Serotyping and detection of pathogenecity loci of environmental isolates of Legionella pneumophila using MALDI-TOF MS

Abstract

Background: The majority of Legionnaires' disease cases is attributed to Legionella pneumophila serogroup 1 (Lp1). Moreover, pathogenicity loci lvh and rtxA were associated with the ability of Lp strains to cause the disease. Consequently, except from serogroup assignment the detection of the aforementioned virulence genes during Legionella detection in water samples, could help environmental risk assessment and the implementation of targeted control measures.

Aim: To establish and validate a rapid and robust MALDI-TOF MS-based method for the assignment of Lp isolates to serogroup, and identify distinct peak biomarkers for the detection of lvh and rtxA loci during environmental investigations.

Method: Fifteen reference strains and 150 Lp environmental isolates (70 Lp1 and 80 Lp2-15 strains) were used. All strains were PCR-tested for the presence of lvh and rtxA loci. Independent training and validation strain sets were constituted and all strains were protein-extracted and submitted to MALDI-TOF MS analysis. The raw spectra of the training set strains obtained, were introduced into the Mass-Up software platform for biomarker detection, for both serogroup assignment and pathogenicity loci detection. Validation of the assigned biomarkers followed using the validation set strains.

Results: For serogroup assignment, the Mass-up analysis indicated five potential discriminating peaks and correctly classified 115 out of 132 validation set strains, displaying sensitivity of 87.5%, specificity of 86.7% and 87.1% accuracy. Concerning the pathogenicity loci detection, the Mass-up analysis indicated two ion peaks for rtxA locus discrimination and one peak for lvh locus discrimination. Concerning the lvh virulence gene, the algorithm correctly classified 113 out of 137 positive and all negative strains 14 in total-showing sensitivity of 82.5%, specificity of 100.% and 84.1% accuracy. For rtxA locus, 134 out of 134 positive and 14 out of 17 negative strains were correctly classified with sensitivity of 100%, specificity of 76.5% and 97.4% accuracy.

Conclusion: MALDI-TOF MS displayed good performance for Lp serogroup assignment and detection of the lvh and rtxA virulence genes. These findings could contribute to the rapid, inexpensive and comprehensive case investigation and risk assessment. Further studies are needed to standardize and evaluate the method using the direct target plate protein profiling instead of protein extraction in order to simplify the protocol. .

Keywords: Legionella pneumophila; Legionella typing; MALDI-TOF MS; Pathogenicity loci; Serogroup.