Sebastien P. Faucher

Microbial communities were cooling tower-specific, with higher bacterial diversity in biofilm than in water. L. pneumophila was detected in both biofilm and water samples, with Echinamoeba uniquely present in L. pneumophila culture-positive systems.

Legionella anisa has been frequently isolated from engineered water systems; however, its population structure remains understudied compared to Legionella pneumophila. Here, we generated complete genome sequences for four L. anisa isolates recovered from a healthcare facility in Rimouski, Canada. Further the population structure of this species was investigated by performing comparative genomic analyses of the genomes generated in this study together with publicly available L. anisa genomes. Genome-wide phylogenetic analysis revealed the presence of three distinct clades separated by substantial genetic divergence (∼500 single nucleotide polymorphisms), with the Rimouski isolates forming a tightly clustered group, suggesting a clonal lineage. Comparative pangenome analysis indicated moderate core genome conservation accompanied by a highly variable accessory genome (∼50%). The isolates characterized in this study harbored multiple plasmids encoding genes associated with conjugation, heavy metal resistance, and other stress-related functions, suggesting potential roles in environmental persistence. Previous studies have shown that L. anisa can proliferate within protozoan host cells, although outcomes vary depending on the host species. Our isolates showed efficient proliferation within Acanthamoeba castellanii, but not within Vermamoeba vermiformis, under the conditions tested. Together, these findings underscore the genomic diversity of this understudied Legionella species and provide a framework for future investigations regarding environmental persistence and potential pathogenicity.

Botrytis cinerea is a pathogen infecting Cannabis sativa L. plants, causing economic losses, and can develop resistance to chemical fungicides, the use of which is restricted in cannabis production. Thus, developing biocontrol methods is imperative. Seven bacterial strains were isolated from hemp seed oil, characterized, and examined for the potential to control a Botrytis cinerea isolate from cannabis. Three isolates, Bacillus mojavensis HOB3, Paenibacillus sp. HOB6, and Bacillus subtilis HOB7 exhibited significant inhibition of Botrytis cinerea. These isolates were further evaluated for their biosurfactant activity using two liquid media, Lysogeny Broth (LB) and hydrocarbon-amended Bushnell and Haas (BH). The oil-spreading and drop-collapse assays revealed growth-medium-dependent variation in surface activity associated with biosurfactant presence. The BH cell-free extract (BH-CFE) of Bacillus subtilis HOB7 showed the highest estimated biosurfactant presence and antifungal activity against Botrytis cinerea, but both activities were absent when using the LB cell-free extract (LB-CFE) of Bacillus subtilis HOB7. Thus, a potential relationship between antifungal activity and biosurfactant production was suggested. Genome mining of the strains identified gene clusters encoding compounds with antifungal activity, including the biosurfactants polymyxin B, fusaricidin B, fengycin, and surfactin. To our knowledge, this is the first report of the isolation of hemp seed oil bacteria with potential biocontrol properties against fungal phytopathogens.

Eight cases of Legionnaires’ disease caused by Legionella pneumophila serogroup 1 sequence type (ST) ST2858 were detected within 4 years in Montréal, Canada. Most cases were associated with a single healthcare facility, and one of them presented with a co-infection, from which ST378 was isolated as well. The source of ST2858 was not identified, despite extensive environmental sampling. The goal of this study was to determine the diversity of both STs and confirm the source of each by matching clinical to environmental isolates. Comparative genome analysis was performed to investigate the genetic relatedness of the isolates. Long- and short-read hybrid assembly was used to produce high-quality closed genomes. A PCR assay, amplifying a unique gene of ST2858, was also developed. None of the 599 environmental isolates screened by PCR was ST2858. The ST2858 clinical isolates had a maximum of two single nucleotide polymorphisms in pairwise comparison, suggesting a common source. ST378 isolates had higher genomic diversity, and the isolate from the co-infection was similar to environmental ST378 from the healthcare facility’s hot water distribution system. The isolate from the co-infection harbored a plasmid conferring increased copper resistance. Understanding genomic diversity could help identify potential sources and should be considered for matching clinical with environmental isolates. Phenotypic diversity may be relevant for outbreak investigation, surveillance, and management. IMPORTANCE: Legionnaires’ disease (LD) is transmitted to humans by inhalation of aerosols contaminated with Legionella . When an outbreak occurs, identification of the source allows public officials to make sure the source is controlled to prevent further cases. In this study, whole genome sequencing was used to investigate the relatedness between clinical and environmental isolates collected during the epidemiological investigation of cases of LD centered around a single healthcare facility, providing valuable information about the diversity of Legionella within water systems and similarity thresholds for matching clinical and environmental strains. The genomic data were also used to design a methodology to rapidly screen hundreds of historical isolates and DNA extracts, which could benefit source identification in other outbreaks. Furthermore, Legionella isolates may differ in their ability to resist disinfection methods and potentially acquire novel genetic determinants, and water system characteristics may select for specific Legionella strains.

The One Health (OH) approach recognizes the interconnectedness of the health of people, animals, plants/crops and ecosystems, and is central to addressing antimicrobial resistance (AMR). The 7th Environmental Dimension of Antimicrobial Resistance Conference (EDAR7), held in Montreal in May 2024, exemplified this approach by convening international experts and stakeholders to discuss AMR research and policy progress. EDAR7 workshop #8 focused on 1) barriers to establishing effective OH AMR research programs, 2) gaps in OH AMR research priorities, and 3) potential solutions/approaches or ‘tools’ to ensure programs develop in accordance with OH principles and generate insightful data that maximizes limited resources. Key workshop outcomes included identifying critical principles for OH AMR research programs and highlighting the pivotal role of sustainable data management strategies. Additionally, the importance of considering AMR policy and risk assessment needs when planning and designing research was emphasized. Discussions explored specific tools and approaches that support the standardized and harmonized collection and analysis of data, and associated challenges of integrating genomics data into current risk assessments and models. Synthesis of the workshop’s discussions outlined critical considerations that interdisciplinary OH AMR research programs and networks should prioritize to enhance the impact of their outputs.

The disease burden from Legionella spp. infections has been increasing in many industrialized countries and, despite decades of scientific advances, ranks amongst the highest for waterborne diseases. We review here several key research areas from a multidisciplinary perspective and list critical research needs to address some of the challenges of Legionella spp. management in engineered environments. These include: (i) a consideration of Legionella species diversity and cooccurrence, beyond Legionella pneumophila only; (ii) an assessment of their environmental prevalence and clinical relevance, and how that may affect legislation, management, and intervention prioritization; (iii) a consideration of Legionella spp. sources, their definition and prioritization; (iv) the factors affecting Legionnaires’ disease seasonality, how they link to sources, Legionella spp. proliferation and ecology, and how these may be affected by climate change; (v) the challenge of saving energy in buildings while controlling Legionella spp. with high water temperatures and chemical disinfection; and (vi) the ecological interactions of Legionella spp. with other microbes, and their potential as a biological control strategy. Ultimately, we call for increased interdisciplinary collaboration between multiple research domains, as well as transdisciplinary engagement and collaboration across government, industry, and science as the way toward controlling and reducing Legionella-derived infections.

The human pathogen Campylobacter jejuni can be packaged within multilamellar bodies (MLB), also called fecal pellets, produced by ciliates such as Tetrahymena pyriformis when these microorganisms are cocultivated. This packaging increases the survival of C. jejuni in oxygenic conditions and potentially protects it against other stressors. Traditional methods for detecting and quantifying these pellets, such as transmission electron microscopy (TEM) and fluorescence microscopy, are time-consuming and labor intensive. In this study, we devised an approach for utilizing flow cytometry to distinguish and quantify C. jejuni-containing pellets produced by both T. pyriformis and T. thermophila. Cocultures of each Tetrahymena species with four different C. jejuni strains, along with monoculture controls, were incubated for 24 h, stained with SYTO9, and analysed using flow cytometry. The results revealed ciliate species-specific and bacterial strain-specific differences in the number of pellets and their fluorescence intensity. TEM confirmed that this variability in fluorescence corresponds to differences in the number of bacteria per pellet. Our method provides a rapid and efficient means of quantifying bacteria-containing MLBs, which would facilitate the screening and comparison of a large quantity of C. jejuni strains and different conditions for studying the packaging of C. jejuni by ciliates.