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      Presence and Persistence of Viable, Clinically Relevant Legionella pneumophila Bacteria in Garden Soil in the Netherlands

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          ABSTRACT

          Garden soils were investigated as reservoirs and potential sources of pathogenic Legionella bacteria. Legionella bacteria were detected in 22 of 177 garden soil samples (12%) by amoebal coculture. Of these 22 Legionella-positive soil samples, seven contained Legionella pneumophila. Several other species were found, including the pathogenic Legionella longbeachae (4 gardens) and Legionella sainthelensi (9 gardens). The L. pneumophila isolates comprised 15 different sequence types (STs), and eight of these STs were previously isolated from patients according to the European Working Group for Legionella Infections (EWGLI) database. Six gardens that were found to be positive for L. pneumophila were resampled after several months, and in three gardens, L. pneumophila was again isolated. One of these gardens was resampled four times throughout the year and was found to be positive for L. pneumophila on all occasions.

          IMPORTANCE Tracking the source of infection for sporadic cases of Legionnaires' disease (LD) has proven to be hard. L. pneumophila ST47, the sequence type that is most frequently isolated from LD patients in the Netherlands, is rarely found in potential environmental sources. As L. pneumophila ST47 was previously isolated from a garden soil sample during an outbreak investigation, garden soils were investigated as reservoirs and potential sources of pathogenic Legionella bacteria. The detection of viable, clinically relevant Legionella strains indicates that garden soil is a potential source of Legionella bacteria, and future research should assess the public health implication of the presence of L. pneumophila in garden soil.

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          Most cited references40

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          Legionella and Legionnaires' disease: 25 years of investigation.

          There is still a low level of clinical awareness regarding Legionnaires' disease 25 years after it was first detected. The causative agents, legionellae, are freshwater bacteria with a fascinating ecology. These bacteria are intracellular pathogens of freshwater protozoa and utilize a similar mechanism to infect human phagocytic cells. There have been major advances in delineating the pathogenesis of legionellae through the identification of genes which allow the organism to bypass the endocytic pathways of both protozoan and human cells. Other bacteria that may share this novel infectious process are Coxiella burnetti and Brucella spp. More than 40 species and numerous serogroups of legionellae have been identified. Most diagnostic tests are directed at the species that causes most of the reported human cases of legionellosis, L. pneumophila serogroup 1. For this reason, information on the incidence of human respiratory disease attributable to other species and serogroups of legionellae is lacking. Improvements in diagnostic tests such as the urine antigen assay have inadvertently caused a decrease in the use of culture to detect infection, resulting in incomplete surveillance for legionellosis. Large, focal outbreaks of Legionnaires' disease continue to occur worldwide, and there is a critical need for surveillance for travel-related legionellosis in the United States. There is optimism that newly developed guidelines and water treatment practices can greatly reduce the incidence of this preventable illness.
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            Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila.

            A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of > 0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.
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              Addition of neuA, the gene encoding N-acylneuraminate cytidylyl transferase, increases the discriminatory ability of the consensus sequence-based scheme for typing Legionella pneumophila serogroup 1 strains.

              The standard sequence-based method for the typing of Legionella pneumophila serogroup 1 strains was extended by using the gspA and neuA alleles. The use of neuA as a seventh allele for typing significantly increased the index of discrimination calculated for a panel of unrelated strains (from 0.932 to 0.963) and subdivided some known large common complexes (e.g., 1,4,3,1,1,1). This modification to the standard method is proposed as the method of choice in the epidemiological investigation of L. pneumophila infections.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                Appl Environ Microbiol
                Appl. Environ. Microbiol
                aem
                aem
                AEM
                Applied and Environmental Microbiology
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                0099-2240
                1098-5336
                17 June 2016
                15 August 2016
                1 September 2016
                15 August 2016
                : 82
                : 17
                : 5125-5131
                Affiliations
                [a ]National Institute for Public Health and the Environment, Bilthoven, the Netherlands
                [b ]Regional Public Health Laboratory Kennemerland, Haarlem, the Netherlands
                [c ]Institute for Risk Assessment Sciences, Utrecht University, Utrecht, the Netherlands
                Washington State University
                Author notes
                Address correspondence to A. M. de Roda Husman, ana.maria.de.roda.husman@ 123456rivm.nl .

                Citation van Heijnsbergen E, van Deursen A, Bouwknegt M, Bruin JP, de Roda Husman AM, Schalk JAC. 2016. Presence and persistence of viable, clinically relevant Legionella pneumophila bacteria in garden soil in the Netherlands. Appl Environ Microbiol 82:5125–5131. doi: 10.1128/AEM.00595-16.

                Article
                00595-16
                10.1128/AEM.00595-16
                4988192
                27316958
                a5f040d1-8daa-410c-9c59-fa36562713f8
                Copyright © 2016 van Heijnsbergen et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                History
                : 23 February 2016
                : 1 June 2016
                Page count
                Figures: 0, Tables: 4, Equations: 0, References: 51, Pages: 7, Words: 6873
                Funding
                Funded by: Dutch Ministry of Health, Welfare and Sport
                Award ID: V/150017
                Award Recipient : Eri van Heijnsbergen Award Recipient : Anne van Deursen Award Recipient : Martijn Bouwknegt Award Recipient : Jacob Bruin Award Recipient : Ana Maria de Roda Husman Award Recipient : Johanna Schalk
                Categories
                Environmental Microbiology

                Microbiology & Virology
                Microbiology & Virology

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