The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water

Current models to study Legionella pathogenesis include the use of primary macrophages and monocyte cell lines, various free-living protozoan species and murine models of pneumonia. However, there are very few studies of Legionella spp. pathogenesis aimed at associating the role of biofilm colonization and parasitization of biofilm microbiota and release of virulent bacterial cell/vacuoles in drinking water distribution systems. Moreover, the implications of these environmental niches for drinking water exposure to pathogenic legionellae are poorly understood. This review summarizes the known mechanisms of Legionella spp. proliferation within Acanthamoeba and mammalian cells and advocates the use of the amoeba model to study Legionella pathogenicity because of their close association with Legionella spp. in the aquatic environment. The putative role of biofilms and amoebae in the proliferation, development and dissemination of potentially pathogenic Legionella spp. is also discussed. Elucidating the mechanisms of Legionella pathogenicity development in our drinking water systems will aid in elimination strategies and procedural designs for drinking water systems and in controlling exposure to Legionella spp. and similar pathogens.     

Survival and multiplication of Legionella pneumophila in municipal drinking water systems

Studies were conducted to investigate the survival and multiplication of Legionella spp. in public drinking water supplies. An attempt was made, over a period of several years, to isolate legionellae from a municipal system. Sampling sites included the river water supply, treatment plant, finished water reservoir system, mains, and distribution taps. Despite the use of several isolation techniques, Legionella spp. could not be detected in any of the samples other than those collected from the river. It was hypothesized that this was due to the maintenance of a chlorine residual throughout the system. To investigate the potential for Legionella growth, additional water samples, collected from throughout the system, were dechlorinated, pasteurized, and inoculated with Legionella pneumophila. Subsequent growth indicated that many of these samples, especially those collected from areas affected by an accumulation of algal materials, exhibited a much greater ability to support Legionella multiplication than did river water prior to treatment. Chemical analyses were also performed on these samples. Correlation of chemical data and experimental growth results indicated that the chemical environment significantly affects the ability of the water to support multiplication, with turbidity, organic carbon, and certain metals being of particular importance. These studies indicate that the potential exists for Legionella growth within municipal systems and support the hypothesis that public water supplies may contaminate the plumbing systems of hospitals and other large buildings. The results also suggest that useful methods to control this contamination include adequate treatment plant filtration, maintenance of a chlorine residual throughout the treatment and distribution network, and effective covering of open reservoirs.     

Survival and multiplication of Legionella pneumophila in municipal drinking water systems.

Studies were conducted to investigate the survival and multiplication of Legionella spp. in public drinking water supplies. An attempt was made, over a period of several years, to isolate legionellae from a municipal system. Sampling sites included the river water supply, treatment plant, finished water reservoir system, mains, and distribution taps. Despite the use of several isolation techniques, Legionella spp. could not be detected in any of the samples other than those collected from the river. It was hypothesized that this was due to the maintenance of a chlorine residual throughout the system. To investigate the potential for Legionella growth, additional water samples, collected from throughout the system, were dechlorinated, pasteurized, and inoculated with Legionella pneumophila. Subsequent growth indicated that many of these samples, especially those collected from areas affected by an accumulation of algal materials, exhibited a much greater ability to support Legionella multiplication than did river water prior to treatment. Chemical analyses were also performed on these samples. Correlation of chemical data and experimental growth results indicated that the chemical environment significantly affects the ability of the water to support multiplication, with turbidity, organic carbon, and certain metals being of particular importance. These studies indicate that the potential exists for Legionella growth within municipal systems and support the hypothesis that public water supplies may contaminate the plumbing systems of hospitals and other large buildings. The results also suggest that useful methods to control this contamination include adequate treatment plant filtration, maintenance of a chlorine residual throughout the treatment and distribution network, and effective covering of open reservoirs.     

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)     

Long-term survival of Legionella pneumophila serogroup 1 under low-nutrient conditions and associated morphological changes

Abstract Extended survival of Legionella pneumophila , using both a clinical and an environmental isolate, was studied in drinking water, creek water, and estuarine water microcosms. Legionella populations were monitored by acridine orange direct counts (AODC) and viable count on buffered charcoal yeast extract agar amended with alpha-ketoglutarate (BCYEα). Initial colony counts of the clinical isolate in drinking and creek water microcosms were 2 × 10⁸ cfu/ml and, after incubation for 1.5 years, the plate counts decreased to 3 × 10⁶ cfu/ml. The AODC counts, however, did not change significantly. The clinical isolate in estuarine water decreased in plate counts to 10² (cfu/ml) over the same period. After incubation for 1.5 years at 15°C in the microcosms, Legionella plate counts of creek and drinking water decreased by two logs. Direct microscopic examination of aliquots removed from all microcosms revealed the presence of small bacilli, large bacilli and rare filamentous cells. The environmental isolate demonstrated only one colony morphology upon culture on BCYEα. Interestingly, after four months incubation in the microcosm, upon plating the clinical isolate on BCYEα, two distinct colony types were evident. Examination by immunofluorescent staining employing a monoclonal antibody against L. pneumophila revealed both bacillus and filamentous forms. The total cellular proteins of both morphotypes were examined by sodium dodecyl sulfate polyacrylyamide gel electrophoresis (SDS-PAGE), demonstrating identical protein patterns. Those Legionella cells remaining culturable during 1.5 years of incubation grew rapidly when transferred to BCYEα. Incubation was continued and it was found that some strains of L. pneumophila serogroup 1 can remain viable for longer than 2.4 years under low-nutrient conditions.     

Effects of Disinfection on Legionella spp., Eukarya, and Biofilms in a Hot Water System

Legionella species are frequently detected in hot water systems, attached to the surface as a biofilm. In this work, the dynamics of Legionella spp. and diverse bacteria and eukarya associated together in the biofilm, coming from a pilot scale 1 system simulating a real hot water system, were investigated throughout 6 months after two successive heat shock treatments followed by three successive chemical treatments. Community structure was assessed by a fingerprint technique, single-strand conformation polymorphism (SSCP). In addition, the diversity and dynamics of Legionella and eukarya were investigated by small-subunit (SSU) ribosomal cloning and sequencing. Our results showed that pathogenic Legionella species remained after the heat shock and chemical treatments (Legionella pneumophila and Legionella anisa, respectively). The biofilm was not removed, and the bacterial community structure was transitorily affected by the treatments. Moreover, several amoebae had been detected in the biofilm before treatments (Thecamoebae sp., Vannella sp., and Hartmanella vermiformis) and after the first heat shock treatment, but only H. vermiformis remained. However, another protozoan affiliated with Alveolata, which is known as a host cell for Legionella, dominated the eukaryal species after the second heat shock and chemical treatment tests. Therefore, effective Legionella disinfection may be dependent on the elimination of these important microbial components. We suggest that eradicating Legionella in hot water networks requires better study of bacterial and eukaryal species associated with Legionella in biofilms.     

Introduction of monochloramine into a municipal water system: impact on colonization of buildings by Legionella spp

Legionnaires' disease (LD) outbreaks are often traced to colonized potable water systems. We collected water samples from potable water systems of 96 buildings in Pinellas County, Florida, between January and April 2002, during a time when chlorine was the primary residual disinfectant, and from the same buildings between June and September 2002, immediately after monochloramine was introduced into the municipal water system. Samples were cultured for legionellae and amoebae using standard methods. We determined predictors of Legionella colonization of individual buildings and of individual sampling sites. During the chlorine phase, 19 (19.8%) buildings were colonized with legionellae in at least one sampling site. During the monochloramine phase, six (6.2%) buildings were colonized. In the chlorine phase, predictors of Legionella colonization included water source (source B compared to all others, adjusted odds ratio [aOR], 6.7; 95% confidence interval [CI], 2.0 to 23) and the presence of a system with continuously circulating hot water (aOR, 9.8; 95% CI, 1.9 to 51). In the monochloramine phase, there were no predictors of individual building colonization, although we observed a trend toward greater effectiveness of monochloramine in hotels and single-family homes than in county government buildings. The presence of amoebae predicted Legionella colonization at individual sampling sites in both phases (OR ranged from 15 to 46, depending on the phase and sampling site). The routine introduction of monochloramine into a municipal drinking water system appears to have reduced colonization by Legionella spp. in buildings served by the system. Monochloramine may hold promise as community-wide intervention for the prevention of LD.     

Concentration and diversity of uncultured Legionella spp. in two unchlorinated drinking water supplies with different concentrations of natural organic matter

Two unchlorinated drinking water supplies were investigated to assess the potential of water treatment and distribution systems to support the growth of Legionella spp. The treatment plant for supply A distributed treated groundwater with a low concentration (<0.5 ppm of C) of natural organic matter (NOM), and the treatment plant for supply B distributed treated groundwater with a high NOM concentration (8 ppm of C). In both supplies, the water temperature ranged from about 10°C after treatment to 18°C during distribution. The concentrations of Legionella spp. in distributed water, analyzed with quantitative PCR (Q-PCR), averaged 2.9 (± 1.9) × 10(2) cells liter(-1) in supply A and 2.5 (± 1.6) × 10(3) cells liter(-1) in supply B. No Legionella was observed with the culture method. A total of 346 clones (96 operational taxonomical units [OTUs] with ≥97% sequence similarity) were retrieved from water and biofilms of supply A and 251 (43 OTUs) from supply B. The estimation of the average value of total species richness (Chao1) in supply A (153) was clearly higher than that for supply B (58). In each supply, about 77% of the sequences showed <97% similarity to described species. Sequences related to L. pneumophila were only incidentally observed. The Legionella populations of the two supplies are divided into two distinct clusters based on distances in the phylogenetic tree as fractions of the branch length. Thus, a large variety of mostly yet-undescribed Legionella spp. proliferates in unchlorinated water supplies at temperatures below 18°C. The lowest concentration and greatest diversity were observed in the supply with the low NOM concentration.     

Molecular Survey of the Occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa, and Amoeba Hosts in Two Chloraminated Drinking Water Distribution Systems

The spread of opportunistic pathogens via public water systems is of growing concern. The purpose of this study was to identify patterns of occurrence among three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa) relative to biotic and abiotic factors in two representative chloraminated drinking water distribution systems using culture-independent methods. Generally, a high occurrence of Legionella (≥69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (≤20%) and M. avium (≤33.3%), and rare detection of Pseudomonas aeruginosa (≤13.3%) were observed in both systems according to quantitative PCR. Also, Hartmanella vermiformis was more prevalent than Acanthamoeba, both of which are known hosts for opportunistic pathogen amplification, the latter itself containing pathogenic members. Three-minute flushing served to distinguish distribution system water from plumbing in buildings (i.e., premise plumbing water) and resulted in reduced numbers of copies of Legionella, mycobacteria, H. vermiformis, and 16S rRNA genes (P < 0.05) while yielding distinct terminal restriction fragment polymorphism (T-RFLP) profiles of 16S rRNA genes. Within certain subgroups of samples, some positive correlations, including correlations of numbers of mycobacteria and total bacteria (16S rRNA genes), H. vermiformis and total bacteria, mycobacteria and H. vermiformis, and Legionella and H. vermiformis, were noted, emphasizing potential microbial ecological relationships. Overall, the results provide insight into factors that may aid in controlling opportunistic pathogen proliferation in real-world water systems.     

Occurrence and genetic diversity of uncultured Legionella spp. in drinking water treated at temperatures below 15 degrees C

Representatives of the genus Legionella were detected by use of a real-time PCR method in all water samples collected directly after treatment from 16 surface water (SW) supplies prior to postdisinfection and from 81 groundwater (GW) supplies. Legionella concentrations ranged from 1.1 x 10(3) to 7.8 x 10(5) cells liter(-1) and were significantly higher in SW treated with multiple barriers at 4 degrees C than in GW treated at 9 to 12 degrees C with aeration and filtration but without chemical disinfection. No Legionellae (<50 CFU liter(-1)) were detected in treated water by the culture method. Legionella was also observed in untreated SW and in untreated aerobic and anaerobic GW. Filtration processes in SW and GW treatment had little effect or increased the Legionella concentration, but ozonation in SW treatment caused about 1-log-unit reduction. A phylogenetic analysis of 16S rRNA gene sequences of 202 clones, obtained from a selection of samples, showed a high similarity (>91%) with Legionella sequences in the GenBank database. A total of 40 (33%) of the 16S rRNA gene sequences obtained from treated water were identified as described Legionella species and types, including L. bozemanii, L. worsleiensis, Legionella-like amoebal pathogen types, L. quateirensis, L. waltersii, and L. pneumophila. 16S rRNA gene sequences with a similarity of below 97% from described species were positioned all over the phylogenetic tree of Legionella. Hence, a large diversity of yet-uncultured Legionellae are common members of the microbial communities in SW and GW treated at water temperatures of below 15 degrees C.     

Dynamics of Legionella spp. and bacterial populations during the proliferation of L. pneumophila in a cooling tower facility

The dynamics of Legionella spp. and of dominant bacteria were investigated in water from a cooling tower plant over a 9-month period which included several weeks when Legionella pneumophila proliferated. The structural diversity of both the bacteria and the Legionella spp. was monitored by a fingerprint technique, single-strand conformation polymorphism, and Legionella spp. and L. pneumophila were quantified by real-time quantitative PCR. The structure of the bacterial community did not change over time, but it was perturbed periodically by chemical treatment or biofilm detachment. In contrast, the structure of the Legionella sp. population changed in different periods, its dynamics at times showing stability but also a rapid major shift during the proliferation of L. pneumophila in July. The dynamics of the Legionella spp. and of dominant bacteria were not correlated. In particular, no change in the bacterial community structure was observed during the proliferation of L. pneumophila. Legionella spp. present in the cooling tower system were identified by cloning and sequencing of 16S rRNA genes. A high diversity of Legionella spp. was observed before proliferation, including L. lytica, L. fallonii, and other Legionella-like amoebal pathogen types, along with as-yet-undescribed species. During the proliferation of L. pneumophila, Legionella sp. diversity decreased significantly, L. fallonii and L. pneumophila being the main species recovered.     

Microbial ecology of drinking water distribution systems

The supply of clean drinking water is a major, and relatively recent, public health milestone. Control of microbial growth in drinking water distribution systems, often achieved through the addition of disinfectants, is essential to limiting waterborne illness, particularly in immunocompromised subpopulations. Recent inquiries into the microbial ecology of distribution systems have found that pathogen resistance to chlorination is affected by microbial community diversity and interspecies relationships. Research indicates that multispecies biofilms are generally more resistant to disinfection than single-species biofilms. Other recent findings are the increased survival of the bacterial pathogen Legionella pneumophila when present inside its protozoan host Hartmannella vermiformis and the depletion of chloramine disinfectant residuals by nitrifying bacteria, leading to increased overall microbial growth. Interactions such as these are unaccounted for in current disinfection models. An understanding of the microbial ecology of distribution systems is necessary to design innovative and effective control strategies that will ensure safe and high-quality drinking water.     

橡树岭军团菌的分离及用16S rDNA测序法的鉴定

军团菌属（Ｌｅｇｉｏｎｅｌａ）自１９７１年确定以来,现已有３９个成员。军团菌属主要分布于供水系统中,如空调冷却塔水和浴池淋浴水中。我们曾于１９９４年从北京某宾馆冷却塔水中分离出一株可疑军团菌,但因缺乏标准抗血清而未能定种。由于核酸分析技术和细菌鉴定观念上进步,使我们能根据１６ＳｒＤＮＡ序列比较推测细菌间进化关系,并进而对未知细菌进行鉴定和检测。我们用以１６ＳｒＤＮＡ为靶序列的ＰＣＲ和测定鉴定,确定了该分离株为橡树岭军团菌（Ｌ．ｏａｋｒｉｄｇｅｎｓｉｓ）。     

Expression of a plant-derived peptide harboring water-cleaning and antimicrobial activities

Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment-friendly alternative, due to their traditional use for the clarification of drinking water. However, the precise nature of the active components of the extract and whether they may be produced in recombinant form are unknown. Here we show that recombinant or synthetic forms of a cationic seed polypeptide mediate efficient sedimentation of suspended mineral particles and bacteria. Unexpectedly, the polypeptide was also found to possesses a bactericidal activity capable of disinfecting heavily contaminated water. Furthermore, the polypeptide has been shown to efficiently kill several pathogenic bacteria, including antibiotic-resistant isolates of Staphylococcus, Streptococcus, and Legionella species. Thus, this polypeptide displays the unprecedented feature of combining water purification and disinfectant properties. Identification of an active principle derived from the seed extracts points to a range of potential for drinking water treatment or skin and mucosal disinfection in clinical settings.     

Interaction of <Emphasis Type="Italic">legionella pneumophila</Emphasis> and <Emphasis Type="Italic">helicobacter pylori</Emphasis> with bacterial species isolated from drinking water biofilms

Background  

It is well established that Legionella pneumophila is a waterborne pathogen; by contrast, the mode of Helicobacter pylori transmission remains unknown but water seems to play an important role. This work aims to study the influence of five microorganisms isolated from drinking water biofilms on the survival and integration of both of these pathogens into biofilms.     

A membrane filter procedure for assaying cytotoxic activity in heterotrophic bacteria isolated from drinking water

Cytotoxic activity assays of Gram-negative, heterotrophic bacteria are often laborious and time consuming. The objective of this study was to develop in situ procedures for testing potential cytotoxic activities of heterotrophic bacteria isolated from drinking water systems. Water samples were passed through 0.45 microns membrane filters which were then placed upon appropriate media incubated. After incubation, each membrane filter was transferred to the surface of Y-1 mouse adrenal cells overlaid with 1% agar. The filters were removed after exposure for 15 min. The Y-1 cells were then incubated at 37 degrees C in 2.5% CO2 for an additional 24 h. The release of putative cytotoxic and cytotonic products from the bacterial colonies was recognized by zones of cellular lysis and injury of Y-1 cells that appeared immediately beneath the membrane. Cytotoxic strains of Aeromonas, Vibrio, Escherichia, and Legionella spp. were readily recognized by this method. About 1% of the bacteria isolated from drinking water also released cytotoxic products. This frequency was dependent upon the primary medium used and the density of bacteria present. The majority of cytotoxic strains isolated from drinking water also expressed protease activity (95%) and haemolytic activity (70%). This in situ membrane filter procedure is a facile method for simultaneously testing many different bacterial colonies.     

Legionella: from environmental habitats to disease pathology, detection and control

Studies on Legionella show a continuum from environment to human disease. Legionellosis is caused by Legionella species acquired from environmental sources, principally water sources such as cooling towers, where Legionella grows intracellularly in protozoa within biofilms. Aquatic biofilms, which are widespread not only in nature, but also in medical and dental devices, are ecological niches in which Legionella survives and proliferates and the ultimate sources to which outbreaks of legionellosis can be traced. Invasion and intracellular replication of L. pneumophila within protozoa in the environment play a major role in the transmission of Legionnaires' disease. Protozoa provide the habitats for the environmental survival and reproduction of Legionella species. L. pneumophila proliferates intracellularly in various species of protozoa within vacuoles studded with ribosomes, as it also does within macrophages. Growth within protozoa enhances the environmental survival capability and the pathogenicity (virulence) of Legionella . The growth requirements of Legionella , the ability of Legionella to enter a viable non-culturable state, the association of Legionella with protozoa and the occurrence of Legionella within biofilms complicates the detection of Legionella and epidemiological investigations of legionellosis. Polymerase chain reaction (PCR) methods have been developed for the molecular detection of Legionella and used in environmental and epidemiological studies. Various physical and chemical disinfection methods have been developed to eliminate Legionella from environmental sources, but gaining control of Legionella in environmental waters, where they are protected from disinfection by growing within protozoa and biofilms, remains a challenge, and one that must be overcome in order to eliminate sporadic outbreaks of legionellosis.     

Isolation of Legionella species from drinking water

Three different species of Legionella were recovered from samples of water taken from chlorinated public water supplies where no coliform bacteria were simultaneously detected. Five of 856 samples yielded Legionella isolates. Three isolates were identified as Legionella pneumophila serogroup 1, the fourth was identified as Legionella dumoffii, and the fifth was identified as Legionella jordanis. Studies to determine the survival of L. pneumophila Flint 1 serogroup 1 in tap water at various temperatures and in tap water with added sodium hypochlorite were done. These organisms were found to survive for 299 days in tap water at 24 and 5 degrees C but not at 35 degrees C. A concentration of at least 0.2 mg of residual chlorine per ml was required to eliminate at least 90% of L. pneumophila and Escherichia coli inocula in 2 h.     

Isolation of Legionella species from drinking water.

Three different species of Legionella were recovered from samples of water taken from chlorinated public water supplies where no coliform bacteria were simultaneously detected. Five of 856 samples yielded Legionella isolates. Three isolates were identified as Legionella pneumophila serogroup 1, the fourth was identified as Legionella dumoffii, and the fifth was identified as Legionella jordanis. Studies to determine the survival of L. pneumophila Flint 1 serogroup 1 in tap water at various temperatures and in tap water with added sodium hypochlorite were done. These organisms were found to survive for 299 days in tap water at 24 and 5 degrees C but not at 35 degrees C. A concentration of at least 0.2 mg of residual chlorine per ml was required to eliminate at least 90% of L. pneumophila and Escherichia coli inocula in 2 h.     

An alkaline approach to treating cooling towers for control of Legionella pneumophila

Earlier field and laboratory studies have shown that Legionella species survive and multiply in the pH range 5.5 to 9.2. Additionally, the technical feasibility of operating cooling towers at elevated alkalinities and pH has previously been documented by published guidelines. The guidelines indicate that these conditions facilitate corrosion control and favor chlorine persistence which enhances the effectiveness of continuous chlorination in biofouling control. This information suggests that control of Legionella species in cooling towers can be accomplished by operating the towers under alkaline conditions. To test this possibility, we collected water samples over a period of months from a hospital cooling tower. The samples were analyzed for a variety of chemical parameters. Subsamples were pasteurized and inoculated with non-agar-passaged Legionella pneumophila which had been maintained in tap water. Correlation of subsequent Legionella growth with corresponding pH and alkalinity values revealed statistically significant inverse associations. These data support the hypothesis that operating cooling towers outside of the optimal conditions for Legionella growth (e.g., at elevated alkalinities and a pH greater than 9) may be a useful approach to controlling growth in this habitat.