Non-culturable Legionella Research Articles

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.

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.

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.

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.

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.

Insights into the long-term persistence of Legionella in facilities from whole-genome sequencing

We investigated the value of whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) analyses in determining the relationships among and evolutionary rates of Legionella species with long-term persistence in three healthcare facilities. We examined retrospective clinical and environmental isolates of Legionella micdadei and Legionella pneumophila serogroup 1 isolates with identical PFGE DNA fingerprints sampled over the course of up to 18 years. WGS analyses demonstrated that heterogeneous populations of Legionella were present within each facility despite displaying the same PFGE profiles. Additionally, clustering of some clinical isolates with those from a separate but related institution exposed a source of infection not previously detected, underscoring the importance of considering phylogenetic relationships when assessing epidemiological links. The data supported an average substitution rate of 0.80 SNPs per genome per year for L. micdadei but a reliable estimate for L. pneumophila serogroup 1 could not be obtained due to complicating factors such as non-chronological links among isolates and inadequate sampling depths. While the substitution rate for L. micdadei is consistent with previous estimates for L. pneumophila, the lack of a temporal signal in our sequence data for L. pneuomphila serogroup 1 isolates suggests either insufficient change to provide an estimate or variable evolutionary rates, which could reflect the presence of both actively dividing and viable but non-culturable Legionella spp. in the built environment. This study highlights the increased discriminatory power of WGS SNP analysis as compared to PFGE, emphasizes the need for extended sampling, and provides insight into the evolution of Legionella from longitudinal investigations.

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.

Detection of culturable and nonculturableLegionellaspecies from hot water systems of public buildings in Japan

To investigate the prevalence of culturable and nonculturable Legionella species in hot water systems of public buildings in Japan and assess the risk factors associated with Legionella contamination in hot water systems. Legionella species were detected by conventional culture and molecular methods in 130 water samples collected from 40 buildings. A total of 26 (20.0%) water samples from 17 (42.5%) buildings were positive by culture, qualitative PCR or both methods: Legionella pneumophila and Leg. anisa were detected in four samples by a culture method, whereas 23 samples were positive by qualitative PCR, with the presence of various Legionella species confirmed by sequencing. Of these 23 samples, bacterial counts were quantifiable in 21 by real-time PCR (from 1.7 x 10(5) to 2.6 x 10(11) cells per litre). Phylogenetic analysis of amplified partial 16S rRNA gene showed close relations to various species of Legionella, including Leg. anisa and Leg. micdadei, all of which have been associated with respiratory diseases or increased antibody titres in human sera. Assessment of risk factors showed that turbidity, free chlorine concentration, iron concentration and heterotrophic plate count (HPC) were significantly associated with Legionella contamination (P < 0.05). Contamination of hot water systems of public buildings with culturable and nonculturable Legionella species may be a potential risk factor for Legionella infection in Japan. Adequate levels of chlorine, low levels of iron and HPC are important maintenance measures in the reduction of Legionella contamination in hot water systems. More than 40% of hot water systems in the Japanese public buildings examined were contaminated by not only culturable Leg. pneumophila and Leg. anisa but also by nonculturable pathogenic species. To our knowledge, this is the first report of both culturable and nonculturable Legionella contamination in hot water systems of public buildings in Japan.

Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii.

Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a non-replicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model.

Study of nonculturable Legionella pneumophila cells during multiple-nutrient starvation

The nonculturable form of Legionella pneumophila in multiple-nutrient starvation culture was studied. During extended starvation, the total direct counts (TDC) of L. pneumophila did not change significantly, but no colonies were detected on day 50. Quantitative PCR detection of L. pneumophila DNA demonstrated that nonculturable cells retained PCR-detectable DNA even after starvation for 300 days, and part of the nonculturable population possessed nonspecific esterase activity. However, resuscitation trials of nonculturable L. pneumophila on day 100 of starvation recovered no colony-forming units, and electrophoresis of nucleic acids extracted from nonculturable cells revealed rRNA subunit (23S, 16S and 5S) degradation. When legionellae have lost the ability to multiply, at least some DNA and enzyme functions may be retained for prolonged periods.

Study of nonculturable Legionella pneumophila cells during multiple-nutrient starvation

The nonculturable form of Legionella pneumophila in multiple-nutrient starvation culture was studied. During extended starvation, the total direct counts (TDC) of L. pneumophila did not change significantly, but no colonies were detected on day 50. Quantitative PCR detection of L. pneumophila DNA demonstrated that nonculturable cells retained PCR-detectable DNA even after starvation for 300 days, and part of the nonculturable population possessed nonspecific esterase activity. However, resuscitation trials of nonculturable L. pneumophila on day 100 of starvation recovered no colony-forming units, and electrophoresis of nucleic acids extracted from nonculturable cells revealed rRNA subunit (23S, 16S and 5S) degradation. When legionellae have lost the ability to multiply, at least some DNA and enzyme functions may be retained for prolonged periods.

Legionella contamination of dental-unit waters

Water samples collected from 28 dental facilities in six U.S. states were examined for the presence of Legionella pneumophila and other Legionella spp. by the PCR-gene probe, fluorescent-antibody microscopic, and viable-plate-count detection methods. The PCR and fluorescent-antibody detection methods, which detect both viable and viable nonculturable Legionella spp., gave higher counts and rates of detection than the plate count method. By the PCR-gene probe detection method, Legionella spp. were detected in 68% of the dental-unit water samples and L. pneumophila was detected in 8%. Concentrations of Legionella spp. in dental-unit water reached 1,000 organisms per ml or more in 36% of the samples, and 19% of the samples were in the category of 10,000/ml or above. L. pneumophila, when present in dental-unit water, never reached concentrations of 1,000/ml or more. Microscopic examination with fluorescent-antibody staining indicated that the contamination was in the dental-unit water lines rather than in the handpieces. Legionella spp. were present in 61% of potable water samples collected for comparative analysis from domestic and institutional faucets and drinking fountains; this percentage was not significantly different from the rate of detection of Legionella spp. in dental-unit water. However, in only 4% of the potable water samples did Legionella spp. reach concentrations of 1,000 organisms per ml, and none was in the 10,000 organisms-per-ml category, and so health-threatening levels of Legionella spp. in potable water were significantly lower than in dental-unit water. L. pneumophila was found in 2% of the potable water samples, but only at the lowest detectable level.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Heat Shock on Recovery of Escherichia coli from Drinking Water

Recent studies on the survival of bacteria in the aquatic environment have suggested that bacteria in water may enter a ‘viable non-culturable' phase in response to stress. Such bacteria are a cause of concern if pathogenic, because they cannot be detected using traditional culture methods. It has been found that mild heat shock causes viable non-culturable Legionella pneumophila to grow on laboratory media. The survival and culturability of Escherichia coli in sterile drinking water was investigated. The isolates used were from the environment rather than clinical. As expected, the count by culture on nutrient agar declined with time while the microscopy count remained approximately constant. Under laboratory conditions the E. coli isolates survived up to three months or more. After three months a portion of each suspension was heat shocked at 35°C for 20 minutes and then assayed by culture and microscopy immediately. An average increase of three log cycles was noted in the count by culture. There was no corresponding increase in the count by microscopy. Thus E. coli appears to exhibit viable non-culturable behaviour. Heat shock causes non-culturable bacteria to regain their ability to grow on artificial media. Such a finding may have several implications for the water industry and microbiology in general.