Confirmed and Potential Sources of <i>Legionella</i> Reviewed

Heijnsbergen, Eri van; Schalk, J.A.C.; Euser, Sjoerd M.; Brandsema, Petra; Boer, Jeroen W. Den; Husman, Ana Maria de Roda

doi:10.1021/acs.est.5b00142

Cited by 133 publications

(99 citation statements)

References 236 publications

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“…In situ determination of specificity of the primer pair Lgsp17F/Lgsp28R was performed using NGS libraries of triplicates of 7 water samples (two cold drinking water (samples 1 and 2), two hot drinking water [3, 4] and three cooling tower water samples [5–7]). Across these samples, we obtained a total of 1,738,388 sequences after quality filtering and removal of genera with a single read, with the median number of quality sequences in the sample set being 60,455.…”

Section: Resultsmentioning

confidence: 99%

“…The infections caused by almost all non- L. pneumophila species have been associated with freshwater systems in hospital settings [3] . Natural aquatic environments are the major reservoir for Legionella species, with aerosol-generating man-made water systems such as cooling towers, drinking water supply systems (DWSS) and recreational waters being the main sources of human exposure to Legionella species [5]. Exceptionally, L. longbeachae infections are commonly linked with the presence of the bacteria in potting soils, instead of freshwater environments, with a high frequency of reported cases in Australia [6].…”

Section: Introductionmentioning

confidence: 99%

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Development of a genus-specific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems

Pereira¹,

Peplies²,

Brettar³

et al. 2017

BMC Microbiol

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BackgroundNext Generation Sequencing (NGS) has revolutionized the analysis of natural and man-made microbial communities by using universal primers for bacteria in a PCR based approach targeting the 16S rRNA gene. In our study we narrowed primer specificity to a single, monophyletic genus because for many questions in microbiology only a specific part of the whole microbiome is of interest. We have chosen the genus Legionella, comprising more than 20 pathogenic species, due to its high relevance for water-based respiratory infections.MethodsA new NGS-based approach was designed by sequencing 16S rRNA gene amplicons specific for the genus Legionella using the Illumina MiSeq technology. This approach was validated and applied to a set of representative freshwater samples.ResultsOur results revealed that the generated libraries presented a low average raw error rate per base (<0.5%); and substantiated the use of high-fidelity enzymes, such as KAPA HiFi, for increased sequence accuracy and quality. The approach also showed high in situ specificity (>95%) and very good repeatability. Only in samples in which the gammabacterial clade SAR86 was present more than 1% non-Legionella sequences were observed. Next-generation sequencing read counts did not reveal considerable amplification/sequencing biases and showed a sensitive as well as precise quantification of L. pneumophila along a dilution range using a spiked-in, certified genome standard. The genome standard and a mock community consisting of six different Legionella species demonstrated that the developed NGS approach was quantitative and specific at the level of individual species, including L. pneumophila. The sensitivity of our genus-specific approach was at least one order of magnitude higher compared to the universal NGS approach. Comparison of quantification by real-time PCR showed consistency with the NGS data. Overall, our NGS approach can determine the quantitative abundances of Legionella species, i. e. the complete Legionella microbiome, without the need for species-specific primers.ConclusionsThe developed NGS approach provides a new molecular surveillance tool to monitor all Legionella species in qualitative and quantitative terms if a spiked-in genome standard is used to calibrate the method. Overall, the genus-specific NGS approach opens up a new avenue to massive parallel diagnostics in a quantitative, specific and sensitive way.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-017-0987-5) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development of a genus-specific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems

Pereira¹,

Peplies²,

Brettar³

et al. 2017

BMC Microbiol

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“…36 With an ideal growth range between 28 and 40 °C, 37 heated water systems are thought to serve as the primary source for Legionella occurrence and transmission, although a wide variety of sources have been linked to legionellosis. 38 Many engineered water systems tend to have physical conditions conducive to Legionella , 39 biofilms they associate with, 40 and eukaryotic hosts they replicate within, 41 all factors commonly found and amplified in premise plumbing (i.e., that found in buildings such as homes, schools, and hospitals). 42 …”

Section: Introductionmentioning

confidence: 99%

Ten questions concerning the aerosolization and transmission of Legionella in the built environment

Prussin

Schwake

Marr

2017

Building and Environment

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Legionella is a genus of pathogenic Gram-negative bacteria responsible for a serious disease known as legionellosis, which is transmitted via inhalation of this pathogen in aerosol form. There are two forms of legionellosis: Legionnaires' disease, which causes pneumonia-like symptoms, and Pontiac fever, which causes influenza-like symptoms. Legionella can be aerosolized from various water sources in the built environment including showers, faucets, hot tubs/swimming pools, cooling towers, and fountains. Incidence of the disease is higher in the summertime, possibly because of increased use of cooling towers for air conditioning systems and differences in water chemistry when outdoor temperatures are higher. Although there have been decades of research related to Legionella transmission, many knowledge gaps remain. While conventional wisdom suggests that showering is an important source of exposure in buildings, existing measurements do not provide strong support for this idea. There has been limited research on the potential for Legionella transmission through heating, ventilation, and air conditioning (HVAC) systems. Epidemiological data suggest a large proportion of legionellosis cases go unreported, as most people who are infected do not seek medical attention. Additionally, controlled laboratory studies examining water-to-air transfer and source tracking are still needed. Herein, we discuss ten questions that spotlight current knowledge about Legionella transmission in the built environment, engineering controls that might prevent future disease outbreaks, and future research that is needed to advance understanding of transmission and control of legionellosis.

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“…In water, L. pneumophila grows primarily as an intracellular parasite of amoebae (7)(8)(9)(10)(11). Human infection occurs mainly by the inhalation of contaminated water droplets originating from a wide range of aerosolgenerating devices (12,13). Yet the first case of person-to-person spread was reported in 2016 (14).…”

mentioning

confidence: 99%

Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms

White

Cianciotto

2016

Infect Immun

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Previously, we documented that type II secretion (T2S) promotes intracellular infection of macrophages by Legionella pneumophila. In the present study, we identified infection events that are modulated by T2S by comparing the behaviors of wild-type and T2S mutant bacteria in murine bone marrow-derived macrophages and human U937 cells. Although the two strains behaved similarly for entry into the host cells and evasion of lysosomal fusion, the mutant was impaired in the ability to initiate replication between 4 and 8 h postentry and to grow to large numbers in the Legionella-containing vacuole (LCV), as evident at 12 h. At 4 h postinoculation, mutant LCVs had a significantly reduced association with Rab1B, a host GTPase that facilitates the tethering of endoplasmic reticulum (ER)-derived vesicles to LCVs. The mutant did not lose expression or translocation of six type IV secretion effectors (e.g., SidM) that are well known for mediating Rab1B association with the LCV, indicating that T2S promotes the interaction between the LCV and Rab1B via a novel mechanism. Interestingly, the mutant's growth defect was exacerbated in macrophages that had been depleted of Rab1B by short hairpin RNA (shRNA) treatment, indicating that T2S also potentiates events beyond Rab1B association. In support of this, a sidM lspF double mutant had an intracellular growth defect that was more dramatic than that of the lspF mutant (and a sidM mutant) and showed a growth difference of as much as a 400-fold compared to the wild type. Together, these data reveal a new role for T2S in intracellular infection that involves both Rab1B-dependent and Rab1B-independent processes. L egionella pneumophila is widespread in natural and man-made water systems and is best known as the etiologic agent of Legionnaires' disease (1-3). Recently, the incidence of pneumonias caused by Gram-negative L. pneumophila has roughly tripled in the United States and elsewhere (4-6). In water, L. pneumophila grows primarily as an intracellular parasite of amoebae (7-11). Human infection occurs mainly by the inhalation of contaminated water droplets originating from a wide range of aerosolgenerating devices (12,13). Yet the first case of person-to-person spread was reported in 2016 (14). In the lungs, L. pneumophila invades and grows in the resident macrophage population and ultimately triggers severe inflammation (3). In both amoebae and macrophages, the bacterium evades the host's degradative lysosomal pathway and replicates to large numbers within a membrane-bound vacuole, the Legionella-containing vacuole (LCV) (15, 16). The Dot/Icm type IV secretion (T4S) system of L. pneumophila plays a major role in pathogenesis (17-19). Indeed, the Dot/Icm apparatus translocates myriad proteins across the LCV membrane and into the host cell cytoplasm, and then the delivered effectors proceed to modulate a wide variety of host processes. One of the earliest and most notable events that has been ascribed to the Dot/Icm system is the recruitment of host GTPases and endoplasmic reticulu...

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Confirmed and Potential Sources of Legionella Reviewed

Cited by 133 publications

References 236 publications

Development of a genus-specific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems

Development of a genus-specific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems

Ten questions concerning the aerosolization and transmission of Legionella in the built environment

Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms

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