Dataset Information

Multiplication of Legionella pneumophila Sequence Types 1, 47, and 62 in Buffered Yeast Extract Broth and Biofilms Exposed to Flowing Tap Water at Temperatures of 38°C to 42°C.

ABSTRACT: Legionella pneumophila proliferates in freshwater environments at temperatures ranging from 25 to 45°C. To investigate the preference of different sequence types (ST) for a specific temperature range, growth of L. pneumophila serogroup 1 (SG1) ST1 (environmental strains), ST47, and ST62 (disease-associated strains) was measured in buffered yeast extract broth (BYEB) and biofilms grown on plasticized polyvinyl chloride in flowing heated drinking water originating from a groundwater supply. The optimum growth temperatures in BYEB were approximately 37°C (ST1), 39°C (ST47), and 41°C (ST62), with maximum growth temperatures of 42°C (ST1) and 43°C (ST47 and ST62). In the biofilm at 38°C, the ST47 and ST62 strains multiplied equally well compared to growth of the environmental ST1 strain and an indigenous L. pneumophila non-SG1 strain, all attaining a concentration of approximately 10⁷ CFU/cm^-2 Raising the temperature to 41°C did not impact these levels within 4 weeks, but the colony counts of all strains tested declined (at a specific decline rate of 0.14 to 0.41 day^-1) when the temperature was raised to 42°C. At this temperature, the concentration of Vermamoeba vermiformis in the biofilm, determined with quantitative PCR (qPCR), was about 2 log units lower than the concentration at 38°C. In columns operated at a constant temperature, ranging from 38 to 41°C, none of the tested strains multiplied in the biofilm at 41°C, in which also V. vermiformis was not detected. These observations suggest that strains of ST47 and ST62 did not multiply in the biofilm at a temperature of ≥41°C because of the absence of a thermotolerant host.

Importance

Growth of Legionella pneumophila in tap water installations is a serious public health concern. The organism includes more than 2,100 varieties (sequence types). More than 50% of the reported cases of Legionnaires' disease are caused by a few sequence types which are very rarely detected in the environment. Strains of selected virulent sequence types proliferated in biofilms on surfaces exposed to warm (38°C) tap water to the same level as environmental varieties and multiplied well as pure culture in a nutrient-rich medium at temperatures of 42 and 43°C. However, these organisms did not grow in the biofilms at temperatures of ≥41°C. Typical host amoebae also did not multiply at these temperatures. Apparently, proliferation of thermotolerant host amoebae is needed to enable multiplication of the virulent L. pneumophila strains in the environment at elevated temperatures. The detection of these amoebae in water installations therefore is a scientific challenge with practical implications.

SUBMITTER: van der Kooij D

PROVIDER: S-EPMC5086563 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Legionella pneumophila in potable water installations poses a potential health risk, but quantitative information about its replication in biofilms in relation to water quality is scarce. Therefore, biofilm formation on the surfaces of glass and chlorinated polyvinyl chloride (CPVC) in contact with tap water at 34 to 39°C was investigated under controlled hydraulic conditions in a model system inoculated with biofilm-grown L. pneumophila The biofilm on glass (average steady-state concentration, 23 ± 9 pg ATP cm-2) exposed to treated aerobic groundwater (0.3 mg C liter-1; 1 ?g assimilable organic carbon [AOC] liter-1) did not support growth of the organism, which also disappeared from the biofilm on CPVC (49 ± 9 pg ATP cm-2) after initial growth. L. pneumophila attained a level of 4.3 log CFU cm-2 in the biofilms on glass (1,055 ± 225 pg ATP cm-2) and CPVC (2,755 ± 460 pg ATP cm-2) exposed to treated anaerobic groundwater (7.9 mg C liter-1; 10 ?g AOC liter-1). An elevated biofilm concentration and growth of L. pneumophila were also observed with tap water from the laboratory. The Betaproteobacteria Piscinibacter and Methyloversatilis and amoeba-resisting Alphaproteobacteria predominated in the clones and isolates retrieved from the biofilms. In the biofilms, the Legionella colony count correlated significantly with the total cell count (TCC), heterotrophic plate count, ATP concentration, and presence of Vermamoeba vermiformis This amoeba was rarely detected at biofilm concentrations of <100 pg ATP cm-2 A threshold concentration of approximately 50 pg ATP cm-2 (TCC = 1 × 106 to 2 × 106 cells cm-2) was derived for growth of L. pneumophila in biofilms.IMPORTANCELegionella pneumophila is the etiologic agent in more than 10,000 cases of Legionnaires' disease that are reported annually worldwide and in most of the drinking water-associated disease outbreaks reported in the United States. The organism proliferates in biofilms on surfaces exposed to warm water in engineered freshwater installations. An investigation with a test system supplied with different types of warm drinking water without disinfectant under controlled hydraulic conditions showed that treated aerobic groundwater (0.3 mg liter-1 of organic carbon) induced a low biofilm concentration that supported no or very limited growth of L. pneumophila Elevated biofilm concentrations and L. pneumophila colony counts were observed on surfaces exposed to two types of extensively treated groundwater, containing 1.8 and 7.9 mg C liter-1 and complying with the microbial water quality criteria during distribution. Control measures in warm tap water installations are therefore essential for preventing growth of L. pneumophila.

Project description:Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoan, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known virulence determinant used by L. pneumophila to infect host cells is a Type IVb translocation system named Icm/Dot, which is used to modify the host cell functions to the benefit of the bacteria. To date the Icm/Dot systeme is known to translocate more than 100 effectors. While the transcriptional response of Legionella to the intracellular environement of A. castelannii as already been investigated, much less is known of how Legionella reacts transcriptionnally inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth and during infection of human cultured macrophages by using microarray and a RNA amplification procedure called SCOTS to allow for the study of conditions of low bacterial loads. Among the genes induced intracellularly are those involved in amino acid synthesis pathway leading to L-arginine, L-histidne and L-proline as well as many transport system involved in amino acid and iron uptake. The Icm/Dot systems is not differentially expressed inside cells compare to the E phase control but the effectors are strongly induced. The intracellular transcriptome was further used to identify putative new Icm/Dot effectors and translocation was show to occur for 3 of them. This study provides a comprehensive view of how L. pneumophila react to the human macrophages intracellular environment. The aim of this work was to study the gene expression profile of Legionella during infection of macrophages infected at a low multiplicity of infection (MOI). SCOTS is a method that allows amplification of small amounts of bacterial RNA from infected host cells, while discarding host cell transcripts and ribosomal RNA. Before infection, macrophages derived from the THP-1 monocyte cell line were pre-treated with antibodies against the L. pneumophila major outer membrane protein, which increases the efficiency of bacterial entry into host cells . After 2 hours of infection, the macrophages were washed and treated with gentamicin for 1 hour, to synchronize the infection and kill extracellular bacteria, and cells were washed 3 times and fresh medium was added. Samples for the first time point (T0) were collected after the gentamicin treatment. Samples were also collected after 6h (T6) and 18h (T18). Samples from all conditions, including growth in AYE broth to exponential (E) or post exponential (PE) phase, were treated with three consecutive rounds of SCOTS and the resulting cDNA was labeled and hybridized to the microarray slides. As a reference channel, labeled gDNA was also hybridized. For each condition studied, three independent biological replicates and two technical replicates were analyzed, resulting in six replicates for each condition. The data was background subtracted and normalized by calculating the contribution of each spot to the total intensity and the ratio to gDNA was recorded. A one tailed student's T test was used for statistical analysis and the ratio between test conditions (PE, T0, T6 and T18) and the control conditions (E phase or T0) was calculated.

Project description:ObjectiveThe diagnostic accuracy of cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) must be improved before widespread clinical use. This study aimed to determine whether CSF A?42/A?40 and A?42/A?38 ratios are better diagnostic biomarkers of AD during both predementia and dementia stages in comparison to CSF A?42 alone.MethodsThe study comprised three different cohorts (n = 1182) in whom CSF levels of A?42, A?40, and A?38 were assessed. CSF A?s were quantified using three different immunoassays (Euroimmun, Meso Scale Discovery, Quanterix). As reference standard, we used either amyloid ((18)F-flutemetamol) positron emission tomography (PET) imaging (n = 215) or clinical diagnosis (n = 967) of well-characterized patients.ResultsWhen using three different immunoassays in cases with subjective cognitive decline and mild cognitive impairment, the CSF A?42/A?40 and A?42/A?38 ratios were significantly better predictors of abnormal amyloid PET than CSF A?42. Lower A?42, A?42/A?40, and A?42/A?38 ratios, but not A?40 and A?38, correlated with smaller hippocampal volumes measured by magnetic resonance imaging. However, lower A?38, A?40, and A?42, but not the ratios, correlated with non-AD-specific subcortical changes, that is, larger lateral ventricles and white matter lesions. Further, the A?42/A?40 and A?42/A?38 ratios showed increased accuracy compared to A?42 when distinguishing AD from dementia with Lewy bodies or Parkinson's disease dementia and subcortical vascular dementia, where all A?s (including A?42) were decreased.InterpretationThe CSF A?42/A?40 and A?42/A?38 ratios are significantly better than CSF A?42 to detect brain amyloid deposition in prodromal AD and to differentiate AD dementia from non-AD dementias. The ratios reflect AD-type pathology better, whereas decline in CSF A?42 is also associated with non-AD subcortical pathologies. These findings strongly suggest that the ratios rather than CSF A?42 should be used in the clinical work-up of AD.

Dataset Information

Multiplication of Legionella pneumophila Sequence Types 1, 47, and 62 in Buffered Yeast Extract Broth and Biofilms Exposed to Flowing Tap Water at Temperatures of 38°C to 42°C.

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