Molecular typing of Legionella pneumophila isolates from environmental water samples and clinical samples using a five-gene sequence typing and standard Sequence-Based Typing.

Xiao-Yong Zhan,Qing-Yi Zhu,Annamaria Bevivino

doi:10.1371/journal.pone.0190986

Abstract

Inadequate discriminatory power to distinguish between L. pneumophila isolates, especially those belonging to disease-related prevalent sequence types (STs) such as ST1, ST36 and ST47, is an issue of SBT scheme. In this study, we developed a multilocus sequence typing (MLST) scheme based on two non-virulence loci (trpA, cca) and three virulence loci (icmK, lspE, lssD), to genotype 110 L. pneumophila isolates from various natural and artificial water sources in Guangdong province of China, and compared with the SBT. The isolates were assigned to 33 STs of the SBT and 91 new sequence types (nSTs) of the MLST. The indices of discrimination (IODs) of SBT and MLST were 0.920 and 0.985, respectively. Maximum likelihood trees of the concatenated SBT and MLST sequences both showed distinct phylogenetic relationships between the isolates from the two environments. More intragenic recombinations were detected in nSTs than in STs, and they were both more abundant in natural water isolates. We found out the MLST had a high discriminatory ability for the disease-associated ST1 isolates: 22 ST1 isolates were assigned to 19 nSTs. Furthermore, we assayed the discrimination of the MLST for 29 reference strains (19 clinical and 10 environmental). The clinical strains were assigned to eight STs and ten nSTs. The MLST could also subtype the prevalent clinical ST36 or ST47 strains: eight ST36 strains were subtyped into three nSTs and two ST47 strains were subtyped into two nSTs. We found different distribution patterns of nSTs between the environmental and clinical ST36 isolates, and between the outbreak clinical ST36 isolates and the sporadic clinical ST36 isolates. These results together revealed the MLST scheme could be used as part of a typing scheme that increased discrimination when necessary.

Highlights

Legionella pneumophila (L. pneumophila) is a gram-negative bacterium worldwide in rivers and lakes as well as in many artificial water systems [1]
NST98 (C7_O) was situated on its own distinct branch, separated from other four new sequence types (nSTs) (S1 Fig). These results suggested that the multilocus sequence typing (MLST) scheme could subtype the prevalent ST36 isolates, and the phylogenetic relationships among ST36 isolates from clinical and environmental sources might be different, which was supported by Mercante and colleague [51]
We reported a five-gene MLST scheme for genotyping of L. pneumophila isolates from environmental water samples and clinical samples, and compared with the Sequence-Based Typing (SBT)

Summary

Introduction

Legionella pneumophila (L. pneumophila) is a gram-negative bacterium worldwide in rivers and lakes as well as in many artificial water systems [1]. Several molecular typing schemes have been used to investigate L. pneumophila epidemiology These schemes included amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) and Sequence-Based Typing (SBT). The SBT, a scheme analogous to multilocus sequence typing (MLST), was proposed by the European Working Group for Legionella Infections (EWGLI, now is the ESCMID Study Group for Legionella Infections, ESGLI) It is an essentially seven-locus sequence typing method performed by sequencing and comparing seven loci (flaA, pilE, asd, mip, mompS, proA, and neuA), and appears to be a powerful tool for global epidemiology [9, 10]. Research and improvement of molecular typing methods for L. pneumophila are desirable

Objectives

Methods

Results

Conclusion