Microbial diversity and geochemistry of groundwater impacted by steel slag leachates.

To understand long-term impacts of steel slag material on aquifer geochemistry and microbial communities, we conducted four sampling campaigns in the Gier alluvial groundwater (Loire, France). In its northern part, the aquifer flows under a 200,000 m3 steel slag exhibiting high levels of chromium and molybdenum. Geochemical analyses of the water table revealed the existence of water masses with different chemical signatures. They allowed us to identify an area particularly contaminated by leachates from the slag heap, whatever the sampling period. Water samples from this area were compared to non-contaminated samples, with geochemical characteristics similar to the river samples. To follow changes in microbial communities, the V3-V4 region of 16 s rRNA gene was sequenced. Overall, we observed lower diversity indices in contaminated areas, with higher relative abundances of Verrucomicrobiota and Myxococcota phyla, while several Proteobacteria orders exhibited lower relative abundances. In particular, one single genus among the Verrucomicrobiota, Candidatus Omnitrophus, represented up to 36 % of total taxon abundance in areas affected by steel slag leachates. A large proportion of taxa identified in groundwater were also detected in the upstream river, indicating strong river-groundwater interactions. Our findings pave the way for future research work on C. Omnitrophus remediation capacities.

Risk Exposure to Legionella pneumophila during Showering: The Difference between a Classical and a Water Saving Shower System.

The increase in legionellosis incidence in the general population in recent years calls for a better characterization of the sources of infection, such as showering. Water-efficient shower systems that use water-atomizing technology have been shown to emit slightly more inhalable particles in the range of bacterial sizes than the traditional systems; however, the actual rate of bacterial emission remains poorly documented. The aim of this study was to assess the aerosolisation rate of the opportunistic water pathogen Legionella pneumophila during showering with one shower system representative of each technology. To achieve this objective, we performed controlled experiments inside a glove box and determined the emitted dose and viability of airborne Legionella. The bioaerosols were sampled with a Coriolis® Delta air sampler and the total number of viable (cultivable and noncultivable) Legionella was determined by flow cytometry and culture. We found that the rate of viable and cultivable Legionella aerosolized from the water jet was similar between the two showerheads: the viable fraction represents 0.02% of the overall bacteria present in water, while the cultivable fraction corresponds to only 0.0005%. The two showerhead models emitted a similar ratio of airborne Legionella viable and cultivable per volume of water used. Therefore, the risk of exposure to Legionella is not expected to increase significantly with the new generation of water-efficient showerheads.

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.

Bacterial diversity on an abandoned, industrial wasteland contaminated by polychlorinated biphenyls, dioxins, furans and trace metals.

Most former industrial sites are contaminated by mixtures of trace elements and organic pollutants. Levels of pollutants do not provide information regarding their biological impact, bioavailability and possible interactions between substances. There is genuine interest in combining chemical analyses with biological investigations. We studied a brownfield where several industrial activities were carried out starting in the 1970s, (incineration of pyralene transformers, recovery of copper by burning cables in the open air). Four representative plots showing different levels of polychlorobiphenyls were selected. Organic and trace metal levels were measured together with soil pedological characteristics. The bacterial community structure and functional diversity were assessed by 16S metagenomics with deep sequencing and community-level physiological profiling. Additionally, a vegetation survey was performed. Polychlorobiphenyls (8 mg.kg-1 to 1500 mg.kg-1) were from 2.4 × 103-fold to 6 × 105-fold higher than the European background level of 2.5 µg.kg-1. Polychlorinated dibenzo-p-dioxins and dibenzofurans ranged from 0.5 to 8.0 µg.kg-1. The soil was also contaminated with trace metals, i.e., Cu > 187, Zn > 217 and Pb > 372 mg.kg-1. Location within the study area, trace metal content and soil humidity were stronger determinants than organic pollutants of bacterial community structures and activities. Thus, the highest biological activity and the greatest bacteriological richness were observed in the plot that was less contaminated with trace metals, despite the high level of organic pollutants in the plot. Moreover, trace element pollution was associated with a relatively low presence of Actinobacteria and Rhizobia. The plot with the highest metal contamination was rich in metal-resistant bacteria such as Sphingomonadales, Geodermatophilaceae and KD4-96 (Chloroflexi phylum). Acidobacteria and Sphingomonadales, capable of resisting trace metals and degrading persistent organic pollutants, were dominant in the plots that had accumulated metal and organic contamination, but bacterial activity was lower in these plots than in the other plots.

Fungal and bacterial outbreak in the wine vinification area in the Saint-Marcel show cave.

In the Saint-Marcel cave (France), wood barrels and thousands of bottles containing red wine were stored for vinification. After storage began, a fungal and bacterial outbreak occurred, and the area was invaded by numerous types of mold colonizing the cave ceilings and all objects related to human activities (the stairwell and oenological materials). In this study, using the metabarcoding approach, we have studied the microbial outbreak and have linked the identified microorganisms to oenological activity. Both 16S and ITS primers were used to sequence the samples collected from the cave. The results showed that the dominant microorganisms proliferating in the cave were related to wine vinification. For instance, Zasmidium cellare, a strain known for living in dark and ethanol-rich environments, was the dominant fungus on the cave stairwell. Furthermore, Guehomyces pullulans, a cold-adapted yeast used for juice clarification, was recorded as the major species on the blackened limestone ceilings. These findings reveal a complex community structure in the studied cave based on the assembly of bacteria and fungi. Finally, our results demonstrate that oenological activities could seriously affect cave preservation, changing the natural microbial communities populating cave environments.

Legionella pneumophila sg1-sensing signal enhancement using a novel electrochemical immunosensor in dynamic detection mode.

This work presents a comparison between static and dynamic modes of biosensing using a novel microfluidic assay for continuous and quantitative detection of Legionella pneumophila sg1 in artificial water samples. A self-assembled monolayer of 16-amino-1-hexadecanethiol (16-AHT) was covalently linked to a gold substrate, and the resulting modified surface was used to immobilize an anti-Legionella pneumophila monoclonal antibody (mAb). The modified surfaces formed during the biosensor functionalization steps were characterized using electrochemical measurements and microscopic imaging techniques. Under static conditions, the biosensor exhibited a wide linear response range from 10 to 108 CFU/mL and a detection limit of 10 CFU/mL. Using a microfluidic system, the biosensor responses exhibited a linear relationship for low bacterial concentrations ranging from 10 to 103 CFU/mL under dynamic conditions and an enhancement of sensing signals by a factor of 4.5 compared to the sensing signals obtained under static conditions with the same biosensor for the detection of Legionella cells in artificially contaminated samples.

A valuable experimental setup to model exposure to Legionella's aerosols generated by shower-like systems.

The mechanism underlying Legionella aerosolization and entry into the respiratory tract remains poorly documented. In previous studies, we characterized the aerodynamic behaviour of Legionella aerosols and assessed their regional deposition within the respiratory tract using a human-like anatomical model. The aim of this study was to assess whether this experimental setup could mimic the exposure to bioaerosols generated by showers. To achieve this objective we performed experiments to measure the mass median aerodynamic diameter (MMAD) as well as the emitted dose and the physiological state of the airborne bacteria generated by a shower and two nebulizers (vibrating-mesh and jet nebulizers). The MMADs of the dispersed bioaerosols were characterized using a 12-stage cascade low-pressure impactor. The amount of dispersed airborne bacteria from a shower was quantified using a Coriolis® Delta air sampler and compared to the airborne bacteria reaching the thoracic region in the experimental setup. The physiological state and concentration of airborne Legionella were assessed by qPCR for total cells, culture for viable and cultivable Legionella (VC), and flow cytometry for viable but non-cultivable Legionella (VBNC). In summary, the experimental setup developed appears to mimic the bioaerosol emission of a shower in terms of aerodynamic size distribution. Compared to the specific case of a shower used as a reference in this study, the experimental setup developed underestimates by 2 times (when the jet nebulizer is used) or overestimates by 43 times (when the vibrating-mesh nebulizer is used) the total emitted dose of airborne bacteria. To our knowledge, this report is the first showing that an experimental model mimics so closely an exposure to Legionella aerosols produced by showers to assess human lung deposition and infection in well-controlled and safe conditions.

Deposition pattern of aerosolized Legionella using an ex vivo human-porcine respiratory model.

Legionella are bacteria responsible for severe lung pathologies. However how they enter and are deposited within the respiratory tract remains poorly documented. Data using animal testing led to the establishment of mathematical models allowing the estimation of aerosol dispersion risks. But direct extrapolation to humans is questionable and experimental models more physiologically representative of the inhalation route are welcome. The aim of this study was to develop a model as close as possible to the human anatomy and physiology allowing determining the deposition pattern of aerosolized Legionella while limiting in vivo experiments. To that purpose, we adapted the chimeric respiratory tract model we previously developed. This original model consisted of a replica of the human upper respiratory airways made by additive manufacturing connected to ex vivo porcine lungs ventilated by passive expansion, as for humans in physiological conditions. These experiments didn't imply specific animal sacrifices as pigs were bred for human consumption and lungs were considered as wastes by the slaughterhouse. Fluorescent Legionella were aerosolized and visualized using Cellvizio® Lab (probe-based confocal fluorescence microscope). Legionella were found in the whole respiratory tract. Broncho-alveolar lavages were also performed and the amount of Legionella reaching the thoracic region was quantified by culture and qPCR. Legionella were found preferentially in the left upper lobe compared to the right lower lobe. To our knowledge, it is the first time that experiments mimicking so closely human exposure by inhalation are performed while limiting animal experiments and providing a model for further Legionella infectious risk assessment.

Experimental human-like model to assess the part of viable Legionella reaching the thoracic region after nebulization.

The incidence of Legionnaires' disease (LD) in European countries and the USA has been constantly increasing since 1998. Infection of humans occurs through aerosol inhalation. To bridge the existing gap between the concentration of Legionella in a water network and the deposition of bacteria within the thoracic region (assessment of the number of viable Legionella), we validated a model mimicking realistic exposure through the use of (i) recent technology for aerosol generation and (ii) a 3D replicate of the human upper respiratory tract. The model's sensitivity was determined by monitoring the deposition of (i) aerosolized water and Tc99m radio-aerosol as controls, and (ii) bioaerosols generated from both Escherichia coli and Legionella pneumophila sg 1 suspensions. The numbers of viable Legionella prior to and after nebulization were provided by culture, flow cytometry and qPCR. This study was designed to obtain more realistic data on aerosol inhalation (vs. animal experimentation) and deposition at the thoracic region in the context of LD. Upon nebulization, 40% and 48% of the initial Legionella inoculum was made of cultivable and non-cultivable cells, respectively; 0.7% of both populations reached the filter holder mimicking the thoracic region in this setup. These results are in agreement with experimental data based on quantitative microbial risk assessment methods and bring new methods that may be useful for preventing LD.

Characterization of aerosols containing Legionella generated upon nebulization.

Legionella pneumophila is, by far, the species most frequently associated with Legionnaires' disease (LD). Human infection occurs almost exclusively by aerosol inhalation which places the bacteria in juxtaposition with alveolar macrophages. LD risk management is based on controlling water quality by applying standardized procedures. However, to gain a better understanding of the real risk of exposure, there is a need (i) to investigate under which conditions Legionella may be aerosolized and (ii) to quantify bacterial deposition into the respiratory tract upon nebulization. In this study, we used an original experimental set-up that enables the generation of aerosol particles containing L. pneumophila under various conditions. Using flow cytometry in combination with qPCR and culture, we determined (i) the size of the aerosols and (ii) the concentration of viable Legionella forms that may reach the thoracic region. We determined that the 0.26-2.5 µm aerosol size range represents 7% of initial bacterial suspension. Among the viable forms, 0.7% of initial viable bacterial suspension may reach the pulmonary alveoli. In conclusion, these deposition profiles can be used to standardize the size of inoculum injected in any type of respiratory tract model to obtain new insights into the dose response for LD.

Prospective evaluation of RT-PCR on sputum versus culture, urinary antigens and serology for Legionnaire's disease diagnosis.

OBJECTIVES: Legionnaires' disease (LD) is a severe disease associated with community and hospital-acquired pneumonia, frequently under diagnosed. The main aim of our study was to determine the value of PCR for the diagnosis of LD in routine clinical practice. METHODS: In a prospective study, from March 2007 to April 2010, the value of PCR on non-invasive respiratory specimens (NIRS) was compared to those of the other available tools for LD diagnosis in patients hospitalized for pneumonia. RESULTS: Among 254 consecutive cases of pneumonia included, 24 cases were LD (19 confirmed and 5 probable) representing the first documented microbiological etiology. Molecular diagnosis of LD was performed on NIRS by using 16S rRNA PCR, and secondarily mip PCR, with no discrepant results between the 2 methods: it was found positive in 14 cases and led to identify 2 supplementary probable cases of LD. Based on clinical and at least 2 positive LD tests, PCR yielded a better diagnostic value than antigen urinary test (12 vs 10 cases). CONCLUSION: These results revealed that molecular diagnosis of LD on NIRS is reliable and may contribute to better identify cases of LD.

Monitoring of Legionella pneumophila viability after chlorine dioxide treatment using flow cytometry.

The viability of three Legionella pneumophila strains was monitored after chlorine dioxide (ClO2) treatment using a flow cytometric assay. Suspensions of L. pneumophila cells were submitted to increasing concentrations of ClO2. Culturable cells were still detected when using 4 mg/L, but could no longer be detected after exposure to 6 mg/L of ClO2, although viable but not culturable (VBNC) cells were found after exposure to 4-5 mg/L of ClO2. When testing whether these VBNC were infective, two of the strains were resuscitated after co-culture with Acanthamoeba polyphaga, but neither of them could infect macrophage-like cells.

Viable but not culturable forms of Legionella pneumophila generated after heat shock treatment are infectious for macrophage-like and alveolar epithelial cells after resuscitation on Acanthamoeba polyphaga.

Legionella pneumophila, the causative agent of legionellosis is transmitted to human through aerosols from environmental sources and invades lung's macrophages. It also can invade and replicate within various protozoan species in environmental reservoirs. Following exposures to various stresses, L. pneumophila enters a non-replicative viable but non-culturable (VBNC) state. Here, we evaluated whether VBNC forms of three L. pneumophila serogroup 1 strains (Philadelphia GFP 008, clinical 044 and environmental RNN) infect differentiated macrophage-like cell lines (U937 and HL-60), A549 alveolar cells and Acanthamoeba polyphaga. VBNC forms obtained following shocks at temperatures ranging from 50 to 70 °C for 5 to 60 min were quantified using a flow cytometric assay (FCA). Their loss of culturability was checked on BCYE agar medium. VBNC forms were systematically detected upon a 70 °C heat shock for 30 min. When testing their potential to resuscitate upon amoebal infection, VBNC forms obtained after 30 min at 70 °C were re-cultivated except for the clinical strain. No resuscitation or cell lysis was evidenced when using U937, HL-60, or A549 cells despite the use of various contact times and culture media. None of the strains tested could infect A. polyphaga, macrophage-like or alveolar epithelial cells after a 60-min treatment at 70 °C. However, heat-treated VBNC forms were able to infect macrophage-like or alveolar epithelial cells following their resuscitation on A. polyphaga. These results suggest that heat-generated VBNC forms of L. pneumophila (i) are not infectious for macrophage-like or alveolar epithelial cells in vitro although resuscitation is still possible using amoeba, and (ii) may become infectious for human cell lines following a previous interaction with A. polyphaga.

Longitudinal evaluation of the efficacy of heat treatment procedures against Legionella spp. in hospital water systems by using a flow cytometric assay.

Legionella spp. are frequently isolated in hospital water systems. Heat shock (30 min at 70°C) is recommended by the World Health Organization to control its multiplication. The aim of the study was to evaluate retrospectively the efficacy of heat treatments by using a flow cytometry assay (FCA) able to identify viable but nonculturable (VBNC) cells. The study included Legionella strains (L. pneumophila [3 clusters] and L. anisa [1 cluster]) isolated from four hot water circuits of different hospital buildings in Saint-Etienne, France, during a 20-year prospective surveillance. The strains recovered from the different circuits were not epidemiologically related, but the strains isolated within a same circuit over time exhibited an identical genotypic profile. After an in vitro treatment of 30 min at 70°C, the mean percentage of viable cells and VBNC cells varied from 4.6% to 71.7%. The in vitro differences in heat sensitivity were in agreement with the observed efficacy of preventive and corrective heating measures used to control water contamination. These results suggest that Legionella strains can become heat resistant after heating treatments for a long time and that flow cytometry could be helpful to check the efficacy of heat treatments on Legionella spp. and to optimize the decontamination processes applied to water systems for the control of Legionella proliferation.

Use of flow cytometry to monitor Legionella viability.

Legionella viability was monitored during heat shock treatment at 70 degrees C by a flow cytometric assay (FCA). After 30 min of treatment, for 6 of the 12 strains tested, the FCA still detected 10 to 25% of cells that were viable but nonculturable (VBNC). These VBNC cells were able to produce ATP and to be resuscitated after culture on amoebae.

Implementation of the EGFP-K562 flow cytometric NK test: determination of NK cytotoxic activity in healthy elderly volunteers before and after feeding.

BACKGROUND: Natural Killer (NK) cells are key actors of innate immunity that supervise the organism's cells, and fight against viral infections and cancer development through their cytotoxic activity. This cytotoxic activity is modulated by cytokines and hormones and could be influenced by physiological or pathological conditions. New techniques for measuring NK cytotoxic activity by flow-cytometry have recently been developed, and they correlated strongly with the standard chromium ((51)Cr) release assay. Our aim was to implement a previously published enhanced green fluorescent protein (EGFP)-K562 flow cytometric method and use it to evaluate NK cytotoxic activity under different nutritional conditions. METHODS: NK effector cells were isolated from peripheral blood mononuclear cells, and a K562 cell line stably transfected by EGFP was used as target cells. Different analytical parameters, including cell ratios and incubation times, were studied to improve the EGFP-K562 flow cytometric NK test conditions. RESULTS: The optimized test was then used to determine the effect of fasting and refeeding on NK cell numbers and activity in a physiological situation. NK cytotoxic activity in fasted conditions (30.4 +/- 4.4%) increased by a factor 1.7 +/- 0.2 (P = 0.0025) in nourished conditions (45.0 +/- 4.6%) in healthy elderly people. CONCLUSION: Therefore, this method provides a reliable, reproducible and rapid test for analyzing NK cytotoxicity under various conditions.

A 7.1 kbp beta-myosin heavy chain promoter, efficient for green fluorescent protein expression, probably induces lethality when overexpressing a mutated transforming growth factor-beta type II receptor in transgenic mice.

The roles of transforming growth factor-beta (TGFbeta) in heart or skeletal muscle development and physiology are still the subject of controversies. Our aim was to block, in transgenic mice, the TGFbeta signalling pathway by a dominant negative mutant of the TGFbeta type II receptor fused to the enhanced green fluorescent protein (TbetaRII-KR-EGFP) under the control of a 7.1 kbp mouse beta-myosin heavy chain (betaMHC) promoter to investigate the roles of TGFbeta in the heart and slow skeletal muscles. First, we generated two transgenic lines overexpressing EGFP under the control of the 7.1 kbp betaMHC promoter. In embryos, EGFP was detectable as early as 7.5 days post coitum. In embryos, newborns and adults, EGFP was expressed mainly in the cardiac ventricles and in slow skeletal muscles. EGFP expression was intense in the bladder but weak in the intestines. In contrast to the endogenous betaMHC promoter, the activity of the 7.1 kbp betaMHC promoter in the transgene was not repressed after birth and remained high in adult transgenic mice. We obtained two founders with the transgene comprising the TbetaRII-KR-EGFP sequence under the control of the 7.1 kbp betaMHC promoter. These founders were generated at a very low frequency and expressed barely detectable levels of TbetaRII-KR-EGFP mRNA. Our failure to obtain transgenic lines overexpressing the dominant negative receptor suggests that the blocking of the TGFbeta signalling pathway in the heart and slow skeletal muscles could be embryonically lethal. To conclude, the 7.1 kbp betaMHC promoter directs high levels of transgene expression in the cardiac ventricles and in slow skeletal muscles of the mouse. Analysis of the consequences of the blocking of the TGFbeta signalling pathway in the heart will require the use of tissue specific means of conditional gene invalidation.

Terminal differentiation of Sol 8 myoblasts is retarded by a transforming growth factor-beta autocrine regulatory loop.

In DM (differentiation medium), Sol 8 myoblasts spontaneously form myotubes and express the betaMHC (beta-myosin heavy chain), their main marker of terminal differentiation. This marker is detectable at 24 h, and increases up to 72 h. Our aim was to define temporal effects of TGFbeta (transforming growth factor beta) on betaMHC expression in Sol 8 cells. TGFbeta1 (1 ng/ml) added at time zero to DM decreased MyoD expression and completely inhibited betaMHC expression in Sol 8 cells. This inhibition of betaMHC expression was progressively lost when TGFbeta1 was added from 8 to 34 h. After 34 h, the cells were irreversibly differentiated, and TGFbeta1 did not inhibit betaMHC accumulation any longer. Two independent approaches showed that a TGFbeta autocrine regulatory loop retarded and partially impaired Sol 8 cell terminal differentiation. First, permanent immunoneutralization of the active TGFbetas released by the cells into DM increased betaMHC levels at 72 h compared with controls. Secondly, a dominant-negative mutant of the TGFbeta type II receptor was overexpressed in Sol 8 cells under the control of the betaMHC promoter. Both the dominant-negative receptor and the betaMHC gene were expressed after 24 h in DM. The delayed blocking of the TGFbeta signalling pathway by the dominant-negative receptor was as effective as permanent immunoneutralization to promote betaMHC expression. To conclude, TGFbeta inhibits Sol 8 cell terminal differentiation within a narrow time interval (24-34 h) that coincides with the onset of betaMHC expression.