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Rapid culture-based detection of Legionella pneumophila using isothermal microcalorimetry with an improved evaluation method.

ABSTRACT: The detection and quantification of Legionella pneumophila (responsible for legionnaire's disease) in water samples can be achieved by various methods. However, the culture-based ISO 11731:2017, which is based on counts of colony-forming units per ml (CFU·ml-1 ) is still the gold standard for quantification of Legionella species (spp.). As a powerful alternative, we propose real-time monitoring of the growth of L. pneumophila using an isothermal microcalorimeter (IMC). Our results demonstrate that, depending on the initial concentration of L. pneumophila, detection times of 24-48 h can be reliably achieved. IMC may, therefore, be used as an early warning system for L. pneumophila contamination. By replacing only visual detection of growth by a thermal sensor, but otherwise maintaining the standardized protocol of the ISO 11731:2017, the new procedure could easily be incorporated into existing standards. The exact determination of the beginning of metabolic heat is often very difficult because at the beginning of the calorimetric signal the thermal stabilization and the metabolic heat development overlap. Here, we propose a new data evaluation based on the first derivation of the heat flow signal. The improved evaluation method can further reduce detection times and significantly increase the reliability of the IMC approach.

SUBMITTER: Fricke C

PROVIDER: S-EPMC7264898 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Rapid culture-based detection of Legionella pneumophila using isothermal microcalorimetry with an improved evaluation method.

Fricke Christian C Xu Juan J Jiang Feng-Lei FL Liu Yi Y Harms Hauke H Maskow Thomas T

Microbial biotechnology 20200325 4

The detection and quantification of Legionella pneumophila (responsible for legionnaire's disease) in water samples can be achieved by various methods. However, the culture-based ISO 11731:2017, which is based on counts of colony-forming units per ml (CFU·ml<sup>-1</sup> ) is still the gold standard for quantification of Legionella species (spp.). As a powerful alternative, we propose real-time monitoring of the growth of L. pneumophila using an isothermal microcalorimeter (IMC). Our results dem ...[more]

PMID: 32212253

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Project description:Legionella pneumophila is a gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to test the hypothesis that sRNAs play a similar role in L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the L. pneumophila genome. A 50-mer probe microarray was constructed to test the expression of predicted sRNAs in bacteria grown under a variety of conditions. This strategy successfully identified 22 expressed RNAs, out of which six were confirmed by northern blot and RACE. One of the identified sRNAs is highly expressed when the bacteria enter post exponential phase and computational prediction of its secondary structure reveals a striking similarity to the structure of 6S RNA, a widely distributed prokaryotic sRNA, known to regulate the activity of Ï70-containing RNAP. A 70-mer probe microarray was used to identify genes affected by L. pneumophila 6S RNA in stationary phase. The 6S RNA encoded by the ssrS gene positively regulates expression of genes encoding type IVB secretion system effectors, stress response genes such as groES and recA as well as many genes with unknown or hypothetical functions. Deletion of 6S RNA significantly reduced L. pneumophila intracellular multiplication in both protist and mammalian host cells, but had no detectable effect on growth in rich media. A microarray was designed for the detection of 101 predicted sRNAs by using OligiWiz version 2.1.3 (Nielsen et al., 2003, Wernersson & Nielsen, 2005). The prokaryotic setting was used to design 50-mers probes located, when possible, at the 3â end of the predicted genes. Probes for 10 negative controls, representing 10 genes of the L. pneumophila Paris plasmid, were also designed. Probes (Illumina) were dissolved in 50% DMSO to a final concentration of 30 uM and printed in triplicate on UltraGAPS coated glass slides (Corning) using a PerkinElmer SpotArray microarray printer. Fifteen micrograms of total RNA was labelled during cDNA synthesis using Superscript II reverse transcriptase (Invitrogen) and amino-allyl dUTP (Sigma). Bacterial genomic DNA was used as the reference channel on each slide to allow comparison of each time point and different samples (Talaat et al., 2002). Five micrograms of genomic DNA (gDNA) was labelled with amino-allyl dUTP by using Klenow fragment and random primers (Invitrogen) at 37 Â°C for 18 h. DNA was subsequently coupled to the succinimidyl ester fluorescent dye AlexaFluor 546 (for cDNA) or Alexa Fluor 647 (for gDNA) (Invitrogen) following manufacturerâs protocols. Hybridization and data acquisition were performed as previously described (Hovel-Miner et al., 2009).Very low density array, like the sRNA microarray used here, cannot be normalised with common procedure like total intensity or lowess. Local background was removed from spot signal intensity and the noise signal was estimated by recording the average signal intensity of 10 negative controls printed on the chip. Normalisation of signal intensity was carried out by calculating the fold increase over the noise signal value. Correlation of replicates using this normalisation procedure was â¥ 0.95.

Dataset Information

Rapid culture-based detection of Legionella pneumophila using isothermal microcalorimetry with an improved evaluation method.

Publications

Rapid culture-based detection of Legionella pneumophila using isothermal microcalorimetry with an improved evaluation method.

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