Biology subjects

Faucher, S. P.

Publications and source records attributed to Faucher, S. P..

8 recordsLinked to original sources

bioRxiv2024

Copper resistance in Legionella pneumophila: role of genetic factors and host cells.

Copper is frequently found in drinking water due to its presence in the natural environment and the widespread usage of copper pipes. This toxic metal has a well-known antimicrobial activity, an activity harnessed in copper-silver ionization (CSI) to eliminate the opportunistic pathogen Legionella pneumophila from engineered water systems. Despite utilizing the antimicrobial properties of copper in Legionella control, little is known about how copper containing environments affect L. pneumophila populations. The goal of this study is to understand how L. pneumophila responds to copper within a hot water distribution system (HWDS) environment. To answer this question, different sequence types and regulatory mutants were exposed to copper to compare their survival. L. pneumophila isolates of 4 sequence types from 3 different HWDSs exhibited a wide diversity of phenotypes after copper stress. The {Delta}letA and {Delta}letS mutants were sensitive to copper, indicating that the LetAS two component system is important for copper resistance. Additionaly, transmissive phase cultures were more resistant to copper than replicative phase cultures. Therefore, the regulation of entry into transmissive phase by the LetAS system is essential for L. pneumophilas ability to survive copper stress. In a water system, L. pneumophila replicates within eukaryotic hosts. When cocultured with the host ciliate Tetrahymena pyriformis, L. pneumophila was more resistant to copper than when the bacteria were in a monoculture. No difference in L. pneumophila replication inside of hosts in cocultures with or without copper was observed. This result confirms that the presence of host cells protects L. pneumophila from copper stress. Therefore, presence of host cells in water system may limit the efficacy of copper-based control strategies.

microbiology↗

bioRxiv2023

Development of heat shock resistance in L. pneumophila modeled by experimental evolution

Because it can grow in buildings with complex hot water distribution systems (HWDS), healthcare facilities recognize the waterborne bacterium Legionella pneumophila as a major nosocomial infection threat and often try to clear the systems with a pasteurization process known as superheat-and-flush. After this treatment, many facilities find that the contaminating populations slowly recover, suggesting the possibility of in situ evolution favouring increased survival in high temperature conditions. To mimic this process in a controlled environment, an adaptive laboratory evolution (ALE) model was used to select a wild-type strain of L. pneumophila for survival to transient exposures to temperatures characteristic of routine hot water use or failed pasteurization processes in HWDS. Over their evolution, these populations became insensitive to exposure to 55 {degrees}C and innovated the ability to survive short exposures to 59 {degrees}C heat shock. Heat-adapted lineages maintained a higher expression of heat shock genes during low-temperature incubation in freshwater, suggesting a pre-adaptation to heat stress. Although there were distinct mutation profiles in each of the heat-adapted lineages, each acquired multiple mutations in the DnaJ/DnaK/ClpB disaggregase complex, as well as mutations in chaperone htpG and protease clpX. These mutations were specific to heat shock survival and were not seen in control lineages included in the ALE without exposure to heat shock. This study supports in situ observations of adaptation to heat stress and demonstrate the potential of L. pneumophila to develop resistance to control measures. ImportanceAs a bacterium that thrives in warm water ecosystems, Legionella pneumophila is a key factor motivating regulations on hot water systems. Two major measures intended to control Legionella are the maintenance of high circulating temperatures to curtail growth and the use of superheat-and-flush pasteurization processes to eliminate established populations. Although hospitals are particularly vulnerable to nosocomial pneumoniae caused by Legionella, they recurrently experience recolonization of their hot water systems after treatment. To understand these long-term survivors, we have used an experimental evolution model to replicate this process. We find major differences between the mutational profiles of heat-adapted and heat-naive L. pneumophila populations, including mutations in major heat shock genes like chaperones and proteases. This model demonstrates the value of appropriate heat treatment of L. pneumophila contaminated systems and - in an analogue to antibiotic resistance - the importance of complete eradication of the resident population to prevent selection for more persistent bacteria.

microbiology↗

bioRxiv2022

Toxoflavin secreted by Pseudomonas alcaliphila inhibits growth of Legionella pneumophila and its host Vermamoeba vermiformis

Legionella pneumophila is a natural inhabitant of water systems. From there, it can be transmitted to humans by aerosolization resulting in severe pneumonia. Most large outbreaks are caused by cooling towers contaminated with L. pneumophila. The resident microbiota of the cooling tower is a key determinant for the colonization and growth of L. pneumophila. The genus Pseudomonas correlates negatively with the presence of L. pneumophila, but it is not clear which species is responsible. Therefore, we identified the Pseudomonas species inhabiting 14 cooling towers using a Pseudomonas-specific 16S rRNA amplicon sequencing strategy. Cooling towers free of L. pneumophila contained a high relative abundance of members from the Pseudomonas alcaliphila/oleovorans phylogenetic cluster. In vitro, P. alcaliphila JCM 10630 inhibited the growth of L. pneumophila on agar plates. Analysis of the P. alcaliphila genome revealed the presence of a genes cluster predicted to produce toxoflavin. L. pneumophila growth was inhibited by pure toxoflavin and by extract from P. alcaliphila culture found to contain toxoflavin by LC-ESI-MS. In addition, toxoflavin inhibits growth of Vermameoba vermiformis, a host cell of L. pneumophila. Our study indicates that P. alcaliphila may be important to restrict growth of L. pneumophila in water systems through the production of toxoflavin. A sufficiently high concentration is likely not achieved in the bulk water but might have a local inhibitory effect such as in biofilm.

microbiology↗

bioRxiv2020

Legionella pneumophila's Tsp is important for surviving thermal stress in water and inside amoeba

Legionella pneumophila (Lp) is an inhabitant of natural and man-made water systems where it replicates within amoebae and ciliates and survives within biofilms. When Lp-contaminated aerosols are breathed in, Lp will enter the lungs and infect human alveolar macrophages, causing a severe pneumonia known as Legionnaires Disease. Lp is often found in hot water distribution systems (HWDS), which are linked to nosocomial outbreaks. Heat treatment is used to disinfect HWDS and reduce the concentration of Lp. However, Lp is often able to recolonize these water systems, indicating an efficient heat-shock response. Tail-specific proteases (Tsp) are typically periplasmic proteases implicated in degrading aberrant proteins in the periplasm and important for surviving thermal stress. In this paper, we show that Tsp, encoded by the lpg0499 gene in Lp Philadelphia-1, is important for surviving thermal stress in water and for optimal infection of amoeba when a shift in temperature occurs during intracellular growth. Tsp is expressed in the post-exponential phase but repressed in the exponential phase. The cis-encoded small regulatory RNA Lpr17 shows opposite expression, suggesting that it represses translation of tsp. In addition, tsp is regulated by CpxR, a major regulator in Lp, in a Lpr17-independent manner. Deletion of CpxR also reduced the ability of Lp to survive heat shock. In conclusion, this study shows that Tsp is an important factor for the survival and growth of Lp in water systems. IMPORTANCELegionella pneumophila (Lp) is a major cause of nosocomial and community-acquired pneumonia. Lp is found in water systems including hot water distribution systems. Heat treatment is a method of disinfection often used to limit Lps presence in such systems; however, the benefit is usually short term as Lp is able to quickly recolonize these systems. Presumably, Lp respond efficiently to thermal stress, but so far not much is known about the genes involved. In this paper, we show that the Tail-specific protease (Tsp) and the two-component system CpxRA are required for resistance to thermal stress, when Lp is free in water and when it is inside host cells. Our study identifies critical systems for the survival of Lp in its natural environment under thermal stress.

microbiology↗

bioRxiv2020

Local adaptation of Legionella pneumophila within a hospital hot water system increases tolerance to copper

In large-building water systems, Legionella pneumophila is exposed to common environmental stressors such as copper. The aim of this study was to evaluate the susceptibility to copper of L. pneumophila isolates recovered from various sites: two clinical and seven environmental from hot water systems biofilm & water, and from cooling tower water. After one-week acclimation in simulated drinking water, strains were exposed to various copper concentrations (0.8 to 5 mg/L) for over 672 hours. Complete loss of culturability was observed for three isolates, following copper exposure to 5 mg/L for 672h. Two ST1427-like isolates were highly sensitive to copper, while the other two, isolated from biofilm samples, were resistant. The expression of the copper resistance gene copA evaluated by RT-qPCR was significantly higher for the biofilm isolates. All four ST1427-like isolates were recovered from the same water system during an outbreak. Whole genome sequencing results confirmed that the four isolates are very close phylogenetically, differing by only 29 single nucleotide polymorphisms, suggesting in situ adaptation to microenvironmental conditions, possibly due to epigenetic regulation. These results indicate that the immediate environment within a building water distribution system influences the tolerance of L. pneumophila to copper. Increased contact of L. pneumophila biofilm strains with copper piping or copper alloys in the heat exchanger might lead to local adaptation. The phenotypic differences observed between water and biofilm isolates from the hot water system of a healthcare facility warrants further investigation to assess the relevance of evaluating disinfection performances based on water sampling alone. ImportanceLegionella pneumophila is a pathogen indigenous to natural and large building water systems in the bulk and the biofilm phases. The immediate environment within a system can impact the tolerance of L. pneumophila to environmental stressors, including copper. In healthcare facilities, copper levels in water can vary, depending on water quality, plumbing materials and age. This study evaluated the impact of the isolation site (water vs biofilm, hot water system vs cooling tower) within building water systems. Closely related strains isolated from a healthcare facility hot water system exhibited variable tolerance to copper stress shown by differential expression of copA, with biofilm isolates displaying highest expression and tolerance. Relying on the detection of L. pneumophila in water samples following exposure to environmental stressor such as copper may underestimate the prevalence of L. pneumophila, leading to inappropriate risk management strategies and increasing the risk of exposure for vulnerable patients.

microbiology↗

bioRxiv2019

Impact of temperature on Legionella pneumophila, its protozoan host cells, and the microbial diversity of the biofilm community of a pilot cooling tower

Legionella pneumophila (Lp) is a waterborne bacterium known for causing Legionnaires Disease, a severe pneumonia. Cooling towers are a major source of outbreaks, since they provide ideal conditions for Lp growth and produce aerosols. In such systems, Lp typically grow inside protozoan hosts. Several abiotic factors such as water temperature, pipe material and disinfection regime affect the colonization of cooling towers by Lp. The local physical and biological factors promoting the growth of Lp in water systems and its spatial distribution are not well understood. Therefore, we built a lab-scale cooling tower to study the dynamics of Lp colonization in relationship to the resident microbiota and spatial distribution. The pilot was filled with water from an operating cooling tower harboring low levels of Lp. It was seeded with Vermamoeba vermiformis, a natural host of Lp, and then inoculated with Lp. After 92 days of operation, the pilot was disassembled, the water was collected, and biofilm was extracted from the pipes. The microbiome was studied using 16S rRNA and 18S rRNA genes amplicon sequencing. The communities of the water and of the biofilm were highly dissimilar. The relative abundance of Legionella in water samples reached up to 11% whereas abundance in the biofilm was extremely low ([≤]0.5 %). In contrast, the host cells were mainly present in the biofilm. This suggest that Lp grows in host cells associated with biofilm and is then released back into the water following host cell lysis. In addition, water temperature shaped the bacterial and eukaryotic community of the biofilm, indicating that different parts of the systems may have different effects on Legionella growth.

microbiology↗

bioRxiv2019

Identification of two aptamers binding to Legionella pneumophila with high affinity and specificity

Legionella pneumophila (Lp) is a water borne bacterium causing Legionnaires Disease (LD) in humans. Rapid detection of Lp in water system is essential to reduce the risk of LD outbreaks. The methods currently available require expert skills and are time intensive, thus delaying intervention. In situ detection of Lp by biosensor would allow rapid implementation of control strategies. To this end, a biorecognition element is required. Aptamers are considered promising biorecognition molecules for biosensing. Aptamers are short oligonucleotide sequence folding into a specific structure and are able to bind to specific molecules. Currently no aptamer and thus no aptamer-based technology exists for the detection of Lp. In this study, Systemic Evolution of Ligands through EXponential enrichment (SELEX) was used to identify aptamers binding specifically to Lp. Ten rounds of positive selection and two rounds of counter-selection against two Pseudomonas species were performed. Two aptamers binding strongly to Lp were identified with KD of 116 and 135 nM. Binding specificity of these two aptamers to Lp was confirmed by flow cytometry and fluorescence microscopy. Therefore, these two aptamers are promising biorecognition molecules for the detection of Lp in water systems.

microbiology↗

bioRxiv2019

Presence of Legionella spp. in cooling towers: the role of microbial diversity, Pseudomonas, and continuous chlorine application.

Legionnaires Disease (LD) is a severe pneumonia caused by Legionella pneumophila. Cooling towers are the main source of L. pneumophila during large outbreaks. Colonization, survival, and proliferation of L. pneumophila in cooling towers are necessary for outbreaks to occur. These steps are affected by chemical and physical parameters of the cooling tower environment. We hypothesize that the bacterial community residing in the cooling tower could also affect the presence of L. pneumophila. A 16S rRNA targeted amplicon sequencing approach was used to study the bacterial community of cooling towers and its relationship with the Legionella spp. and L. pneumophila communities. The results indicated that the water source shaped the bacterial community of cooling towers. Several taxa were enriched and positively correlated with Legionella spp. and L. pneumophila. In contrast, Pseudomonas showed a strong negative correlation with Legionella spp. and several other genera. Most importantly, continuous chlorine application reduced microbial diversity and promoted the presence of Pseudomonas creating a non-permissive environment for Legionella spp. This suggests that disinfection strategies as well as the resident microbial population influences the ability of Legionella spp. to colonize cooling towers.

microbiology↗