Table 1.
Potential protozoan hosts of Legionella pneumophila isolated from and hospital and potable water systems.
| Isolation Source (Temperature at Time of Sampling) |
Water Treatment Method | L. pneumophila | Potential Protozoan Host | Comments | Country of Origin (Sampling Site) | Reference | ||
|---|---|---|---|---|---|---|---|---|
| Identification Method | Serogroup Sequence-Type |
Genus/Species | Identification Method | |||||
| Hospital Settings | ||||||||
| Hot (45–52 °C) water tanks | - | Culturing, co-culture assay and serological identification | SG1 |
Hartmannella cantabrigiensis
Vermamoeba vermiformis Echinamoeba exudans |
Culturing, light and transmission electron microscopy | Nosocomial legionellosis investigation | USA | [31] |
| Potable water sites (39–40 °C) | - | Culturing and monoclonal antibody based serotyping | SG1 |
Acanthamoeba hatchetti
Hartmannella cantabrigiensis Vermamoeba vermiformis Vahlkampfia Filamoeba nolandi Comandonia operculata Paravahlkampfia ustiana |
Culturing and light microscopy | Nosocomial legionellosis investigation Thermal treatment (70 °C) and chlorination (1.5–2.0 mg/L) controlled the bacteria for 6 months but not amoebae. The treatment reduced incidence of legionellosis |
South Dakota, USA | [35] |
| Cooling tower, humidifier, hot water tank and supply | - | Culturing and co-culture assay | - |
Vermamoeba vermiformis
Naegleria |
Culturing, light and transmission electron microscopy | - | Paris, France | [32] |
| Hot (39–60 °C) and cold water supply | - | Culturing (ODR: 1 × 103–9.7 × 104 CFU/L), direct fluorescent antibody and monoclonal antibody based serotyping | SG1 SG5 |
Hartmannella (Hartmannellidae/limax amoebae) |
Culturing and light microscopy | Post nosocomial outbreak surveillance | Halifax, Nova Scotia, Canada | [36] |
| Organ transplant unit hot (mean 56.2 °C) and cold water (mean 16.6 °C) supplies | - | Culturing and serological assay | SG1 |
Acanthamoeba
Hartmannella Echinamoeba Vahlkampfia Tetrahymena Vannella |
Culturing and light microscopy | Population density of amoebae was greater in hot water supplies than cold water supplies Along amoebae other diverse eukaryotic microbes were detected as well |
UK | [37] |
| Water supplies | Thermal treatment (60 and 70 °C) | Culturing (Legionella ODR: 2.89–6.74 × 105 CFU/L), co-culture, latex agglutination, indirect and immunofluorescence assays, and PFGE | SG1 SG2 |
Acanthamoeba
Vahlkampfia Mayorella |
Culturing and light microscopy | Thermal treatment (70 °C) only controlled bacterial contamination for 3 months SG1 is more thermotolerant than SG2 at 60 °C |
Germany | [38] |
| Water network system (mean 56 °C) | - | Amoebae co-culture assay, PCR and sequencing | - | Vermamoeba vermiformis | Culturing, PCR and sequencing | Detection of thermotolerant Vermamoeba vermiformis | Lausanne, Switzerland | [39] |
| Water distribution system (18.9–32.6 °C) | Chlorine dioxide treatment Thermal treatment (<50 °C) |
Culturing (ODR: L. pneumophila SG1: 1 × 102–3.5 × 104 CFU/L and L. pneumophila SG2-14: 1 × 102–4 × 104 CFU/L) and latex agglutination assay | SG1 SG2-14 |
Acanthamoeba
Hartmannella |
Culturing and light microscopy | - | Messina, Italy | [40] |
| Tap water | Chloramine (1.93 ± 1.04 mg/L) treatment | Culturing (protocol: ISO 11731-2:2004, LOD: 1 CFU/100 mL, ODR: 100–1.4 × 105 ± 1.3 × 105 CFU/L), qPCR (LOD: 5 GU, LOQ: 25 GU, Legionella ODR: 100–109 gu/L) and EMA-qPCR | ST269 | Acanthamoeba polyphaga | Culturing, light microscopy and PCR | Italy | [27] | |
| Cold (14.9 °C) and warm (45.1 °C) potable water | Thermal treatment, chlorination (hypochlorates, chloramine), bacterial filters and chlorine dioxide treatment | Culturing (protocols: ISO 11731:1998 and ISO 11731-2:2004, LOD: 1 CFU/100 mL, ODR: 0–3 × 103 CFU/100 mL) and MALDI-TOF MS | - |
Acanthamoeba
Vermamoeba vermiformis |
Culturing and light microscopy | - | Bratislava, Slovakia | [41] |
| Cold water system (20–27.3 °C) | Chlorine contents 0.01–0.32 mg/L | qPCR (protocol: ISO/TS 12869:2012, LOD: 5 GU, LOQ: 25 GU, ODR: 2.7–3.8 × 102 gu/L) | - |
Acanthamoeba
Vermamoeba vermiformis |
Culturing and light microscopy | Johannesburg, South Africa | [42] | |
| Dental unit waterlines | H2O2 treatment (occasionally) | Heterotrophic plate counts, culturing (protocol: ISO 11731-2:2004, LOD: 1 CFU/100 mL, ODR: 0–2700 CFU/L) and agglutination test | - | Vermamoeba vermiformis | Culturing, light microscopy, PCR and sequencing | Italy | [43] | |
| Potable Water System | ||||||||
| Unchlorinated water supplies (9.5–13.5 °C) | - | qPCR | - |
Acanthamoeba
Acanthamoeba polyphaga Vermamoeba vermiformis |
qPCR (LOD: 1 cell/reaction), T-RFLP, cloning and sequencing | Along amoebae other diverse eukaryotic microbes were detected as well | Netherlands | [44] |
| Ground water supplies (5–39 °C) | Aeration, lime stone, granular activated carbon slow sand and rapid sand filtration, ozonisation and pellet softening | Culturing, biofilm batch test and qPCR | - |
Acanthamoeba
Vermamoeba vermiformis |
18S rDNA sequencing, PCR, T-RFLP and sequencing | Along amoebae other eukaryotic microbes were detected as well | Netherlands | [45] |
| Water supplies (mean 30 °C) | Reverse osmosis, distillation (82%), chlorination (<0.005–0.2 mg/L), dolomite, limestone and granular activated carbon filtration, fluoride addition (0.3–0.7 mg/L), UV treatment (7.5–35.99 mJ/cm2) | Culturing (LOD: 250 CFU/L, Legionella ODR: 2.5 × 102–2.5 × 105 CFU/L) and latex agglutination assay | - |
Acanthamoeba
Vermamoeba vermiformis Echinamoeba exundans Echinamoeba thermarum Neoparamoeba |
qPCR (LOD: 2 C/L, ODR: Acanthamoeba < 2–56 C/L and V. vermiformis < 2–1670 C/L) | - | Caribbean islands, Leeward Antilles | [46] |
| Water distribution systems (mean 37.3 ± 8.4 °C) | Chloramine treatment (Chlorine contents 1.8 mg/L), flocculation, sedimentation, and dual-medium filtration | Culturing, qPCR (LOQ: 1–10 copies/reaction, maximum ODR: 13.7 ± 5.1 gc/mL) and T-RFLP | - |
Acanthamoeba
Vermamoeba vermiformis |
qPCR (LOQ: 1–10 copies/reaction, maximum ODR: Acanthamoeba 6.8 ± 2.9 gc/mL and V. vermiformis 7.1 × 104 ± 4.4 × 103 gc/mL) | High concentration of chloramine is unable to disinfect water | Southwest Virginia, USA | [47] |
| Water treatment plant (7–21 °C) | - | Multiplex PCR | - | Vermamoeba vermiformis | Culturing, light microscopy, PCR and sequencing | Amoebae were frequently detected at 17 °C | Aragon, Spain | [33] |
| Water treatment facility (25 ± 3.4–28.2 ± 1.1 °C) | - | PCR (Legionella ODR: 1.2 × 104–2.4 × 105 gc/L) and sequencing | - |
Acanthamoeba
Vermamoeba vermiformis Naegleria |
Culturing, PCR, qPCR (ODR: Acanthamoeba 2.1 × 102–7.7 × 102 gc/L and Naegleria 7.6 × 102–9.4 × 102 gc/L) and sequencing | - | Kaoping River, Taiwan | [48] |
| Sediments of municipal water storage tank (2.2–28.9 °C) | Chlorination (<4 mg/L) | qPCR (LOD: 2 CE/reaction, Legionella ODR: 51 ± 114–7.98 × 104 ± 2.49 × 104 CE/g), cloning and sequencing | SG1 |
Acanthamoeba
Vermamoeba vermiformis |
qPCR (LOD: 2 CE/reaction, ODR: Acanthamoeba 22 ± 50–391 ± 243 CE/g and V. vermiformis 17 ± 23 CE/g), cloning and sequencing | - | Northeast, East Coast, Midwest, South and West Coast, USA | [49] |
| Water distribution system | - | qPCR (LOD: 2 CE/reaction, Legionella ODR: 2 ± 4–391 ± 17 CE/L), cloning and sequencing | - |
Acanthamoeba
Acanthamoeba castellanii Vermamoeba vermiformis |
qPCR (LOD: 2 CE/reaction, ODR: Acanthamoeba 1 ± 2–16 ± 2 * CE/L and V. vermiformis 1 ± 1–9 ± 11 * CE/L), cloning and sequencing | - | USA | [50] |
| Domestic water systems (mean 20.6 ± 3.8 °C) | - | Culturing, co-culture assay, PCR and sequencing | - | Vermamoeba vermiformis | Culturing, light microscopy, PCR and sequencing | - | Geneva, Lausanne and Sion, Switzerland | [51] |
| Sediments of water storage tank | - | qPCR (ODR: 25 ± 51–300 ± 38 gn/g) and NGS | - |
Acanthamoeba
Vermamoeba vermiformis |
qPCR (ODR: Acanthamoeba 3–7 gn/g, V. vermiformis 99 ± 43–120 ± 60 gn/g) and NGS | - | Ohio, West Virginia and Texas, USA | [52] |
| Potable water | Polyaluminium chloride coagulation, sedimentation, sand and biologically activated carbon filtration and chlorination | qPCR (LOQ: 1–10 copy/reaction, minimum ODR: 3.5 log(gc)/mL) | - |
Acanthamoeba
Vermamoeba vermiformis |
qPCR (LOD: 1–10 copy/reaction, minimum ODR: 2 log(gc)/mL for V. vermiformis and 4 log(gc)/mL for Acanthamoeba) and sequencing | Antibiotics (sulfadiazine and ciprofloxacin) promote growth of both bacterium and amoebae | Northern China | [53] |
| Potable water | Polyaluminium chloride coagulation, sedimentation, sand and biologically activated carbon filtration, chlorination and ozonisation | qPCR (LOQ: 1–10 copies/reaction, minimum ODR ≈ 1 log(gc)/g) | - |
Acanthamoeba
Naegleria |
qPCR (LOQ: 1–10 copies/reaction, minimum ODR: ≈ 0.5 log(gc)/g for Naegleria and ≈ 1 log(gc)/g for Acanthamoeba) | Combined chlorination and ozonisation are effective than chlorination only | Northern China | [54] |
| Potable water | Coagulation, ozonisation, pellet softening, granular activated carbon filtration, rapid and slow sand filtration | Heterotrophic plate counts, culturing (protocol: NEN 6275, LOD: 1 log(CFU)/cm2) epifluorescence microscopy, bioluminescence assay, PCR and sequencing | - | Vermamoeba vermiformis | qPCR (ODR: 0.7–384 CE/cm2) | - | Netherlands | [55] |
| Residential secondary water supply systems (13.9 ± 4.0–17.4 ± 2.9 °C) | Chloramine treatment (Chlorine contents 0.19–0.89 mg/L) |
qPCR (LOQ: 10 copies/reaction, maximum ODR: ≈ 102 gc/mL) and sequencing | - |
Acanthamoeba
Vermamoeba vermiformis |
qPCR (LOQ: 10 copies/reaction, ODR: 101–103 gc/mL for both Acanthamoeba and V. vermiformis) and sequencing | - | Shanghai, China | [56] |
| Water treatment facility | Coagulation, sedimentation, chlorination, ozonisation, granular activated carbon and sand filtration | qPCR (LOQ: 10 copies/reaction, minimum ODR: 102 log(gc)/mL) and sequencing | - | Vermamoeba vermiformis | qPCR (LOQ: 10 copies/reaction) and sequencing | Sand filtration after granular activated carbon treatment improves water quality | Southeast China | [57] |
| Water from private wells after flood | - | Culturing (protocol: ISO 11731, LOD: 1 CFU/100 mL) and qPCR (LOQ: 9.5 gc/mL, maximum ODR: 52.4 gc/mL) | - | Naegleria fowleri | qPCR (ODR: 11–610 gc/mL) | - | Louisiana, USA | [58] |
| Potable water | - | Culturing and DVC-FISH | - |
Acanthamoeba
Vermamoeba vermiformis |
Culturing and PCR | - | Valencia, Spain | [34] |
Vermamoeba vermiformis was previously known as Hartmannella vermiform. Paravahlkampfia ustiana was previously known as Vahlkampfia ustiana. ODR: Observed detection range, the amount of bacteria/amoebae/DNA experimentally determined from the samples; CFU/L: colony forming unit/liter; PFGE: pulsed-field gel electrophoresis; PCR: polymerase chain reaction; ISO: International organization for standardization; MALDI-TOF MS: matrix assisted laser desorption ionization-time of flight mass spectrometry; qPCR: quantitative PCR; gu/L: genome unit/liter; LOQ: limit of quantification; LOD: limit of detection; EMA-qPCR: ethidium monoazide-qPCR; T-RFLP: terminal-restriction fragment length polymorphism; C/L: cells/liter; gc/mL: gene copy/milliliter; gc/L: gene copy/liter; CE/reaction: cell equivalent/reaction; CE/g: cell equivalent/gram; CE/L: cell equivalent/liter; * CE/L: cyst equivalent/liter; gn/g: genome copy number/gram; gc/g: gene copy/gram; NGS: next generation sequencing; NEN: Nederlands normalisatie instituut; CE/cm2: cell equivalent/cm2; DVC-FISH: direct viable count combined with fluorescence in situ hybridization.