm, and 32% of finished water samples. Therefore, Acanthamoeba spp. plays an essential role in the multiplication, persistence, and spread of Legionella species in the environment. The first case of Legionnaires' disease caused by L. feeleii in Spain is described in this study. The case was diagnosed in an older woman through PCR and sequencing from urine and sputum samples. A respiratory infection could be linked with health care procedures, and the patient presented several risk factors (age, insulin-dependent diabetes, and heart disease). The detection of non-L. pneumophila, such as L. feeleii, is a factor that must be considered when establishing or reviewing measures for the control and prevention of legionellosis.

Active and adaptive Legionella CRISPR-Cas reveals a recurrent challenge to the pathogen.

Clustered regularly interspaced short palindromic repeats with CRISPR-associated gene (CRISPR-Cas) systems are widely recognized as critical genome defense systems that protect microbes from external threats such as bacteriophage infection. Several isolates of the intracellular pathogen Legionella pneumophila possess multiple CRISPR-Cas systems (type I-C, type I-F and type II-B), yet the targets of these systems remain unknown. With the recent observation that at least one of these systems (II-B) plays a non-canonical role in supporting intracellular replication, the possibility remained that these systems are vestigial genome defense systems co-opted for other purposes. Our data indicate that this is not the case. Using an established plasmid transformation assay, we demonstrate that type I-C, I-F and II-B CRISPR-Cas provide protection against spacer targets. We observe efficient laboratory acquisition of new spacers under 'priming' conditions, in which initially incomplete target elimination leads to the generation of new spacers and ultimate loss of the invasive DNA. Critically, we identify the first known target of L. pneumophila CRISPR-Cas: a 30 kb episome of unknown function whose interbacterial transfer is guarded against by CRISPR-Cas. We provide evidence that the element can subvert CRISPR-Cas by mutating its targeted sequences - but that primed spacer acquisition may limit this mechanism of escape. Rather than generally impinging on bacterial fitness, this element drives a host specialization event - with improved fitness in Acanthamoeba but a reduced ability to replicate in other hosts and conditions. These observations add to a growing body of evidence that host range restriction can serve as an existential threat to L. pneumophila in the wild.

Virulence factor rtx in Legionella pneumophila, evidence suggesting it is a modular multifunctional protein.

BACKGROUND: The repeats in toxin (Rtx) are an important pathogenicity factor involved in host cells invasion of Legionella pneumophila and other pathogenic bacteria. Its role in escaping the host immune system and cytotoxic activity is well known. Its repeated motives and modularity make Rtx a multifunctional factor in pathogenicity. RESULTS: The comparative analysis of rtx gene among 6 strains of L. pneumophila showed modularity in their structures. Among compared genomes, the N-terminal region of the protein presents highly dissimilar repeats with functionally similar domains. On the contrary, the C-terminal region is maintained with a fashionable modular configuration, which gives support to its proposed role in adhesion and pore formation. Despite the variability of rtx among the considered strains, the flanking genes are maintained in synteny and similarity. CONCLUSION: In contrast to the extracellular bacteria Vibrio cholerae, in which the rtx gene is highly conserved and flanking genes have lost synteny and similarity, the gene region coding for the Rtx toxin in the intracellular pathogen L. pneumophila shows a rapid evolution. Changes in the rtx could play a role in pathogenicity. The interplay of the Rtx toxin with host membranes might lead to the evolution of new variants that are able to escape host cell defences.

[Community outbreak of pneumonia due to Legionella pneumophila: importance of monitoring hospital cooling towers]. / Brote comunitario de neumonía por Legionella pneumophila: importancia del control de las torres de refrigeración en los centros sanitarios.

OBJECTIVE: Description of an outbreak of legionnaires' disease originating in one of the cooling towers of a hospital. PATIENTS AND METHODS: This study included patients with confirmed pneumonia caused by Legionella pneumophila serogroup 1 and related to the Vallcarca neighborhood of Barcelona (Spain) in August 2004. Exposure was determined by a standardized questionnaire. An environmental investigation was carried out to identify the source of the outbreak. A descriptive analysis including incidence rates estimation was performed, as well as molecular study to document the genetic identity among human and environmental strains. RESULTS: Thirty-three cases of L. pneumophila pneumonia were detected. Median age was 68 years and 70% of the affected patients were men. Incidence rate among residents in less than 200 meters of the source and older than 65 was 888.9 cases/100,000 inhabitants. Lethality rate was 6%. Four seasonal cooling towers that were not registered with the authorities were identified in a health care center. L. pneumophila was isolated from all four and at least one colony in each tower had the same genetic profile as the strains isolated from patients. CONCLUSIONS: An association was demonstrated between a community outbreak of legionellosis and unregistered seasonal cooling towers located in a hospital. All risk facilities should be registered and inspected to ensure that they fulfill current legislation requirements.

Brote comunitario de neumonía por Legionella pneumophila: importancia del control de las torres de refrigeración en los centros sanitarios / Outbreak of legionnaires’ disease linked to hospital cooling towers

Objetivo. Describir la investigación de un brote comunitario de legionelosis originado en unas torres de refrigeración de un hospital. Pacientes y métodos. Pacientes diagnosticados de neumonía por Legionella pneumophila serogrupo 1 (L. pneumophila) asociados con el barrio de Vallcarca (Barcelona) en agosto de 2004. La exposición se determinó mediante una encuesta estandarizada. Se llevó a cabo una investigación ambiental para identificar el foco emisor. Se realizó un análisis descriptivo con cálculo de tasas de incidencia, así como el estudio molecular para documentar la identidad genética entre las cepas humanas y ambientales aisladas. Resultados. Se detectaron 33 casos de neumonía por L. pneumophila. La edad media fue de 68 años y el 70% de los afectados eran varones. La tasa de incidencia en los mayores de 65 años residentes a una distancia menor o igual a 200 m del foco emisor fue de 888,9 casos/100.000 habitantes. La tasa de letalidad fue del 6%. Se identificaron cuatro torres de refrigeración estacionales no censadas ubicadas en un centro sanitario. En todas se aisló L. pneumophila y al menos una colonia de cada instalación compartía perfil genético con las cepas aisladas en los pacientes. Conclusiones. Se muestra la asociación de un brote comunitario de neumonía por Legionella y las torres de refrigeración de un centro sanitario que no estaban censadas. Se remarca la necesidad de notificar cualquier instalación de riesgo y de realizar un seguimiento para asegurar que cumplen con la legislación (AU)

Objective. Description of an outbreak of legionnaires’ disease originating in one of the cooling towers of a hospital. Patients and methods. This study included patients with confirmed pneumonia caused by Legionella pneumophila serogroup 1 and related to the Vallcarca neighborhood of Barcelona (Spain) in August 2004. Exposure was determined by a standardized questionnaire. An environmental investigation was carried out to identify the source of the outbreak. A descriptive analysis including incidence rates estimation was performed, as well as molecular study to document the genetic identity among human and environmental strains. Results. Thirty-three cases of L. pneumophila pneumonia were detected. Median age was 68 years and 70% of the affected patients were men. Incidence rate among residents in less than 200 meters of the source and older than 65 was 888.9 cases/100,000 inhabitants. Lethality rate was 6%. Four seasonal cooling towers that were not registered with the authorities were identified in a health care center. L. pneumophila was isolated from all four and at least one colony in each tower had the same genetic profile as the strains isolated from patients. Conclusions. An association was demonstrated between a community outbreak of legionellosis and unregistered seasonal cooling towers located in a hospital. All risk facilities should be registered and inspected to ensure that they fulfill current legislation requirements (AU)

Acanthamoeba polyphaga resuscitates viable non-culturable Legionella pneumophila after disinfection.

Amoebae are the natural hosts for Legionella pneumophila and play essential roles in bacterial ecology and infectivity to humans. When L. pneumophila colonizes an aquatic installation, it can persist for years despite repeated treatments with disinfectants. We hypothesized that freshwater amoebae play an important role in bacterial resistance to disinfectants, and in subsequent resuscitation of viable non-culturable (VNC) L. pneumophila that results in re-emergence of the disease-causing strain in the disinfected water source. Our work showed that in the absence of Acanthamoeba polyphaga, seven L. pneumophila strains became non-culturable after treatment by 256 p.p.m. of sodium hypochlorite (NaOCl). In contrast, intracellular L. pneumophila within A. polyphaga was resistant to 1024 p.p.m. of NaOCl. In addition, L. pneumophila-infected A. polyphaga exhibited increased resistance to NaOCl. When chlorine-sterilized water samples were co-cultured with A. polyphaga, the non-culturable L. pneumophila were resuscitated and proliferated robustly within A. polyphaga. Upon treatment by NaOCl, uninfected amoebae differentiated into cysts within 48 h. In contrast, L. pneumophila-infected A. polyphaga failed to differentiate into cysts, and L. pneumophila was never detected in cysts of A. polyphaga. We conclude that amoebic trophozoites protect intracellular L. pneumophila from eradication by NaOCl, and play an essential role in resuscitation of VNC L. pneumophila in NaOCl-disinfected water sources. Intracellular L. pneumophila within trophozoites of A. polyphaga block encystation of the amoebae, and the resistance of both organisms to NaOCl is enhanced. To ensure long-term eradication and complete loss of the VNC state of L. pneumophila, we recommend that Legionella-protozoa co-culture should be an important tool to ensure complete loss of the VNC state of L. pneumophila.

Legionnaires' disease outbreak in Murcia, Spain.

An explosive outbreak of Legionnaires' disease occurred in Murcia, Spain, in July 2001. More than 800 suspected cases were reported; 449 these cases were confirmed, which made this the world's largest outbreak of the disease reported to date. Dates of onset for confirmed cases ranged from June 26 to July 19, with a case-fatality rate of 1%. The epidemic curve and geographic pattern from the 600 competed epidemiologic questionnaires indicated an outdoor point-source exposure in the northern part of the city. A case-control study matching 85 patients living outside the city of Murcia with two controls each was undertaken to identify to outbreak source; the epidemiologic investigation implicated the cooling towers at a city hospital. An environmental isolate from these towers with an identical molecular pattern as the clinical isolates was subsequently identified and supported that epidemiologic conclusion.

El laboratorio de microbiología clínica en el brote de Legionella SPP, en la comarca de Alcoy: rentabilidad de las diferentes técnicas diagnósticas / The role of the clinical microbiology laboratory during the outbreak of Legionella spp., in the municipality of Alcoy: the effectiveness of different diagnosis methods

FUNDAMENTO. La rentabilidad de diferentes técnicas de laboratorio en el diagnóstico de Legionella spp. en muestras clínicas varía según el contexto epidemiológico. En este trabajo se evalúa la utilidad de los métodos de laboratorio en un brote aparecido en la comarca de Alcoy (Alicante). MATERIAL Y MÉTODOS. Se estudian 222 casos comunitarios de infección por Legionella pneumophila serogrupo I, subtipo Pontiac-Knoxville, genotipos I y II, diagnosticados por el laboratorio de Microbiología, en el período comprendido entre enero de 1999 y diciembre del año 2000, correspondientes a pacientes residentes en la comarca de Alcoy (Alicante). Las técnicas empleadas han sido la detección directa de antígeno en muestras respiratorias por inmunofluorescencia, la detección directa de antígeno en orina, el cultivo y la serología. RESULTADOS. El antígeno en orina permitió diagnosticar 201 casos (90,5 por ciento). La inmunofluorescencia directa dio una alta tasa de resultados falsamente positivos (n=24). El cultivo fue esencial para confirmar la etiología del brote (25 cepas de esputos de 22 enfermos). La serología complementó al resto de técnicas y ayudó a diagnosticar retrospectivamente a 21 enfermos (9 por ciento) en los que el resto de pruebas no se solicitaron o resultaron negativas. CONCLUSIONES. El diagnóstico rápido es esencial para la evaluación de los pacientes y para el control de los brotes epidémicos, a lo que ayuda en gran medida la detección de antígeno urinario, pero debe ser complementado por otras técnicas. El cultivo de las muestras respiratorias y posterior tipado de las cepas permite establecer de forma certera la etiología y ayuda a precisar la/s fuente/s de la infección. La serología complementó el diagnóstico en el 9 por ciento de casos (AU)

A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results of a pan-European study.

OBJECTIVES: To compare genotypic methods for epidemiologic typing of Legionella pneumophila serogroup (sg) 1, in order to determine the best available method within Europe for implementation and standardization by members of the European Working Group on Legionella Infections. METHODS: Coded isolates (114) of L. pneumophila sg 1 comprising one epidemiologically 'unrelated' (79) and one 'related' panel of isolates (35) were sent to 12 laboratories in 11 European countries. Analysis was undertaken in each laboratory using one or more of the following methods: ribotyping, restriction fragment length polymorphism analysis, restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), PCR using arbitrary/repeat sequence primers (AP-, AP/rep-PCR), and amplified fragment length polymorphism (AFLP) analysis. Results were analyzed visually or using gel analysis software. Each method was assessed for its: index of discrimination (D), epidemiologic concordance (E), speed of application and ease of use. In addition, phenotypic analysis was performed in two laboratories using monoclonal antibodies (mAbs). RESULTS: The D of each of the genotypic methods ranged from 0.840 for ribotyping to 0.990 for PFGE using Sfil: E ranged from 0.06 for AP- and AP/rep-PCR to 1.00 for ribotyping using Pstl/EcoRI and AFLP: in general, E was inversely related to D. Although offering only limited discrimination (D=0.838), mAb typing was both rapid and highly epidemiologically concordant (E=1.00). CONCLUSIONS: Two methods, PFGE using Sfil and AFLP, were selected for further study. AFLP is rapid and highly epidemiologically concordant (E=1.00), but is not highly discriminatory. This method will be developed as a rapid screening tool. PFGE using Sfil is highly discriminatory but, in the present study, yielded low values of E (0.12-0.71). Attempts will be made to rigorously standardize this method for use as the reference method. Primary screening of isolates by mAb subgrouping is recommended.