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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.

International audience; 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.

Legionella are bacteria responsible for severe lung pathologies but how they are aerosolized and enter the respiratory tract remains poorly documented. Previous data using animal experimentations (especially through intratracheal instillations) led to the establishment of mathematical models allowing the estimation of aerosol dispersion risks. But direct extrapolation to humans is questionable and a model more representative of a physiological route (for example inhalation of aerosols) should be developed. The aim of this study was to determine the deposition pattern of aerosolized Legionella in a model as close as possible to the human anatomy and to the physiological respiratory function while limiting in vivo experiments. To that purpose, we used a human-porcine respiratory tract model previously published. This chimeric model consists of a replica of the upper respiratory airways made by additive manufacturing connected to ex vivo porcine lungs ventilated by passive expansion, as for humans in physiological conditions. In 6 independent experiments, fluorescent Legionella were aerosolized in the model and visualized using the Cellvizio Lab technology (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 quantitative PCR. 0.3 to 21% of aerosolized Legionella reached the left upper lobe while only 0.003 to 0.06% the right lower lobe. To the best of our knowledge, it is the first time that experiments mimicking a real human physiological exposure are performed while limiting animal experiments.

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.

The microscopic surface molecular structures and properties of monoclonal anti-Legionella pneumophila antibodies on an indium-tin oxide (ITO) electrode surface were studied to elaborate an electrochemical immunosensor for Legionella pneumophila detection. A monoclonal anti-Legionella pneumophila antibody (MAb) has been immobilized onto an ITO electrode via covalent chemical bonds between antibodies amino-group and the ring of (3-Glycidoxypropyl) trimethoxysilane (GPTMS). The functionalization of the immunosensor was characterized by atomic force microscopy (AFM), water contact angle measurement, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)₆](3-/4-) as a redox probe. Specific binding of Legionella pneumophila sgp 1 cells onto the antibody-modified ITO electrode was shown by confocal laser scanning microscopy (CLSM) imaging and EIS. AFM images evidenced the dense and relatively homogeneous morphology on the ITO surface. The formation of the complex epoxysilane-antibodies acting as barriers for the electron transfer between the electrode surface and the redox species in the solution induced a significant increase in the charge transfer resistance (Rct) compared to all the electric elements. A linear relationship between the change in charge transfer resistance (ΔRct=Rct after immunoreactions - Rct control) and the logarithmic concentration value of L. pneumophila was observed in the range of 5 × 10(1)-5 × 10(4) CFU mL(-1) with a limit of detection 5 × 10(1)CFU mL(-1). The present study has demonstrated the successful deposition of an anti-L. pneumophila antibodies on an indium-tin oxide surface, opening its subsequent use as immuno-captor for the specific detection of L. pneumophila in environmental samples.

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.

Aims: Legionella isolation from environmental samples is often difficult because of the presence of heterotrophic-associated bacteria that frequently overgrow when using standard culture (ISO 11731, 1998; NF T90-431, 2003) methods. To improve Legionella pneumophila recovery from complex water samples (water from cooling towers, biofilms), we evaluated an immunomagnetic separation (IMS) assay using a monoclonal antibody raised against the lipopolysaccharide of Leg. pneumophila sg1 in combination with culture. Methods and Results: This study was conducted on 51 environmental specimens. The comparison between IMS-culture and standard culture (ISO 11731, 1998; NF T90-431, 2003) methods was made using ISO 17994, 2004 criteria for establishing equivalence between microbiological methods based on the upper and lower (XH and XL) values of the relative difference (95% confidence limit) and D as maximum acceptable deviation (value of the confidence limit). Conclusions: We found that the average performance of IMS culture was higher than the reference method. Significance and Impact of the Study: Thus, this IMS-culture assay is particularly well adapted to the detection of Leg. pneumophila sg1 in environmental samples with high levels of interfering microflora.

The development of a preventive vaccine against human immunodeficiency virus type-1 (HIV-1) provides hope for control of the pandemic over the coming years. Nevertheless, it is clear that one of the greatest difficulties in achieving this vaccine is the high mutation rate of the virus, which enables it to evade the host's immune response. The production of neutralizing antibodies (NAb) against the HIV-1 envelope proteins is believed to play an important role in controlling the infection and in providing effective protection following vaccination. Several studies have shown that the V1/V2 domain of the HIV-1 gp120 envelope protein is involved in viral tropism during infection, in masking conserved neutralizing epitopes, in the conformational changes occurring after coreceptor binding, and in NAb induction. Nonetheless, this domain has been poorly investigated. However, because the V1/V2 domain is highly glycosylated, numerous studies have determined the influence of carbohydrates on NAb production. The present review focuses on the importance of NAb directed against epitopes of the variable regions, mainly V1/V2, their importance in protecting against HIV-1 infection, and the role these regions play in evading the immune response. Lastly, we will discuss the importance of NAb in the search for an effective vaccine against HIV-1.

International audience; 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.

Legionella pneumophila may cause a fatal pneumonia in humans known as Legionnaires’ disease (LD). The strategies of L. pneumophila to adapt to and resist stressful environmental conditions include the ability to enter into a VBNC (viable but not culturable) state. The detection of L. pneumophila in environmental samples benefits from the use of standardised methods: for detection and enumeration following membrane filtration (AFNOR T90-431, ISO 11731) and detection and quantification by polymerase chain reaction PCR (AFNOR T90-471, ISO 12869). Culture is hampered by its inability to detect VBNC forms and PCR is unable to discriminate between live and dead bacteria. The present immunosensor was obtained by the immobilisation of a monoclonal anti-L. pneumophila antibody (MAb) on an indium-tin oxide (ITO) electrode by the self-assembled monolayers (SAMs) method using an aminosilane. The immunosensor was characterised by wettability (contact angle measurement), atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM), and electrochemical impedance spectroscopy (EIS). A limit of detection of 10 bacteria per mL was observed on artificial samples.

An erratum to this article can be found at http://dx.doi.org/10.1007/s00248-015-0580-0.; International audience; 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.

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.

Neutralizing activity of secretory immunoglobulin A (S-IgA) directed against the V1/V2 domain of HIV-1 was studied in parotid saliva of HIV-1- infected patients in Colombian and French cohorts. Purified V1/V2-specific S-IgA antibodies were found to neutralize clades A, B and C primary isolates in five out 76 and 82 patients from each cohort, respectively. These results suggest that neutralizing S-IgA antibodies targeting the V1/V2 domain may provide protection against HIV-1 infection in vivo and may be beneficial in mucosal vaccines.

Legionella viability was monitored during heat shock treatment at 70°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.

The V1/V2 domain of the HIV-1 gp120 envelope protein has been shown to contribute to viral cell tropism during infection and also to viral recognition by neutralizing monoclonal antibodies. However, this domain has been poorly investigated. Carbohydrates have been demonstrated to dramatically influence immune reactivity of antisera to viral glycoprotein antigens. In this study, DNA sequences coding for V1/V2 domains from HIV-1 primary isolates of three subtypes (A, B, and C) were subcloned into a secretion vector and used to transfect CHO cells that are able to achieve the glycosylation of proteins. The structure of purified recombinant V1/V2 proteins was tested using two anti-V1/V2 monoclonal antibodies directed against either a linear or a conformational and glycosylation-dependent epitope (8.22.2 and 697-D). Serum or saliva of 14/82 seropositive patients with anti-V1/V2 reactivity demonstrated good recognition of the recombinant proteins. Deglycosylation of the recombinant proteins was found to increase the reactivity of the serum IgG to the clade A and C but not to clade B V1/V2 domain demonstrating that the recognition of glycosylation sites by serum IgG is clade dependent. When considering SIgA from parotid saliva, deglycosylation of all recombinant proteins tested decreased the reactivity, suggesting that glycosylation plays an important role in the recognition of V1/V2 domain target epitopes by this class of antibodies. In conclusion, these results suggest the influence of carbohydrate moieties on the specificity of the antibodies to the V1/V2 domain produced during HIV infection and the potential importance of viral glycans in vaccine responses after mucosal administration.

The development of a preventive vaccine against human immunodeficiency virus type-1 (HIV-1) provides hope for control of the pandemic over the coming years. Nevertheless, it is clear that one of the greatest difficulties in achieving this vaccine is the high mutation rate of the virus, which enables it to evade the host's immune response. The production of neutralizing antibodies (NAb) against the HIV-1 envelope proteins is believed to play an important role in controlling the infection and in providing effective protection following vaccination. Several studies have shown that the V1/V2 domain of the HIV-1 gp120 envelope protein is involved in viral tropism during infection, in masking conserved neutralizing epitopes, in the conformational changes occurring after coreceptor binding, and in NAb induction. Nonetheless, this domain has been poorly investigated. However, because the V1/V2 domain is highly glycosylated, numerous studies have determined the influence of carbohydrates on NAb production. The present review focuses on the importance of NAb directed against epitopes of the variable regions, mainly V1/V2, their importance in protecting against HIV-1 infection, and the role these regions play in evading the immune response. Lastly, we will discuss the importance of NAb in the search for an effective vaccine against HIV-1.

This study focuses on the interest of the hypervariable 23S-5S ribosomal intergenic spacer region (ISR) of the genus Legionella to analyze the phylogenic diversity of Legionella at the species and subspecies levels and to identify isolates directly from clinical specimens. The method, using a real-time PCR assay with a single primer pair followed by sequencing, was able to identify correctly 49 reference strains of Legionella belonging to 37 different species, including those implicated in human infections, and to clearly differentiate the three subspecies of L. pneumophila. Based on sequence similarities, the 23S-5S ISR sequences were much more variable than the rpoB and mip sequences (P

A group of 42 Legionella-like organisms reacting specifically with Legionella spiritensis serogroup 1 antisera were collected throughout Europe by the Centre National de Reference (French National Reference Centre) for Legionella. This group of isolates differed somewhat from L. spiritensis in terms of biochemical reactions, ubiquinone content and protein profile. The latter two analyses revealed that one of these L. spiritensis-like isolates, Turin I no. 1T, was highly related, but not identical to any of the red autofluorescent species of Legionella. In fact, this strain was the first of these particular isolates recognized to emit a red autofluorescence when exposed to UV light. Profile analysis of randomly amplified polymorphic DNA established that the red autofluorescent L. spiritensis-like isolates constituted a homogeneous group distinct from Legionella rubrilucens and Legionella erythra. DNA-DNA hybridization studies involving the use of S1 nuclease confirmed that the indicated group of isolates are a new species of Legionella, for which the name Legionella taurinensis is proposed with strain Turin I no. 1T (deposited as ATCC 700508T) as the type strain.

The demetalation reaction of (5,10,15,20 tetraphenylporphinato)mercury(II) in CHCl 3 , containing imidazole, 0.1 M TBAP was studied by means of stopped-flow rapid-scanning spectroscopy. Addition of imidazole to TPPHg yielded first the formation of a mono ligand adduct, TPPHg(Im). This rapid step was followed by a slower demetalation reaction to produce TPPH 2 . The demetalation reaction was first-order in both imidazole and metalloporphyrin and gave a rate constant of 0.63 M −1 s −1 at 25 °C. A mechanism for the overall reaction is discussed which involves a proposed transient six coordinate TPPHg(Im) 2 species.

Legionella pneumophila, the causative agent of legionellosis is transmitted to human through aerosols from environmental sources and invades lung’s macrophages. It also can replicate within various protozoan species in environmental reservoirs. Following exposures to various stresses, L. pneumophila enters a Viable Non Cultivable state (VBNC) which is likely to be a survival strategy. The objective of our work was to study the VBNC forms of several strains of L. pneumophila serogroup 1 obtained after thermal and chemical treatments and to evaluate the infectivity of these VBNC forms against macrophages and alveolar epithelial cells. First we studied the physiological patterns of the three different strains (Philadelphia GFP 008, 044 clinical and environmental RNN). For all strains we observed the presence of VBNC bacteria in the native (non stressed) state. The results show that for each strain, three populations of Legionella can be identified: viable and culturable, VBNC and dead cells. Once submitted to the various stresses, we observed that each strain had its own physiological pattern and the presence (or not) of VBNC bacteria was dependent on the applied treatment and the strain used. The second part was related to the study of the pathogenicity of these VBNC forms against macrophages or epithelial cells. The study focused on heat shock treatment at 70°C for 30 min and chlorine dioxide treatment at 4, 6 and 7 mg/L for 60 min at room temperature. The results show that no Legionella VBNC forms were able to grow within the cells and no growth on BCYE medium was observed after co-culture. Then we investigated the behavior of L. pneumophila resuscitated after culture on ameba within macrophages. The results shows that Legionella VBNC induced by heat shock treatment and resuscitated by Acanthamoeba polyphaga co-culture are able to infect macrophages. In conclusion, these results suggest that: (i) the VBNC forms of L. pneumophila are not infectious for macrophages and alveolar epithelial cells in vitro and; (ii) they can be pathogenic for human cells after revivification by an amoeba (A. polyphaga), Legionella pneumophila, l’agent responsable de la légionellose est transmissible à l’Homme par les aérosols environnementaux et infecte les macrophages pulmonaires. Après l’exposition à différents stress L. pneumophila est capable de d’entrer dans un état Viable Non Cultivable (VBNC) qui semble être une stratégie de survie. L’objectif de nos travaux était d’étudier l’état VBNC de différentes souches de L. pneumophila après des traitements thermique et chimique et d’évaluer le pouvoir infectieux des formes VBNC envers les macrophages et les cellules épithéliales alvéolaires. Nous avons étudié les profils physiologiques de L. pneumophila de trois souches différentes. Les résultats montrent que pour chaque souche 3 populations peuvent être identifiées, les légionelles viables cultivables, les VBNC et les bactéries mortes. Lorsque soumises aux stress, chaque souche possède un profil physiologique propre et la présence ou non de bactéries VBNC était dépendante du traitement appliqué et de la souche utilisée. La deuxième partie fut relative à l’étude des traitements thermiques de 70°C pendant 30 min et des chocs au dioxyde de chlore de 4, 6 et 7 mg/L pendant 60 min à température ambiante sur ces VBNC. Aucune légionelle VBNC n’est capable de se développer au sein des cellules et aucune croissance sur milieu BCYE n’a été observée après co-culture. La suite de notre étude a été d’étudier le comportement, envers les macrophages, de L. pneumophila revivifiées après culture sur amibes. Les résultats montrent que les légionelles VBNC induites par choc thermique et revivifiées par co-culture sur Acanthamoeba polyphaga sont capables d’infecter de nouveau les macrophages. En conclusion, ces résultats suggèrent que: (i) les formes VBNC de L. pneumophila ne sont pas spontanément infectieuses pour les macrophages et les cellules épithéliales alvéolaires in vitro et (ii) elles peuvent devenir pathogènes pour les cellules humaines après revivification préalable sur A. polyphaga