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- Published: 09 December 2021
Population analysis of Legionella pneumophila reveals a basis for resistance to complement-mediated killing
- Bryan A. Wee ORCID: orcid.org/0000-0001-6135-5903 1 na1 ,
- Joana Alves ORCID: orcid.org/0000-0003-3131-9834 1 na1 ,
- Diane S. J. Lindsay ORCID: orcid.org/0000-0003-1378-0926 2 ,
- Ann-Brit Klatt 3 ,
- Fiona A. Sargison ORCID: orcid.org/0000-0001-5160-4208 1 ,
- Ross L. Cameron 4 ,
- Amy Pickering ORCID: orcid.org/0000-0003-2154-224X 1 ,
- Jamie Gorzynski ORCID: orcid.org/0000-0002-4921-9789 1 ,
- Jukka Corander 5 , 6 ,
- Pekka Marttinen ORCID: orcid.org/0000-0001-7078-7927 7 ,
- Bastian Opitz ORCID: orcid.org/0000-0003-1276-836X 3 ,
- Andrew J. Smith ORCID: orcid.org/0000-0003-0580-4078 2 , 8 &
- J. Ross Fitzgerald ORCID: orcid.org/0000-0002-9233-8468 1
Nature Communications volume 12 , Article number: 7165 ( 2021 ) Cite this article
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- Bacterial genomics
- Population genetics
Legionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires’ disease. However, the microorganism is typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 902 L. pneumophila isolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We find that the capacity for human disease is representative of the breadth of species diversity although some clones are more commonly associated with clinical infections. We identified a single gene ( lag-1 ) to be most strongly associated with clinical isolates. lag-1 , which encodes an O -acetyltransferase for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades of L. pneumophila by frequent recent recombination events. The gene confers resistance to complement-mediated killing in human serum by inhibiting deposition of classical pathway molecules on the bacterial surface. Furthermore, acquisition of lag-1 inhibits complement-dependent phagocytosis by human neutrophils, and promoted survival in a mouse model of pulmonary legionellosis. Thus, our results reveal L. pneumophila genetic traits linked to disease and provide a molecular basis for resistance to complement-mediated killing.
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Introduction.
Legionella pneumophila is a γ-proteobacterial species that parasitises free-living amoeba in freshwater environments 1 , 2 . L. pneumophila hijacks the phagocytic process in amoebae and human alveolar macrophages by subverting host cellular mechanisms to promote intracellular replication 3 . Legionella infections are a global public health concern presenting as either a severe pneumonia known as Legionnaires’ disease or Pontiac fever, a self-limiting flu-like syndrome 4 , 5 , 6 , 7 . Importantly, recent surveillance studies have indicated a steady increase of legionellosis incidences globally 8 , 9 .
L. pneumophila infection in humans is considered to be the result of accidental environmental exposure and the selection for pathogenic traits among L. pneumophila is likely to be driven by co-selective pressures that exist in its natural habitat 10 . The pivotal mechanism required for intracellular replication is the type IV secretion system (T4SS) that is conserved across all known members of the genus Legionella 11 , 12 . A very large repertoire of effector proteins in different combinations is encoded by Legionella species and can be secreted by this system to mediate critical host-pathogen interactions 11 . In addition, the ability to infect eukaryotic cells has evolved independently many times 11 . Despite this, less than half of all currently described Legionella species have been reported to cause human disease 13 . Furthermore, there is an over-representation of a single serogroup (Sg-1) of L. pneumophila in human infections, which is responsible for more than 85% of all reported cases of legionellosis 14 . Sg-1 strains can be further subdivided phenotypically using monoclonal antibodies (mAbs) that recognise various components of the lipopolysaccharide (LPS). The most prevalent mAb subtype in human infections is associated with an LPS O- acetyltransferase enzyme encoded by the lag-1 gene ( lpg0777 ), which confers an LPS epitope recognised by the mAb 3/1 from the Dresden mAb panel 15 , 16 . lag-1 has previously been reported to be associated with clinical isolates of L. pneumophila but its role in pathogenicity remains a mystery 17 , 18 , 19 , 20 , 21 .
Recently, it was shown that a very limited number ( n = 5) of L. pneumophila sequence types (ST)s are responsible for almost half of all human infections and that these clones have undergone very recent emergence and expansion 22 . In addition, it was shown that recombination between the dominant STs has led to sharing of alleles that may be beneficial for human pathogenicity 22 . However, the genetic basis for the enhanced human pathogenic potential of these STs is unknown. Early studies identified the capacity for some L. pneumophila strains to resist killing in human serum, a phenotype that may correlate with increased virulence 23 . However, the molecular mechanism of strain-dependent serum resistance by L. pneumophila has remained elusive for over 30 years.
Here, we employ a population genomic analysis of L. pneumophila isolates from clinical and environmental sources to investigate the diversity of genotypes associated with human disease. Further, we employ genome-wide association analyses to identify genetic traits associated with human pathogenic strains revealing lag-1 to be the most strongly associated determinant of human pathogenic potential in L. pneumophila . We demonstrate that acquisition of lag-1 has occurred widely across the species by recent horizontal gene transfer and recombination, leading to strains that have enhanced resistance to complement-mediated killing, neutrophil phagocytosis and survival in a murine model of pneumonia.
Results and discussion
The potential for human infection is distributed across the l. pneumophila species phylogeny.
In order to examine the diversity of L. pneumophila associated with human clinical infection in comparison to those from environmental sources, we carried out whole-genome sequencing of 397 clinical and environmental L. pneumophila subsp. pneumophila isolates from an archived collection (1984 to 2015) of Legionella species held at the Scottish Haemophilus, Legionella, Meningococcus & Pneumococcus Reference Laboratory, Scottish Microbiology Reference Laboratory, Glasgow (SHLMPRL) as detailed in the Methods section. The dataset included 166 clinical isolates from infected patients, primarily from sputum and bronchoalveolar lavages (BAL). The proportion of clinical isolates from the less severe Pontiac fever form of legionellosis is unknown but is presumed to be very limited due to low hospitalisation rates for these infections. In addition, we included sequences from 231 environmental isolates of L. pneumophila subsp. pneumophila from water sources such as cooling towers and plumbing systems sent to the reference laboratory for routine testing and surveillance (Supplementary Data 1 ). Some of the isolates classified as environmental in our dataset were sampled as part of an outbreak investigation and therefore may in some cases be related to clinical isolates. Accordingly, the power to identify traits that differentiate clinical and environmental groups may be negatively affected. To place the SHLMPRL isolates into context of the known global diversity of L. pneumophila subsp. pneumophila isolates, we included 500 assembled whole genomes that were available in the public database (NCBI Genbank). We constructed a Maximum-Likelihood phylogenetic tree from 139,142 core genome single nucleotide polymorphisms (SNP), which indicates the segregation of the L. pneumophila subsp. pneumophila population into seven major clades (Fig. 1 ) each supported by a minimum of one and a maximum of two sub-clusters defined by Bayesian Analysis of Population Structure (BAPS) analysis (Supplementary Fig. 1 ). Isolates from the SHLMPRL collection were distributed among all major clades indicating that the isolates are representative of the global species diversity (Fig. 1 ). Although recombination has played an important role in diversification of the species, complicating the accurate reconstruction of the phylogeny 24 , the major phylogenetic clusters are consistent with those identified in previous population studies employing fewer isolates 25 , 26 . Recombination is more likely between phylogenetically related (and genetically similar) isolates, which may amplify the phylogenetic signal that defines these major groups 27 . Of the 166 clinical isolates, 80 belonged to the five most common sequence types (ST) implicated in human infections (ST1, 23, 36/37, 47 and 62). The remaining 86 clinical isolates were from STs distributed across all seven major phylogenetic clusters (Fig. 1 ). These data indicate that although only 5 STs are responsible for almost half of all infections, the other half of human infection isolates come from diverse genetic backgrounds distributed across the species. Of note, five clusters from distinct outbreaks of Legionnaires’ disease in Scotland between 1985 and 2012 28 , 29 , 30 originated from different clades (Fig. 1 ). Similarly, the 231 environmental isolates are also distributed across all major phylogenetic groups (Fig. 1 ). Notably, 35% (81 of 231) of environmental isolates belong to one of the five clinically dominant STs suggesting that at least a third of all environmental L. pneumophila from the sampled sources in Scotland have human pathogenic potential. Overall, although some STs appear to have higher human pathogenicity, our findings support the understanding that the capacity for causing human disease is widely distributed across diverse L. pneumophila genetic backgrounds.
A maximum-likelihood phylogeny of 902 isolates based on 139,142 core genome SNPs divides the subspecies into seven major clades that are also supported by BAPS clustering. Isolates linked to five major outbreaks that have occurred in Scotland, UK between 1985 and 2012 originated from different lineages are indicated. In addition, isolates associated with sporadic cases of human infection have also emerged from diverse genetic backgrounds across all major clades. For reference, the position of five major disease associated clones (ST1, 23, 36/37, 47 and 62) and well-characterised reference genomes are indicated. The tree scale indicates the number of subtitutions per site.
Genome-wide association analysis of L. pneumophila reveals genetic traits associated with human pathogenicity
The factors that contribute to the enhanced human infectivity or transmissibility of some L. pneumophila clones are unknown. Our strategy, to address this gap in understanding, involved sequencing of large numbers of genetically diverse environmental isolates, affording for the first time, a large-scale genome-wide association study (GWAS) of clinical vs environmental isolates to explore the bacterial genetic basis for human clinical disease. Initially, we performed systematic subsampling on our dataset to reduce the number of closely related isolates originating from the same type of source, either clinical or environmental, while retaining genetic diversity. This step removes over-represented endemic or epidemic clonal lineages in the dataset that arise from opportunistic, convenience sampling and also represents an additional control for a stratified bacterial population structure. From this reduced dataset ( n = 452), we used the programme SEER to identify a total of 1737 k- mers that were enriched (significantly associated, p < 0.05) among clinical isolates in comparison to environmental isolates. Mapping to the L. pneumophila Philadelphia 1 Sg-1 reference genome (Accession number: AE017354) [ https://www.ncbi.nlm.nih.gov/nuccore/AE017354.1 ] revealed that 39% ( n = 673) of the k- mers aligned to a region of the genome spanning between loci lpg0748 and lpg0781 representing an 18 kb cluster of genes involved in LPS biosynthesis and modification (Fig. 2 ) 31 . A total of 22 genes in this cluster each had at least one significantly enriched k -mer that mapped to it: namely, lpg0751, lpg0752, lpg0755, lpg0758, lpg0759, lpg0760, lpg0761, lpg0762, lpg0766, lpg0767, lpg0768, lpg0769, lpg0771, lpg0772, lpg0773, lpg0774, lpg0775, lpg0777 (lag-1), lpg0778, lpg0779, lpg0780 and lpg0781 (Fig. 2 and Supplementary Table 1 ). Two additional conservative SEER analyses using more stringently subsampled datasets resulted in a smaller number of significantly enriched k- mers (205 and 61, respectively), that converge on a single gene within the LPS cluster ( lpg 0777, lag-1 ) that encodes an O -acetyltransferase involved in modification of the O-antigen of the L. pneumophila Sg-1 LPS (Supplementary Fig. 2 ). To account for the random nature of the subsampling algorithm, multiple iterations were performed at the middle threshold, and the distribution of significant k-mers was shown to be consistent across all (Supplementary Fig. 2b ).
Manhattan plot showing the genomic position of k -mers significantly associated with clinical isolates. The LPS biosynthesis and modification locus (822,150bp–855,010 bp: lpg0751 - lpg0781 ) in the Philadelphia 1 reference genome (AE017354) [ https://www.ncbi.nlm.nih.gov/nuccore/AE017354.1 ] is represented and genes with significant k- mer associations coloured in red, with lag-1 in blue.
To corroborate the initial findings of the k -mer based SEER approach, we employed a different GWAS method (SCOARY) that examined the distribution of orthologous genes using the pan-genome pipeline ROARY 32 , 33 . Consistent with SEER, this approach also indicated that the Sg-1 LPS biosynthesis genes were enriched among clinical isolates with lag-1 exhibiting the strongest statistical support over other Sg-1-associated LPS genes (Supplementary Table 1 ). The corrected (Benjamini–Hochberg) p -value for lag-1 is several orders of magnitude lower (9.74E-11) than other Sg-1 LPS genes that were above the significance threshold ( lpg0779: 2.35E-05, lpg0780: 2.35E-05, lpl0815/lpg0774: 3.84E-05 and lpg0767 : 6.92E-05). As mentioned, lag-1 has previously been reported to be prevalent among clinical isolates 17 , 18 , 19 , 20 , 21 . However, our large objective pangenome-wide analysis of the L. pneumophila species indicates that of all 11198 accessory genes, lag-1 has the strongest association with clinical isolates, suggesting a major role in human disease. In total, 80.1% of clinical isolates contained lag-1 compared to 30.8% of environmental isolates (Supplementary Fig. 3 ). Sg-1 LPS genes were found in high-frequency throughout the population, and in each major lineage indicating species-wide gene transfer. The Sg-1 LPS cluster is also found in other subspecies of L. pneumophila that can infect humans, such as subspecies fraseri and pascullei but has not been reported in other species of Legionella 34 , 35 . Here, we observed that lag-1 can be associated with different combinations of Sg-1 LPS genes and still express the expected lag-1 phenotype represented by the mAb 3/1-specific epitope (Dresden mAb scheme) (Supplementary Data 1 ) 36 , 37 . However, genetic instability and phase variation has been reported to affect the mAb phenotype between closely related strains 38 , 39 . One mechanism of phase variation is the excision of a 30 kb genetic element that results in a change in mAb specificity manifested by a loss of virulence in guinea pigs and loss of resistance to complement 40 , 41 .
Recombination has mediated the dissemination of three dominant lag-1 alleles across the L. pneumophila species
It has been reported since the 1980s that isolates expressing the epitope recognised by the mAb 3/1 are more frequently associated with isolates from community-acquired and travel-associated infections 37 , 42 , 43 . However, the pathogenic basis for this association remains a mystery. In order to further investigate the role of the clinically-associated gene lag-1 , we examined its diversity and distribution across the 902 L. pneumophila genomes employed in the current study. In total, three major allelic variants of lag-1 that had been previously identified to be representative of reference strains, Philadelphia, Arizona, and Corby, respectively, were identified 43 . In our dataset, variant 1 (Philadelphia), was present in 195 (22%), variant 2 (Arizona) in 195 (22%) and variant 3 (Corby) in 178 (20%) of the 902 isolates examined (Fig. 3a ). Each variant was distributed across the phylogeny, with variant 1 found in 3 major clades (1, 4 and 7) and variants 2 and 3 identified among isolates of all 7 major clades (Fig. 3a ). Of note, clade 2 has the lowest frequency (12%) of isolates encoding the lag-1 gene and is also characterised by an under-representation of clinical isolates (31%). In addition to the three major lag-1 alleles, we identified a relatively small number ( n = 40) of derived minor allelic variants that differ by <1% nucleotide identity from any of the 3 major variants (Supplementary Table 2 ). Of these, only 10 are predicted to encode for full-length proteins suggesting most are likely to be non-functional pseudogenes. The occurrence of these three allelic variants of lag-1 gene and their widespread distribution across the species phylogeny indicates frequent horizontal dissemination of recently acquired lag-1 alleles driven by a strong selection pressure for the conservation of lag-1 sequence and function. The nucleotide sequence identity of 89%–95% between the three allelic variants is lower than the average nucleotide sequence identity for genes across the species, which is typically >95% (Supplementary Table 2 ). The closest homologue of lag-1 in the NCBI non-redundant protein database shares only 45% amino acid sequence identity and encodes a putative acyltransferase present in an environmental species of the genus Pseudomonas . The selective advantage for L. pneumophila to maintain lag-1 in the environment is still unclear. It has been proposed that the increased hydrophobicity of LPS when acetylated by O -acetyltransferase may enhance L. pneumophila survival in amoebae vacuolar compartments 44 . An alternative hypothesis is that L. pneumophila is evolving the capacity to transmit between humans or from humans to the environment as proposed by David et al. 22 . In which case, the recent spread of lag-1 across the phylogeny may have been driven by selection in the human host and contributed to the recent expansion of successful clones. Horizontal transfer and recombination events involving the 18 kb LPS biosynthesis gene cluster has been reported previously, including between Philadelphia-1 (ST36) and Paris (ST1) 45 , 46 . In addition, a microarray-based comparative genomic study identified this genomic region to be associated with human disease 47 . To investigate the potential role of recombination in the distribution of the LPS genes including lag-1 , we carried out a split network analysis based on the concatenated alignment of 10 LPS biosynthesis genes that were present in 23 representative isolates from across the phylogeny encoding different lag-1 variants (Fig. 3b ). This analysis revealed extensive reticulation consistent with recombination across the gene cluster and identified horizontal transfer of LPS genes between three major clinical sequence types, ST23, ST47 and ST62, respectively (Fig. 3b ). This network analysis revealed that even highly similar LPS biosynthesis gene clusters, such as those found in isolates 4454, Corby and Alcoy or 4616 and 4718, encode different variants of lag-1 , consistent with recent gene conversion of lag-1 . We also identified three examples of closely related genomes (average nucleotide identity across the genome of >99.8%) that exhibited a signature of homologous recombination affecting lag-1 and the surrounding genomic region (Supplementary Fig. 4 ). These findings expand on a previously proposed mechanism of lag-1 gene deletion by Kozak and colleagues 43 and demonstrate that recombination can restore a disrupted or missing lag-1 gene or replace one functional lag-1 variant with another (Supplementary Fig. 4 ).
a Core-genome SNP-based phylogenetic tree indicating the distribution of the 3 major allelic variants of lag-1 across Sg-1 strains. Variant 1 (Philadelphia, Red), Variant 2 (Arizona, Green) and Variant 3 (Corby, Blue). Each major clade is associated with at least two different lag-1 alleles. Minor alleles that are within >99% nucleotide sequence identity to a major variant are shown with lighter colours. Isolates not containing lag-1 are coloured grey. The tree scale indicates the number of subtitutions per site. b A neighbour-net phylogenetic network indicating recombination of Sg-1 LPS genes including lag-1 . The network was drawn using uncorrected P-distances with the equal angle method in Splitstree from a concatenated alignment of 10 conserved Sg-1 LPS genes that were significantly associated with clinical isolates (9405 positions). Orthologs of 10 LPS cluster genes were extracted from 16 phylogenetically representative isolates coding 3 different variants of lag-1 , 7 isolates missing lag-1 and 9 reference genomes (Lens, HL06041035, Philadelphia-1 [ST36], Paris [ST1], Corby, Alcoy, Pontiac [ST62], Lorraine [ST47], ST23).
A comparison of re-assortment rates across major phylogenetic lineages showed no significant differences between LPS and non-LPS genes across the L. pneumophila genome ( p = 0.6275). This suggests that recombination is active on a genome-wide scale, in agreement with earlier analyses of rates of recombination across the genome 45 , 46 (Supplementary Fig. 5 and Supplementary Data 2 ). Taken together, these findings highlight the wide dissemination of lag-1 and other LPS genes by recombination.
lag-1 confers resistance to killing by human plasma
Our population data indicate that lag-1 gene has the highest statistical support of all accessory genes for a role in human pathogenicity. Consistent with this, previous studies have reported an epidemiological correlation between lag-1 or its mAb 3/1-recognised epitope and clinical disease 17 , 18 , 19 , 20 , 21 but a mechanistic understanding of its role in pathogenesis has proved elusive. Of note, an early study described an increased ability of an endemic nosocomial L. pneumophila strain to survive complement killing when compared to an environmental strain collected from the same medical facility 23 . More recently, resistance to complement-mediated killing has been proposed as a key fitness advantage that enables the Philadelphia Sg-1 strain to establish infections compared to other serogroups 48 . To investigate this phenotype in the context of lag-1 genotype, we examined resistance to killing in human plasma for the aforementioned 23 Sg-1 L. pneumophila strains selected to represent the diversity of lag-1 genotypes from across the phylogeny. We observed that all strains lacking a lag-1 gene were susceptible to killing in plasma, whereas groups of isolates containing lag-1 variants exhibited enhanced resistance, independent of the lag-1 variant encoded (Fig. 4a ).
a Representative L. pneumophila isolates containing allelic variants 1, 2, and 3 of lag-1 (depicted in red, green, and blue, respectively) or lag-1 -negative (depicted in grey) were incubated with human plasma (coloured bars and dots) or heat-inactivated plasma (open bars and dots) for 1 h at 37 °C. Each dot represents an average cfu count in plasma from a single donor ( n = 7 donors for strain 4470, n = 6 for strains 4454, 4471, 4472, 4616, 4537, 4597; n = 5 for strains 4310, 4682, 4449, 4403, 4415, 4312; n = 4 for strains 4552, 4438, 4718, 4562, 4821, 4450 and n = 3 for strains 4546, 4819, 4719, 4813). Mean +/− SEM, One-way Anova with Holm-Sidak’s multiple comparisons test between Plasma and Heat-inactivated plasma for the same strain. One-way Anova with Tukey’s multiple comparisons test for differences between the variants. * p < 0.05, ** p < 0.01, **** p < 0.0001. Source data and exact p -values are provided in the Source Data file. b Schematic representation of the multiple natural lag-1 independent deactivating mutations in epidemiologically-related isolates. In blue—functional lag-1 , with dots— lag-1 with His28Leu mutation, with lines— lag-1 truncated by an insertion sequence in aa 251, in grey— lag-1 truncated by a premature stop codon. c Isolates with lag-1 mutations show increased susceptibility to killing in human plasma. Closely related isolates from the same hospital-associated cluster were incubated with human plasma (coloured and patterned bars and dots) or heat-inactivated plasma (open bars and dots) for 1 h at 37 °C. Each dot represent an average cfu count in plasma from a single donor ( n = 9 donors for strain 3956; n = 7 for strain 4443; n = 6 for strain 4681; n = 4 for strains 3947, 3959, 4567, 4451, 4474, 4568, 4595 and n = 3 for all other strains). Mean +/− SEM, One-way Anova with Holm-Sidak’s multiple comparisons test between Plasma and Heat-inactivated plasma for the same strain, * p < 0.05, ** p < 0.01, **** p < 0.0001. Source data and exact p -values are provided in the Source Data file.
Within our dataset, we identified a group of epidemiologically-related isolates from a single Scottish healthcare facility typed as ST5 (a single locus variant of ST1), which were predicted to vary with regard to lag-1 functionality. ST5 has only been found in this location, to date. The earliest isolates obtained from a nosocomial outbreak in 1984/1985 30 contained variant 3 (Corby) of the lag-1 gene and all were found to express the mAb 3/1 epitope (Supplementary Data 1 ). In contrast, 16 of 19 environmental isolates from the same healthcare facility 12 to 21 years later (1997 to 2006) contained multiple independent mutations in lag-1 predicted to disrupt functionality. Specifically, a nonsense mutation (L48*), an insertion of a transposase, and the acquisition of a deleterious substitution (H28L) (Fig. 4b ), each correlated with a lack of reactivity with mAb 3/1 from the Dresden panel classification (Supplementary Data 1 ). Consistent with our previous findings (Fig. 4a ), the presence of a functional LPS O -acetyltransferase in this epidemiological cluster also correlated with resistance to killing in human plasma, whereas isolates with a non-functional lag-1 were susceptible to killing, independent of the type of deactivating mutation (Fig. 4c ). Of note, no clinical episodes of disease were identified to be caused by this epidemiological cluster after 1985, and all clinical isolates contained a functional lag-1 gene. The identification of multiple independent mutations associated with loss of lag-1 function suggests a selection pressure that drives the inactivation of the lag-1 gene and LPS O -acetylation in this environment. The trend of lag-1 gene loss or deactivation was not observed among longitudinal ST1 healthcare facility-associated isolates from a cluster sequenced in a recent study by David and colleagues 24 . However, a study on starvation of L. pneumophila in ultrapure water showed that in a short-term period, the viable cell numbers of all mAb 3/1-positive strains decreased strongly compared to the other strains suggesting a negative selection for lag-1 function in some water environments 49 . Overall, these data indicate that the presence of a functional lag-1 correlates with enhanced resistance to serum killing.
To investigate the role of lag-1 in mediating resistance to serum killing, we introduced each of three major lag-1 variants encoded on an expression plasmid (pMMB207) into the strain 4681 that contains a non-functional lag-1 gene due to the insertion of a transposase. Introduction of each lag-1 variant led to expression and LPS 8- O -acetylation as confirmed by flow cytometric detection of the mAb 3/1 epitope (Fig. 5a, b ). Strikingly, complementation of the strains with each lag-1 variant resulted in resistance to killing in human serum (Fig. 5d ). In order to test this phenomenon in a distinct strain background, we introduced the lag-1 variant 1 into the lag-1 -negative strain 4312, which contains a 1 bp deletion resulting in a frameshift at position 156. Similarly, lag-1 expression conferred resistance to killing in human serum (Supplementary Fig. 6 ). Consistent with this, we observed a decrease in human C3 deposition at the surface of the lag-1 expressing bacteria when compared to the isogenic lag-1 -negative strain, at levels similar to the wild-type lag-1 -positive isolate 3656 (Supplementary Fig. 7 ). Of note, it was previously reported that a wild-type lag-1 -positive isolate and a spontaneously derived lag-1 mutant demonstrated similar levels of resistance to serum killing 20 . We were unable to obtain the relevant strains, but we speculate that the discordance with our findings could be due to phase-variable expression of LPS 19 or differences in transcriptional levels of lag-1 gene that have been previously observed depending on culture conditions 50 .
a and b Introduction of any lag-1 gene variant leads to mAb 3/1 epitope expression. Detection of mAb 3/1 epitope by flow cytometry in ( a ) WT and ( b ) isogenic mutants of L. pneumophila isolates expressing the lag-1 variant 1, 2 or 3. c and d lag-1 expression confers serum complement resistance to L. pneumophila strains with non-functional lag-1 gene. Isolates were incubated with human serum or heat-inactivated serum for 1 h at 37 °C. Each point represents an average of triplicate CFU counts of a single sera donor ( n = 3). Bars represent mean + SEM. One-way ANOVA, Tukey’s multiple comparisons test, * p < 0.05, **** p < 0.0001. Comparison to Empty vector isogenic strain represented on the top of the bars. Comparison to Empty vector isogenic strain represented on the top of the bars. Source data and exact p -values are provided in the Source Data file.
lag-1 confers resistance to killing by the classical complement pathway
We next investigated if a specific complement pathway was responsible for the serum killing of the Sg-1 mAb 3/1-negative L. pneumophila . As a similar inhibitory effect of lag-1 on bacterial killing was observed in both serum and plasma, we employed serum for these experiments to facilitate the use of commercially available depleted serum samples. EDTA can be used to inhibit the classical, lectin and alternative pathways via chelation of both Ca 2+ and Mg 2+ , whereas chelation with EGTA/Mg 2+ inhibits the Ca 2+ -dependent classical and lectin pathways only, leaving the alternative pathway unaffected (Fig. 6a ). Killing assays in human serum in the presence of either EDTA or EGTA/Mg 2+ resulted in complete abrogation of lag-1- negative strains susceptibility to complement, and no difference in cell viability compared to incubation in heat-inactivated serum, suggesting the alternative complement pathway is insufficient to kill L. pneumophila (Fig. 6b ). To distinguish the role of the classical from the lectin pathway, killing assays were performed in the presence of mannose that competes for the association of mannose binding lectin (MBL) with the bacterial surface and blocks the lectin complement pathway 51 . Using this approach, there was no effect on bacteria viability, indicating that the lectin pathway is not required for L. pneumophila Sg-1 killing, consistent with the previous report that MBL polymorphisms are not associated with a higher risk for legionellosis 52 . The exclusion of the lectin pathway suggests the essential role of the classical pathway in the complement killing of L. pneumophila . To confirm this finding we depleted serum of C1q, required for the classical pathway, resulting in loss of serum-mediated killing (Fig. 6b ). It is noteworthy that we also observed an increase in bacterial viability in the presence of Factor B-depleted serum (Fig. 6b ), implying a possible role for the alternative pathway in amplification of classical pathway activation as previous described for Streptococcus pneumoniae 53 . The impact of this pathway in L. pneumophila serum killing may be strain-dependent as indicated by the difference in the impact of Factor-B depletion on the 2 strains examined (Fig. 6b ). Previously, purified L. pneumophila LPS was reported to activate both classical and alternate pathways, primarily through the activation of the classical pathway dependent on natural IgM antibodies 54 . In another study, complement C1q protein was demonstrated to bind to the major outer membrane protein (MOMP) of L. pneumophila , activating complement in an antibody-independent way 55 .
a Simplified schematic representation of the complement pathways and the inhibitors used in this study. Made using Lucidchart. b Inhibition of both classical and alternative pathways confers L. pneumophila resistance to complement killing in human serum. 3956, 4451, 4681:: Empty vector isolates were incubated with 90% heat-inactivated serum, serum, serum with EDTA, EGTA/Mg 2+ or mannose, Factor B- or C1q-depleted serum for a 1 h at 37 °C. Each point represents an average of three technical replicates for a single biological replicate ( n = 8 biological replicates for heat-inactivated serum and serum; n = 6 for serum with EDTA and with EGTA/Mg; n = 4 for serum with Manose, Factor B- and C1q-depleted serum). Statistical analysis when compared to serum on top of the bars. One-way ANOVA, Tukey’s multiple comparisons test, ** p < 0.01 **** p < 0.0001. Source data and exact p -values are provided in the Source Data file.
lag-1 expression enhances resistance to neutrophil phagocytosis, and survival in a murine model of pulmonary legionellosis
Classical pathway complement activity exists in healthy human BAL, despite the relatively low concentration of some complement proteins 56 , and exposure to aerosolized LPS leads to a rapid increase of the level of these proteins in the lung of human volunteers 57 . The importance of this innate immune mechanism in lung health is supported by the observation that many patients with deficiencies in complement proteins or complement receptors have recurrent respiratory infections 58 . The role of the complement system in lung immune defences extends beyond the proteolytic cascade associated with bacterial lysis, as opsonisation with complement C3b protein induces phagocytosis of opsonized targets by neutrophils and macrophages 59 . Accordingly, to test the effect of lag-1 expression on phagocytosis of L. pneumophila by neutrophils, human blood purified neutrophils were incubated with fluorescein isothiocyanate (FITC)-labelled L. pneumophila strains in the presence of non-immune human serum (Fig. 7a to d ). We examined naturally occurring lag-1 -positive ( n = 3) and lag-1 -negative ( n = 3) strains from diverse phylogenetic groups, and observed elevated FITC mean fluorescence intensity (MFI) in neutrophils infected with lag-1 -negative strains compared to lag-1 -positive strains indicating a lag-1 -dependent reduction in internalisation (Fig. 7a ). Further, introduction of the lag-1 gene on the pMMB207 expression plasmid significantly reduced neutrophil phagocytosis (Fig. 7c and Supplementary Fig. 6 ). The same effect was observed in neutrophils infected with the naturally occurring strains expressing DSred via pSW001 (Supplementary Figs. 8 and 9 ). Consistent with these findings, confocal microscopy of neutrophils infected with lag-1 expressing strains had lower numbers of internalised bacteria when compared to neutrophils infected with lag-1 -negative strains (Fig. 7b, d ). Taken together, these data demonstrate a role for lag-1 in resistance to neutrophil phagocytosis. It was previously shown that lag-1 expression is associated with an increase in ability of L. pneumophila to adhere and, consequently, penetrate into Acanthamoeba castelani membranes 60 . However, the ability of LPS to inhibit phagosome maturation in A. castellanii was reported to be independent of a functional lag-1 61 . Our data suggest that that internalisation by neutrophils is inhibited by lag-1 and a requirement for serum suggests an effect that is complement-dependent (Supplementary Fig. 9 ). Therefore, the ability of lag-1 -positive L. pneumophila strains to escape complement deposition correlates with the capacity to escape phagocytosis. These data are consistent with the findings of early L. pneumophila studies, where the L. pneumophila strain Philadelphia 1 ( lag-1 -positive) was demonstrated to be resistant to complement and neutrophil killing in the absence of specific antibodies 62 , 63 . Of note, there is increasing evidence for the pivotal role of neutrophils in the resolution of L . pneumophila lung infections, and either neutrophil depletion or blockage of recruitment to infected lungs renders mice susceptible to L. pneumophila 64 , 65 , 66 , 67 , 68 .
a to d lag-1 confers resistance to human neutrophil phagocytosis. WT lag-1 -positive and -negative strains expressing DsRed fluorescent protein, as well as isogenic mutants of L. pneumophila isolates expressing the lag-1 variant 1, 2 or 3, were stained with FITC and pre-incubated with 10% human serum 15 min prior incubation with human neutrophils for 30 min. a and b Phagocytosis was evaluated by measuring neutrophils FITC fluorescence by flow cytometry. Each point represents a technical replicate for a single donor. Data are representative of three independent experiments. L. pneumophila strains expressing lag-1 variant 1, 2, or 3 are represented in red, green, or blue, respectively. Strains that do not express a functional lag-1 gene are represented in grey. Non-infected (NI) neutrophils are represented in black. Bars represent mean +/− SEM. One-way ANOVA, Dunnett’s multiple comparisons test to non-infected control or to Empty vector , ** p < 0.01, **** p < 0.0001. Source data and exact p -values are provided in the Source Data file. c and d Representative images of L. pneumophila- infected human neutrophils obtained by confocal microscopy and maximum intensity algorithm. Bacteria are represented in green, cytoplasmic membrane in red and nucleus in blue. e lag-1 expression increases L. pneumophila lung survival. C57BL/6J mice were intranasally infected with 1 × 10 6 L. pneumophila cells in 40 μL of PBS and bacterial numbers in the lungs evaluated 96 h post infection. Each point represents an individual animal ( n = 4 in each group). Data are presented as mean +/− SEM. Two-tailed Mann–Whitney t- test * p < 0.0286. Source data are provided as a Source Data file.
In order to test the role of lag-1 in the pathogenesis of legionellosis in vivo we used an established murine model of pneumonia 69 . C56BL/6 mice were intranasally infected with L. pneumophila strain 4681 containing either the empty plasmid or plasmid expressing the lag-1 variant 3. Mice infected with the lag-1- expressing strain exhibited higher bacterial burden in the lungs at 96 h post infection compared to mice infected with the lag-1- negative isogenic strain (Fig. 7e ). These data indicate that acquisition and expression of lag-1 enhances L. pneumophila survival during infection of the lung.
Taken together, our data indicate that L. pneumophila lag-1 mediated inhibition of complement deposition, disrupts the innate immune response via inhibition of complement-mediated lysis and inhibition of neutrophil phagocytosis, promoting survival in vivo.
The steady global increase in L. pneumophila infections is worrisome and studies that explore the evolutionary basis of increased pathogenicity can provide insights into the nature of the public health threat posed. Here we have employed a combined large-scale population level study of clinical and environmental isolates, along with functional ex vivo and in vivo analyses to reveal the evolutionary and functional basis for serum resistance in L. pneumophila . The apparent selective advantage conferred by lag-1 for disease in humans is intriguing in the context of its environmental reservoir. It is feasible that lag-1 provides a functional advantage in its natural amoebal host as reported by Palusinska-Szysz et al., and that co-selection occurs for enhanced pathogenicity in humans 60 . Alternatively, David et al previously proposed that the recent expansion of L. pneumophila in man-made water systems and global spread of selected clones is consistent with possible human-to-human or human-to-environmental dissemination 22 . If indeed some L. pneumophila clones are evolving towards human colonisation and transmission, lag-1 may represent a key human host-adaptive trait. Finally, we suggest that the identification of a specific modification of LPS that is required for resistance to classical complement-mediated killing, inhibits phagocytosis by neutrophils and promotes survival in vivo, could inform the design of novel therapeutic approaches that subvert the capacity of L. pneumophila to resist innate immune killing during severe human infection.
Bacterial culture
L. pneumophila Sg-1 isolates from the Scottish Haemophilus, Legionella, Meningococcus & Pneumococcus Reference Laboratory (SHLMPRL), Scottish Microbiology Reference Laboratories (SMiRL), Glasgow collection were grown on buffered charcoal yeast extract plates (Legionella CYE agar base-Oxoid, BCYE Growth Supplement SR0110-Oxoid) for 3 days at 37 °C. Three colonies were inoculated is of supplemented yeast extract broth (YEB, Yeast extract—Fluka, BCYE Growth Supplement SR0110-Oxoid) for 24 h at 37 °C 200 rpm. Strains were subcultured by transferring 150 µL of the previous inoculation into 15 mL of YEB at 37 °C 200 rpm until it reaches O.D. 600 nm ≈ 0.8. When necessary, media was supplemented with 10 µg/mL of chloramphenicol (Sigma-Aldrich) for plasmid maintenance. After submission of the first version of the manuscript, the BCYE Growth Supplement SR0110 from Oxoid was discontinued. The data from Supplementary Fig. 10 was obtained during revision with bacteria cultured with the alternative BCYE Growth Supplement LS0053 (E&O Laboratories Limited) to post-exponential phase O.D. 600 nm ≈ 1.4, and on Legionella BCYE with GVPC pre-poured plates (PP0870, E&O Laboratories Limited). Escherichia coli cells were cultured at 37 °C in Luria-Bertani (LB) broth or agar (Melford) supplemented with 10 µg/mL of chloramphenicol (Sigma-Aldrich) or 100 µg/mL of ampicillin (Sigma-Aldrich). For blue-white colony screening, LB agar plates were spread with 2% X-gal (Melford).
Whole-genome sequencing
Four-hundred seventy-four Legionella isolates from the SHLMPRL isolate collection were recovered from frozen stocks and sent to MicrobesNG (microbesng.uk, Birmingham, United Kingdom) for DNA extraction and whole-genome sequencing as follows: plated cultures of each isolate were inoculated into a suspension of plastic beads in a cryopreservative (Microbank™, Pro-Lab Diagnostics UK, United Kingdom). Three beads were washed with extraction buffer containing lysozyme (final concentration 0.1 mg/mL) and RNase A (ITW Reagents, Barcelona, Spain) (final concentration 0.1 mg/mL), incubated for 25 min at 37 °C. Proteinase K (VWR Chemicals, Ohio, USA) (final concentration 0.1 mg/mL) and SDS (Sigma-Aldrich, Missouri, USA) (final concentration 0.5% v/v) were added and incubated for 5 min at 65 °C. Genomic DNA was purified using an equal volume of SPRI beads and resuspended in EB buffer (Qiagen, Germany).
DNA was quantified with the Quant-iT dsDNA HS kit (ThermoFisher Scientific) assay in an Eppendorf AF2200 plate reader (Eppendorf UK Ltd, United Kingdom). Genomic DNA libraries were prepared using the Nextera XT Library Prep Kit (Illumina, San Diego, USA) following the manufacturer’s protocol with the following modifications: 2 ng of DNA were used as input, and PCR elongation time was increased to 1 min from 30 s. DNA quantification and library preparation were carried out on a Hamilton Microlab STAR automated liquid handling system (Hamilton Bonaduz AG, Switzerland). Pooled libraries were quantified using the Kapa Biosystems Library Quantification Kit for Illumina on a Roche light cycler 96 qPCR machine. Libraries were sequenced with the Illumina HiSeq using a 250 bp paired end protocol.
Sequence processing and analysis
Reads were trimmed with Trimmomatic (v0.36) using default settings and de novo assembly was performed using SPAdes (v3.10.0) and contigs were reordered with Mauve Contig Reorderer (Muave v2.4.0) 70 . Gene annotation and functional prediction for each assembly was generated using Prokka (v1.12). The quality of whole-genome sequencing was assessed using Quast (v4.5) and genomes that exceeded the following metrics were selected as high-quality assemblies for analyses. (i) Minimum 1.5 Mbp aligned to the L. pneumophila Philadelphia reference (total aligned length), (ii) Maximum duplication ratio of 1.03 (based on distribution), (iii) Minimum total length of contigs larger than 50 Kbp is 1 Mbp. To maximise the fraction of core genome that can be aligned, isolates belonging to the minor subspecies, ( L. pneumophila subsp. fraseri , L. pneumophila subspecies pascullei and L. pneumophila subspecies raphaeli ) were not included in downstream analyses. Whole-genome sequences are available on the European Nucleotide Archive (ENA) database under Bioproject number PRJEB31628 [ https://www.ebi.ac.uk/ena/browser/view/PRJEB31628 ]. Functional prediction of non-synonymous mutations in lag-1 were predicted using PROVEAN (v1.1.3) 71 .
Twelve genes from the LPS cluster, specific to Sg-1 and overlapped by k -mers enriched in clinical isolates ( lpg0762 , lpg0766 , lpg0767 , lpg0768 , lpg0771 , lpg0772 , lpg0773 , lpg0777 , lpg0778 , lpg0779 , lpg0780 , lpg0781 ). Orthologs of these 12 genes were identified. Genes that were not conserved in all 23 genomes analysed were removed ( lpg0771 and lpg0777 ). Paralogs of lpg0778 were also removed. Genes were translated and aligned at the codon level using TranslatorX and Mafft. Alignments for all 12 genes were concatenated. Phylogenetic networks were generated using uncorrected P-distances with the equal angle method in Splitstree v4.14.6 72 .
Sequence alignments were visualised in Artemis Comparison Tool v.17.0.1 and figures generated with Easyfig v2.2.2 73 , 74 .
Genome-wide association analyses
Five-hundred five high-quality publicly available genome assemblies of L. pneumophila subsp. pneumophila were downloaded on the 2nd of March 2017 for inclusion in these analyses. Quality criteria for inclusion of published genomes: fewer than 200 contigs of 1 kb or longer, less than 1.03 duplication ratio, not a subspecies or taxonomic outlier (bright green or grey clade), contigs >50 Kb add up to at least 1 Mbp, not >700 uncalled bases per 100 Kb. At least 3.2 Mbp in length.
From the combined dataset of 902 isolates, we were able to extract metadata describing the isolation source for 758 isolates (environmental or clinical). A ML phylogenetic tree was then constructed using RAxML (v8.2.10) from the 1.8 Mbp core genome alignment generated using Parsnp v1.2 ( https://github.com/marbl/parsnp ). This phylogenetic tree was used as input for the subsampling approach. An automated subsampling algorithm was used to reduce the redundancy of the dataset by iteratively removing one of each pair of isolates, which had the same source type. We implemented this algorithm in a modified version of a phylogeny-based dataset reduction tool called Treemmer (downloaded: 8th March 2018) 75 . The modified subsampling script is available on github.com/bawee/Treemmer. Specifically, we added the ability to take into account phenotypic categories (i.e., clinical or environmental) before iteratively removing one taxon from each pair of the most closely related taxa in the phylogeny. This was repeated until the minimum distance between the pairs of isolates with the same phenotype reached a user-defined threshold. This approach was performed with increasing minimum patristic distance thresholds between pairs on the tree, ranging from 0.0001 (≈180 SNPs), 0.001 (≈1800) and 0.01 (≈18,000 SNPs). The subsampled datasets contained 452 (60%), 382 (50%) and 353 (47%) of the original isolates, respectively.
GWAS analysis based on k -mer enrichment was performed using SEER (v1.1.3alpha) 76 . The frequency of k- mers between 12 and 100 bp long were counted with fsm-lite v1.0 (-m 12 -M 100). Only k -mers present in 5% and 95% of the total number of isolates (-s < 5%> -S < 95%>) were used and -maf 0.05 (minimum allele frequency) was used when performing the SEER analysis. The frequency and distribution in clinical and environmental isolates were evaluated individually for each k- mer. Pan-GWAS was performed using the homologue clusters generated by ROARY (v3.12.0) using the following settings (-i 95 -s) 32 . The association analyses were calculated using SCOARY (v1.6.9) 33 . The following thresholds were used to identify the highest-scoring associations: Naive p -value < 0.05, p -value after Bonferroni correction <0.05, Benjamini–Hochberg p -value < 0.05 and Empirical p -value after 500 label-switching permutations <0.05 (-e 500).
Molecular biology
For lag-1 cloning, primers (Variant 1: Fwd- GGC C GA ATT C GT AAG GAA AAA TAA TTT ATC, Rev- CCC GGA TCC TTA TGT TGA ATA AGC TAA CTT GTT TGA TGT; Variant 2: Fwd – GGC C GA ATT C GT AAG GAA AAA TAA TTT ATC, Rev – AT G GAT CC T TAC ATC ATC ACC ATC ATC ATT GTT GAA TAA GCC AAC; Variant 3: Fwd – GGC C GA ATT C AC ATG CAA GAA TAA TTTA, Rev – CCC GGA TCC GCT TAC GTA ATA TAA GCT AAC TTA TTT GAT GTG Eco RI and Bam HI restriction sites in bold) were used to amplify a 1221 bp fragment of chromosomal DNA from L. pneumophila strains 4454, 4449 and 4471, encoding the variant 1, 2 and 3 of lag-1 allele and upstream region, respectively. These fragments were blunt cloned with StrataClone Blunt PCR Cloning kit (Agilent) into vector pSC-B and transformed into E. coli . Plasmids from transformants were purified with Monarch Plasmid DNA Miniprep Kit and digested with Eco RI and Bam HI enzymes (New England BioLabs). lag-1 gene was ligated to pMMB207 using T4 DNA ligase (New England BioLabs) and both pMMB207:: lag-1 and empty plasmid were electroporated into the electrocompetent mAb 3/1-negative strains 4681 and 4312 using a Gene Pulser II (Bio-Rad) following published protocols 77 . To confirm O -acetyltransferase activity of the lag-1 cloned bacteria, transformed and positive control strains were fixed in 10% formalin (VWR) for 20 min and incubated with 1:100 of mouse mAb 3/1 (Dresden panel) for 30 min in ice prior incubation with 1:100 polyclonal AlexaFluor 488 conjugated donkey anti-mouse IgG (H + L) (Cat no. A21202, ThermoFisher Scientific). After incubation on ice for 30 min, the bacteria were washed and resuspended in PBS for flow cytometry analysis. DsRed fluorescent protein expression was introduced into WT lag-1 -positive and -negative strains by electroporation with pSW001 plasmid (pMMB207C, ΔlacIq, constitutive dsred ) 78 . DsRed expression was confirmed by flow cytometry. Formaldehyde-fixed samples were analysed on a BD LSRFortessa X-20 flow cytometer (BD biosciences) and data analysed using FlowJo software (v10).
Ethics statement
Ethical approval for the collection of blood from anonymous donors was granted by the University of Edinburgh Research Ethics Committee. This study was reviewed by the University of Edinburgh, College of Medicine Ethics Committee (2009/01) and subsequently renewed by the Lothian Research Ethics Committee (11/AL/0168). Written informed consent was received from all volunteers participating in the study. No compensation was provided to the volunteers. All animal experiments were approved by governmental animal welfare committees by the LaGeSo (Landesamt für Gesundheit und Soziales) Berlin. All experiments are assigned to project G0334/17.
Plasma and serum killing assays
Human blood was obtained from healthy volunteers in syringes treated with anticoagulant citrate dextrose. Plasma was obtained by centrifuging whole blood for 15 min at 25 °C at 1200 × g without the centrifuge brake and collecting the layer above the buffy coat. Normal serum was collected from human blood obtained from healthy volunteers in BD Vacutainer serum tubes. Tubes were centrifuged for 10 min at 25 °C 1200 × g and the supernatant collected. Plasma and serum were stored following immediate freezing at –80 °C. Samples used were negative for anti- L. pneumophila antibodies as determined by standard diagnostic serology methods (immune fluorescence test). The ability of L. pneumophila strains to resist killing by human plasma and serum was determined by incubating 1 × 10 5 bacterial cells in 90% plasma or serum at 37 °C for 60 min. Serial dilutions were plated in duplicate on BCYE media, incubated at 37 °C for 3 days before enumeration. Heat inactivation of plasma was carried out by incubation at 56 °C for 30 min in a water bath. Both plasma, serum, heat-inactivated plasma and heat-inactivated serum were centrifuged at 4000 × g for 20 min at 4 °C prior use. Inhibition of complement pathways was carried out by adding 12.5 mM EDTA (Sigma-Aldrich), 12.5 mM EGTA/Mg 2+ (Sigma-Aldrich) or 100 mM mannose (Acros Organics) to normal human serum or by using C1q- and Factor B-depleted serum (Pathway Diagnostics). GraphPad prism v7 was used for statistical analysis.
C3 binding assays
For analysis of C3 deposition on the surface of L. pneumophila , bacteria were grown to post-exponential phase and fixed in 10% formalin (VWR) for 20 min. Nunc maxisorp ELISA plates were coated with 1 × 10 7 bacteria per well overnight at 4 °C, blocked with 1% w/v bovine serum albumin (Sigma-Aldrich) in PBS for 2 h and incubated with serial dilution of human serum for 2 h at room temperature. Binding of C3 at the surface of the bacteria was determined by incubating with 1:100 FITC conjugated goat F(ab’)2 anti-human C3 complement (Protos Immunoresearch, Cat no. 365) for 2 h at room temperature and fluorescence detected in using the CLARIOstar fluorescence plate reader (BMG Labtech). GraphPad prism v7 was used for statistical analysis.
Neutrophil phagocytosis assay
Human venous blood was obtained from healthy volunteers in syringes treated with anticoagulant citrate dextrose and the isolation of neutrophils was performed by Ficoll/Histopaque centrifugation 79 . L. pneumophila -DsRed strains and L. pneumophila isogenic strains were labelled with 0.5 mg/mL of FITC (Sigma) solution for 30 min on ice in the dark. After several washes with PBS to remove any non-associated FITC, the bacteria were incubated with 10% normal human serum for 15 min at 37 °C before incubation with 2.5 × 10 5 purified neutrophils (MOI 10). Infection was maintained at 37 °C, 5% CO 2 with orbital shaking at 600 rpm for 30 min. Cells were then fixed in 10% formalin (VWR) for 20 min and kept in PBS for flow cytometry analysis. Samples were analysed on a BD LSRFortessa X-20 flow cytometer using the FACSDiva software v9 (BD biosciences) and data analysed using FlowJo software (v10). Neutrophils were selected by their FSC/SSC profile and doublets excluded using FSC-H/FSC-A ratios. MFI was calculated in the total single cell population (Supplementary Fig. 9 ). GraphPad prism v7 was used for statistical analysis.
Confocal microscopy
L. pneumophila -infected neutrophils were incubated for 20 min with WGA-AF555 (Invitrogen) and with Hoechst 33342 (Thermo Scientific) for 5 min for cytoplasmatic membrane and nucleus staining, respectively. Cells were attached to a microscope slide using cytospins and analysed using the Zeiss LSM 710 inverted confocal. The images were obtained using a maximum intensity projection algorithm.
Murine pulmonary infection
All mice used were 12–14 weeks old, female WT on a C57BL/6J background. Mice were either purchased from Charles River or from institutional breeding stock at the Charité-University Medicine, Berlin. Upon transfer to the animal unit, mice were kept in ventilated cages at a conditioned room temperature at 22 °C +/− 2 °C and humidity at 55% +/− 5%. A dark/light cycle of 12 h/12 h was maintained. All experiments were approved by the LaGeSo (landesamt für Gesundheit und Soziales) Berlin. Anaesthetised mice were intranasally infected with 1 × 10 6 L. pneumophila in 40 µL of PBS. 96 h post infection, mice were anesthetised and euthanized through final blood withdrawal. Lungs were flushed via the pulmonary artery with sterile 0.9% NaCl, subsequently removed and homogenised using a cell strainer (100 µM, BD Bioscience). For determination of bacterial counts, the lung homogenates were lysed with 0.2% Triton X-100. Serial dilutions were plated on BCYE agar and incubated for 3 days at 37 °C.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Data availability
The sequence data that support the findings of the study have been deposited in the European Nucleotide Archive (ENA) database under Bioproject number PRJEB31628 [ https://www.ebi.ac.uk/ena/browser/view/PRJEB31628 ]. Accession numbers of each genome used is listed in Supplementary Data S1 . A source data file is provided with this paper. Source data are provided with this paper.
Code availability
We have implemented a modified version of the subsampling software Treemmer ( https://github.com/fmenardo/Treemmer ) 75 , which is available at http://github.com/bawee/Treemmer .
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Acknowledgements
This work was supported by funding to JRF from the Chief Scientists Office Scotland (Grant No ETM/421), the Wellcome Trust Collaborative award (Grant No. 201531/Z/16/Z), the Medical Research Council (UK) award MR/N02995X/1, and Biotechnology and Biological Sciences Research Council institute strategic grant funding (ISP2) (Grant no. BB/P013740/1). Computing resources were supported in part by MRC CLIMB (Grant Number: MR/L015080/1). A.B.K. was supported by the International Max-Planck Research School (IMPRS-IDI), and B.O. by the German Research Foundation (OP 86/12-1 and SFB-TR84A1/A5). We are grateful to Prof. Carmen Buchrieser for providing plasmids pMMB207 and pSW001. Thanks also to Prof. Kenneth Baillie, Dr. Sara Clohisey-Hendry and Dr. Clark Russell for organising the human blood donation study and the volunteers from the Roslin Institute who provided blood samples for the serum and plasma killing assays and neutrophil assays.
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The Roslin Institute, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, Scotland, UK
Bryan A. Wee, Joana Alves, Fiona A. Sargison, Amy Pickering, Jamie Gorzynski & J. Ross Fitzgerald
Bacterial Respiratory Infections Service (Ex Mycobacteria), Scottish Microbiology Reference Laboratory, Glasgow, Scotland, UK
Diane S. J. Lindsay & Andrew J. Smith
Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité Universitätsmedizin Berlin, Berlin, Germany
Ann-Brit Klatt & Bastian Opitz
NHS National Services Scotland, Health Protection Scotland, Glasgow, Scotland, UK
Ross L. Cameron
Department of Mathematics and Statistics, University of Helsinki, Helsinki, Finland
Jukka Corander
Department of Biostatistics, University of Oslo, Oslo, Norway
Helsinki Institute for Information Technology, Department of Computer Science, Aalto University, Aalto, Finland
Pekka Marttinen
College of Medical, Veterinary & Life Sciences, Glasgow Dental Hospital & School, University of Glasgow, Glasgow, UK
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B.A.W. designed and performed the population genomic analysis and wrote the paper and J.A. designed and performed the functional experiments, analysed data and wrote the paper; D.S.J.L. provided the strains, carried out bacterial subtyping and conceptualised the study, A.B.K. performed the mouse experiments, F.A.S performed functional experiments, A.P. provided scientific and technical support for cloning, J.G. analysed data, R.L.C., J.C. and P. M. developed analytical tools, B.O. designed the murine infection studies, A.J.S. provided strains and conceptualised the study, J.R.F. conceptualised the study, analysed data and wrote the paper.
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Wee, B.A., Alves, J., Lindsay, D.S.J. et al. Population analysis of Legionella pneumophila reveals a basis for resistance to complement-mediated killing. Nat Commun 12 , 7165 (2021). https://doi.org/10.1038/s41467-021-27478-z
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Legionnaires’ Disease: Update on Diagnosis and Treatment
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Legionellosis is the infection caused by bacteria of the genus Legionella , including a non-pneumonic influenza-like syndrome, and Legionnaires’ disease is a more serious illness characterized by pneumonia. Legionellosis is becoming increasingly important as a public health problem throughout the world; although it is an underreported disease, studies have consistently documented a high incidence. In addition, health costs associated with the disease are high. Diagnosis of Legionnaires’ disease is based mainly on the detection of Legionella pneumophila serogroup 1 antigen in urine. However, there have been advances in detection tests for patients with legionellosis. New methodologies show greater sensitivity and specificity, detect more species and serogroups of Legionella spp., and have the potential for use in epidemiological studies. Testing for Legionella spp. is recommended at hospital admission for severe community-acquired pneumonia, and antibiotics directed against Legionella spp. should be included early as empirical therapy. Inadequate or delayed antibiotic treatment in Legionella pneumonia has been associated with a worse prognosis. Either a fluoroquinolone (levofloxacin or moxifloxacin) or a macrolide (azithromycin preferred) is the recommended first-line therapy for Legionnaires’ disease; however, little information is available regarding adverse events or complications, or about the duration of antibiotic therapy and its association with clinical outcomes. Most published studies evaluating antibiotic treatment for Legionnaires’ disease are observational and consequently susceptible to bias and confounding. Well-designed studies are needed to assess the usefulness of diagnostic tests regarding clinical outcomes, as well as randomized trials comparing fluoroquinolones and macrolides or combination therapy that evaluate outcomes and adverse events.
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Avoid common mistakes on your manuscript.
Legionellosis is becoming an important public health threat. The incidence of legionellosis is rising and the health costs associated with the disease are high. |
Diagnosis of Legionnaires’ disease is based mainly on the detection of serogroup 1 antigen in urine. |
Other diagnostic methods such as culture and PCR allow the detection of cases due to most species and serogroups but have some drawbacks. |
Most guidelines recommend the use of diagnostic tests in patients with severe community-acquired pneumonia or whenever Legionnaires’ disease is suspected based on epidemiological or clinical features. |
Either a fluoroquinolone (levofloxacin or moxifloxacin) or a macrolide (azithromycin preferred) is the recommended first-line therapy for Legionnaires’ disease. |
Delayed antibiotic treatment in pneumonia is a factor associated with a worse prognosis. |
Randomized trials comparing fluoroquinolones with macrolides and evaluating adverse events in the treatment of pneumonia are now needed. |
The Scale of the Problem
Legionella is a genus of intracellular, aerobic, non-sporing, Gram-negative bacteria. Legionella species have been found worldwide, mainly from soil and natural or artificial aqueous reservoirs including freshwater streams, lakes, showers, pools, sprinklers, or cooling towers [ 1 , 2 ]. Most human infections are caused by Legionella pneumophila , but other disease-causing species reported include L. longbeachae in Australia and New Zealand. Numerous serogroups of L. pneumophila and other Legionella spp. have been discovered; currently around 65 species of Legionella have been described [ 1 , 3 , 4 , 5 , 6 ].
The term legionellosis refers to the infection caused by bacteria of the genus Legionella . The clinical spectrum includes a non-pneumonic, influenza-like syndrome known as Pontiac fever, and Legionnaires’ disease, which is a more severe presentation characterized by pneumonia. Legionella pneumophila associated with human infection was first described in 1977, after an outbreak of severe pneumonia at the American Legion Convention held in 1976 in Philadelphia, USA [ 1 , 2 ]. Legionnaires’ disease has clinical manifestations like those of other types of pneumonia, and as it lacks a particular pattern, microbiological tests are the key element for its diagnosis. Most cases of Legionnaires’ disease are sporadic, and more than 70% are community-acquired, though some are associated with travel or health care [ 7 ]. Legionella spp. are estimated to be the causative agent of 2–10% of community-acquired pneumonia (CAP) cases, with a higher incidence in severe disease [ 8 , 9 , 10 , 11 ]. However, studies have shown that adhering to guidelines and recommendations for diagnostic testing results in poor sensitivity for identifying patients with Legionnaires’ disease [ 12 ]. Predisposing conditions for legionellosis include advanced age, male sex, immunosuppression, chronic lung disease, alcohol abuse, malignancies, iron overload, anti-tumor necrosis factor (TNF)-alpha treatment and smoking (current or past) [ 1 , 2 , 7 , 13 ].
In the present study, we performed a narrative review to update information regarding the diagnosis and treatment of Legionnaires’ disease. This article is based on previously published work and does not contain novel data or information related to human or animal studies; no permission was required from the institutional ethics review board.
Morbidity and mortality in Legionnaires' disease remains high. Intensive care unit (ICU) admission rates reported in studies range from 20 to 27% [ 1 , 7 , 14 , 15 , 16 ]. Andrea et al. [ 14 ] also found elevated frequency of invasive mechanical ventilation, septic shock, acute respiratory distress syndrome, and acute kidney injury among those patients requiring ICU admission. Moreover, Legionnaires' disease has an overall mortality rate of 4–18% [ 7 , 14 , 15 , 16 , 17 , 18 ]. However, mortality is higher in patients with nosocomial Legionella pneumonia, immunocompromised individuals, and those requiring ICU admission. Regueiro-Mira et al. [ 15 ] found that mortality was 4.6% in medical wards compared with 23.1% in patients transferred to the ICU. Other studies have documented mortality rates close to 40% in transplant recipients or cases of hospital-acquired infection [ 1 , 13 , 19 , 20 , 21 ].
Although information on legionellosis has improved in recent decades, its incidence remains unknown, mainly because it is underdiagnosed and underreported. Countries differ in terms of their level of surveillance, diagnostic methods, and investigation efforts [ 1 , 2 , 22 , 23 ]. An increase in the incidence of the condition has been documented, although the causes are not entirely clear. The growth may be due to an increase in infections or an aging population, or perhaps to changes in climate, reporting criteria, or testing practices (for example, the increase in the use of urine antigen testing or polymerase chain reaction), or to a combination of these factors [ 23 , 24 ]. A study of all cases of legionellosis reported to the US Centers for Disease Control and Prevention from 1990 through 2005 found an increase in reported cases from a mean of 1268 yearly cases before 2003 to more than 2000 from 2003 through 2005 [ 25 ]. More recently, 6141 cases were reported in 2016 and 7458 in 2017 [ 26 ]. Another retrospective study evaluated Legionnaires’ disease-associated hospitalizations using the US National Hospital Discharge Survey (NHDS) data from 2006 to 2010, and found significant increases in Legionnaires’ over the 5-year study period (from 5.37 per 100,000 population in 2006 to 9.66 in 2010, with a peak in 2009 of 17.07) [ 22 ]. Likewise, the European Legionnaires’ disease Surveillance Network (ELDSNet) reported that the notification rates for Legionnaires’ disease have nearly doubled in the past few years, from 1.4 in 2015 to 2.2 per 100,000 population in 2019, and mostly involved men aged 65 years and above. Four countries (France, Germany, Italy, and Spain) accounted for nearly 70% of all reported cases [ 27 ]. Similar increases in the incidence of Legionnaires’ disease in recent years have been reported in other studies [ 28 , 29 ].
Estimates of the costs associated with Legionnaires’ disease could also help to draw attention to the scale of the problem and to the need for prevention and treatment efforts. The studies estimate direct medical costs incurred due to hospitalizations and emergency department visits, and those related to productivity losses caused by absenteeism and premature deaths. Legionnaires’ disease is the second most expensive waterborne disease reported in the United States, with the cost per hospital stay ranging from US$ 7950 to $149,000 in terms of direct health care costs derived from emergency room visits and hospitalizations [ 30 ]. Similarly, Baker-Goering et al. [ 31 ] assessed productivity losses combined with existing estimates of medical costs of Legionnaires’ disease in the United States for 2014. The economic burden of Legionnaires’ disease was more than double when lifetime productivity losses were added to medical costs and was approximately US$ 835 million, including US$ 21 million caused by absenteeism and US$ 412 million caused by premature deaths. Moreover, a study of Legionnaires’ disease in Belgium evaluated disability-adjusted life year (DALY) rates, with DALYs being the sum of the years of life lost due to premature mortality and the years lived with a disability due to a disease or health condition in a population. One DALY is the loss of the equivalent of 1 year of full health. Legionnaires’ disease caused 3.05 DALYs per case and 8147 total DALYs in Belgium in 2017, which corresponds to 71.9 (95% uncertainty interval: 39.33–109.75) DALYs per 100,000 persons [ 29 ].
Given the rising incidence and high costs associated with the disease, legionellosis is gradually becoming a major public health threat. Data on the burden of the disease show the value of studying the epidemiology and of increasing investment in measures to prevent Legionnaires’ disease, such as water management programs and investigations of the outbreaks.
Diagnosis of Legionnaires’ Disease
The diagnosis of legionellosis is based on a combination of the presence of clinical and/or radiological symptoms and laboratory tests. These tests are not routinely performed by the clinical microbiology laboratory and therefore must be specifically requested (Table 1 ). According to a 2019 European Centre for Disease Prevention and Control (ECDC) report, most cases in Europe (90%) are diagnosed using the urine antigen test (UAT) method. This has been a consistent finding over the past decade. Polymerase chain reaction (PCR) detects 9% of the total number of reported cases, and the proportion of cases diagnosed or confirmed by culture is 10% [ 32 ]. The species L. pneumophila alone is responsible for more than 90% of cases of Legionnaires’ disease diagnosed worldwide. In Europe, most human cases (80%) are caused solely by L. pneumophila serogroup 1 (Lp1), with other serogroups and species accounting for 16% and 3% of infections, respectively [ 27 ]. In Australia and New Zealand, L. longbeachae is the predominant species and in 2019 accounted for up to 60% of the reported cases [ 4 , 5 , 33 ].
Culture of respiratory samples is still considered the gold standard for diagnosis of legionellosis, but it is a very demanding test requiring considerable expertise and growth for several days on complex media. Legionella does not grow on the standard media used in microbiology laboratories, and a specific medium containing yeast extract and activated charcoal (buffered charcoal yeast extract, BCYE) is needed. Legionella has been more successfully isolated from lower respiratory tract samples than from nasopharyngeal or throat swabs, and culture positivity in samples outside the respiratory tract is extremely rare [ 34 ]. The main advantage of the culture approach is that it enables isolation of the bacterial strain, which can be used for antibiotic sensitivity testing and for epidemiological typing analyses [ 35 ]. The availability of the clinical strain for comparison with environmental strains is one of the key points in epidemiological investigations for understanding the spread of legionellosis—hence the importance of culture in the diagnosis of legionellosis [ 36 ]. The disadvantages are of course the slowness of the analysis (which can take up to 14 days), poor sensitivity compared with other techniques, and the restricted availability of suitable clinical material (preferably from the lower respiratory tract) [ 37 ]. Some guidelines have recommended the culture of Legionella in patients with severe CAP or whenever Legionnaires’ disease is suspected based on epidemiological or clinical features. In addition, Legionella cultures should be routinely performed on invasive respiratory samples or for patients who have a positive Legionella UAT [ 38 ].
The introduction in the late 1990s of enzyme immunoassays for the detection of L. pneumophila antigen in urine made it possible to speed up the diagnosis of Legionnaires’ disease [ 39 ]. The antigen detected is a bacterial lipopolysaccharide (LPS), on whose diversity of structure and antigenicity the identification of L. pneumophila serogroups is based. The antigen is detectable in most patients as early as 1–3 days after the onset of symptoms but may persist for several weeks or months [ 40 ]. The development and spread of rapid urinary antigen detection kits such as lateral flow immunochromatographic assays or fluorescent immunoassays has revolutionized the diagnosis of legionellosis and allows for early adaptation of the antibiotic therapy. These methods can be implemented in a point-of-care format and today constitute the first-line diagnostic tests for Legionnaires' disease. Commercially available kits mainly detect Lp1 LPS and do not perform as well with strains belonging to other serogroups. The sensitivity therefore depends on the serotype causing the infection and may vary from 86% for Lp1 to 74–79% when considering all serogroups [ 41 ].
There are some differences in UAT use criteria between countries. In most guidelines, routine use of the Legionella UAT is not recommended. In this regard, American and European guidelines advise the use of a UAT for Legionnaires' disease only for adults with severe CAP or in patients with risk factors, such as recent travel or a link to a Legionella outbreak. In contrast, other guidelines recommend Legionella UAT for all patients admitted with CAP [ 42 , 43 ]. An important drawback of UAT is the occurrence of non-specific signals, possibly due to the presence in urine of immunocomplexes that interact with the test and give false positive results [ 44 , 45 ]. This phenomenon has been known since the 1980s and can be resolved by heating the urine for 5 min at 100 °C [ 46 , 47 ]. Since the Legionella LPS is heat-stable, heating of the urine sample allows the release of bacterial polysaccharides from antibody complexes and eliminates the nonspecific interferences. This simple heating procedure is recommended for the confirmation of any positive test [ 39 , 48 , 49 ]. Another factor that can cause false positive results is the persistence of Legionella antigen in the urine. Even recent studies have shown that the antigen can still be detected several months (up to 1 year) after the onset of symptoms. This occurs in about 10% of cases and mainly in patients with severe underlying diseases or in immunocompromised individuals [ 50 , 51 ].
The advantages of urinary antigen detection over other diagnostic methods are considerable. Urine samples are easily obtained, the antigen is detectable very early in the course of the disease, and the test is quick and easy to perform. For the clinician, the test’s usefulness lies in its high positive predictive value. In the presence of suggestive symptoms, while a positive result strongly suggests legionellosis, a negative test does not exclude Legionella spp. as the cause of the pneumonia [ 52 , 53 ]. In 2019, approximately 90% of reported cases in Europe were diagnosed by urinary antigen. This percentage has increased over the years and has certainly contributed to better diagnosis of the disease [ 27 , 32 ]. Nevertheless, experts wonder about the importance of cases that are not detected by UAT because they are due to other species or other serogroups for which these tests do not seem to perform as well as for Lp1 [ 54 , 55 ]. Most commercially available UAT tests are based on lipopolysaccharide detection, which is why they are mainly able to diagnose pneumonia due to L. pneumophila serogroup 1 [ 41 , 56 ]. Recently, UAT tests based on the detection of other antigens such as ribosomal protein L7/L12 have been developed and launched on the market. Although scientific studies are so far limited, these tests seem very promising and have the capacity to detect other serogroups and even other species [ 57 , 58 ].
PCR is a method that has the potential to detect all known Legionella species. It is a rapid test with good specificity and sensitivity for Legionella spp., especially when performed on respiratory tract samples (bronchial secretions, bronchoalveolar lavage [BAL], biopsies, or sputum) [ 59 ]. The possibility of also performing this assay in materials such as urine or serum would avoid the problem of having to obtain a respiratory sample. Unfortunately, the few studies available that have evaluated PCR in serum and urine have shown low sensitivity [ 60 , 61 , 62 , 63 ]. Numerous panels are commercially available and allow the amplification of L. pneumophila and/or Legionella spp. The design of specific PCRs also makes it possible to design specific assays for the detection of particular serogroups [ 64 , 65 , 66 ] and specific sequence types [ 67 , 68 ]. In general, Legionella nucleic acid-based detection offers significant advantages in terms of sensitivity and speed. However, there are several disadvantages and limitations. PCR may not be ideal for testing non-lower respiratory tract samples such as urine and serum. In addition, one drawback of all nucleic acid amplification methods is the difficulty in assessing bacterial viability (i.e., after antibiotic treatment) and causing false positive results. Finally, nucleic acid amplification technologies still require specially trained personnel and sophisticated machines, although thanks to the technological advances we have witnessed in recent years, they are increasingly accessible to a wider range of laboratories with a moderate budget [ 35 , 38 ]. Compared with UAT, PCR offers higher sensitivity. One study revealed an additional diagnosis of 18–30% of LD cases [ 69 ]. Similarly, an Italian study showed that the use of real-time PCR in addition to UAT and culture resulted in an increase of 18% of the LD cases [ 70 ].
In Europe, the use of PCR seems to have remained quite restricted (< 10%) despite its many advantages [ 32 ]. With the increasing use of multi-syndromic PCR panels targeting multiple microorganisms simultaneously, the use of PCR to detect Legionella is expected to increase in the diagnosis of CAP. Some of these PCR panels have been developed for use with BALs, while other tests can also be performed with samples from the upper respiratory tract such as nasopharyngeal smears and sputum [ 71 ]. The growth in the popularity of whole-genome sequencing techniques reflects their high diagnostic potential, and they are becoming increasingly affordable. Recent studies also demonstrate their value for microbial identification and in epidemiological studies [ 72 , 73 , 74 ].
Antimicrobial Treatment
Delayed therapy and prognosis.
The literature on the association between the time of the onset of therapy and the prognosis in Legionnaires’ disease is scarce [ 75 , 76 , 77 , 78 ]. In a retrospective study, Heath et al. [ 75 ] reported that the delay in antibiotic initiation after hospital admission and the total delay in appropriate antibiotic initiation after symptoms onset were both factors related to higher mortality in Legionella pneumonia. Following the diagnosis of pneumonia, the median delay before starting therapy was 5 days (range, 1–10 days) for patients who died, and 1 day for those who survived (range, 1–5 days) ( p < 0.001). In addition, the total delay after symptom onset in starting therapy ranged from 1 to 12 days (median, 6 days) for survivors and from 8 to 23 days (median, 11 days) for non-survivors ( p < 0.001). Another study compared the outcomes of patients with severe Legionella pneumonia requiring ICU care according to the delays in initiating fluoroquinolones and macrolides [ 76 ]. After logistic regression analysis, fluoroquinolone administration within 8 h of ICU admission was associated with reduced mortality. Moreover, a recent single-center, retrospective study carried out in Pisa, Italy, showed that delayed macrolide/levofloxacin therapy (> 24 h of hospital admission) was associated with a higher need for ICU admission [ 77 ]. The frequency of ICU admission was 54.2% among those who received macrolides/levofloxacin therapy within 24 h of admission and 80.4% in those who initiated therapy later. Interestingly, the investigators also found that the delay in the administration of antibiotics against Legionella was directly correlated with a delay in the performance of the UAT. Similarly, in a study that reported 23% inadequate empirical coverage during the first 2 days of hospitalization in patients diagnosed with Legionella pneumonia, factors related to inadequate coverage were older age, renal replacement therapy, chronic heart failure, nonsmoking status, and having risk for hospital-acquired pneumonia or multidrug-resistant pathogens [ 9 ].
These findings indicate that it is necessary to test for Legionella spp. at the time of hospital admission in severe CAP and to decide promptly whether to withdraw or continue antibiotic treatment according to the test results. This strategy improves antimicrobial prescription and prognosis in patients. If the UAT is not available at the time of CAP diagnosis, initial antimicrobial therapy should include a drug with activity against Legionella spp. It is recommended that antibiotics directed against Legionella spp. be included promptly in the empirical therapy of severe cases of CAP and in cases of immunocompromised patients. Inadequate or delayed antibiotic treatments in Legionella pneumonia have been associated with worse prognosis [ 75 , 76 , 77 ].
Fluoroquinolones or Macrolides
Several studies have evaluated the role of fluoroquinolones or macrolides in Legionella pneumonia. Most of them are observational, and the number of randomized trials is small. The current guidelines for CAP recommend either a fluoroquinolone (levofloxacin or moxifloxacin) or a macrolide (preferably azithromycin) as first-line therapy for Legionnaires’ disease [ 79 , 80 ].
In this regard, a recent systematic review and meta-analysis compared the effectiveness of fluoroquinolone versus macrolide monotherapy in Legionella pneumonia [ 81 ]. A total of 3525 patients from 21 publications were included in this meta-analysis. No difference in mortality was found between patients treated with fluoroquinolones and those treated with macrolides (6.9% vs 7.4%, pooled OR 0.94, 95% CI 0.71–1.25, p = 0.66). Similar results were reported for comparison of fluoroquinolones versus azithromycin and for fluoroquinolones versus clarithromycin. The mortality rates for fluoroquinolones and macrolides in studies that were solely ICU-based was also similar. Moreover, the authors also found no differences in the effect of these antibiotics on clinical cure, length of hospital stay, or the occurrence of complications. Likewise, Kato et al. [ 82 ] performed a meta-analysis comparing these treatment groups in terms of their efficacy and safety in Legionella pneumonia, including studies published until January 2020. Seventeen publications met the inclusion criteria. Clinical cure was comparable between the treatment groups, but overall and 30-day mortality was significantly higher for macrolides than for fluoroquinolones. However, in the subgroup analyses, levofloxacin significantly reduced the length of hospital stay compared with two specific macrolides (azithromycin and clarithromycin), although mortality did not differ significantly between the treatment groups. Nor were significant differences found in other outcomes such as time to apyrexia and adverse events.
The results of these systematic reviews and meta-analyses support the present guidelines regarding the use of a fluoroquinolone or a macrolide for the treatment of Legionella pneumonia. However, certain limitations of the current data should be recognized [ 81 , 82 , 83 ]. First, the usefulness of these antibiotics has not been extensively evaluated in some subgroups of patients, such as immunocompromised patients or those requiring admission to the ICU. Second, the reporting of adverse events or complications varies widely in the currently available studies. Third, the duration of antibiotic therapy and its association with clinical outcomes has not been sufficiently assessed. Fourth, most published studies are observational and are therefore susceptible to bias and confounders. Ideally, randomized trials comparing fluoroquinolones with macrolides for the treatment of Legionella pneumonia should be conducted. Finally, future studies need to evaluate and monitor antibiotic susceptibility data on Legionella spp. Some studies have documented, in general, low values of minimum inhibitory concentrations in environmental and clinical strains, although determinants of resistance to macrolides, such as the LpeAB active efflux system, have also rarely been found [ 84 , 85 , 86 ].
Combination Therapy
Several in vitro studies suggest a synergistic effect of combination therapy in isolates of Legionella spp . [ 87 , 88 , 89 , 90 ]. Martin et al. [ 87 ] tested the synergy of erythromycin, clarithromycin, and azithromycin, each in combination with ciprofloxacin and levofloxacin, against 41 isolates of Legionella . The authors did not report any antagonism. Synergy occurred mainly for the clarithromycin-levofloxacin and azithromycin-levofloxacin combinations. Similarly, another study evaluated the susceptibilities of 56 L. pneumophila isolates to levofloxacin, ofloxacin, erythromycin, and rifampin, finding that only levofloxacin plus rifampin demonstrated synergy [ 88 ]. Moreover, evaluating the intracellular susceptibility of several antibiotic combinations against L. pneumophila in infected macrophages, Descours et al. [ 89 ] found that macrolides, especially azithromycin, were synergistic with rifampicin against Legionella in this in vitro model.
Observational studies have also assessed the synergistic effect of combination therapy in patients with Legionella pneumonia. In a literature review evaluating the role of rifampin in the combination treatment of L. pneumophila pneumonia, Varner et al. [ 91 ] concluded that the data on the clinical advantage of rifampin combination therapy are unclear because of the possibility for patient selection bias and the absence of consistent comparators. In an observational cohort study, Grau et al. [ 92 ] found that patients who received rifampicin in addition to clarithromycin to treat Legionnaires’ disease had a 50% longer hospital stay and a trend towards elevated bilirubin levels. Combination therapy of clarithromycin and rifampicin had no additional benefit in prognosis compared with clarithromycin monotherapy. Similarly, Blázquez-Garrido et al. [ 93 ] compared macrolides versus levofloxacin, in addition to rifampicin, in a prospective, non-randomized study involving 292 patients with Legionella pneumonia. Although the use of levofloxacin was associated with fewer complications and shorter hospital stays compared with macrolides, the addition of rifampicin to the treatment regimen provided no additional benefit. Moreover, the efficacy of combination therapy with macrolides plus quinolones in patients with severe Legionella pneumonia has been reported [ 94 , 95 ]. In some of the cases described, combination therapy was used because of initial failure with a monotherapy. All the patients survived. Finally, a retrospective study compared the clinical effects of single-agent therapies, fluoroquinolones and macrolides, with combination therapy in the treatment of 22 patients with L. pneumophila pneumonia [ 96 ]. A fluoroquinolone combined with a macrolide may be able to improve the inflammation caused by L. pneumophila pneumonia, although there were no significant differences in outcomes.
Based on the literature reviewed here, combination therapy has most often been suggested for use in patients with treatment failure or severe disease, but the data available are limited. Only observational studies evaluating combination therapies in Legionella pneumonia have been performed, with small sample sizes; no randomized trials have been conducted. Nor have the adverse effects and possible drug-drug interactions been adequately evaluated in the studies carried out to date.
Duration of Treatment
According to the guidelines, the duration of antibiotic therapy in CAP should continue until the patient reaches clinical stability and for no less than a total of 5 days [ 80 ]. Several meta-analyses have demonstrated the efficacy of shorter courses of antibiotic therapy of 3–7 days [ 97 , 98 , 99 ]. However, most randomized trials evaluating the duration of antibiotic therapy had a low number of patients with Legionella pneumonia [ 100 , 101 , 102 ]. In a multicenter, noninferiority randomized clinical trial to validate Infectious Diseases Society of America/American Thoracic Society guidelines for the duration of antibiotic treatment in hospitalized patients with CAP, Uranga et al. [ 102 ] found clinical success to be similar between study groups (antibiotics for a minimum of 5 days or according to physicians). Of the 312 patients included in the study, 11 (3.5%) had Legionella pneumonia. Similarly, the number of patients with Legionella pneumonia was zero or not reported in other randomized trials evaluating short courses of antibiotic therapy in CAP [ 100 , 101 ]. In a retrospective study, Kuzman et al. [ 103 ] assessed the clinical usefulness of azithromycin in the treatment of serologically confirmed L. pneumophila pneumonia. The mean age of 16 patients was 42.8 years and they did not have severe chronic cardiac or pulmonary diseases or immunodeficiency. Azithromycin was administered orally for 5 days (first day 500 mg and remaining days 250 mg, once daily) or for 3 days (500 mg once daily). All patients were effectively cured with azithromycin.
The optimal duration of antibiotic therapy for Legionnaires’ disease has not been established, and it may vary according to the antimicrobial agent used, disease severity, and response to therapy. Of note, there are no randomized studies evaluating the duration of antibiotic therapy specifically in patients with Legionella pneumonia. The recommended total duration of antibiotic treatment in mild disease is 3–7 days, and until the patient is clinically stable and afebrile for at least 48 h. However, extended courses of therapy (10–14 days or longer based on clinical response) are reserved for immunocompromised patients, those with complications (e.g., empyema, or extrapulmonary infection), and patients with severe pneumonia or chronic comorbidities [ 104 , 105 , 106 ]. Levofloxacin or azithromycin for 7–10 days is recommended in cases of moderate to severe Legionella pneumonia. For immunocompromised hosts, a 21-day course of levofloxacin or a 10-day course of azithromycin is usually recommended [ 105 , 106 , 107 ].
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Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC; CB21/13/00009), Instituto de Salud Carlos III, Madrid, Spain funded this study. No funding or sponsorship was received for the publication of this article.
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Viasus, D., Gaia, V., Manzur-Barbur, C. et al. Legionnaires’ Disease: Update on Diagnosis and Treatment. Infect Dis Ther 11 , 973–986 (2022). https://doi.org/10.1007/s40121-022-00635-7
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Uncovering gaps in knowledge: a survey of belgian general practitioners’ awareness of legionnaires’ disease diagnostic testing.
1. Introduction
2. materials and methods, 2.1. study design, 2.2. research tool, 2.3. sample selection, 2.4. survey administration, 2.5. statistical methods, 3.1. response rate and nonresponse bias, 3.2. knowledge on legionnaires’ disease, 3.3. diagnostic tool availability in legionnaires’ disease, 3.4. antibiotic treatment in legionnaires’ disease, 3.5. additional information, 4. discussion, 4.1. summary of key findings, 4.2. interpretation of study findings, 4.3. study strengths and limitations, 5. conclusions, supplementary materials, author contributions, institutional review board statement, informed consent statement, data availability statement, acknowledgments, conflicts of interest.
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Click here to enlarge figure
| Trainees 35 (28%) | <10 years 26 (21%) | 10–19 years 17 (14%) | 20–29 years 13 (10%) | 30–40 years 19 (15%) | >40 years 15 (12%) | |||||
| Walloon Brabant 34 (27%) | Brussel-capital 21 (17%) | Hainaut 20 (16%) | Namur 12 (10%) | Liege 10 (8%) | Flemish Brabant 8 (6%) | West Flanders 6 (5%) | Luxemburg 6 (5%) | Antwerp 4 (3%) | Limburg 2 (2%) | East Flanders 2 (2%) |
| GPs Association 42 (34%) | Individual practice 34 (27%) | Fee-for-service practice 30 (24%) | Fixed-fee practice 19 (15%) | |||||||
| Urban 56 (45%) | Semi-urban 44 (35%) | Rural 25 (20%) |
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Moretti, M.; Van Nedervelde, J.; Vanstokstraeten, R.; Seyler, L.; Echahidi, F.; Prevost, B.; Martiny, D.; Wybo, I.; Michel, C. Uncovering Gaps in Knowledge: A Survey of Belgian General Practitioners’ Awareness of Legionnaires’ Disease Diagnostic Testing. Infect. Dis. Rep. 2024 , 16 , 820-827. https://doi.org/10.3390/idr16050063
Moretti M, Van Nedervelde J, Vanstokstraeten R, Seyler L, Echahidi F, Prevost B, Martiny D, Wybo I, Michel C. Uncovering Gaps in Knowledge: A Survey of Belgian General Practitioners’ Awareness of Legionnaires’ Disease Diagnostic Testing. Infectious Disease Reports . 2024; 16(5):820-827. https://doi.org/10.3390/idr16050063
Moretti, Marco, Julien Van Nedervelde, Robin Vanstokstraeten, Lucie Seyler, Fedoua Echahidi, Benoit Prevost, Delphine Martiny, Ingrid Wybo, and Charlotte Michel. 2024. "Uncovering Gaps in Knowledge: A Survey of Belgian General Practitioners’ Awareness of Legionnaires’ Disease Diagnostic Testing" Infectious Disease Reports 16, no. 5: 820-827. https://doi.org/10.3390/idr16050063
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Legionnaires' disease: clinical, epidemiological, and public health perspectives
Affiliation.
- 1 Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
- PMID: 9643385
Legionnaires' disease is a modern environmental infectious disease. It stems from the capacity of the causative agent, Legionella, to multiply within amoebae in warm water and the use, during the 20th century, of devices that maintain water at warm temperatures and produce aerosols. When contaminated with Legionella, aerosols consisting of respirable droplets place the bacteria in juxtaposition with alveolar macrophages, which, as with amoebae, they may parasitize, resulting in illness in susceptible persons. The disease is much more common than previously appreciated with at least 13,000 cases estimated to occur per year in the United States, based on prospective studies. Two highly specific tests, urinary antigen detection and sputum culture, are available for diagnosis during illness. With 60% to 80% sensitivity, urinary antigen tests rapidly detect antigens of Legionella pneumophila serogroup 1, which are responsible for 70% of the cases of legionnaires' disease; results can be available within a few hours. Culture of sputum is 50% to 60% sensitive, but several days are required for growth, and many patients do not produce sputum. Serologic testing, although useful for epidemiologic studies when convalescent-phase antibody titers can be compared with acute-phase titers, is not helpful for clinical decision making because of the low positive predictive value of commercially available acute-phase serologic tests. Erythromycins, intravenous azithromycin, and levofloxacin are currently approved by the US Food and Drug Administration for treatment of legionnaires' disease. However, clarithromycin and several other fluoroquinolones are active against Legionella and may also provide effective therapy. Recent recommendations from the Centers for Disease Control and Prevention's Hospital Infection Control Practices Advisory Committee should be helpful in reducing nosocomial legionnaires' disease. Recommendations are in place or are being developed to minimize the risk of disease in a variety of other settings.
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Legionnaires’ disease in the time of COVID-19
Kelsie cassell.
1 Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT USA
J Lucian Davis
2 Pulmonary, Critical Care, and Sleep Medicine Section, Yale School of Medicine, New Haven, CT USA
Ruth Berkelman
3 Rollins School of Public Health, Emory University, Atlanta, GA USA
Associated Data
The datasets used to generate Fig. 1 are available from CDC National Notifiable Diseases Surveillance System, 2000–2018.
Due to similarities in initial disease presentation, clinicians may be inclined to repeatedly test community-acquired pneumonia cases for COVID-19 before recognizing the need to test for Legionnaires’ disease. Legionnaires’ disease is an illness characterized by pneumonia that has a summer/early fall seasonality due to favorable conditions for Legionella growth and exposure. Legionella proliferate in warm water environments and stagnant sections of indoor plumbing and cooling systems. During the ongoing pandemic crisis, exposures to aerosolized water from recently reopened office or retail buildings should be considered as an epidemiologic risk factor for Legionella exposure and an indication to test. The majority of Legionnaires’ disease cases occurring each year are not diagnosed, and some experts recommend that all patients hospitalized with community-acquired pneumonia without a known etiology be tested for Legionella infection. Proper diagnosis can increase the likelihood of appropriate and timely antibiotic treatment, identify potential clusters of disease, and facilitate source attribution.
As SARS-CoV-2 continues to sweep through the world’s population, healthcare providers should be on heightened alert for another potential cause of pneumonia with similar symptoms: Legionnaires’ disease. Public health professionals have recognized that due to the similarities in initial disease presentation, clinicians may repeatedly test for coronavirus disease 2019 (COVID-19) before recognizing the need to test for Legionnaires’ disease. Legionnaires’ disease is a common cause of community-acquired pneumonia with ~ 10% mortality; most patients require hospitalization with some progressing to acute respiratory failure leading to intensive care unit admission, similar to COVID-19 [ 1 ]. Infections are due to inhalation of aerosols containing Legionella . As buildings reopen and previously stagnant plumbing and cooling systems return to use, many additional cases could present to emergency departments in the coming months. Over the past two decades, U.S. incidence of Legionnaires’ disease has increased over five-fold to more than 3.0 cases per 100,000 population in 2018, with most cases occurring in the summer and early autumn (Fig. 1 ). Yet, Legionnaires’ disease remains vastly underdiagnosed with the true number of cases estimated to be more than 50,000 per year [ 2 ].
Incidence of Legionnaires’ disease in the United States, 2000 – 2018. Note: Data from the CDC National Notifiable Diseases Surveillance System, 2000 – 2018
Several reports have underscored the need for caution when re-opening buildings [ 3 ]. Aerosolization of waterborne Legionella can occur from both indoor and outdoor sources, including showers, gardening hoses, fountains, hot tubs, and cooling towers. Biofilms proliferate in water pipes under low-flow conditions, especially where disinfectant levels are inadequate or when building occupancy is low. Garrison et al. [ 4 ] noted that building-related Legionnaires’ disease outbreaks investigated between 2000 and 2014 in North America included outbreaks attributed to low occupancy, closures of hospital wards, and water stagnation due to water flow disruptions (e.g. construction, water main break) [ 4 ]. As states relax lockdowns and physical distancing measures in response to COVID-19, the reopening of commercial buildings – and more importantly, the taps of their dormant plumbing systems – has the potential to expose large numbers of people to stagnant water containing Legionella spp. Additionally, these reopenings coincide with the annual seasonal peak of Legionnaires’ disease cases in the Northern hemisphere [ 5 ]. Over the last few months, the CDC, the EPA, and multiple professional societies (e.g. AIHA, ASHRAE) have issued guidance on safely reopening buildings in order to prevent Legionella growth and transmission linked to pandemic response measures [ 3 , 6 – 8 ].
Pneumonia is the most common manifestation of both Legionnaires’ disease and COVID-19, and initial presentation for both may include fever, headache, confusion, dyspnea, nausea, and diarrhea. Individual risk factors for both Legionnaires’ disease and severe COVID-19 include older age, diabetes, and chronic lung disease. The incubation period for Legionnaires’ disease is about 5 to 6 days but may range from 2 to 14 days, similar to COVID-19. Travel with overnight stays and healthcare exposure (e.g. hospitals, long-term care facilities) are known major risk factors for Legionnaires’ disease [ 1 ]. During the ongoing pandemic crisis, exposures to aerosolized water from recently reopened office or retail buildings should also be considered as new risk factors for Legionella exposure. Additionally, changing behaviors in the home and for recreational activities during COVID-19 should be considered (e.g. gardening).
Because clinical manifestations may be indistinguishable between COVID-19 and Legionnaires’ disease, targeted microbiologic testing for both Legionella and SARS-CoV2 are essential. The American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA) guidelines for community-acquired pneumonia recommend Legionella urinary antigen testing (UAT) for severe disease in adults or those with epidemiological indications, in addition to sending sputum and other lower respiratory tract specimens for PCR and culture [ 9 ]. It is important to note that the UAT has a sensitivity around 70% and a 99% specificity but this is for Legionella pneumophila serogroup 1 and cannot reliably detect other pathogenic species and serogroups of Legionella that cause disease [ 10 ]. Historically, epidemiological indication referred to recent travel (e.g. hotels, cruises) or history of recent hospitalization or residence in a long-term care facility. Currently, it is important to consider other risk factors as well, including returning to work in reopened office buildings or patronizing businesses or other buildings that had been shuttered. The Centers for Disease Control and Prevention in Atlanta closed multiple buildings in August due to presence of Legionella in the buildings water systems, potentially linked to the long term building closure during the pandemic [ 11 ].
Some experts recommend that all patients hospitalized with pneumonia and without a known etiology be tested with UAT [ 2 ], however, IDSA/ITS guidelines recommend only severe CAP be tested with the UAT. This test is widely considered to have relatively low cost, with the ability to reduce discordant antibiotic therapy, and is of greater applicability in higher prevalence areas of the US [ 12 , 13 ]. Of note, coinfection with Legionella and SARS-CoV2 has been documented [ 14 ]. Because the UAT cannot detect Legionella caused by non- Legionella pneumophila serogroup 1, and because morbidity and mortality due to Legionella are high, ATS/IDSA guidelines also recommend early empiric antimicrobial therapy with a fluoroquinolone or macrolide such as azithromycin and levofloxacin [ 1 ].
Because empiric treatment of Legionnaires’ disease is not always effective, early testing including UAT could improve the clinical and public health response. Like COVID-19, public health officials require a laboratory diagnosis to investigate cases, which could lead to identification of the exposure source. Such actions will reduce morbidity and mortality from this severe and increasingly common disease.
Acknowledgements
The authors would like to acknowledge the CDC Legionella Epidemiology Team, including, but not limited to, Jessica Smith, MPH, Elizabeth Hannapel, MPH, and Chris Edens, PhD.
Abbreviations
COVID-19 | Coronavirus disease 2019 |
CDC | Centers for Disease Control and Prevention |
EPA | Environmental Protection Agency |
AIHA | American Industrial Hygiene Association |
ASHRAE | American Society of Heating, Refrigerating, and Air Conditioning Engineers |
UAT | Legionella urinary antigen testing |
ATS/IDSA | The American Thoracic Society (ATS)/The Infectious Diseases Society of America |
Authors' contributions
KC, JLD, and RB all contributed equally to the design and drafting of this manuscript. The author(s) read and approved the final manuscript.
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The authors declare that they have no competing interests
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Legionellosis is the collective term for the clinical syndromes caused by members of the genus Legionella that can present as either Legionnaires' disease (LD) or Pontiac fever. Since the ...
Legionnaires disease is a significant worldwide public health concern worldwide; the bacteria are spread through aspiration or inhalation of contaminated aerosolized water particles. Legionnaires disease may be particularly severe in older patients and those who are immunocompromised. ... Ongoing research seeks to elucidate the complex ...
Legionnaires disease is an atypical pneumonia frequently clinically different from other bacterial pneumonia. The predominant symptoms of Legionnaires disease include fever, cough, dyspnea, headache, and fatigue. ... Ongoing research seeks to elucidate the complex interactions between legionellae, their environmental niches, and human ...
Legionellosis is the infection caused by bacteria of the genus Legionella, including a non-pneumonic influenza-like syndrome, and Legionnaires' disease is a more serious illness characterized by pneumonia.Legionellosis is becoming increasingly important as a public health problem throughout the world; although it is an underreported disease, studies have consistently documented a high incidence.
Legionnaires' disease (LD) is a severe pneumonia caused by Legionella spp. bacteria. Approximately 95% of patients require hospitalization, and 10% die ().Risk factors include older age (>50 years), smoking, a weakened immune system, and chronic lung conditions ().Pontiac fever (a self-limited, influenza-like illness) and extrapulmonary legionellosis (Legionella infection with a primary ...
Legionella species are environmental gram-negative bacteria able to cause a severe form of pneumonia in humans known as Legionnaires' disease. Since the identification of Legionella pneumophila in 1977, four decades of research on Legionella biology and Legionnaires' disease have brought important insights into the biology of the bacteria and the molecular mechanisms that these intracellular ...
The exact incidence of Legionnaires' disease worldwide is unknown, mainly because countries differ in awareness levels, diagnostic methods, and reporting. Legionnaires' disease accounts for 2-9% of cases of community-acquired pneumonia. 44. Data from the USA indicate a 192% increase in the crude national incidence of Legionnaires' disease ...
The disease is caused by any species of the Gram-negative aerobic bacteria belonging to the genus Legionella; Legionella pneumophila serogroup 1 is the causative agent of most cases in Europe. In this Review we outline the global epidemiology of Legionnaires' disease, summarise its diagnosis and management, and identify research gaps and ...
Legionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires' disease. However, the microorganism is typically a symbiont of free-living amoeba, and ...
Legionella is an opportunistic premise plumbing pathogen and causative agent of a severe pneumonia called Legionnaires' Disease (LD). Cases of LD have been on the rise in the U.S. and globally. Although Legionella was first identified 45 years ago, it remains an 'emerging pathogen."Legionella is part of the normal ecology of a public water system and is frequently detected in regulatory ...
In addition, health costs associated with the disease are high. Diagnosis of Legionnaires' disease is based mainly on the detection of Legionella pneumophila serogroup 1 antigen in urine. However, there have been advances in detection tests for patients with legionellosis. New methodologies show greater sensitivity and specificity, detect more ...
In this Review we outline the global epidemiology of Legionnaires' disease, summarise its diagnosis and management, and identify research gaps and priorities. Early clinical diagnosis and prompt initiation of appropriate antibiotics for Legionella spp in all patients with community-acquired or hospital-acquired pneumonias is a crucial measure ...
Introduction. Legionnaires' disease (LD), a type of pneumonia that is often severe and has a 7-10% mortality rate, is the leading cause of drinking water disease outbreaks in the United States (USA) [1-3].Legionellosis, caused by infection with Legionella bacteria, includes both LD and the flu-like illness, Pontiac fever. LD is especially severe in older people, smokers, and those with ...
significant increases in Legionnaires' over the 5-year study period (from 5.37 per 100,000 population in 2006 to 9.66 in 2010, with a peak in 2009 of 17.07) [22]. Likewise, the European Legionnaires' disease Surveillance Network (ELDSNet) reported that the notification rates for Legionnaires' disease have nearly doubled in
Legionnaires' disease is a severe respiratory illness resulting from breathing water droplets or aspirating water containing Legionella bacteria. Legionnaires' disease is a public health challenge, with an estimated annual hospitalization cost of $433 million in the United States. 1 In 2018, 9933 Legionnaires' disease cases were reported to the Centers for Disease Control and Prevention ...
We investigated the genomic epidemiology of Legionnaires' disease over 36 years in Scotland, comparing genome sequences for all clinical L pneumophila isolates (1984-2020) with a sequence dataset of 3211 local and globally representative isolates. We used a stratified clustering approach to capture epidemiological relationships by core genome Multi-locus Sequence Typing, followed by high ...
1. Introduction. Legionnaires disease is a form of pneumonia caused by gram-negative bacteria, Legionella spp., which are found in freshwater environments worldwide and infect people via inhalation of contaminated aerosols. [1,2] Incidence rates have been increasing annually, with 1.30 to 1.89 cases per 100,000 reported in surveillance studies in Europe [] and the United States [] in 2015.
Legionellosis is the infection caused by bacteria of the genus Legionella, including a non-pneumonic influenza-like syndrome, and Legionnaires' disease is a more serious illness characterized by pneumonia. Legionellosis is becoming increasingly important as a public health problem throughout the world; although it is an underreported disease, studies have consistently documented a high ...
Feature papers represent the most advanced research with significant potential for high impact in the field. A Feature Paper should be a substantial original Article that involves several techniques or approaches, provides an outlook for future research directions and describes possible research applications. ... Legionnaires' disease ...
Legionnaires' disease (LD) is a type of pneumonia predominantly caused by Legionella pneumophila serogroup 1 (LpS1), which is responsible for 82% of cases worldwide [].The epidemiological data from Europe reveal a consistent rise in LD cases over the last decade [].Similar trends were observed in Belgium, with incidences of LD of 3.1 and 3.2/100.000 inhabitants in 2021 and 2022, respectively ...
Abstract. Legionnaires' disease is a modern environmental infectious disease. It stems from the capacity of the causative agent, Legionella, to multiply within amoebae in warm water and the use, during the 20th century, of devices that maintain water at warm temperatures and produce aerosols. When contaminated with Legionella, aerosols ...
Summary. Legionellosis or Legionnaires' disease is an emerging and often-fatal form of pneumonia that is most severe in elderly and immunocompromised people, an ever-increasing risk group for infection. In recent years, the genomics of Legionella spp. has significantly increased our knowledge of the pathogenesis of this disease by providing ...
Serologic data appear in the paper by McDade et al. 3 In summary, 91 per cent (101 of 111) of patients with Legionnaires' disease from whom adequate serum specimens were obtained either showed ...
Legionnaires' disease is an illness characterized by pneumonia that has a summer/early fall seasonality due to favorable conditions for Legionella growth and exposure. Legionella proliferate in warm water environments and stagnant sections of indoor plumbing and cooling systems. During the ongoing pandemic crisis, exposures to aerosolized ...