Legionella Pneumophila In Water Research Articles

Identification of Genes Required for Long-Term Survival of Legionella pneumophila in Water.

Long-term survival of Legionella pneumophila in aquatic environments is thought to be important for facilitating epidemic outbreaks. Eliminating bacterial colonization in plumbing systems is the primary strategy that depletes this reservoir and prevents disease. To uncover L. pneumophila determinants facilitating survival in water, a Tn-seq strategy was used to identify survival-defective mutants during 50-day starvation in tap water at 42°C. The mutants with the most drastic survival defects carried insertions in electron transport chain genes, indicating that membrane energy charge and/or ATP synthesis requires the generation of a proton gradient by the respiratory chain to maintain survival in the presence of water stress. In addition, periplasmically localized proteins that are known (EnhC) or hypothesized (lpg1697) to stabilize the cell wall against turnover were essential for water survival. To test that the identified mutations disrupted water survival, candidate genes were knocked down by CRISPRi. The vast majority of knockdown strains with verified transcript depletion showed remarkably low viability after 50-day incubations. To demonstrate that maintenance of cell wall integrity was an important survival determinant, a deletion mutation in lpg1697, in a gene encoding a predicted l,d-transpeptidase domain, was analyzed. The loss of this gene resulted in increased osmolar sensitivity and carbenicillin hypersensitivity relative to the wild type, as predicted for loss of an l,d-transpeptidase. These results indicate that the L. pneumophila envelope has been evolutionarily selected to allow survival under conditions in which the bacteria are subjected to long-term exposure to starvation and low osmolar conditions. IMPORTANCE Water is the primary vector for transmission of L. pneumophila to humans, and the pathogen is adapted to persist in this environment for extended periods of time. Preventing survival of L. pneumophila in water is therefore critical for prevention of Legionnaires' disease. We analyzed dense transposon mutation pools for strains with severe survival defects during a 50-day water incubation at 42°C. By tracking the associated transposon insertion sites in the genome, we defined a distinct essential gene set for water survival and demonstrate that a predicted peptidoglycan cross-linking enzyme, lpg1697, and components of the electron transport chain are required to ensure survival of the pathogen. Our results indicate that select characteristics of the cell wall and components of the respiratory chain of L. pneumophila are primary evolutionary targets being shaped to promote its survival in water.

Surface acoustic wave-based lab-on-a-chip for the fast detection of Legionella pneumophila in water

Surface acoustic wave (SAW) -based immuno-biosensors are used for several applications, thanks to their versatility and faster response than conventional analytical methods. SAW immuno-biosensors can be usefully applied to promptly detect bacteria and prevent bacterial infections that can lead to severe diseases. Here, we present a SAW immuno-biosensor to detect Legionella pneumophila in water. Our device, working at ultra-high frequency (740 MHz), is functionalized with an anti-L. pneumophila antibody to maximize the specificity. We report the characteristic curve of the sensor, calculated measuring bacterial samples at known densities, and its related parameters. We also measure L. pneumophila samples contaminated with different Gram-positive and Gram-negative bacterial species (Escherichia coli and Enterococcus faecium) and samples diluted in mains waters. The proposed device is able to detect L. pneumophila in the range from 1·106 to 1·108 CFU/mL, with a limit of blank of 1.22·106 CFU/mL and a limit of detection of 2.01·106 CFU/mL. The nonspecific signal due to contaminant bacteria is very limited and measurements of L. pneumophila are not affected by contamination. We obtain a good detection also in mains water, representing a realistic matrix for L. pneumophila. Our results are encouraging and pave the way to the use of fast, easy-to-use, reliable and precise sensors to prevent bacterial infections in human activities.

Legionella pneumophila recovery using Legiolert and a traditional culture method

AbstractMethods for recovery and identification of Legionella spp. in water have not been improved significantly since the traditional culture method using buffered charcoal yeast extract (BCYE) agar was developed in 1979. Variations to the BCYE method include sample concentration techniques, acid and heat treatment of samples, and inclusions of antibiotics and dyes in the medium to improve Legionella recovery. A new culture method—Legiolert—was developed and is targeted specifically for recovery and enumeration of Legionella pneumophila, the pathogen responsible for >97% of Legionella pneumonia cases. This study assessed the performance of the Legiolert and BCYE methods in water samples. A total of 247 potable water samples were collected and analyzed using both methods. Mean recoveries in reagent water were similar for both methods (~70%), while Legiolert was more likely than BCYE to detect L. pneumophila when it is present in low numbers.Article Impact StatementThe Legiolert method for enumeration of Legionella pneumophila in water is a significant improvement over the buffered charcoal yeast extract method and should be adopted.

Risks of legionella pneumonia in patients receiving immunosuppressive therapy

The legionellosis infection associated with medical care is usually registered for patients with reduced immunity, including those receiving immunosuppressive therapy. Prevention of nosocomial legionellosis in patients is essential due to high mortality. The aim . Analyze of the potential risks of legionellosis infection in patients of surgical departments. Materials and methods . Isolation of Legionella pneumophila from water supply systems in Moscow hospitals and serotyping of the isolated microorganism was performed using bacteriological, molecular genetic and enzyme immunoassay methods. Results and discussion . The study showed that there are potential risks of disease in surgical departments of hospitals that provide medical care, including immunocompromised patients. The content of Legionella pneumophila in water reached 10 5 CFU/l. The share of Legionella pneumophila SG 1, which causes up to 80 % of cases of legionellosis in the world, is 13 % in the water of the surveyed hospitals. The only action prevention of legionellosis is the biological safety of water. Conclusion . Due to the presence of potential risks, prevention of legionellosis is a necessary component of ensuring the safety of the treatment process for patients with immunosuppression.

Dynamic modelling of Legionella pneumophila thermal inactivation in water

A predictive mathematical model describing the effect of temperature on the inactivation of Legionella pneumophila in water was developed. Thermal inactivation of L. pneumophila was monitored under isothermal conditions (51 – 61°C). A primary log-linear model was fitted to the inactivation data and the estimated D values ranged from 0.23 to 25.31 min for water temperatures from 61 to 51°C, respectively. The effect of temperature on L. pneumophila inactivation was described using a secondary model, and the model parameters z value and Dref (D-value at 55°C) were estimated at 5.54°C and 3.47 min, respectively. The developed model was further validated under dynamic temperature conditions mimicking various conditions of water thermal disinfection in plumbing systems. The results indicated that the model can satisfactorily predict thermal inactivation of the pathogen at dynamic temperature environments and effectively translate water temperature profiles to cell number reduction. The application of the model in combination with effective temperature monitoring could provide the basis of an integrated preventive approach for the effective control of L. pneumophila in plumbing systems.

Electrically heatable carbon nanotube point-of-use filters for effective separation and in-situ inactivation of Legionella pneumophila

Despite municipal chlorination and secondary disinfection, opportunistic waterborne pathogens (e.g., Legionella spp.) persist in public and private water distribution systems. As a potential source of healthcare-acquired infections, this warrants development of novel pathogen removal and inactivation systems. In this study, electrically heatable carbon nanotube (CNT) point-of-use (POU) filters have been designed to remove and inactivate Legionella pneumophila in water. The CNT/polymer composite membranes effectively removed Legionella (>99.99%) (i.e., below detection limit) and were able to inactive them on the membrane surface at 100% efficiency within 60 s using ohmic heating at 20 V. The novel POU filters could be used as a final barrier to provide efficient rejection of pathogens and thereby simultaneously eliminate microorganisms in public and private water supplies.

A fast and sensitive real-time PCR assay to detect Legionella pneumophila with the ZEN™ double-quenched probe

Legionella pneumophila is a waterborne pathogen that causes respiratory ailments including Pontiac fever and Legionnaires’ disease. A culture-free, fast and sensitive detection technique is very important for detection of the pathogen. The present study describes the implementation of rapid cycle real-time PCR in the detection of Legionella pneumophila in water through design and development of a real-time qPCR assay based on the ZENTM probe chemistry. The assay targeted the mip gene for the fast and specific detection of Legionella pneumophila. The novel assay was very specific and fast as the amplification was obtained within 30 minutes. Sensitivity of the assay as evaluated in terms of its limit of detection (LoD) was as low as 100 cells/reaction with the quantification range between 1x103 and 1x107 cells/reaction. The assay has been confirmed for repeatability and reproducibility with approximately less than 1% mean intra- and inter-assay variations (CV%). Therefore, the assay reported can be used for a fast, sensitive and specific culture-free detection and quantification of Legionella pneumophila in water.

The LetA/S two-component system regulates transcriptomic changes that are essential for the culturability of Legionella pneumophila in water

Surviving the nutrient-poor aquatic environment for extended periods of time is important for the transmission of various water-borne pathogens, including Legionella pneumophila (Lp). Previous work concluded that the stringent response and the sigma factor RpoS are essential for the survival of Lp in water. In the present study, we investigated the role of the LetA/S two-component signal transduction system in the successful survival of Lp in water. In addition to cell size reduction in the post-exponential phase, LetS also contributes to cell size reduction when Lp is exposed to water. Importantly, absence of the sensor kinase results in a significantly lower survival as measured by CFUs in water at various temperatures and an increased sensitivity to heat shock. According to the transcriptomic analysis, LetA/S orchestrates a general transcriptomic downshift of major metabolic pathways upon exposure to water leading to better culturability, and likely survival, suggesting a potential link with the stringent response. However, the expression of the LetA/S regulated small regulatory RNAs, RsmY and RsmZ, is not changed in a relAspoT mutant, which indicates that the stringent response and the LetA/S response are two distinct regulatory systems contributing to the survival of Lp in water.

The Membrane Protein LasM Promotes the Culturability of Legionella pneumophila in Water.

The water-borne pathogen Legionella pneumophila (Lp) strongly expresses the lpg1659 gene in water. This gene encodes a hypothetical protein predicted to be a membrane protein using in silico analysis. While no conserved domains were identified in Lpg1659, similar proteins are found in many Legionella species and other aquatic bacteria. RT-qPCR showed that lpg1659 is positively regulated by the alternative sigma factor RpoS, which is essential for Lp to survive in water. These observations suggest an important role of this novel protein in the survival of Lp in water. Deletion of lpg1659 did not affect cell morphology, membrane integrity or tolerance to high temperature. Moreover, lpg1659 was dispensable for growth of Lp in rich medium, and during infection of the amoeba Acanthamoeba castellanii and of THP-1 human macrophages. However, deletion of lpg1659 resulted in an early loss of culturability in water, while over-expression of this gene promoted the culturability of Lp. Therefore, these results suggest that lpg1659 is required for Lp to maintain culturability, and possibly long-term survival, in water. Since the loss of culturability observed in the absence of Lpg1659 was complemented by the addition of trace metals into water, this membrane protein is likely a transporter for acquiring essential trace metal for maintaining culturability in water and potentially in other metal-deprived conditions. Given its role in the survival of Lp in water, Lpg1659 was named LasM for Legionella aquatic survival membrane protein.

Comparison of pulsed corona plasma and pulsed electric fields for the decontamination of water containing Legionella pneumophila as model organism

Pulsed corona plasma and pulsed electric fields were assessed for their capacity to kill Legionella pneumophila in water. Electrical parameters such as in particular dissipated energy were equal for both treatments. This was accomplished by changing the polarity of the applied high voltage pulses in a coaxial electrode geometry resulting in the generation of corona plasma or an electric field. For corona plasma, generated by high voltage pulses with peak voltages of +80kV, Legionella were completely killed, corresponding to a log-reduction of 5.4 (CFU/ml) after a treatment time of 12.5min. For the application of pulsed electric fields from peak voltages of −80kV a survival of log 2.54 (CFU/ml) was still detectable after this treatment time. Scanning electron microscopy images of L. pneumophila showed rupture of cells after plasma treatment. In contrast, the morphology of bacteria seems to be intact after application of pulsed electric fields. The more efficient killing for the same energy input observed for pulsed corona plasma is likely due to induced chemical processes and the generation of reactive species as indicated by the evolution of hydrogen peroxide. This suggests that the higher efficacy and efficiency of pulsed corona plasma is primarily associated with the combined effect of the applied electric fields and the promoted reaction chemistry.

Rapid quantification method for Legionella pneumophila in surface water.

World-wide legionellosis outbreaks caused by evaporative cooling systems have shown that there is a need for rapid screening methods for Legionella pneumophila in water. Antibody-based methods for the quantification of L. pneumophila are rapid, non-laborious, and relatively cheap but not sensitive enough for establishment as a screening method for surface and drinking water. Therefore, preconcentration methods have to be applied in advance to reach the needed sensitivity. In a basic test, monolithic adsorption filtration (MAF) was used as primary preconcentration method that adsorbs L. pneumophila with high efficiency. Ten-liter water samples were concentrated in 10 min and further reduced to 1 mL by centrifugal ultrafiltration (CeUF). The quantification of L. pneumophila strains belonging to the monoclonal subtype Bellingham was performed via flow-based chemiluminescence sandwich microarray immunoassays (CL-SMIA) in 36 min. The whole analysis process takes 90 min. A polyclonal antibody (pAb) against L. pneumophila serogroup 1-12 and a monoclonal antibody (mAb) against L. pneumophila SG 1 strain Bellingham were immobilized on a microarray chip. Without preconcentration, the detection limit was 4.0 × 10(3) and 2.8 × 10(3) CFU/mL determined by pAb and mAb 10/6, respectively. For samples processed by MAF-CeUF prior to SMIA detection, the limit of detection (LOD) could be decreased to 8.7 CFU/mL and 0.39 CFU/mL, respectively. A recovery of 99.8 ± 15.9% was achieved for concentrations between 1-1000 CFU/mL. The established combined analytical method is sensitive for rapid screening of surface and drinking water to allow fast hygiene control of L. pneumophila.

A regulatory feedback loop between RpoS and SpoT supports the survival of Legionella pneumophila in water.

Legionella pneumophila is a waterborne pathogen, and survival in the aquatic environment is central to its transmission to humans. Therefore, identifying genes required for its survival in water could help prevent Legionnaires' disease outbreaks. In the present study, we investigate the role of the sigma factor RpoS in promoting survival in water, where L. pneumophila experiences severe nutrient deprivation. The rpoS mutant showed a strong survival defect compared to the wild-type strain in defined water medium. The transcriptome of the rpoS mutant during exposure to water revealed that RpoS represses genes associated with replication, translation, and transcription, suggesting that the mutant fails to shut down major metabolic programs. In addition, the rpoS mutant is transcriptionally more active than the wild-type strain after water exposure. This could be explained by a misregulation of the stringent response in the rpoS mutant. Indeed, the rpoS mutant shows an increased expression of spoT and a corresponding decrease in the level of (p)ppGpp, which is due to the presence of a negative feedback loop between RpoS and SpoT. Therefore, the lack of RpoS causes an aberrant regulation of the stringent response, which prevents the induction of a successful response to starvation.

Validation of the Legipid® Bioalarm Legionella Assay

Legipid Bioalarm Legionella is a test based on combined magnetic immunocapture and enzyme-immunoassay (CEIA) for the detection of Legionella pneumophila in water. Anti-L. pneumophila antibodies are immobilized on magnetic microspheres. Immunomagnetic analysis is applied to preconcentrated water samples in a final test volume of 9 mL. The method was compared with the standard culture method on both spiked and naturally contaminated water samples. The test was evaluated in potable, industrial, and natural water matrixes, according to the scope of the ISO 11731 reference method. These waters were tested with the target at levels ranging from low (10-99 CFU/mL) to high (100-999 CFU/mL); a Chi-square value of 1.8 indicated that there was no significant difference between the test and the reference method. The false-positive rate was 7%, and the false-negative rate 2%. For the inclusivity study, all 17 strains of L. pneumophila of different serogroups reacted with the test. For the exclusivity study, 17 strains of other Legionella species and 16 non-Legionella strains were tested. There were no cross-reactions with non-Legionella strains. L. beliardensis, L. adelaidensis, and one environmentally isolated Legionella sp. produced a positive result at high concentrations of 1800, 230, and 3900 CFU/mL, respectively. Agreement between the two methods was 95.9%.

Detection of Legionella pneumophila in water and biofilm samples by culture and molecular methods from man-made systems in São Paulo - Brazil

Legionella pneumophila is a pathogenic bacteria associated to aquatic habitat of natural and artificial environments. Clinical cases of legionellosis have been reported in Brazil but there is a lack of information about the incidence and concentration of this bacterium in environmental sources. Thus, the present study was designed to evaluate the occurrence of legionellae in Sao Paulo city, Brazil, using different methods of detection and identification. Sixty-seven water and biofilm samples from natural reservoirs and man-made systems were collected and analyzed for the presence of Legionella spp by culturing onto a selective medium, coculture in axenic free-living amoebae and direct fluorescent antibody (DFA) assay. Results showed that freshwater of reservoirs did not contain legionellae, Legionella pneumophila was isolated from man-made systems, with predominance of Legionella pneumophila serogroup 1 strains. Although there was no statistical difference among the proposed detection methods, the plate culture method yielded a higher number of L. pneumophila positive samples, followed by amoebic coculture procedure and direct fluorescent antibody assay. Results of PCR and sequencing reactions revealed that application of macrophage infectivity potentiator gene as a molecular marker was an important tool for the identification of environmental isolates of L. pneumophila. The agreement among the three detection methods-when all methods yielded similar results- and the prevalence of a single Legionella species in the sampled man-made systems could suggest that the occurrence of this bacterium had been influenced by the higher concentration of metallic ions dissociated in water of those systems than in natural reservoirs. Thus, the results of this study revealed that the water of man-made systems in Sao Paulo may serve as a reservoir for L. pneumophila and other microorganism, including free-living protozoans.

Deja Vu All Over Again: Rapid Enumeration of Legionella pneumophila in Water

Delgado-Viscogliosi and colleagues recently reported a rapid method for detecting viable Legionella pneumophila in water using immunofluorescence microscopy (1). Carl Fliermans and colleagues reported a similar assay in this journal 24 years ago (3) and later reported an assay for viability using a tetrazolium dye (4). Both the papers published in the 1980s and the recently published paper reported that the immunofluorescence microscopy method detected greater numbers of bacterial cells than did culture, and both argued that the greater sensitivity of the microscopic method was probably due to poor culture recovery of the Legionella bacteria. Another possibility for this poor correlation is nonspecificity of the antibodies used. A number of clinical and environmental bacteria, many of which are deposited at the ATCC, cross-react with L. pneumophila polyvalent antibodies (2). The immunofluorescence microscopy enumeration method was in vogue for a number of years in the 1980s but fell out of favor due to the poor correlation between viable counts and the microscopic method. It is not clear to me that adding an assay for viability will improve the utility of this assay method, but knowing that the antibodies used do not cross-react with known cross-reacting strains would be useful information. It would be of importance for Delgado-Viscogliosi and colleagues to test their antibodies against the known cross-reacting bacteria deposited at ATCC, as well as to acknowledge the seminal work on this method by Fliermans and colleagues.

Detection of Legionella pneumophila DNA in urine and serum samples from patients with pneumonia

albicans strains adhere better to epithelial cells of the oral cavity or the esophagus, but it is accepted that esophageal candidiasis represents a form of invasive candidiasis which requires higher dosages of antifungal drugs compared to OPC [11]. In this patient, replacement of a fluconazole-resistant C. albicans strain by a genetically different fluconazole-sensitive strain was observed; this finally caused further recurrences of OPC, which responded well to intermittent therapy with itraconazole solution. However, it is unclear whether the disappearance of the resistant genotype is due to the temporary increase of the CD4 cell count or because of therapy with itraconazole despite highlevel resistance to azoles in vitro. It may be concluded that serial DNA typing of C. albicans strains, together with antifungal susceptibility testing, may help us to monitor the susceptibility of clinical isolates to azole therapy.

Nested polymerase chain reaction for detection of Legionella pneumophila in water

A highly sensitive nested polymerase chain reaction (PCR) method was evaluated for detection of Legionella pneumophila in water. Two sets of primers homologous to the coding region of the L. pneumophila macrophage infectivity potentiator ( mip) gene were used. Even when starting from minute amounts of L. pneumophila DNA, the double PCR products were readily detected by direct visualization in ethidium-bromide-stained agarose gels. The method was tested on 34 potable water samples from a hospital building and compared with standard culture isolation. L. pneumophila was isolated in only twelve samples, whereas, by nested PCR, 19 samples were positive, 12 of them coincidental with culturing. These results indicate that nested PCR permits detection of L. pneumophila in samples where culturing fails, with the advantage of a rapid turnaround time, simplicity and the ability to detect non-culturable cells, fulfilling the requirements of sensitivity and specificity for routine use in an environmental laboratory.

Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods

Methods using polymerase chain reaction (PCR) and gene probes to detect viable Legionella pneumophila were investigated with cells exposed to biocide or elevated temperature. Exposure to hypochlorite caused viable nonculturable cells to form. Culturable and viable nonculturable cells showed positive PCR amplification, whereas nonviable cells did not. Viable cells were also specifically detected with mip mRNA as the target, reverse transcription (to form cDNA), and PCR amplification. After exposure to elevated temperature, only viable culturable cells were detected, which corresponded with positive PCR amplification.

Species-specific detection of Legionella pneumophila in water by DNA amplification and hybridization.

A sensitive detection system specific for Legionella pneumophila in water was developed. This system is based on amplification of a chromosomal DNA sequence from L. pneumophila by the polymerase chain reaction, followed by detection of the amplified product by hybridization of a radiolabeled oligodeoxynucleotide. After 35 cycles of amplification, a water sample which had been seeded with 35 CFU of L. pneumophila contained sufficient amplified DNA to be detected on dot blots. Bacteria of other genera tested did not generate positive signals under these conditions. Application of this technique to environmental water samples may help identify the natural reservoirs of nosocomial and community-acquired L. pneumophila infections.

A survey of Legionella pneumophila in water in 12 Canadian cities

A survey of 12 cities across Canada was conducted in order to determine the prevalence of Legionella species in potable water and cooling tower water within buildings. Legionellae were detected in 11.9% of the samples overall: 6.7% from potable water sources and 28.9% from cooling tower water. The maximum concentration of the organism was 45,000 l −1 in one shower-water sample by culture methods. A significant difference in the isolation rate of Legionella pneumophila among cities was observed. The organism was isolated from waters at a temperature of 15–41°C and was most frequently isolated in the 20–29°C range. The concentration of free and total available chlorine in the water was not associated with legionellae recovery except that the organisms were never recovered when the free available chlorine residual exceeded 7.5 mg l −1. Although L. pneumophila were in low concentrations or absent in most samples, the isolated organisms were usually serogroups 1 or 6, the same serogroups that are most often implicated in legionellosis cases in Canada.