Epidemiological Investigation of Legionella pneumophila Serogroup 2 to 14 Isolates from Water Samples by Amplified Fragment Length Polymorphism and Sequence-Based Typing and Detection of Virulence Traits

Abstract

The aim of this study is to explore the dispersion, clonality, and virulence of Legionella pneumophila serogroups 2 to 14 in the Greek environment. Eighty L. pneumophila serogroup 2 to 14 strains isolated from water distribution systems of hotels, hospitals, athletic venues, and ferries in Greece were tested by monoclonal antibodies (MAbs) for serogroup discrimination and molecularly by amplified fragment length polymorphism (AFLP) for genetic diversity. Fifty-six of 80 strains were also typed by the sequence-based typing (SBT) method. 〈ll strains were further analyzed for detection of two pathogenicity loci: Legionella vir homologue (lvh) and repeats in structural toxin (rtxA). Thirty-seven strains (46.2%) belonged to serogroup 6, 26 strains (32.5%) to serogroup 3, and 7 (8.8%) to other serogroups (4, 5, 8, and 10). Ten strains (12.5%) were nontypeable (NT) into the known serogroups. Thirty-nine different AFLP types were found among the 80 L. pneumophila serogroup 2 to 14 strains, and 24 different SBT types were found among the 56 strains tested. Among the 80 strains, the lvh locus was present in 75 (93.8%), the rtxA locus was found in 76 (95%), and both loci were found in 73 (91.3%) strains. This study showed that there is genetic variability of L. pneumophila serogroups 2 to 14 in the Greek environment as well as a high percentage of the pathogenicity loci. ⌱ntroducing an effective diagnostic test for L. pneumophila serogroups 2 to 14 in urine and promoting the examination of respiratory specimens from patients hospitalized for pneumonia in Greek hospitals are essential. IMPORTANCEIn this study, the dispersion, clonality, and virulence of environmental isolates of Legionella pneumophila serogroups 2 to 14 (Lp2-14) in Greece were investigated. Genetic variability of Lp2-14 in the Greek environment was identified together with the presence of the pathogenicity loci in a high percentage of the isolates. Despite the high prevalence of Lp2-14 in the Greek environment, no clinical cases were reported, which may be due to underdiagnosis of the disease. Almost all the legionellosis cases are diagnosed in Greece by using the urine antigen test, which is specific for Lp1. There is an urgent need to improve the clinical diagnosis of legionellosis by introducing an effective diagnostic test for Lp2-14 in urine and by promoting the PCR examination of respiratory specimens from patients with compatible clinical symptoms. L egionella species are inhabitants of water and biofilms in both natural and industrial aquatic environments (1). Legionella spp. can cause Legionnaires' disease and the flu-like Pontiac fever in humans, through inhalation of Legionella-contaminated water aerosols (2, 3). In Europe, according to the World Health Organization (WHO), the incidence rate was 10 to 15 cases per million population, and the total case-fatality rate (CFR) was about 12% (4, 5). In the last surveillance report from the European Centre for Disease Prevention and Control (ECDC), for the year 2014, the notification rate of Leg...

Prevalence of Infection-Competent Serogroup 6 Legionella pneumophila within Premise Plumbing in Southeast Michigan

“…2 ) and also present in hospital water systems in Poland ( 31 ). In contrast, a 2-year surveillance study of a larger territory in Greece identified 10 different STs among 28 SG6 L. pneumophila strains isolated ( 51 ). More detailed genetic and phenotypic analyses of SG6 ST367 and ST461 strains from Flint, Detroit, Ontario, and Spain ( 33 , 35 ) can evaluate whether this L. pneumophila lineage is either endemic to Southeast Michigan, more readily recovered from water samples, or more fit in the physiochemical conditions peculiar to the 2014–2015 Flint municipal water system.…”

“…In the water distribution systems of nine hospitals in Poland, SG6 strains comprised 33% of the 11 environmental isolates, whereas SG1 strains accounted for 24% ( 31 ). In the Attica region of Greece, SG6 strains represented 62% of 37 L. pneumophila environmental isolates ( 51 ). In contrast, SG6 strains comprised only 15% of the L. pneumophila strains collected in England and Wales ( 46 ) and 11% of those isolated in France ( 52 ).…”

“…In one hospital in Sweden, SG6 L. pneumophila isolates from three different patients and seven water samples were each of ST1392, as were eight of nine control clinical isolates from unrelated locations ( 53 ). In the Attica region of Greece, among 25 SG6 isolates, 28% were of ST68 and 20% were of ST461 ( 51 ), a type prevalent in Flint premise plumbing ( Fig. 2 ) and also present in hospital water systems in Poland ( 31 ).…”

Molecular Typing of Legionella pneumophila Isolates in the Province of Quebec from 2005 to 2015

“…The standardised SBT scheme for Lp presents an important advancement in the study of the molecular epidemiology of LD. It has been shown that the results obtained by different typing scheme for Legionella , such as PFGE and AFLP, are not necessarily concordant between them and also with SBT [ 13 , 14 ]. Implementation of SBT method has yielded comparable data worldwide and has been shown to be applicable in investigation of legionellosis outbreaks [ 1 ].The genetic variability of Lp has been assessed by SBT for countries such as the United-States, Canada, United-Kingdom, Germany, Spain, Belgium, Portugal, Italy, South Korea, China and Japan [ 3 , 19 – 21 , 43 – 49 ].…”

“…Pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) are two methods based on digestion of genomic DNA with restriction enzyme [ 9 ]. These two methods have been used both in outbreak investigation and for assessing genetic diversity of sporadic cases [ 10 – 14 ]. However, even though information provided is useful, their efficiency varies and most of them are not standardized [ 15 ].…”