Biodiversity of Amoebae and Amoeba-Resisting Bacteria in a Hospital Water Network

Free-living amoebae (FLA) are ubiquitous organisms that have been isolated from various domestic water systems, such as cooling towers and hospital water networks. In addition to their own pathogenicity, FLA can also act as Trojan horses and be naturally infected with amoeba-resisting bacteria (ARB) that may be involved in human infections, such as pneumonia. We investigated the biodiversity of bacteria and their amoebal hosts in a hospital water network. Using amoebal enrichment on nonnutrient agar, we isolated 15 protist strains from 200 (7.5%) samples. One thermotolerant Hartmannella vermiformis isolate harbored both Legionella pneumophila and Bradyrhizobium japonicum. By using amoebal coculture with axenic Acanthamoeba castellanii as the cellular background, we recovered at least one ARB from 45.5% of the samples. Four new ARB isolates were recovered by culture, and one of these isolates was widely present in the water network. Alphaproteobacteria (such as Rhodoplanes, Methylobacterium, Bradyrhizobium, Afipia, and Bosea) were recovered from 30.5% of the samples, mycobacteria (Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium xenopi) were recovered from 20.5% of the samples, and Gammaproteobacteria (Legionella) were recovered from 5.5% of the samples. No Chlamydia or Chlamydia-like organisms were recovered by amoebal coculture or detected by PCR. The observed strong association between the presence of amoebae and the presence of Legionella (P < 0.001) and mycobacteria (P = 0.009) further suggests that FLA are a reservoir for these ARB and underlines the importance of considering amoebae when water control measures are designed.     

Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant

The complex ecology of free-living amoebae (FLA) and their role in spreading pathogenic microorganisms through water systems have recently raised considerable interest. In this study, we investigated the presence of FLA and amoebae-resisting bacteria (ARB) at various stages of a drinking water plant fed with river water. We isolated various amoebal species from the river and from several points within the plant, mostly at early steps of water treatment. Echinamoeba- and Hartmannella-related amoebae were mainly recovered in the drinking water plant whereas Acanthamoeba- and Naegleria-related amoebae were recovered from the river water and the sand filtration units. Some FLA isolates were recovered immediately after the ozonation step, thus suggesting resistance of these microorganisms to this disinfection procedure. A bacterial isolate related to Mycobacterium mucogenicum was recovered from an Echinamoeba-related amoeba isolated from ozone-treated water. Various other ARB were recovered using co-culture with axenic Acanthamoeba castellanii, including mycobacteria, legionella, Chlamydia-like organisms and various proteobacteria. Noteworthy, a new Parachlamydia acanthamoebae strain was recovered from river water and from granular activated carbon (GAC) biofilm. As amoebae mainly multiply in sand and GAC filters, optimization of filter backwash procedures probably offers a possibility to better control these protists and the risk associated with their intracellular hosts.     

Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks,and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata

The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species.     

Mycobacterium avium Bacilli Grow Saprozoically in Coculture with Acanthamoeba polyphaga and Survive within Cyst Walls

Protozoans are gaining recognition as environmental hosts for a variety of waterborne pathogens. We compared the growth of Mycobacterium avium, a human pathogen associated with domestic water supplies, in coculture with the free-living amoeba Acanthamoeba polyphaga with the growth of M. avium when it was separated from amoebae by a 0.1-μm-pore-size polycarbonate membrane (in a parachamber). Although viable mycobacteria were observed within amoebal vacuoles, there was no significant difference between bacterial growth in coculture and bacterial growth in the parachamber. This suggests that M. avium is able to grow saprozoically on products secreted by the amoebae. In contrast, Legionella pneumophila, a well-studied intracellular parasite of amoebae, multiplied only in coculture. A comparison of amoebae infected with L. pneumophila and amoebae infected with M. avium by electron microscopy demonstrated that there were striking differences in the locations of the bacteria within amoebal cysts. While L. pneumophila resided within the cysts, M. avium was found within the outer walls of the double-walled cysts of A. polyphaga. These locations may provide a reservoir for the bacteria when environmental conditions become unfavorable.     

Mycobacterium gilvum Illustrates Size-Correlated Relationships between Mycobacteria and Acanthamoeba polyphaga

Mycobacteria are isolated from soil and water environments, where free-living amoebae live. Free-living amoebae are bactericidal, yet some rapidly growing mycobacteria are amoeba-resistant organisms that survive in the amoebal trophozoites and cysts. Such a capacity has not been studied for the environmental rapidly growing organism Mycobacterium gilvum. We investigated the ability of M. gilvum to survive in the trophozoites of Acanthamoeba polyphaga strain Linc-AP1 by using optical and electron microscopy and culture-based microbial enumerations in the presence of negative controls. We observed that 29% of A. polyphaga cells were infected by M. gilvum mycobacteria by 6 h postinfection. Surviving M. gilvum mycobacteria did not multiply and did not kill the amoebal trophozoites during a 5-day coculture. Extensive electron microscopy observations indicated that M. gilvum measured 1.4 ± 0.5 μm and failed to find M. gilvum organisms in the amoebal cysts. Further experimental study of two other rapidly growing mycobacteria, Mycobacterium rhodesiae and Mycobacterium thermoresistibile, indicated that both measured <2 μm and exhibited the same amoeba-mycobacterium relationships as M. gilvum. In general, we observed that mycobacteria measuring <2 μm do not significantly grow within and do not kill amoebal trophozoites, in contrast to mycobacteria measuring >2 μm (P < 0.05). The mechanisms underlying such an observation remain to be determined.     

Isolation and identification of amoeba-resisting bacteria from water in human environment by using an Acanthamoeba polyphaga co-culture procedure

Amoeba-resisting bacteria (ARB) such as Legionella spp. are currently regarded as potential human pathogens living in the environment. To detect ARB from both human and environmental samples, co-culture with amoebae has been demonstrated as an efficient tool. However, using this procedure, mostly water from cooling towers and hospital water supplies have been investigated as the possible reservoir of ARB. In the present study, we studied ARB population in 77 environmental water samples including rivers, fountains, lakes and domestic wells in the south of France. As a result, a total of 244 isolates corresponding to 89 different species of ARB, but not Legionella spp., were identified. Ability to grow within and/or to be lytic for amoebae was revealed for the first time for several human pathogens . Six isolates are likely to be the members of a new or uncharacterized genus/species. An anaerobic bacterium, Clostridium frigidicarnis was demonstrated to be lytic for amoebae. This preliminary work demonstrates that the water environment in the vicinity of humans is a reservoir of ARB, including well-known pathogens for which amoebae and/or water was not recognized earlier as a possible reservoir.     

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.     

Free-living amoebae (FLA) co-occurring with legionellae in industrial waters

Legionella pneumophila is known as the causative agent of Legionnaires’ disease and free-living amoebae (FLA) can serve as vehicles for legionellae. The aim of this study was to screen industrial waters for the occurrence of FLA and their co-occurrence with legionellae. A total of 201 water samples, including 129 cooling waters and 72 process waters, and 30 cooling lubricants were included in the study. Treated waters were screened periodically, pre and post treatment. Altogether, 72.6% of the water samples were positive for FLA, acanthamoebae being most prevalent (in 23.9% of the samples) followed by Vermamoeba vermiformis (19.4%). Only one cooling lubricant was positive (Acanthamoeba genotype T4). Legionella spp. were detected in 34.8% of the water samples and in 15% in high concentrations (>1000 CFU/100 ml). Altogether, 81.4% of the Legionella-positive samples were positive for FLA by standard methods. By applying a highly sensitive nested PCR to a representative set of random samples it was revealed that Legionella spp. always co-occurred with Acanthamoeba spp. Although the addition of disinfectants did influence amoebal density and diversity, treated waters showed no difference concerning FLA in the interphases of disinfection. It appears that FLA can re-colonize treated waters within a short period of time.     

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.     

Isolation of Legionella pneumophila from pluvial floods by amoebal coculture

Viable Legionella pneumophila bacteria were isolated by amoebal coculture from pluvial floods after intense rainfall and from water collected at sewage treatment plants. Several isolated L. pneumophila strains belonged to sequence types that have been previously identified in patients.     

Prevalence of <Emphasis Type="Italic">Acanthamoeba</Emphasis> from Tap Water in Rio Grande do Sul,Brazil

A total of 136 samples of tap water were collected from state and municipal schools between March and November 2009. The samples were filtered through cellulose nitrate membranes that were seeded at non-nutrient agar 1.5% containing an overlayer of Escherichia coli suspension. Thirty-one (22.79%) tap water samples investigated were found positive for free-living amoebae (FLA). From these, 13 presented as FLA that seems to belong to the genus Acanthamoeba. All samples of FLA were cloned and identified as belonging to the genus Acanthamoeba by the morphology of cysts and trophozoites and by PCR using genus-specific primers that amplify the ASA.S1 region of 18S rDNA gene. Physiological tests of thermotolerance and osmotolerance were used to evaluate the pathogenicity of the isolates. The sequencing analysis by comparing the sequences submitted to GenBank, showed genotype distribution into groups T2, T2/T6, T6, and T4. In tests of thermotolerance and osmotolerance, 50% of the isolates had a low pathogenic potential. The results indicated the presence of Acanthamoeba in tap water in Rio Grande do Sul, Brazil, revealing its importance and the need for more epidemiological studies to determine their distribution in the environment and its pathogenic potential.     

Relationships between Mycobacterium isolates from patients with pulmonary mycobacterial infection and potting soils

High numbers of mycobacteria, including known pathogenic species such as Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium chelonae, were recovered from aerosols produced by pouring commercial potting soil products and potting soil samples provided by patients with pulmonary mycobacterial infections. The dominant mycobacteria in the soil samples corresponded to the dominant species implicated clinically. Profiles of large restriction fragments obtained by pulsed-field gel electrophoresis demonstrated a closely related pair of M. avium isolates recovered from a patient and from that patient's own potting soil. Thus, potting soils are potential sources of infection by environmental mycobacteria. Use of dust-excluding masks should be considered during potting or other activities that generate aerosol with soil.     

Intracellular multiplication ofLegionella pneumophila in amoebae isolated from hospital hot water tanks

We studied the ability ofLegionella to multiply in potable water samples obtained from investigations of nosocomial legionellosis. AutochthonousLegionella multiplied in three of 14 hospital water samples after incubation at 35°C and 42°C. All three samples were from hot water tanks. Multiplication did not occur when a selected sample was filtered through a 0.45-m membrane and reinoculated with indigenousLegionella. We isolated bothLegionella pneumophila and one or more species of free-living amoebae, primarity members of theHartmannellidae, from each of these hot water tank samples. Amoebae from a total of six hot water tank samples were used for cocultivation studies withL. pneumophila. All amoebae supported multiplication ofLegionella in coculture at 35°C. Four of six isolates of amoebae supported multiplication oflegionella at 42°C, while none supported multiplication at 45°C. Gimenez staining and electron microscopy showed thatLegionella multiplied intracellularly in amoebae. Control of these amoebae in potable water may prevent colonization and multiplication ofLegionella in domestic hot water systems.     

Occurrence of mycobacteria in water treatment lines and in water distribution systems

The frequency of recovery of atypical mycobacteria was estimated in two treatment plants providing drinking water to Paris, France, at some intermediate stages of treatment. The two plants use two different filtration processes, rapid and slow sand filtration. Our results suggest that slow sand filtration is more efficient for removing mycobacteria than rapid sand filtration. In addition, our results show that mycobacteria can colonize and grow on granular activated carbon and are able to enter distribution systems. We also investigated the frequency of recovery of mycobacteria in the water distribution system of Paris (outside buildings). The mycobacterial species isolated from the Paris drinking water distribution system are different from those isolated from the water leaving the treatment plants. Saprophytic mycobacteria (present in 41.3% of positive samples), potentially pathogenic mycobacteria (16.3%), and unidentifiable mycobacteria (54.8%) were isolated from 12 sites within the Paris water distribution system. Mycobacterium gordonae was preferentially recovered from treated surface water, whereas Mycobacterium nonchromogenicum was preferentially recovered from groundwater. No significant correlations were found among the presence of mycobacteria, the origin of water, and water temperature.     

Occurrence of Mycobacteria in Water Treatment Lines and in Water Distribution Systems

The frequency of recovery of atypical mycobacteria was estimated in two treatment plants providing drinking water to Paris, France, at some intermediate stages of treatment. The two plants use two different filtration processes, rapid and slow sand filtration. Our results suggest that slow sand filtration is more efficient for removing mycobacteria than rapid sand filtration. In addition, our results show that mycobacteria can colonize and grow on granular activated carbon and are able to enter distribution systems. We also investigated the frequency of recovery of mycobacteria in the water distribution system of Paris (outside buildings). The mycobacterial species isolated from the Paris drinking water distribution system are different from those isolated from the water leaving the treatment plants. Saprophytic mycobacteria (present in 41.3% of positive samples), potentially pathogenic mycobacteria (16.3%), and unidentifiable mycobacteria (54.8%) were isolated from 12 sites within the Paris water distribution system. Mycobacterium gordonae was preferentially recovered from treated surface water, whereas Mycobacterium nonchromogenicum was preferentially recovered from groundwater. No significant correlations were found among the presence of mycobacteria, the origin of water, and water temperature.     

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.     

Presence of Legionella and Free-Living Amoebae in Composts and Bioaerosols from Composting Facilities

Several species of Legionella cause Legionnaires’ disease (LD). Infection may occur through inhalation of Legionella or amoebal vesicles. The reservoirs of Legionella are water, soil, potting soil and compost. Some species of free-living amoebae (FLA) that are naturally present in water and soil were described as hosts for Legionella. This study aimed to understand whether or not the composting facilities could be sources of community-acquired Legionella infections after development of bioaerosols containing Legionella or FLA. We looked for the presence of Legionella (by co-culture) and FLA (by culture) in composts and bioaerosols collected at four composting facilities located in southern Switzerland. We investigated the association between the presence of Legionella and compost and air parameters and presence of FLA. Legionella spp. (including L. pneumophila) were detected in 69.3% (61/88) of the composts and FLA (mainly Acanthamoeba, Vermamoeba, Naegleria and Stenamoeba) in 92.0% (81/88). L. pneumophila and L. bozemanii were most frequently isolated. FLA as potential host for Legionella spp. were isolated from 40.9% (36/88) of the composts in all facilities. In Legionella-positive samples the temperature of compost was significantly lower (P = 0.012) than in Legionella-negative samples. Of 47 bioaerosol samples, 19.1% (9/47) were positive for FLA and 10.6% (5/47) for L. pneumophila. Composts (62.8%) were positive for Legionella and FLA contemporaneously, but both microorganisms were never detected simultaneously in bioaerosols. Compost can release bioaerosol containing FLA or Legionella and could represent a source of infection of community-acquired Legionella infections for workers and nearby residents.     

A Comparative Study of Mycobacteria from Patients' Room Dusts and from Sputa of Tuberculous Patients

The source of mycobacteria other than Mycobacterium tuberculosis occurring in sputa of tuberculous patients as casual isolates was investigated by comparing the occurrence rate of mycobacterial species between patient's room dust and patient's sputum. Almost all species of mycobacteria recovered from sputa could be found in dusts obtained from the rooms. However, the percentage of occurrence of the mycobacterial species in dusts differed from that in sputa. In dusts, Mycobacterium fortuitum (39.6%), Mycobacterium nonchromogenicum (23.5%) and Mycobacterium gordonae (16.7%) occurred in high frequencies, whereas Mycobacterium intracellulare (69.6%), M. gordonae (5.9%), Mycobacterium scrofulaceum (5.2%), and Gordona bronchialis (10.4%) were the main species found in sputa. The patterns of occurrence of mycobacterial species as illustrated above may suggest that pathogenic mycobacteria survive in the respiratory tract while nonpathogenic ones are destroyed there, thus the human body acts as a selective medium for the pathogenic mycobacterial species. Serotype studies on strains of M. intracellular as casual isolates indicated that those from sputa of tuberculous patients were different from those derived from patients with lung diseases due to this species of mycobacteria. These results led us to the conclusion that mycobacteria in patients' room dusts were the source of mycobacteria occurring in sputa as casual isolates, particularly the pathogenic mycobacteria in dusts are more likely to survive in the human respiratory tract, occasionally multiplying and causing disease under favorable circumstances.     

Relationships between Mycobacterium Isolates from Patients with Pulmonary Mycobacterial Infection and Potting Soils

High numbers of mycobacteria, including known pathogenic species such as Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium chelonae, were recovered from aerosols produced by pouring commercial potting soil products and potting soil samples provided by patients with pulmonary mycobacterial infections. The dominant mycobacteria in the soil samples corresponded to the dominant species implicated clinically. Profiles of large restriction fragments obtained by pulsed-field gel electrophoresis demonstrated a closely related pair of M. avium isolates recovered from a patient and from that patient's own potting soil. Thus, potting soils are potential sources of infection by environmental mycobacteria. Use of dust-excluding masks should be considered during potting or other activities that generate aerosol with soil.