Rapid adaptations of Legionella pneumophila to the human host.

Legionella pneumophila are host-adapted bacteria that infect and reproduce primarily in amoeboid protists. Using similar infection mechanisms, they infect human macrophages, and cause Legionnaires' disease, an atypical pneumonia, and the milder Pontiac fever. We hypothesized that, despite the similarities in infection mechanisms, the hosts are different enough that there exist high-selective value mutations that would dramatically increase the fitness of Legionella inside the human host. By comparing a large number of isolates from independent infections, we identified two genes, mutated in three unrelated patients, despite the short duration of the incubation period (2-14 days). One is a gene coding for an outer membrane protein (OMP) belonging to the OmpP1/FadL family. The other is a gene coding for an EAL-domain-containing protein involved in cyclic-di-GMP regulation, which in turn modulates flagellar activity. The clinical strain, carrying the mutated EAL-domain-containing homologue, grows faster in macrophages than the wild-type strain, and thus appears to be better adapted to the human host. As human-to-human transmission is very rare, fixation of these mutations into the population and spread into the environment is unlikely. Therefore, parallel evolution - here mutations in the same genes observed in independent human infections - could point to adaptations to the accidental human host. These results suggest that despite the ability of L. pneumophila to infect, replicate in and exit from macrophages, its human-specific adaptations are unlikely to be fixed in the population.

Toxoflavin secreted by Pseudomonas alcaliphila inhibits the growth of Legionella pneumophila and 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 colonized with L. pneumophila. The resident microbiota of the cooling tower is a key determinant for the colonization and growth of L. pneumophila. In our preceding study, the genus Pseudomonas correlated negatively with the presence of L. pneumophila in cooling towers, but it was not clear which species was responsible. Therefore, we identified the Pseudomonas species inhabiting 14 cooling towers using a Pseudomonas-specific 16S rRNA amplicon sequencing strategy. We found that cooling towers that are free of L. pneumophila contained a high relative abundance of members from the Pseudomonas alcaliphila/oleovorans phylogenetic cluster. P. alcaliphila JCM 10630 inhibited the growth of L. pneumophila on agar plates. Analysis of the P. alcaliphila genome revealed the presence of a gene cluster predicted to produce toxoflavin. L. pneumophila growth was inhibited by pure toxoflavin and by extracts from P. alcaliphila culture found to contain toxoflavin by liquid chromatography coupled with mass spectrometry. In addition, toxoflavin inhibits the 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 of toxoflavin is likely not achieved in the bulk water but might have a local inhibitory effect such as near or in biofilms.

Bacterial Antagonistic Species of the Pathogenic Genus Legionella Isolated from Cooling Tower.

Legionella pneumophila is the causative agent of Legionnaires' disease, a severe pneumonia. Cooling towers are a major source of large outbreaks of the disease. The growth of L. pneumophila in these habitats is influenced by the resident microbiota. Consequently, the aim of this study was to isolate and characterize bacterial species from cooling towers capable of inhibiting several strains of L. pneumophila and one strain of L. quinlivanii. Two cooling towers were sampled to isolate inhibiting bacterial species. Seven inhibitory isolates were isolated, through serial dilution plating and streaking on agar plates, belonging to seven distinct species. The genomes of these isolates were sequenced to identify potential genetic elements that could explain the inhibitory effect. The results showed that the bacterial isolates were taxonomically diverse and that one of the isolates may be a novel species. Genome analysis showed a high diversity of antimicrobial gene products identified in the genomes of the bacterial isolates. Finally, testing different strains of Legionella demonstrated varying degrees of susceptibility to the antimicrobial activity of the antagonistic species. This may be due to genetic variability between the Legionella strains. The results demonstrate that though cooling towers are breeding grounds for L. pneumophila, the bacteria must contend with various antagonistic species. Potentially, these species could be used to create an inhospitable environment for L. pneumophila, and thus decrease the probability of outbreaks occurring.

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 isolates from hot water system biofilm and water and from cooling tower water. After a 1-week acclimation in simulated drinking water, strains were exposed to various copper concentrations (0.8 to 5 mg/liter) for over 672 h. Complete loss of culturability was observed for three isolates following copper exposure to 5 mg/liter for 672 h. Two sequence type 1427 (ST1427)-like isolates were highly sensitive to copper, while the other two, isolated from biofilm samples, maintained higher culturability. The expression of the copper resistance gene copA evaluated by reverse transcription-quantitative PCR (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 health care 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 health care 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 versus biofilm, hot water system versus cooling tower) within building water systems. Closely related strains isolated from a health care 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 stressors 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.

Unravelling the importance of the eukaryotic and bacterial communities and their relationship with Legionella spp. ecology in cooling towers: a complex network.

BACKGROUND: Cooling towers are a major source of large community-associated outbreaks of Legionnaires' disease, a severe pneumonia. This disease is contracted when inhaling aerosols that are contaminated with bacteria from the genus Legionella, most importantly Legionella pneumophila. How cooling towers support the growth of this bacterium is still not well understood. As Legionella species are intracellular parasites of protozoa, it is assumed that protozoan community in cooling towers play an important role in Legionella ecology and outbreaks. However, the exact mechanism of how the eukaryotic community contributes to Legionella ecology is still unclear. Therefore, we used 18S rRNA gene amplicon sequencing to characterize the eukaryotic communities of 18 different cooling towers. The data from the eukaryotic community was then analysed with the bacterial community of the same towers in order to understand how each community could affect Legionella spp. ecology in cooling towers. RESULTS: We identified several microbial groups in the cooling tower ecosystem associated with Legionella spp. that suggest the presence of a microbial loop in these systems. Dissolved organic carbon was shown to be a major factor in shaping the eukaryotic community and may be an important factor for Legionella ecology. Network analysis, based on co-occurrence, revealed that Legionella was correlated with a number of different organisms. Out of these, the bacterial genus Brevundimonas and the ciliate class Oligohymenophorea were shown, through in vitro experiments, to stimulate the growth of L. pneumophila through direct and indirect mechanisms. CONCLUSION: Our results suggest that Legionella ecology depends on the host community, including ciliates and on several groups of organisms that contribute to its survival and growth in the cooling tower ecosystem. These findings further support the idea that some cooling tower microbiomes may promote the survival and growth of Legionella better than others. Video Abstract.

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 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 L. pneumophila growth and produce aerosols. In such systems, L. pneumophila typically grow inside protozoan hosts. Several abiotic factors such as water temperature, pipe material and disinfection regime affect the colonization of cooling towers by L. pneumophila. The local physical and biological factors promoting the growth of L. pneumophila in water systems and its spatial distribution are not well understood. Therefore, we built a lab-scale cooling tower to study the dynamics of L. pneumophila 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 L. pneumophila. It was seeded with Vermamoeba vermiformis, a natural host of L. pneumophila, and then inoculated with L. pneumophila. 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 suggests that L. pneumophila 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.

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 several species of the genus Legionella, most frequently by Legionella pneumophila. Cooling towers are the most common source for large community-associated outbreaks. Colonization, survival, and proliferation of L. pneumophila in cooling towers are necessary for outbreaks to occur. These steps are affected by the 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 gene 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.

Legionella pneumophila levels and sequence-type distribution in hospital hot water samples from faucets to connecting pipes.

Recent studies have reported increased levels of Legionella pneumophila (Lp) at points of use compared to levels in primary and secondary components of hot water systems, suggesting possible selection by environmental conditions. In this study, concentrations of Lp in a hospital hot water system were evaluated by profile sampling, collecting successive water samples to determine the prevalence at the faucet (distal) and upstream piping before and after a system intervention to increase temperature. Lp strain diversity was compared between different points of use and different areas of the hot water system (i.e., tap, intermediate piping and main upflow piping). In total, 47 isolates were recovered from 32 positive hot water samples collected from designated taps, showers and recirculation loops; these isolates were subsequently analyzed by sequence-based typing (SBT). Lp levels were comparable between first draw (500 mL) and flushed (2 and 5 min) samples, whereas a decrease was observed in the amount of culturable cells (1 log). Two sequence types (STs) were identified throughout the system. ST378 (sg4/10) was present in 91% of samples, while ST154-like (sg1) was present in 41%; both STs were simultaneously recovered in 34% of samples. Isolated STs displayed comparable tolerance to copper (0.8-5 mg/L) and temperature (55 °C, 1 h) exposure. The ability to replicate within THP1 cells and Acanthamoeba castellanii was similar between the two STs and a comparative environmental outbreak strain. The low Lp diversity and the detection of both Lp sequence types in repeated subsequent samples collected from positive faucets in a hospital wing suggest a minimal impact of the distal conditions on strain selection for the sampled points, as well as a possible adaptation to stressors present in the system, leading to the predominance of a few strains.

Energy Conservation and the Promotion of Legionella pneumophila Growth: The Probable Role of Heat Exchangers in a Nosocomial Outbreak.

OBJECTIVE To determine the source of a Legionella pneumophila serogroup 5 nosocomial outbreak and the role of the heat exchanger installed on the hot water system within the previous year. SETTING A 400-bed tertiary care university hospital in Sherbrooke, Canada. METHODS Hot water samples were collected and cultured for L. pneumophila from 25 taps (baths and sinks) within wing A and 9 taps in wing B. Biofilm (5) and 2 L water samples (3) were collected within the heat exchangers for L. pneumophila culture and detection of protists. Sequence-based typing was performed on strain DNA extracts and pulsed-field gel electrophoresis patterns were analyzed. RESULTS Following 2 cases of hospital-acquired legionellosis, the hot water system investigation revealed a large proportion of L. pneumophila serogroup 5 positive taps (22/25 in wing A and 5/9 in wing B). High positivity was also detected in the heat exchanger of wing A in water samples (3/3) and swabs from the heat exchanger (4/5). The outbreak genotyping investigation identified the hot water system as the source of infections. Genotyping results revealed that all isolated environmental strains harbored the same related pulsed-field gel electrophoresis pattern and sequence-based type. CONCLUSIONS Two cases of hospital-acquired legionellosis occurred in the year following the installation of a heat exchanger to preheat hospital hot water. No cases were reported previously, although the same L. pneumophila strain was isolated from the hot water system in 1995. The heat exchanger promoted L. pneumophila growth and may have contributed to confirmed clinical cases. Infect. Control Hosp. Epidemiol. 2016;1475-1480.

Effect of nitrogen regime on microalgal lipid production during mixotrophic growth with glycerol.

Mixotrophic growth of microalgae to boost lipid production is currently under active investigation. Such a process could be of practical importance if a cheap source of organic carbon, such as waste glycerol from biodiesel production, could be used. Several previous studies have already demonstrated that this carbon source can be used by different indigenous strains of microalgae. In this study it is shown that different nitrogen limitation strategies can be applied to further increase lipid production during growth with glycerol. In one strategy, cultures were grown in nitrogen replete medium and then resuspended in nitrogen free medium. In a second strategy, cultures were grown with different initial concentrations of nitrate. Lipid production by the two microalgal strains used, Chlorella sorokiniana (PCH02) and Chlorella vulgaris (PCH05), was shown to be boosted by strategies of nitrogen limitation, but they responded differently to how nitrogen limitation was imposed.

Regulation and production of Tcf, a cable-like fimbriae from Salmonella enterica serovar Typhi.

tcf (Typhi colonization factor) is one of the 12 putative chaperone/usher fimbrial clusters present in the Salmonella enterica serovar Typhi genome. We investigated the production, expression and regulation of tcf as well as its role during interaction with human cells. The tcf gene cluster was cloned and induced in Escherichia coli and S. Typhi, and the production of intertwined fibres similar to the Cbl (cable) pili of Burkholderia cepacia was observed on the bacterial surface by electron microscopy. In S. Typhi, tcf was expressed more after growth in M63 minimal medium than in standard Luria-Bertani medium. Analysis of the promoter region identified putative binding sites for the global regulators RcsB, ArgR and Fur. The expression of tcf was measured in isogenic strains lacking these global regulators. Under the conditions tested, the results showed that tcf expression was higher in the fur mutant and was regulated by iron concentration. Fur may regulate these fimbriae indirectly via the small RNAs RyhB1 and RyhB2. An isogenic mutant harbouring a deletion of the tcf cluster did not demonstrate any defect in adhesion or invasion of human epithelial cells, or in phagocytosis or survival in macrophages, when compared to the WT serovar Typhi strain. However, the tcf cluster contributed to adherence to human epithelial cells when introduced into E. coli. Thus, tcf genes encode functional fimbriae that can act as an adhesin and may contribute to colonization during typhoid fever.

Strain variation in microalgal lipid production during mixotrophic growth with glycerol.

Algal cultivation at high latitudes is challenged by the relatively low annual solar flux. One possible scenario to overcome this limitation is the use of mixotrophic growth to potentially boost biomass and lipid production. Here the effect of glycerol addition on the growth and lipid production by twelve indigenous microalgae was examined. The results show that there is considerable strain dependent variation in the maximum growth rate under mixotrophic conditions with the addition of glycerol causing in some cases up to a 2.4-fold increase in growth rate and a up to a 1.9-fold increase in biomass. In addition, glycerol increased total lipid production 40-60% in some strains. These results also show the value in screening culture collections for desired traits independent of strain identification since here one (PCH02) of the five Chlorella strains showed a large increase in lipid with glycerol.

Utilization of biodiesel-derived glycerol or xylose for increased growth and lipid production by indigenous microalgae.

Microalgae are a promising alternative for sustainable biofuel production, but production yields and costs present a significant bottleneck. Here, the use of glycerol and xylose to boost the lipid yield was evaluated using ten strains from the Université de Montréal collection of microalgae. This report shows that some microalgal strains are capable of mixotrophic and heterotrophic growth on xylose, the major carbon source found in wastewater streams from pulp and paper industries, with an increase in growth rate of 2.8-fold in comparison to photoautotrophic growth, reaching up to µ=1.1/d. On glycerol, growth rates reached as high as µ=1.52/d. Lipid productivity increased up to 370% on glycerol and 180% on xylose for the strain LB1H10, showing the suitability of this strain for further development of biofuels production through mixotrophic cultivation.