Viable Legionella Research Articles

Occurrence of Uncultured Legionella spp. in Treated Wastewater Effluent and Its Impact on Human Health (SCA.Re.S Project).

Wastewater treatment plants (WWTPs) provide optimal conditions for the environmental spread of Legionella. As part of the Evaluation of Sanitary Risk Related to the Discharge of Wastewater to the Ground (SCA.Re.S) project, this study was conducted to evaluate the presence of Legionella in WWTP effluent and in groundwater samples collected from two wells located downstream from the plant. The samples were analyzed to determine the concentrations of Legionella spp using the standard culture-based method and molecular techniques, followed by genomic sequencing analysis. Legionella was detected only with the molecular methods (except in one sample of effluent positive for L. pneumophila serogroup 6), which showed viable Legionella pneumophila and L. non-pneumophila through the use of free DNA removal solution in both the effluent and groundwater, with concentrations that progressively decreased downstream from the plant. Viable L. pneumophila appeared to be slightly more concentrated in warm months. However, no significant differences (p ≥ 0.05) in concentrations between cold and warm months were observed. A genotypic analysis characterized the species present in the samples and found that uncultured Legionella spp, as yet undefined, constituted the prevalent species in all the samples (range 77.15-83.17%). WWTPs play an important role in the hygienic and sanitary quality of groundwater for different uses. The application of Legionella control systems during the purification of effluents is warranted to prevent possible outbreaks of legionellosis.

Persistence of viable but nonculturable Legionella pneumophila state in hospital water systems: A hidden enemy?

There is little evidence of the long-term consequences of maintaining sanitary hot water at high temperatures on the persistence of Legionella in the plumbing system. The aims of this study were to describe the persistence and genotypic variability of L. pneumophila in a hospital building with two entirely independent hot water distribution systems, and to estimate the thermotolerance of the genotypic variants by studying the quantity of VBNC L. pneumophila. Eighty isolates from 55 water samples obtained between the years 2012–2017 were analyzed. All isolates correspond to L. pneumophila serogroup 6. The isolates were discriminated in four restriction patterns by pulsed-field gel electrophoresis. In one installation, pattern A + Aa predominated, accounting for 75.8 % of samples, while the other installation exhibited pattern B as the most frequent (81.8 % of samples; p < 0.001). The mean temperature of the isolates was: 52.6 °C (pattern A + Aa) and 55.0 °C (pattern B), being significantly different. Nine strains were selected as representative among patterns to study their thermotolerance by flow-cytometry after 24 h of thermic treatment. VBNC bacteria were detected in all samples. After thermic treatment at 50 °C, 52.0 % of bacteria had an intact membrane, and after 55 °C this percentage decreased to 23.1 %. Each pattern exhibited varying levels of thermotolerance. These findings indicate that the same hospital building can be colonized with different predominant types of Legionella if it has independent hot water installations. Maintaining a minimum temperature of 50 °C at distal points of the system would allow the survival of replicative L. pneumophila. However, the presence of Legionella in hospital water networks is underestimated if culture is considered as the standard method for Legionella detection, because VBNC do not grow on culture plates. This phenomenon can carry implications for the Legionella risk management plans in hospitals that adjust their control measures based on the microbiological surveillance of water.

Validation of PVT VIABLE® for Detection of Legionella in Potable and Non-Potable Water: AOAC Performance Tested MethodSM 082303.

Phigenics Validation Test (PVT) VIABLE® (Viability Identification Assay by Legionella Enrichment) is a method to detect viable Legionella bacteria in building water systems. To evaluate PVT VIABLE against the ISO 11731:2017 Water Quality-Enumeration of Legionella reference method for the detection of viable Legionella species in potable and non-potable water. PVT VIABLE was tested for inclusivity (n = 50 strains of Legionella) and exclusivity (n = 30 non-target strains), robustness, and stability. A multi-laboratory instrument variation study was performed to evaluate the PCR data. The matrix study was performed on potable and non-potable water samples inoculated with Legionella pneumophila at a low (n = 20) and high fractional level (n = 5). Samples were analyzed using the PVT VIABLE and confirmed using ISO 11731:2017. Statistical analysis showed no difference between PVT VIABLE and ISO 11731:2017 for 100 mL test portions of potable and non-potable water. PVT VIABLE demonstrated high levels of specificity and sensitivity in the inclusivity and exclusivity study. Results of the robustness and stability studies demonstrated the method was sufficiently robust to handle small method changes and met the method claims of stability. PVT VIABLE allows the end user to obtain presumptive results for viable Legionella spp. contamination of potable and non-potable water in 2-3 days. PVT VIABLE provides viable Legionella results in 2-3 days versus 10-14 days for traditional spread plating. This novel diagnostic tool also differentiates between Legionella pneumophila sg1, Legionella pneumophila sg2-15, and Legionella spp. without the need for additional confirmation steps as outlined in ISO 11731:2017.

Stagnation arising through intermittent usage is associated with increased viable but non culturable Legionella and amoeba hosts in a hospital water system.

Hospital water systems are a significant source of Legionella, resulting in the potentially fatal Legionnaires' disease. One of the biggest challenges for Legionella management within these systems is that under unfavorable conditions Legionella transforms itself into a viable but non culturable (VBNC) state that cannot be detected using the standard methods. This study used a novel method (flow cytometry-cell sorting and qPCR [VFC+qPCR] assay) concurrently with the standard detection methods to examine the effect of temporary water stagnation, on Legionella spp. and microbial communities present in a hospital water system. Water samples were also analyzed for amoebae using culture and Vermamoeba vermiformis and Acanthamoeba specific qPCR. The water temperature, number and duration of water flow events for the hand basins and showers sampled was measured using the Enware Smart Flow® monitoring system. qPCR analysis demonstrated that 21.8% samples were positive for Legionella spp., 21% for L. pneumophila, 40.9% for V. vermiformis and 4.2% for Acanthamoeba. All samples that were Legionella spp. positive using qPCR (22%) were also positive for VBNC Legionella spp.; however, only 2.5% of samples were positive for culturable Legionella spp. 18.1% of the samples were positive for free-living amoebae (FLA) using culture. All samples positive for Legionella spp. were also positive for FLA. Samples with a high heterotrophic plate count (HPC ≥ 5 × 103 CFU/L) were also significantly associated with high concentrations of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila (p < 0.01) and V. vermiformis (p < 0.05). Temporary water stagnation arising through intermittent usage (< 2 hours of usage per month) significantly (p < 0.01) increased the amount of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila, and V. vermiformis; however, it did not significantly impact the HPC load. In contrast to stagnation, no relationship was observed between the microbes and water temperature. In conclusion, Legionella spp. (DNA and VBNC) was associated with V. vermiformis, heterotrophic bacteria, and stagnation occurring through intermittent usage. This is the first study to monitor VBNC Legionella spp. within a hospital water system. The high percentage of false negative Legionella spp. results provided by the culture method supports the use of either qPCR or VFC+qPCR to monitor Legionella spp. contamination within hospital water systems.

Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR.

Legionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires' disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC) Legionella. The management of engineered water systems to prevent Legionnaires' disease is hindered by the presence of VBNC Legionella that cannot be detected using the standard culture (ISO11731:2017-05) and quantitative polymerase reaction (ISO/TS12869:2019) methods. This study describes a novel method to quantify VBNC Legionella from environmental water samples using a "viability based flow cytometry-cell sorting and qPCR" (VFC + qPCR) assay. This protocol was then validated by quantifying the VBNC Legionella genomic load from hospital water samples. The VBNC cells were unable to be cultured on Buffered Charcoal Yeast Extract (BCYE) agar; however, their viability was confirmed through their ATP activity and ability to infect amoeba hosts. Subsequently, an assessment of the ISO11731:2017-05 pre-treatment procedure demonstrated that acid or heat treatment cause underestimation of alive Legionella population. Our results showed that these pre-treatment procedures induce culturable cells to enter a VBNC state. This may explain the observed insensitivity and lack of reproducibility often observed with the Legionella culture method. This study represents the first time that flow cytometry-cell sorting in conjunction with a qPCR assay has been used as a rapid and direct method to quantify VBNC Legionella from environmental sources. This will significantly improve future research evaluating Legionella risk management approaches for the control of Legionnaires' disease.

Inaccuracies of the ISO 11731 Method for Environmental Validation of Legionella in Building Water Systems: Opportunities to Improve Sensitivity and Detect Viable but Non-Culturable Legionella

Current environmental diagnostics for the detection of Legionella fail to detect viable but non-culturable Legionella, have sensitivity limitations and are time-consuming (10-14 days to results). The objective of this study was to compare Legionella detection results between the standard ISO 11731 and an innovative Legionella detection method that utilizes a hybrid methodology of traditional microbiology and molecular detection. In this study, four hundred and seventy-six (476) potable building water samples were analyzed with ISO 11731 and the novel method in parallel. Of the 476 total samples that were tested, a discrepancy of 21% was observed when comparing the ISO 11731 method to the novel method. Separating the samples based on hazard control methods yielded a 15.4% discrepancy for chlorinated systems (n = 284) and a 29% discrepancy for monochloraminated systems (n = 192). The data presented here conclusively show inaccuracies in environmental validation for building water systems based on results returned by the standard ISO 11731 method. This is especially evident in systems primarily disinfected with monochloramines. Overall, these data highlight the need for new and innovative methods to overcome the inaccuracies of the traditional ISO 11731 spread plates to prevent disease and injury caused by Legionella.

A Multidisciplinary Approach to Water Management and Legionella Prevention

<h3>Background</h3> Legionella and other waterborne pathogens are of great concern for healthcare systems. The Center for Medicare and Medicaid Services (CMS) issued a memorandum requiring a reduction of Legionella and other opportunistic waterborne pathogens in healthcare facility water systems. A Water Management Team (WMT) was implemented in 2019 in a newly constructed 55-bed acute care facility due to lack of a formal program and to proactively prevent infection in the patient population. <h3>Methods</h3> An acute care facility collaborated with a third-party vendor to initiate a water management program (WMP). Key stakeholders collaborated and performed verification and validation strategies including environmental water sampling, water flushing, and active surveillance in high-risk areas. The first round of testing showed viable Legionella growth in multiple samples. The site implemented several actions to eliminate the viable Legionella without success. It was found that the Ultra-Violet light system that was installed on cold water prior to supplying the hot water loop was dropping chlorine levels from the city very low. A chlorine injection system was installed, and regularly scheduled water sampling was completed. The WMT collaborated to bring concerns and issues to key areas and departments as well as executive leadership to facilitate discussion and decision-making regarding the safety and operation of all potable water systems. <h3>Results</h3> Following the last intervention, the environmental water sampling showed no growth for viable Legionella. There were no reported hospital-acquired Legionella cases reported during this time frame. <h3>Conclusions</h3> A multidisciplinary WMP reduced the site's risk for healthcare associated Legionella as evidenced by results of water surveillance and supported by infection control surveillance. A WMP is a fundamental component to meet CMS mandates and address Legionella prevention.

Propidium monoazide-quantitative PCR to detect viable Legionella spp. in the supply process of tap water

Abstract Legionella are common in the aquatic environment and are responsible for legionellosis including severe pneumonia and Pontiac fever. The culture method has some limitations in quickly detecting viable Legionella. Therefore, we optimized real-time PCR (qPCR) combined with propidium monoazide (PMA) to quantify viable Legionella in the supply process of tap water, considering factors such as PMA concentration, length of the target gene, and turbidity of water samples. Among 30, 50, 100, and 200 μM PMA concentration, 100 μM PMA had the greatest difference in copy number between PMA-treated live and dead cells while minimizing the cytotoxic effect on live cells. Among the various sizes of the target gene (108, 386, 456, and 654 bp), the primer in 386 bp size effectively excluded dead cells without loss of qPCR efficiency. As a result of applying the PMA-qPCR method to samples including river, purified water, and tap water, live and dead cells could be distinguished for samples with turbidity of less than 10 NTU. The optimized PMA-qPCR can be a useful method of rapidly detecting viable Legionella spp. in the process of supplying tap water, and contributing to tap water that is safe from pathogens.

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.

Comparison of BCYE\u03b1+AB agar and MWY agar for detection and enumeration of Legionella spp. in hospital water samples

BackgroundThis study illustrates for the first time the performance (sensitivity and selectivity) of the selective medium BCYEα +AB suggested by the new edition of ISO 11731 for legionella isolation and enumeration. We compared the efficacy of the selective BCYEα +AB medium with that of the highly selective MWY medium.ResultsLegionella spp. was detected in 48.2 and 47.1% of the samples by BCYEα +AB and MWY agar, respectively. For optimal detection of Legionella spp., most protocols recommend using selective media to reduce the number of non-Legionella bacteria. Agreement between the two media was 86.7%.ConclusionsAccording to the results, both media have a very similar performance and they both have advantages and disadvantages over each other. In AB medium there is the risk of being less selective so more interfering microbiota may grow but in MWY medium there is the risk of being too selective. The low selectivity of the AB medium could be resolved if other treatments are applied after filtration, e.g. acid and/or heat treatment, but it must be taken into account that these treatments still reduce the number of viable Legionella. In conclusion, we recommend using MWY as a selective medium for the detection of Legionella spp. as it is easier discern suspected colonies and facilitate the final Legionella spp. count.

Water Stagnation and Flow Obstruction Reduces the Quality of Potable Water and Increases the Risk of Legionelloses

Legionella is an opportunistic waterborne pathogen associated with Legionnaires' disease and Pontiac fever. Despite improved public awareness, the incidence of Legionella associated infections has been increasing. Aerosols generated from engineered potable water systems are a demonstrated cause of both nosocomial and community-acquired legionellosis. The ecology of Legionella in these systems is complex with multiple factors impacting their colonization and persistence. Flow dynamics has been identified as an important factor and stagnation in cooling towers is an accepted risk for increased Legionella growth; however, less is known about the impact of flow dynamic on Legionella in potable water systems. This is especially complex due to the inherent intermittent and variable usage observed within outlets of a potable water system. This systematic literature review examines the role of fluid dynamics and stagnation on the colonization and growth of Legionella in potable water systems. Twenty two of 24 identified studies show a positive association between stagnation zones and increased colonization of Legionella. These zones included dead legs, dead ends, storage tanks, and obstructed water flow (such as intermittent usage or flow restriction). Prolonged stagnation in building plumbing systems also deteriorates the quality of thermally or chemically treated potable water. This stimulates the colonization of Legionella established biofilms. Such biofilms are intrinsically resistant to disinfection procedures and accelerate the rate of decay of chemical disinfectants. Sub-lethal doses of disinfectants and the presence of protozoan hosts in stationary water promote generation of viable but non-culturable Legionella cells. This results in false negatives in surveillance methods that use culture methodology. In conclusion, elimination of temporal and permanent stagnation points can improve the quality of potable water, efficacy of disinfectants, and reduce the risk of legionellosis. Current guidelines and water safety plans recognize the risks associated with permanent stagnation point (dead ends and dead legs); however, there is a need for greater emphasis on controlling temporal stagnation arising from intermittent usage.

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.

DVC-FISH to identify potentially pathogenic Legionella inside free-living amoebae from water sources

Despite all safety efforts, drinking and wastewater can still be contaminated by Legionella and free-living amoebae (FLA) since these microorganisms are capable of resisting disinfection treatments. An amoebae cyst harboring pathogenic Legionella spp. can be a transporter of this organism, protecting it and enhancing its infection abilities. Therefore, the aim of this work is to identify by DVC-FISH viable Legionella spp and Legionella pneumophila cells inside FLA from water sources in a specific and rapid way with the aim of assessing the real risk of these waters.A total of 55 water samples were processed, 30 reclaimed wastewater and 25 drinking water. FLA presence was detected in 52.7% of the total processed water samples. When DVC-FISH technique was applied, the presence of viable internalized Legionella spp. cells was identified in 69.0% of the total FLA-positive samples, concretely in 70.0% and 66.7% of wastewater and drinking water samples, respectively. L. pneumophila was simultaneously identified in 48.3% of the total FLA-positive samples, specifically in 50.0% and 44.4% of wastewater and drinking water samples, respectively.By culture, potentially pathogenic Legionella cells were recovered in 27.6% of the total FLA-positive bacteria, particularly in 35.0% and 11.1% of wastewater and drinking water samples, respectively.These findings demonstrate that FLA may promote resistance of bacteria to the performed disinfection treatments for drinking as well as for wastewater. So, in addition to the risk for the presence of pathogenic FLA in water it is necessary to take into account that these can be transporters of the pathogenic bacteria Legionella, which are able to survive inside them. The DVC-FISH method described here has been proved to be a rapid and specific tool to identify pathogenic Legionella spp. and L. penumophila viable cells harboured by FLA in these water sources, posing particular public health concern.

Viability and infectivity of viable but nonculturable Legionella pneumophila strains induced at high temperatures

Thermal disinfection is commonly used to prevent the proliferation of culturable Legionella in engineered water systems (EWS). In response to such stress, culturable Legionella populations can switch into a viable but nonculturable (VBNC) state. The importance of such VBNC Legionella cells is currently hotly debated. Here, we investigated the stress response patterns and transitions of the bacteria to the VBNC state at 55 °C, 60 °C and 70 °C on two L. pneumophila strains for >80 days using a combination of cell-based viability indicators. Complete loss of culturability at 55 °C, 60 °C and 70 °C occurred after 3–8 h, 60 min and <2 min, respectively. In contrast, L. pneumophila strains required 9 days at 55 °C, 8 h at 60 °C and 20 min at 70 °C to achieve a 2 log reduction in cells with intact membranes and high esterase activity; a 4 log reduction was achieved only after 150, 8–15 and 1–4 days, respectively. In parallel, the presence of diagnostic outer-membrane epitopes (OMEs) and changes in the infectivity patterns of the two strains towards amoebae and THP-1 cells were assessed. OMEs were more persistent than viability indicators, showing their potential as targets for VBNC Legionella detection. L. pneumophila strains infected amoebae and THP-1 cells for at least 85 days at 55 °C and 60 °C and for up to 8 days at 70 °C. However, they did so with reduced efficiency, requiring prolonged co-incubation times with the hosts and higher Legionella cell numbers in comparison to culturable cells. Consequently, infection of amoebae by thermally induced VBNC L. pneumophila with lowered virulence can be expected in EWS. Although the gold standard method cannot detect VBNC Legionella, it provides important information about the most virulent bacterial subpopulations. Our results indicate that a prolonged thermal regime ≥60 °C at the central parts of warm water systems is not only effective against culturable L. pneumophila but in the long run even against VBNC cells.

Investigations on Contamination of Environmental Water Samples by &lt;i&gt;Legionella&lt;/i&gt; using Real-Time Quantitative PCR Combined with Amoebic Co-Culturing

We analyzed the contamination of environmental water samples with Legionella spp. using a conventional culture method, real-time quantitative PCR (qPCR), and real-time qPCR combined with an amoebic co-culture method. Samples (n = 110) were collected from 19 cooling towers, 31 amenity water facilities, and 60 river water sources of tap water in Japan. Legionella was detected in only three samples (3/110, 2.7%) using the culture method. The rate of Legionella detection using amoebic co-culture followed by qPCR was 74.5%, while that using qPCR without amoebic co-culture was 75.5%. A higher than 10-fold bacterial count was observed in 19 samples (19/110, 17.3%) using real-time qPCR subsequent to amoebic co-culture, compared with identical samples analyzed without co-culture. Of these 19 samples, 13 were identified as Legionella spp., including L. pneumophila and L. anisa, and the non-culturable species were identified as L. lytica and L. rowbothamii. This study showed that the detection of Legionella spp., even in those samples where they were not detected by the culture method, was possible using real-time qPCR and an amoebic co-culture method. In addition, this analytical test combination is a useful tool to detect viable and virulent Legionella spp..

Phylogenetic Characterization of Viable but-not-yet Cultured Legionella Groups Grown in Amoebic Cocultures: A Case Study using Various Cooling Tower Water Samples.

Legionella spp. exist naturally in association with amoeba in water environments and are known to be the etiological agent of a severe form of pneumonia. To detect diverse Legionella populations in cooling tower water systems, amoebic coculturing was performed for 15 water samples obtained from five different kinds of facilities in six geographically different locations. The growth of Legionella in coculture with Acanthamoeba sp. cells was monitored by quantitative PCR targeting Legionella-specific 16S rRNA genes. Seven out of the 15 samples were positive for Legionella growth and subjected to clone library analysis. A total of 333 clones were classified into 14 operational taxonomic units composed of seven known species and seven previously undescribed groups. Four of the seven Legionella-growth-positive samples harbored detectable levels of free-living amoeba and were predominated by either L. drozanskii or L. lytica, by both L. bozemanii and L. longbeachae, or by a not-yet-described group named OTU 4. The Legionella-growth- positive samples contained higher ATP levels (>980 pM) than the growth-negative samples (<160 pM) , suggesting that ATP content would be a good indicator of the presence of viable but nonculturable Legionella populations able to grow with amoeba.

Control of legionellae in a new healthcare facility following implementation of a thermal control strategy

Introduction: The management of the Legionella risk in hospitals is essentially related to preventive measures of the hot-water supplies.Aim: To monitor the control of legionellae before and after moving to a new hospital facility.Methods: We implemented a survey program based on the surveillance of the temperature of the hot-water supply and detection and counting of Legionella pneumophila and Legionella spp. by quantitative polymerase chain reaction and culture methods.Results: Our survey program revealed that the hot-water system was colonized by L. pneumophila and Legionella spp. before the arrival of the first patients, despite the implementation of preventive measures. Thus, maintenance on the hot-water production system and subsequent cleaning and superheat disinfection of the hot-water supplies were performed, leading to the eradication of L. pneumophila reservoirs and the decrease of Legionella spp. reservoirs. No reservoirs of L. pneumophila and only rare persistent reservoirs of Legionella spp. were detected after the transfer of hospitalized patients to the new healthcare facility and during the following four years, demonstrating the effectiveness of our corrective measures, without using biocides. L. anisa was identified as the only strain of viable and cultivable Legionella spp. and was undetected during the last year.Conclusions: The strict application of our survey program before and after moving to the new hospital associated with strict implementation of corrective measures allowed us to efficiently manage the Legionella-linked risk during this period.

Starved viable but non-culturable (VBNC) Legionella strains can infect and replicate in amoebae and human macrophages

Legionella infections are among the most important waterborne infections with constantly increasing numbers of cases in industrialized countries, as a result of aging populations, rising numbers of immunocompromised individuals and increased need for conditioned water due to climate change. Surveillance of water systems is based on microbiological culture-based techniques; however, it has been shown that high percentages of the Legionella populations in water systems are not culturable. In the past two decades, the relevance of such viable but non-culturable (VBNC) legionellae has been controversially discussed, and whether VBNC legionellae can directly infect human macrophages, the primary targets of Legionella infections, remains unclear. In this study, it was demonstrated for the first time that several starved VBNC Legionella strains (four L. pneumophila serogroup 1 strains, a serogroup 6 strain and a L. micdadei strain) can directly infect different types of human macrophages and amoebae even after one year of starvation in ultrapure water. However, under these conditions, the strains caused infection with reduced efficacy, as represented by the lower percentages of infected cells, prolonged time in co-culture and higher multiplicities of infection required. Interestingly, the VBNC cells remained mostly non-culturable even after multiplication within the host cells. Amoebal infection by starved VBNC Legionella, which likely occurs in oligotrophic biofilms, would result in an increase in the bacterial concentration in drinking-water systems. If cells remain in the VBNC state, the real number of active legionellae will be underestimated by the use of culture-based standard techniques. Thus, further quantitative research is needed in order to determine, whether and how many starved VBNC Legionella cells are able to cause disease in humans.

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.

Detection of viable but non-culturable legionella in hospital water network following monochloramine disinfection

Prevention of legionellosis remains a critical issue in healthcare settings where monochloramine (MC) disinfection was recently introduced as an alternative to chlorine dioxide in controlling Legionella spp. contamination of the hospital water network. Continuous treatments with low MC doses in some instances have induced a viable but non-culturable state (VBNC) of Legionella spp. To investigate the occurrence of such dormant cells during a long period of continuous MC treatment. Between November 2010 and April 2015, 162 water and biofilm samples were collected and Legionella spp. isolated in accordance with standard procedures. In sampling sites where MC was <1.5mg/L, VBNC cells were investigated by ethidium monoazide bromide (EMA)-real-time polymerase chain reaction (qPCR) and 'resuscitation' test into Acanthamoeba polyphaga CCAP 1501/18. According to the Health Protection Agency protocol, free-living protozoa were researched in 60 five-litre water samples. In all, 136 out of 156 (87.2%) of the samples taken from sites previously positive for L.pneumophila ST269 were negative by culture, but only 47 (34.5%) negative by qPCR. Although no positive results were obtained by EMA-qPCR, four out of 22 samples associated with MC concentration of 1.3±0.5mg/L showed VBNC legionella resuscitation. The presence of the amoeba A.polyphaga in the hospital water network was demonstrated. Our study is the first report evidencing the emergence of VNBC legionella during a long period of continuous MC treatment of a hospital water network, highlighting the importance of keeping an appropriate and uninterrupted MC dosage to ensure the control of legionella colonization in hospital water supplies.