Factors that affect water column hydrocarbon concentrations have minor impacts on microbial responses following simulated diesel fuel spills.

Effects of season and mixing on hydrocarbon concentrations and the microbial community response was explored in a series of mesocosm experiments simulating surface spills of diesel into coastal waters. Mixing of any amount contributed to hydrocarbons entering the water column, but diesel fuel composition had a significant effect on hydrocarbon concentrations. Higher initial concentrations of aromatic hydrocarbons resulted in higher water column concentrations, with minimal differences among seasons due to high variability. Regardless of the concentrations of hydrocarbons, prokaryotes increased and there were higher relative abundances of hydrocarbon affiliated bacteria with indications of biodegradation within 4 d of exposure. As concentrations decreased over time, the eukaryote community shifted from the initial community to one which appeared to be composed of organisms with some resilience to hydrocarbons. This series of experiments demonstrates the wide range of conditions under which natural attenuation of diesel fuel is an effective response.

Effects of marine diesel on microbial diversity and activity in high Arctic beach sediments.

Global warming induced sea ice loss increases Arctic maritime traffic, enhancing the risk of ecosystem contamination from fuel spills and nutrient loading. The impact of marine diesel on bacterial metabolic activity and diversity, assessed by colorimetric assay, 16S rRNA and metagenomic sequencing, of Northwest Passage (Arctic Ocean) beach sediments was assessed with nutrient amendment at environmentally relevant temperatures (5 and 15 °C). Higher temperature and nutrients stimulated microbial activity, while diesel reduced it, with metabolism inhibited at and above 0.01 % (without nutrients) and at 1 % (with nutrients) diesel inclusions. Diesel exposure significantly decreased microbial diversity and selected for Psychrobacter genus. Microbial hydrocarbon degradation, organic compound metabolism, and exopolysaccharide production gene abundances increased under higher diesel concentrations. Metagenomic binning recovered nine MAGs/bins with hydrocarbon degradation genes. We demonstrate a nutrients' rescue-type effect in diesel contaminated microbial communities via enrichment of microorganisms with stress response, aromatic compound, and ammonia assimilation metabolisms.

Long-term biodegradation of crude oil in high-arctic backshore sediments: The Baffin Island Oil Spill (BIOS) after nearly four decades.

With an on-going disproportional warming of the Arctic Ocean and the reduction of the sea ice cover, the risk of an accidental oil spill from ships or future oil exploration is increasing. It is hence important to know how crude oil weathers in this environment and what factors affect oil biodegradation in the Arctic. However, this topic is currently poorly studied. In the 1980s, the Baffin Island Oil Spill (BIOS) project carried out a series of simulated oil spills in the backshore zone of beaches located on Baffin Island in the Canadian High Arctic. In this study two BIOS sites were re-visited, offering the unique opportunity to study the long-term weathering of crude oil under Arctic conditions. Here we show that residual oil remains present at these sites even after almost four decades since the original oiling. Oil at both BIOS sites appears to have attenuated very slowly with estimated loss rates of 1.8-2.7% per year. The presence of residual oil continues to significantly affect sediment microbial communities at the sites as manifested by a significantly decreased diversity, differences in the abundance of microorganisms and an enrichment of putative oil-degrading bacteria in oiled sediments. Reconstructed genomes of putative oil degraders suggest that only a subset is specifically adapted for growth under psychrothermic conditions, further reducing the time for biodegradation during the already short Arctic summers. Altogether, this study shows that crude oil spilled in the Arctic can persist and significantly affect the Arctic ecosystem for a long time, in the order of several decades.

Biodegradation potential of residue generated during the in-situ burning of oil in the marine environment.

The biodegradability of residues derived from in-situ burning, an oil spill response strategy which involves burning an oil slick on the sea surface, has not yet been fully studied. With a growing risk of oil spills, the fate of the persistent burn residue containing potentially toxic substances must be better understood. Microcosms were used to study the microbial community response and potential biodegradability of in-situ burn residues generated from Ultra Low Sulphur (ULS) marine diesel. Microcosm studies were conducted using residues originating from the burning of unweathered and weathered diesel, with the addition of a fertilizer and a dispersant. Burn residues were incubated for 6 weeks at 7 °C in natural seawater with continual agitation in the dark. Samples were subsequently sacrificed for chemistry as well as 16S rRNA gene amplicon and shotgun metagenomic sequencing. Chemistry analyses revealed a reduction in hydrocarbon concentrations. Medium chain-length n-alkanes (nC16-nC24) decreased by 8% in unweathered burn residue microcosms and up to 26% in weathered burn residue microcosms. A significant decrease in polycyclic aromatic hydrocarbon (PAH) concentrations was observed only for naphthalene, fluorene and their alkylated homologs, in the microcosms amended with residue produced from burning weathered diesel. Decreases of 2-24%, were identified depending on the compound. Microcosms amended with burn residues had distinct microbial communities marked by an increase in relative abundance of putative hydrocarbon degraders as well as an increase of known hydrocarbon-degradation genes. These novel results suggest that if in-situ burning is performed on ULS marine diesel, some of the indigenous bacteria would respond to the newly available carbon source and some of the residual compounds would be biodegraded. Future studies involving longer incubation periods could give a better understanding of the fate of burn residues by shedding light on the potential biodegradability of the more recalcitrant residual compounds.

An experimental oil spill at a tidal freshwater wetland along the St. Lawrence River re-visited after 21 years.

In 1999, a tidal wetland located along the St. Lawrence River close to Ste. Croix de Lotbinière (Quebec, Eastern Canada) was the site of an experimental oil spill. Test plots were established and subjected to an experimental crude oil spill to evaluate natural attenuation, nutrient amendment and vegetation cropping as countermeasures. In 2020, this study re-visited the test plots to investigate residual oil and habitat recovery. Only concentrations of mid-chain length n-alkanes (C10-C36), but not of polycyclic aromatic hydrocarbons (PAHs), were significantly above detection limit, and were detected in both test plot and control sediments. Hydrocarbon, total organic carbon, nitrogen and phosphate contents did not differ significantly between test plot and control sediments. Microbial analyses did not detect significant differences in microbial load, microbial diversity or microbial community composition between test plot and control sediments. Key genes for the aerobic and anaerobic degradation of n-alkanes as well as for the aerobic degradation of PAHs were detected in all sediment samples. Associated gene abundances did not differ significantly between test plot and control sediments. This study shows that oil-exposed test plot sediments of the Ste. Croix wetland can be considered completely recovered after 21 years irrespective of the performed countermeasure.

Draft Whole-Genome Sequences of the Polar Cyanobacterium Leptolyngbya sp. Strain Cla-17 and Its Associated Flavobacterium.

Draft whole-genome sequences of a coculture are presented. One component was a polar cyanobacterium, Leptolyngbya sp. strain Cla-17. The second was a heterotrophic bacterium, Flavobacterium saccharophilum, found in the phycosphere of the cyanobacterium.

Microbial community response to simulated diluted bitumen spills in coastal seawater and implications for oil spill response.

Oil spills in coastal waters can have devastating impacts on local ecosystems, from the microscopic base through to mammals and seabirds. Increasing transport of diluted bitumen has led to concerns about how this novel product might impact coastal ecosystems. A mesocosm study determined that the type of diluent and the season can affect the concentrations of hydrocarbons entering the water column from a surface spill. Those same mesocosms were sampled to determine whether diluent type and season also affected the microbial response to a surface spill. Overall, there were no differences in impacts among the three types of diluted bitumen, but there were consistent responses to all products within each season. Although microbial abundances with diluted bitumen rarely differed from unoiled controls, community structure in these organisms shifted in response to hydrocarbons, with hydrocarbon-degrading bacteria becoming more abundant. The relative abundance of heterotrophic eukaryotes also increased with diluted bitumen, with few photosynthetic organisms responding positively to oil. Overall shifts in the microbial communities were minimal relative to spills of conventional oil products, with low concentrations of hydrocarbons in the water column. Oil spill response should focus on addressing the surface slick to prevent sinking or stranding to minimize ecosystem impacts.

Impact of sample collection on prokaryotic and eukaryotic diversity of niche environments of the oil-sand mining impacted Athabasca River.

Microbial communities are an important aspect of overall riverine ecology; however, appreciation of the effects of anthropogenic activities on unique riverine microbial niches, and how the collection of these samples affects the observed diversity and community profile is lacking. We analyzed prokaryotic and eukaryotic communities from surface water, biofilms, and suspended load niches along a gradient of oil sands-related contamination in the Athabasca River (Alberta, Canada), with suspended load or particle-associated communities collected either via Kenney Sampler or centrifugation manifold. At the phylum level, different niche communities were highly similar to each other and across locations. However, there were significant differences in the abundance of specific genera among the different niches and across sampling locations. A generalized linear model revealed that use of the Kenney Sampler resulted in more diverse bacterial and eukaryotic suspended load community than centrifugal collection, though suspended load communities collected by any means remained stably diverse across locations. Although there was an influence of water quality parameters on community composition, all sampled sites support diverse bacterial and eukaryotic communities regardless of the degree of contamination, highlighting the need to look beyond ecological diversity as a means of assessing ecological perturbations, and consider collecting samples from multiple niche environments.

In situ microcosms deployed at the coast of British Columbia (Canada) to study dilbit weathering and associated microbial communities under marine conditions.

Douglas Channel and the adjacent Hecate Strait (British Columbia, Canada) are part of a proposed route to ship diluted bitumen (dilbit). This study presents how two types of dilbit naturally degrade in this environment by using an in situ microcosm design based on dilbit-coated beads. We show that dilbit-associated n-alkanes were microbially biodegraded with estimated half-lives of 57-69 days. n-Alkanes appeared to be primarily degraded using the aerobic alkB, ladA and CYP153 pathways. The loss of dilbit polycyclic aromatic hydrocarbons (PAHs) was slower than of n-alkanes, with half-lives of 89-439 days. A biodegradation of PAHs could not be conclusively determined, although a significant enrichment of the phnAc gene (a marker for aerobic PAH biodegradation) was observed. PAH degradation appeared to be slower in Hecate Strait than in Douglas Channel. Microcosm-associated microbial communities were shaped by the presence of dilbit, deployment location and incubation time but not by dilbit type. Metagenome-assembled genomes of putative dilbit-degraders were obtained and could be divided into populations of early, late and continuous degraders. The majority of the identified MAGs could be assigned to the orders Flavobacteriales, Methylococcales, Pseudomonadales and Rhodobacterales. A high proportion of the MAGs represent currently unknown lineages or lineages with currently no cultured representative.

Draft Whole-Genome Sequence of the Alkane-Synthesizing Polar Cyanobacterium Pseudanabaena biceps Strain O-153.

Alkane biosynthesis by polar cyanobacteria has not yet been reported. We present here the draft whole-genome sequence of an alkane-synthesizing polar cyanobacterium, Pseudanabaena biceps strain O-153. The genes coding for the two key enzymes involved in the alkane biosynthetic pathway were found contiguously in the genome.

Vegetation drives the structure of active microbial communities on an acidogenic mine tailings deposit.

Plant-microbe associations are increasingly recognized as an inextricable part of plant biology and biogeochemistry. Microbes play an essential role in the survival and development of plants, allowing them to thrive in diverse environments. The composition of the rhizosphere soil microbial communities is largely influenced by edaphic conditions and plant species. In order to decipher how environmental conditions on a mine site can influence the dynamics of microbial communities, we characterized the rhizosphere soil microbial communities associated with paper birch, speckled alder, and spruce that had naturally colonized an acidogenic mine tailings deposit containing heavy metals. The study site, which had been largely undisturbed for five decades, had highly variable vegetation density; with some areas remaining almost barren, and others having a few stands or large thickets of mature trees. Using Illumina sequencing and ordination analyses (redundancy analysis and principal coordinate analysis), our study showed that soil bacterial and fungal community structures correlated mainly with vegetation density, and plant species. Tailings without any vegetation were the most different in bacterial community structure, compared to all other areas on the mine site, as well as an adjacent natural forest (comparison plot). The bacterial genera Acidiferrobacter and Leptospirillum were more abundant in tailings without vegetation than in any of the other sites, while Bradyrhizobium sp. were more abundant in areas of the tailings deposit having higher vegetation density. Frankia sp. is equally represented in each of the vegetation densities and Pseudomonas sp. present a greater relative abundance in boreal forest. Furthermore, alder rhizosphere showed a greater relative abundance of Bradyrhizobium sp. (in comparison with birch and spruce) as well as Haliangium sp. (in comparison with birch). In contrast, fungal community structures were similar across the tailings deposit regardless of vegetation density, showing a greater relative abundance of Hypocrea sp. Tailings deposit fungal communities were distinct from those found in boreal forest soils. Alder rhizosphere had greater relative abundances of Hypocrea sp. and Thelephora sp., while birch rhizosphere were more often associated with Mollisia sp. Our results indicate that, with increasing vegetation density on the mine site, the bacterial communities associated with the individual deciduous or coniferous species studied were increasingly similar to the bacterial communities found in the adjacent forest. In order to properly assess and restore disturbed sites, it is important to characterize and understand the plant-microbe associations that occur since they likely improve plant fitness in these harsh environments.

Complete Genome Sequences of Two Marine Biofilm Isolates, Leisingera sp. nov. Strains 201A and 204H, Novel Representatives of the Roseobacter Group.

Here, we report the complete-genome assemblies of biofilm isolates 201A and 204H. They possess six and seven plasmids, respectively, with a size ranging from 44 kb to 159 kb. Genomic comparisons place the two strains into one new species belonging to the genus Leisingera as novel representatives of the Roseobacter group.

Dominant gut Prevotella copri in gastrectomised non-obese diabetic Goto-Kakizaki rats improves glucose homeostasis through enhanced FXR signalling.

AIMS/HYPOTHESIS: Drug and surgical-based therapies in type 2 diabetes are associated with altered gut microbiota architecture. Here we investigated the role of the gut microbiome in improved glucose homeostasis following bariatric surgery. METHODS: We carried out gut microbiome analyses in gastrectomised (by vertical sleeve gastrectomy [VSG]) rats of the Goto-Kakizaki (GK) non-obese model of spontaneously occurring type 2 diabetes, followed by physiological studies in the GK rat. RESULTS: VSG in the GK rat led to permanent improvement of glucose tolerance associated with minor changes in the gut microbiome, mostly characterised by significant enrichment of caecal Prevotella copri. Gut microbiota enrichment with P. copri in GK rats through permissive antibiotic treatment, inoculation of gut microbiota isolated from gastrectomised GK rats, and direct inoculation of P. copri, resulted in significant improvement of glucose tolerance, independent of changes in body weight. Plasma bile acids were increased in GK rats following inoculation with P. copri and P. copri-enriched microbiota from VSG-treated rats; the inoculated GK rats then showed increased liver glycogen and upregulated expression of Fxr (also known as Nr1h4), Srebf1c, Chrebp (also known as Mlxipl) and Il10 and downregulated expression of Cyp7a1. CONCLUSIONS: Our data underline the impact of intestinal P. copri on improved glucose homeostasis through enhanced bile acid metabolism and farnesoid X receptor (FXR) signalling, which may represent a promising opportunity for novel type 2 diabetes therapeutics.

Metagenomic and metatranscriptomic responses of natural oil degrading bacteria in the presence of dispersants.

Oil biodegradation has been extensively studied in the wake of the deepwater horizon spill, but the application of dispersant to oil spills in marine environments remains controversial. Here, we report metagenomic (MG) and metatranscriptomic (MT) data mining from microcosm experiments investigating the oil degrading potential of Canadian west and east coasts to estimate the gene abundance and activity of oil degrading bacteria in the presence of dispersant. We found that the addition of dispersant to crude oil mainly favours the abundance of Thalassolituus in the summer and Oleispira in the winter, two key natural oil degrading bacteria. We found a high abundance of genes related not only to n-alkane and aromatics degradation but also associated with transporters, two-component systems, bacterial motility, secretion systems and bacterial chemotaxis.

Potential for Microbially Mediated Natural Attenuation of Diluted Bitumen on the Coast of British Columbia (Canada).

Western Canada produces large amounts of bitumen, a heavy, highly weathered crude oil. Douglas Channel and Hecate Strait on the coast of British Columbia are two water bodies that may be impacted by a proposed pipeline and marine shipping route for diluted bitumen (dilbit). This study investigated the potential of microbial communities from these waters to mitigate the impacts of a potential dilbit spill. Microcosm experiments were set up with water samples representing different seasons, years, sampling stations, and dilbit blends. While the alkane fraction of the tested dilbit blends was almost completely degraded after 28 days, the majority of the polycyclic aromatic hydrocarbons (PAHs) remained. The addition of the dispersant Corexit 9500A most often had either no effect or an enhancing effect on dilbit degradation. Dilbit-degrading microbial communities were highly variable between seasons, years, and stations, with dilbit type having little impact on community trajectories. Potential oil-degrading genera showed a clear succession pattern and were for the most part recruited from the "rare biosphere." At the community level, dispersant appeared to stimulate an accelerated enrichment of genera typically associated with hydrocarbon degradation, even in dilbit-free controls. This suggests that dispersant-induced growth of hydrocarbon degraders (and not only increased bioavailability of oil-associated hydrocarbons) contributes to the degradation-enhancing effect previously reported for Corexit 9500A.IMPORTANCE Western Canada hosts large petroleum deposits, which ultimately enter the market in the form of dilbit. Tanker-based shipping represents the primary means to transport dilbit to international markets. With anticipated increases in production to meet global energy needs, the risk of a dilbit spill is expected to increase. This study investigated the potential of microbial communities naturally present in the waters of a potential dilbit shipping lane to mitigate the effects of a spill. Here we show that microbial degradation of dilbit was mostly limited to n-alkanes, while the overall concentration of polycyclic aromatic hydrocarbons, which represent the most toxic fraction of dilbit, decreased only slightly within the time frame of our experiments. We further investigated the effect of the oil dispersant Corexit 9500A on microbial dilbit degradation. Our results highlight the fact that dispersant-associated growth stimulation, and not only increased bioavailability of hydrocarbons and inhibition of specific genera, contributes to the overall effect of dispersant addition.

Genomic Recombination Leading to Decreased Virulence of Group B Streptococcus in a Mouse Model of Adult Invasive Disease.

Adult invasive disease caused by Group B Streptococcus (GBS) is increasing worldwide. Whole-genome sequencing (WGS) now permits rapid identification of recombination events, a phenomenon that occurs frequently in GBS. Using WGS, we described that strain NGBS375, a capsular serotype V GBS isolate of sequence type (ST)297, has an ST1 genomic background but has acquired approximately 300 kbp of genetic material likely from an ST17 strain. Here, we examined the virulence of this strain in an in vivo model of GBS adult invasive infection. The mosaic ST297 strain showed intermediate virulence, causing significantly less systemic infection and reduced mortality than a more virulent, serotype V ST1 isolate. Bacteremia induced by the ST297 strain was similar to that induced by a serotype III ST17 strain, which was the least virulent under the conditions tested. Yet, under normalized bacteremia levels, the in vivo intrinsic capacity to induce the production of pro-inflammatory cytokines was similar between the ST297 strain and the virulent ST1 strain. Thus, the diminished virulence of the mosaic strain may be due to reduced capacity to disseminate or multiply in blood during a systemic infection which could be mediated by regulatory factors contained in the recombined region.

Genome and Plasmid Analysis of blaIMP-4-Carrying Citrobacter freundii B38.

Sequencing of the blaIMP-4-carrying C. freundii B38 using the PacBio SMRT technique revealed that the genome contained a chromosome of 5,134,500 bp and three plasmids, pOZ172 (127,005 bp), pOZ181 (277,592 bp), and pOZ182 (18,467 bp). Plasmid pOZ172 was identified as IncFIIY, like pP10164-NDM and pNDM-EcGN174. It carries a class 1 integron with four cassettes (blaIMP-4-qacG2-aacA4-aphA15) and a complete hybrid tni module (tniR-tniQ-tniB-tniA). The recombination of tniR from Tn402 (identical) with tniQBA from Tn5053 (99%) occurred within the res site of Tn402/5053 The Tn402/5053-like integron, named Tn6017, was inserted into Tn1722 at the res II site. The replication, partitioning, and transfer systems of pOZ181 were similar to those of IncHI2 plasmids (e.g., R478) and contained a sul1-type class 1 integron with the cassette array orf-dfrA1-orf-gcu37-aadA5 linked to an upstream Tn1696 tnpA-tnpR and to a downstream 3' conserved sequence (3'-CS) and ISCR1 A Tn2 transposon encoding a blaTEM-1 ß-lactamase was identified on pOZ182. Other interesting resistance determinants encoded on the B38 chromosome included multidrug resistance (MDR) efflux pumps, an AmpC ß-lactamase, and resistances to Cu, Ag, As, and Zn. This is the first report of a complete tni module linked to a blaIMP-4-carrying class 1 integron, which, together with other recently reported non-sul1 integrons, represents the emergence of a distinct evolutionary lineage of class 1 integrons lacking a 3'-CS (qacEΔ1-sul1). The unique cassette array, complete tni module of Tn6017, and incompatibility group of pOZ172 suggest a blaIMP-4 evolutionary pathway in C. freundii B38 different from that for other blaIMP-4 genes found in Gram-negative bacteria in the Western Pacific region.

Envelope-specific B-cell populations in African green monkeys chronically infected with simian immunodeficiency virus.

African green monkeys (AGMs) are natural primate hosts of simian immunodeficiency virus (SIV). Interestingly, features of the envelope-specific antibody responses in SIV-infected AGMs are distinct from that of HIV-infected humans and SIV-infected rhesus monkeys, including gp120-focused responses and rapid development of autologous neutralization. Yet, the lack of genetic tools to evaluate B-cell lineages hinders potential use of this unique non-human primate model for HIV vaccine development. Here we define features of the AGM Ig loci and compare the proportion of Env-specific memory B-cell populations to that of HIV-infected humans and SIV-infected rhesus monkeys. AGMs appear to have a higher proportion of Env-specific memory B cells that are mainly gp120 directed. Furthermore, AGM gp120-specific monoclonal antibodies display robust antibody-dependent cellular cytotoxicity and CD4-dependent virion capture activity. Our results support the use of AGMs to model induction of functional gp120-specific antibodies by HIV vaccine strategies.

Population Structure and Antimicrobial Resistance Profiles of Streptococcus suis Serotype 2 Sequence Type 25 Strains.

Strains of serotype 2 Streptococcus suis are responsible for swine and human infections. Different serotype 2 genetic backgrounds have been defined using multilocus sequence typing (MLST). However, little is known about the genetic diversity within each MLST sequence type (ST). Here, we used whole-genome sequencing to test the hypothesis that S. suis serotype 2 strains of the ST25 lineage are genetically heterogeneous. We evaluated 51 serotype 2 ST25 S. suis strains isolated from diseased pigs and humans in Canada, the United States of America, and Thailand. Whole-genome sequencing revealed numerous large-scale rearrangements in the ST25 genome, compared to the genomes of ST1 and ST28 S. suis strains, which result, among other changes, in disruption of a pilus island locus. We report that recombination and lateral gene transfer contribute to ST25 genetic diversity. Phylogenetic analysis identified two main and distinct Thai and North American clades grouping most strains investigated. These clades also possessed distinct patterns of antimicrobial resistance genes, which correlated with acquisition of different integrative and conjugative elements (ICEs). Some of these ICEs were found to be integrated at a recombination hot spot, previously identified as the site of integration of the 89K pathogenicity island in serotype 2 ST7 S. suis strains. Our results highlight the limitations of MLST for phylogenetic analysis of S. suis, and the importance of lateral gene transfer and recombination as drivers of diversity in this swine pathogen and zoonotic agent.

Active and adaptive Legionella CRISPR-Cas reveals a recurrent challenge to the pathogen.

Clustered regularly interspaced short palindromic repeats with CRISPR-associated gene (CRISPR-Cas) systems are widely recognized as critical genome defense systems that protect microbes from external threats such as bacteriophage infection. Several isolates of the intracellular pathogen Legionella pneumophila possess multiple CRISPR-Cas systems (type I-C, type I-F and type II-B), yet the targets of these systems remain unknown. With the recent observation that at least one of these systems (II-B) plays a non-canonical role in supporting intracellular replication, the possibility remained that these systems are vestigial genome defense systems co-opted for other purposes. Our data indicate that this is not the case. Using an established plasmid transformation assay, we demonstrate that type I-C, I-F and II-B CRISPR-Cas provide protection against spacer targets. We observe efficient laboratory acquisition of new spacers under 'priming' conditions, in which initially incomplete target elimination leads to the generation of new spacers and ultimate loss of the invasive DNA. Critically, we identify the first known target of L. pneumophila CRISPR-Cas: a 30 kb episome of unknown function whose interbacterial transfer is guarded against by CRISPR-Cas. We provide evidence that the element can subvert CRISPR-Cas by mutating its targeted sequences - but that primed spacer acquisition may limit this mechanism of escape. Rather than generally impinging on bacterial fitness, this element drives a host specialization event - with improved fitness in Acanthamoeba but a reduced ability to replicate in other hosts and conditions. These observations add to a growing body of evidence that host range restriction can serve as an existential threat to L. pneumophila in the wild.