Molecular typing of Legionella pneumophila serogroup 1 clinical strains isolated in Italy

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Abstract

Molecular typing methods for discriminating different bacterial isolates are essential epidemiological tools in prevention and control of Legionella infections and outbreaks. A selection of 56 out of 184 Legionella pneumophila serogroup 1 (Lp1) clinical isolates, collected from different Italian regions between 1987 and 2012, and stored at the National Reference Laboratory for Legionella, were typed by monoclonal antibody (MAb) subgrouping, amplified fragment length polymorphism (AFLP) and sequence based typing (SBT). These strains were isolated from 39 community (69.6%), 14 nosocomial (25%) and 3 travel associated (5.4%) Legionnaires’disease cases. MAb typing results showed a prevalence of MAb 3/1 positive isolates (75%) with the Philadelphia subgroup representing 35.7%, followed by Knoxville (23.2%), Benidorm (12.5%), Allentown/France (1.8%), Allentown/France-Philadelphia (1.8%). The remaining 25% were MAb 3/1 negative, namely 11 Olda (19.6%), 2 Oxford (3.6%) and 1 Bellingham (1.8%) subgroups.

AFLP analysis detected 20 different genomic profiles. SBT analysis revealed 32 different sequence types (STs) with high diversity of STs (IODSTs = 0.952) 12 of which were never described before. ST1 and ST23 were most frequently isolated as observed worldwide. A helpful analysis of data from SBT, MAb subgrouping and AFLP is provided, as well as a comparison to the Lp1 types investigated from other countries. This study describes the first Italian Lp1 strains database, providing molecular epidemiology data useful for future epidemiological investigations, especially of travel associated Legionnaires’ diseases (TALD) cases, Italy being the country associated with the highest number of clusters.

Introduction

Legionella pneumophila is a Gram-negative waterborne pathogen causing a severe form of pneumonia known as Legionnaires’ disease (LD), which is commonly acquired by inhalation or micro-aspiration of aerosol particles originating from contaminated man-made water systems (Carratalà and Garcia-Vidal, 2010). Sources of infection were indeed demonstrated to be hot water systems, cooling towers, spa pools, dental unit waterlines, etc. (Rota et al., 2005, Ricci et al., 2012, Lee and Lee, 2013). Lp includes 16 serogroups that are able to cause disease, but serogroup 1 is the most frequently isolated from LD patients. Lp1 can be further divided into several subgroups according to the expression of different epitopes on the lipopolysaccharide (Helbig et al., 1997). MAbs directed against these antigens have differentiated Lp1 strains in MAb 3/1 positive, associated to higher virulence, and MAb 3/1 negative (Helbig et al., 2002). For epidemiological investigations, AFLP genomic typing has been demonstrated to have a greater discriminatory power and to be efficient in establishing the clonal relatedness between clinical and environmental strains of Lp. However several cons were observed when using this method, including the difficulty in determining the fragment size and the consequent trouble in comparing genomic patterns among different laboratories and platforms (Fry et al., 2002).

Afterwards, a new typing scheme based on the partial sequence of 7 genes, named SBT, was developed by the EWGLI and a dedicated database was then created (Gaia et al., 2005, Scaturro et al., 2005, Ratzow et al., 2007). The SBT method is now considered the gold standard for Lp molecular typing and represents a useful tool for easy, rapid and efficient exchange of molecular epidemiological data (Rota et al., 2011).

Typing methods are applied not only for LD epidemiological investigations to determine the sources of infection, but also to investigate the distribution of Lp strains in specific areas (Borchardt et al., 2008, Chasqueira et al., 2009, Harrison et al., 2009, Vekens et al., 2012, Kozak-Muiznieks et al., 2013).

In Italy, notification of legionellosis to health authority has become mandatory since 1983. The Legionellosis National Register was established and managed by Istituto Superiore di Sanità (Italian Institute of Health, ISS). In addition to epidemiological data collection, the National Reference Laboratory for Legionella performs diagnosis confirmation and typing of the isolated strains. Since 2008 more than 1000 cases of legionellosis per year have been notified either as sporadic or clusters/outbreaks associated. Most cases have been diagnosed by detection of Lp urinary antigen, while the isolation of the pathogen occurred only in 2.3% of the cases (Rota et al., 2013a, Rota et al., 2013b).

From 1987 to 2012, 206 Legionella clinical strains were isolated and stored in our laboratory, among them 184 were Lp1. In this study 56 strains were analyzed by SBT after a preliminary selection that took into account molecular typing results, obtained from MAb and AFLP, as well as geographical distribution and year of occurrence. The aim of this investigation was to describe the molecular characteristics and distribution of Lp1 strains isolated in Italy. Results were then compared to those obtained in other countries (Borchardt et al., 2008, Den Boer et al., 2008, Chasqueira et al., 2009, Harrison et al., 2009, Kozak et al., 2009, Amemura-Maekawa et al., 2010, Vekens et al., 2012, Kozak-Muiznieks et al., 2013) and to the ones present in the EWGLI SBT database.

Section snippets

Legionella strains

The 56 clinical strains, isolated during the period 1987–2012, were from 10 out of 21 Italian regions and accompanied by epidemiological data obtained from patients’ medical history. These strains were selected from a collection of 184 Lp1 clinical strains. The selection criteria were the molecular patterns (MAb and AFLP), the year and the region of isolation, epidemiological data, so that the strains chosen were representative of the whole collection.

A LD case was considered as confirmed

Legionella strains

The 56 Lp1 strains were from 39 CA (69.6%), 14 NA (25%) and 3 (5.4%) TA cases. These strains, like the others present in the collection, were isolated in 10 Italian regions, with Lazio (n = 19, 34%) and Lombardia (n = 12, 21.5%) being the most represented. The remaining strains (44.5%) were isolated in regions prevalently of the North of Italy, except for 3 Lp1 isolates originated from the Campania region in Southern Italy (Fig. 1).

On the basis of epidemiological information, present in the Italian

Discussion

This is the first report exploring the molecular characteristics of Lp1 clinical strains that caused LD in Italy in the last 26 years. In this period, cases due to other serogroups and species also occurred, and overall 206 clinical strains were collected. This collection includes 184 Lp1 (89.32%), 5 Lp6 (2.43%), 3 Lp3 (1.45%), 3 Lp8 (1.45%), and few isolates of Lp4, Lp5, Lp7, Lp9 and Lp14 amounting to 4.35%. Two L. micdadei (0.97%), one L. longbeachae and one L. bozemanii (0.48%) were also

Acknowledgments

The authors are grateful to all those who contributed to the realization of this study (Microbiologists, clinicians, etc.) by sending biological samples and strains of Legionella and to Dr. M.Castellani-Pastoris who started and carefully collected Legionella isolates since the first cases of LD occurred in Italy. The authors would also like to thank Prof M.T. Montagna (University of Bari) for kindly supporting the PhD studies of Dr. Stefano Fontana. A particular thank to Dr. Massimo Mentasti

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