Free-living amoebae: Biological by-passes in water treatment

International Journal of Hygiene and Environmental Health 213 (2010) 167–175

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International Journal of Hygiene and Environmental Health journal homepage: www.elsevier.de/ijheh

Free-living amoebae: Biological by-passes in water treatment Jean-Franc¸ois Loret a,∗ , Gilbert Greub b a b

Suez Environnement, CIRSEE, 38 rue du Président Wilson, 78230 Le Pecq, France Centre for Research on Intracellular Bacteria, Institute of Microbiology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland

a r t i c l e

i n f o

Article history: Received 24 November 2009 Received in revised form 24 March 2010 Accepted 24 March 2010 Keywords: Free-living amoebae Amoebae-resisting bacteria Legionella Mycobacteria Drinking water Biofilms

a b s t r a c t Free-living amoebae constitute reservoirs for many bacteria including not only well-known pathogens but also emerging pathogens responsible for respiratory diseases, and contribute to the protection, survival and dissemination of these bacteria in water systems, despite the application of disinfection or thermal treatments. In this article we review the available information on the presence of free-living amoebae and amoebae-resisting bacteria in drinking water systems, on the factors that contribute to their presence in the water and/or the biofilms, on the possible control measures and their effectiveness, and we identify some gaps in current knowledge needing further research. © 2010 Elsevier GmbH. All rights reserved.

Introduction There is a growing evidence that the complex microbial ecosystems that develop in biofilms in contact with water contribute to the survival and protection of pathogenic micro-organisms. Within these microbial communities, free-living amoebae (FLA) are increasingly suspected to favour the multiplication and dissemination of pathogenic bacteria belonging to a large range of families, including Legionellaceae, Mycobacteriaceae, Enterobacteriaceae, Vibrionaceae and many others (Greub and Raoult, 2004; Thomas et al., 2010; Winiecka-Krusnel and Linder, 2001). Intracellular parasitism of FLA by these bacteria allows them not only to survive, but also to proliferate in harsh environments, by using the amoebal host as a food source and as a protective shield against external aggressions (Kilvington and Price, 1990; King et al., 1988; Storey et al., 2004). This mechanism is probably at the origin of the rapid re-colonization of cooling or domestic water systems by Legionella, generally observed immediately after stopping a disinfection programme (Thomas et al., 2004). For some of these pathogens, especially Legionella pneumophila and Mycobacterium avium, it has been demonstrated that intra-amoebal replication enhances virulence (Cirillo et al., 1994, 1997). FLA thus constitute a potential reservoir for these pathogens, allowing the survival, the multiplication, and the dissemination of virulent bacteria in water systems, despite the application of disinfection or thermal treatments. This role of reservoir is supported by the results of recent

studies that demonstrated a significant association between the presence of Legionella spp., Mycobacterium spp., and FLA, in different drinking water systems (Corsaro et al., 2010; Thomas et al., 2006b). In addition to these established pathogenic bacteria, a series of emerging pathogens, responsible for respiratory diseases, including Parachlamydiaceae, Simkaniaceae or new Legionella species, are also able to resist within amoebae (Corsaro and Greub, 2006; Greub and Raoult, 2002, 2004; La Scola et al., 2004). As these bacteria grow poorly or not at all on culture media used routinely for detecting human pathogens from clinical samples, they are suspected to be the causative agents of pneumonia of unknown etiology, which represent 47–55% of community-acquired pneumonia worldwide in adults and 20–75% of nosocomial pneumonia (Greub and Raoult, 2002; Lamoth and Greub, 2010). Therefore, amoebae and amoebae-resisting bacteria (ARB) might represent a significant threat to public health. It is however expected that controlling FLA in artificial water systems should allow a better control of all these associated established and emerging pathogens. Thus, the aim of this article is to review the available information on the presence of FLA and ARB in drinking water systems, on the factors that contribute to their presence, on the possible control measures and their effectiveness, and to identify the main gaps in current knowledge needing further research. Free-living amoebae and amoebae-resisting bacteria

∗ Corresponding author. Tel.: +33 1 34 80 22 76; fax: +33 1 30 53 62 09. E-mail address: [email protected] (J.-F. Loret). 1438-4639/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijheh.2010.03.004

The term amoeba covers a heterogeneous group of diverse unicellular eukaryotes that share common morphological and

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Table 1 Prevalence of free-living amoebae in environmental freshwaters. Examples from recent studies. Country

Volume analysed per sample (mL)

Water source

Germany

1000

River

11

Reservoir Ground water USA

Bulgaria

FLA/L in positive samples

Main genera identified

11 (100%)

200–90000

Acanthamoeba, Hoffmann and Naegleria, Hartmannella Michel (2001)

15 11

15 (100%) 9 (82%)

2–3000 1–3000

330

143 (43%)

ND

39

31 (94%)

ND

31 121

27 (87%) 80 (66%)

Rivers, lakes

28

21 (75%)

50

River

500

Rivers Lakes Reservoirs

The Netherlands

200

Number of samples analysed

Number of positive samples

References

Naegleria, Vannella, Acanthamoeba, Vahlkampfia, Hartmannella Acanthamoeba, Hartmannella

Ettinger et al. (2003)

5–75

Hartmannella (1) Acanthamoeba, Naegleria, Willaertia, Vahlkampfia Acanthamoeba, Naegleria, Hartmannella, Echinamoeba, Vannella, Platyamoeba Acanthamoeba, Vannella, Hartmannella

Kuiper et al. (2006) Declerck et al. (2007)

Belgium

1000

Lakes, ponds, brooks, creeks

37

37 (100%)

ND

France, Spain

3330

Rivers, lakes

30

30 (100%)

15–4500

3330

Ground waters

23

15 (65%)

1–110

Tsvetkova et al. (2004)

Adapted from Thomas et al. (2008); Corsaro et al. (2009, 2010)

(1) Study targeted on this genus only. (ND: not documented).

behavioural characteristics (Winiecka-Krusnel and Linder, 2001). More than 11,300 amoebal species have been identified up to now (http://www.bms.ed.ac.uk/research/others/smaciver/amoebae.htm), of which only very few are recognized human pathogens. Most of them present two developmental stages: a vegetative stage, called trophozoite, and a resting form, the cyst, which allows them to survive in aggressive environments or in the absence of nutrients. FLA are present worldwide and live more especially at the contact of biofilms where they can feed on smaller microorganisms like bacteria, fungi and algae (Rodriguez-Zaragoza, 1994). Digestion normally occurs within phagolysosomes, but some micro-organisms, termed ARB (i.e. amoebae-resisting bacteria), are not internalized, or are able to survive, grow, and exit FLA after internalization. When internalized, some ARB are also able to survive encystation of the amoebae (Molmeret et al., 2005), and thus are protected against external physical or chemical aggressions, until the amoebae excyst when environmental conditions become more favourable. ARB egress their hosts inside vesicles expelled by the amoebae or after lysis of the amoebae, once intracellular replication is terminated, and can then infect new cells (Alli et al., 2002). Many amoebae genera have been identified as potential hosts for ARB. Among these, Acanthamoeba appears to be the most universal host used by most pathogenic ARB identified to date. However, this may be biased because of the common use of this amoeba genus as cell background in most amoebal co-culture protocols. Other potential hosts mainly include the Hartmannella, Naegleria, and Saccamoeba genera (Greub and Raoult, 2004; Thomas et al., 2010; Winiecka-Krusnel and Linder, 2001). If most of the ARB are facultative intracellular bacteria, some are obligate intracellular, whereas some others are extracellular (Greub and Raoult, 2004). In a recent review, a total of 102 recognized and 27 suspected pathogenic bacterial species have been identified as being able to survive or grow within FLA (Thomas et al., 2010). ARB however do not exclusively include pathogenic micro-organisms; this point will be discussed later.

Prevalence and biodiversity of free-living amoebae in raw waters and in treated waters Raw waters Table 1 summarizes recent data from different water resources, some of them being used for drinking water production. Despite the diversity of analytical methods applied, the results from these studies demonstrate the presence of FLA in most samples studied, and in all types of water investigated. Thus, overall, when considering as an example only the data provided in Table 1, 62% of surface water samples and 71% of ground water samples are positive for FLA. Furthermore, higher FLA concentrations are generally observed in surface waters, compared to ground waters. Interestingly, the biodiversity of amoebal genera identified in these studies do not show important differences, whatever the nature (ground or surface) or geographical origin of the water, and amoebal genera known as potential hosts for ARB have been recovered at all these sites. Several factors obviously influence the presence of FLA in environmental waters. High temperatures are known to favour thermophilic amoebae like the pathogenic species Naegleria fowleri (Tyndall et al., 1989). Ettinger et al. observed important seasonal changes in the composition of amoebal populations in the James river (Virginia, USA), with Naegleria and Acanthamoeba being more prevalent in spring and early to mid-summer, whereas Vannella, Vahlkampfia and Hartmannella were more prevalent in late summer and fall (Ettinger et al., 2003). In this study, a higher prevalence of FLA was observed after a rain event leading to increased discharge from combined sewer outflows, thus confirming other observations of higher concentrations of FLA following increased runoff caused by rainfall events (John and Howard, 1995; Kyle and Noblet, 1987). Ettinger et al. and Tsvetkova et al. also mentioned higher isolation rates and higher counts of FLA in sediment and biofilm samples from surface waters (Ettinger et al., 2003; Tsvetkova et al., 2004). More recently, in a survey of eight European surface water sources

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used for drinking water production, Loret et al. did not observe any correlation between water temperature and FLA concentrations, in a range of temperatures of 7–28.5 ◦ C, but found a significant positive correlation between FLA concentrations and dissolved organic matter, as measured by UV absorption (Loret et al., 2008c). Valster et al. also observed such a relationship in a comparison of two treated ground waters and found that the presence of Hartmannella vermiformis was associated with high levels of active biomass and natural organic matter (Valster et al., 2009). All these observations demonstrate that water temperature and the presence of organic matter, biofilms and sediments affect the biodiversity and concentrations of FLA in environmental waters.

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in a distribution system, amoebal concentrations in these cases, as measured by culture methods, were below 100 FLA/L (Corsaro et al., 2010). A concentration of 815 Hartmannella vermiformis/L has been observed in a drinking water distribution system supplied with ground water in The Netherlands (Valster et al., 2009). It is not clear however whether such a high concentration for a single amoebal species is due to the application of a quantitative PCR method, or to the fact that the distributed water in that case is not chlorinated and contains a very high level of natural organic matter. As for environmental waters, much higher concentrations, up to 104 FLA/mL, have been observed in sediments recovered from distribution systems (Loret et al., 2008c).

Drinking water production and distribution Domestic water systems Although the presence of amoebae in environmental waters is well documented, few studies have been dedicated to the investigation of the biodiversity and prevalence of FLA and ARB in drinking water production and distribution systems. Since they are naturally present in raw waters, FLA are also present in drinking water treatment lines. In a series of drinking water treatment plants operated in Germany and France, Hoffmann and Michel and Thomas et al. observed in all cases a significant decrease in amoebal concentration (up to 2 log) and biodiversity after the clarification and filtration steps (Hoffmann and Michel, 2001; Thomas et al., 2008). In both studies however, occasional releases of FLA, likely due to filter colonization, were observed at the outlet of the filters. This colonization may alter the filtration performance (see below). Thomas et al. observed that Hartmannella vermiformis and Echinamoeba exudans were largely predominant after the sand and granular activated carbon filtration stages, including in ozonated water (Thomas et al., 2008). In both studies, a significant increase in amoebal density and diversity was observed in the distributed water, at increasing distances from the treatment plant. Table 2 summarizes the amoebal species recovered along the treatment line of a French drinking water treatment plant supplied with water from the River Seine, as observed during a one-year monitoring (Thomas et al., 2008). These results demonstrate that despite the application of an effective full treatment chain, FLA are still present at the outlet of the treatment lines, and that amoebal re-growth occurs in the distribution systems. Hoffmann and Michel and Loret et al. both observed a presence of FLA in approximately one third of treated and distributed drinking waters, both studies representing a total of 14 treatment plants supplied with surface and ground waters in Germany, Spain and France (Hoffmann and Michel, 2001; Loret et al., 2008c). At the exception of one sample obtained from a dead leg

Domestic water systems offer even better conditions for amoebal re-growth, especially when retention time of the water in the system is important. This is the case in the United Kingdom where water storage tanks are in use and where, in an investigation of 27 households, Kilvington et al. isolated FLA from 89% of them (Kilvington et al., 2004). Reported concentrations in domestic systems can reach several thousands of FLA/L (Barbeau and Buhler, 2001; Ménard-Szczebara et al., 2008). Interestingly, in a survey of 36 domestic water systems, Ménard-Szczebara et al. observed a higher prevalence of FLA at the taps, compared to water at the inlet (water meters) or within the domestic water network (Ménard-Szczebara et al., 2008). A lower prevalence of FLA in hot water, compared to cold water, has also been observed, and reported prevalence of FLA is significantly lower for temperatures above 60 ◦ C (Kilvington et al., 2004; Ménard-Szczebara et al., 2008; Thomas et al., 2006b). FLA recovered from domestic water systems are generally similar to those recovered from environmental waters and drinking water distribution systems. Most reported genera include Hartmannella, Vannella, Vahlkampfia, Naegleria, Acanthamoeba, Echinamoeba or Saccamoeba. An unusual predominance of Vexillifera has been reported form a survey of 283 domestic water systems in Florida, USA (Shoff et al., 2008), but this study is the only one based on an analysis of aged biofilm, recovered from the cistern tanks serving the toilets, and it is not clear whether the age of the biofilm can influence FLA diversity. As expected, similarly to what has been observed for environmental waters, high temperatures favour thermophilic species, and Hartmannella vermiformis has been reported as the major component of FLA populations in hot water systems (Rohr et al., 1998; Thomas et al., 2006b).

Table 2 Amoebal species recovered from a drinking water treatment plant (number of isolates in parentheses), adapted from Thomas et al. (2008). Raw surface water

A. castellanii (2) A. palestinensis (1) A.polyphaga (1) A. quina (1)

Biofilm from sand filter

A. castellanii (2)

E. exudans (2) Glaeseria related (1) H. vermiformis (3)

N. clarki (1) N. fultoni (1) N. gruberi (2)

P. placida (1) V. miroides (2) V. persistens (1)

N. andersoni (3) N. clarki (1) N. fultoni (1) N. gruberi (2)

V. miroides (1)

E. exudans (4) H. vermiformis (1)

N. clarki (1)

V. persistens (1)

Sand filtered water

H. vermiformis (4)

Ozonated water

E. exudans (2) H. vermiformis (1)

Biofilm from GAC filter

E. exudans (4)

GAC filtered water

E. exudans (1) H. vermiformis (1)

Distribution system

A. polyphaga (1)

Amoebozoa sp. (1)

A. = Acanthamoeba; N. = Naegleria; E. = Echinamoeba; P. = Platyamoeba; H. = Hartmannella; V. = Vannella.

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Table 3 Infected free-living amoebae recovered from different drinking water treatment steps. Sampling point

Infected FLA

ARB identified

Reference

Raw surface water

Platyamoeba sp. Acanthamoeba castellanii Naegleria australiensis

Parachlamydia acanthamoeba Protochlamydia sp. Legionella sp.

Loret et al. (2008b) Corsaro et al. (2010)

Biofilm from sand filters

Naegleria gruberi

Thomas et al. (2008)

Naegleria andersoni Echinamoeba exudans Hartmannella vermiformis Echinamoeba exudans Echinamoeba exudans Echinamoeba exudans Acanthamoeba castellanii Acanthamoeba castellanii

Acidovorax temperans Legionella anisa Flavobacterium johnsoniae Unclassified Chlamydiales Legionella micdadei Caulobacter vibrioides Neochlamydia sp. Mycobacterium mucogenicum Legionella londiniensis Legionella donaldsonii Legionella sp. Neochlamydia sp.

Acanthamoeba sp. Acanthamoeba sp. Hartmannella vermiformis

Cytophaga johnsonae Unidentified Gram-negative bacterium Legionella pneumophila, Bradyrhizobium japonicum

Hoffmann and Michel (2001)

Naegleria andersoni

Sand filtered water Ozonated water Biofilm from GAC filters GAC filtered water Biofilm from distribution system Sediment from distribution system Tap water

Amoebae-resisting bacteria in raw waters and in treated waters Amoebae-resisting bacteria recovered from infected amoebae FLA known as potential hosts for ARB are normal components of the microbial flora present in both environmental water and drinking water. Infected amoebae have been recovered from different drinking water systems, and the associated ARB have been identified in a few instances, confirming that some FLA can really vehicle ARB within these systems. These results (summarized in Table 3) reveal a large biodiversity of ARB infecting a large number of amoebal species. Although ARB identified in these studies include common environmental bacteria, for which a pathogenic role has not yet been recognized, their capacity to resist destruction by FLA however rises question of their potential infectivity for other phagocytic cells, including human macrophages. Interestingly, more than half of the ARB identified in these studies belong to Legionellaceae or Parachlamydiaceae, two families including recognized or emerging pathogens causing respiratory diseases (Corsaro and Greub, 2006; Greub and Raoult, 2004; Lamoth and Greub, 2010). In a systematic analysis of FLA at each treatment step of a drinking water treatment plant, Thomas et al. observed that infected amoebae were mainly recovered from filter media and filtered water (Thomas et al., 2008). They also observed an important colonization of filter media by FLA. As filter media are known to contain important quantities of fixed biofilm on which

Corsaro et al. (2010)

Thomas et al. (2006b)

amoebae can feed, they hypothesized that biofilms favour the contact between FLA and bacteria and thus constitute a major source of infected amoebae in drinking water. These conclusions are in agreement with the results from Berk et al. who observed a significantly higher rate of infected amoebae in biofilms from cooling towers, compared to environmental waters or sediments (Berk et al., 2006). They are also consistent with the observation of Acanthamoeba species infected with Legionella pneumophila in floating biofilms from artificial or natural water systems (Declerck et al., 2007).

Legionella species In a survey of eight drinking water treatment plants, representing ten different surface water treatment chains in France and Spain, Loret et al. observed that Legionella prevalence followed the same trends as for FLA (Loret et al., 2008c), with a progressive decrease along the treatment process, from raw to ozonated water, an increase after granular activated carbon (GAC) filtration, and again a decrease after chlorination (Table 4). Legionella spp. were detected by real-time PCR in 70% of samples immediately after the chlorination step, with maximum concentrations of about 105 Legionella spp./L, and in 47% of samples from the distribution systems supplied by these treatment plants. This suggests that Legionella spp. take advantage of the presence of amoebae to replicate within GAC filters, and that a large part of them survive final disinfection. As for FLA, high concentrations, up to 104

Table 4 Positive samples for amoebae, Legionella spp., and L. pneumophila in raw and treated waters from ten treatment lines, adapted from Loret et al. (2008c). Sample volumes were 3.33 L for amoebae, 1 L for Legionella spp. and L. pneumophila. Sampling point

Water

Raw water Flotation Sedimentation Filtration Ozonation GAC filtration Chlorination Distribution system

Biofilm/sediments Sludge/biofilm from clarifiers Biofilm from filters Sediments from distribution Biofilm from distribution

Number of samples

Free-living amoebae

Legionella spp.

L. pneumophila

Positive samples

%

Positive samples

%

Positive samples

%

25 3 18 41 14 15 23 34

25 3 14 24 9 10 7 10

100 100 78 59 64 67 30 29

20 2 11 28 7 12 16 16

80 67 61 68 50 80 70 47

1 0 0 5 0 0 0 1

4 0 0 12 0 0 0 3

9 11 12 11

9 10 11 3

100 91 92 27

4 6 10 3

44 55 83 27

1 2 0 1

11 18 0 9

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Table 5 Amoebae-resisting bacteria recovered by amoebal co-culture from a drinking water treatment plant and distribution system, adapted from Thomas et al. (2008). Raw water

Acinetobacter haemolyticus Acinetobacter baumannii Aeromonas hydrophila Agromyces subbeticus Bacillus pumilus Bacillus luciferensis Bosea sequanensis Bosea vestrisii Brevundimonas bullata Citrobacter freundii Criblamydia sequanensis Kocuria kristinae Legionella anisa Microbacterium oxydans

Microbacterium takaoensis Micromonospora carbonacea Mycobacterium insubricum Mycobacterium frederiksbergense Mycobacterium gordonae Mycobacterium kansasii Mycobacterium terrae Mycobacterium vaccae Mycobacterium vanbaalenii Paenibacillus ginsengagri Parachlamydia acanthamoebae Pseudomonas fluorescens Rhodococcus equi Stenotrophomonas acidaminiphila

Biofilm from sand filters

Bacillus cereus Bacillus licheniformis Bosea lascolae Bosea minatitlanensis Criblamydia sequanensis Enterobacter cloacae Flavobacterium aquatile Flavobacterium kamogawaensis

Legionella LLAP2 Microbacterium oxydans Mycobacterium insubricum Mycobacterium gordonae Mycobacterium poriferae Mycobacterium septicum Rhodococcus equi Rhodococcus erythropolis

Distribution system

Acinetobacter johnsonii Acinetobacter lwoffii Acinetobacter radioresistens Arthrobacter davidanieli Brevundimonas mediterranea Chryseobacterium daeguense Chryseobacterium defluvium Microbacterium oxydans

Microbacterium phyllosphaerae Mycobacterium anthracenicum Mycobacterium frederiksbergense Mycobacterium gadium Mycobacterium neglectum Mycobacterium vanbaalenii Pseudomonas fluorescens Pseudomonas proteolytica

Table 6 Inactivation of Naegleria cysts by different disinfectants.

*

Disinfectant

Naegleria species (strain)

Temperature (◦ C)

pH

Ct 99% (mg min/L)

Reference

Chlorine

N. fowleri (HB1) N. gruberi (1518/1e) N. spp. (HB1, TY, A1) N. gruberi (NEG)

25 25 25 25

7.3–7.4 7.3–7.4 7.2–7.3 7

9–24* 9–30* 28* 12.1

De Jonckheere and Van de Voorde (1976)

Chlorine dioxide

N. gruberi N. fowleri (2 strains) N. lovaniensis

25 25 25

7 8–9 8–9

5.5 0.9–1.2* 1.7–2.7*

Chen et al. (1985) Pringuez et al. (2001)

Monochloramine

N. fowleri (2 strains) N. lovaniensis N. lovaniensis (ATCC 3011)

25 25 25

8–9 8–9 7–9

44.5–51.6* 23–30* 118–237*

Pringuez et al. (2001)

Ozone

N. gruberi (NEG) N. gruberi (1518/1d) N. gruberi (Echirolles) N. spp. (MO5) N. spp. (Cl10) N. spp. (An24) N. fowleri (0359)

25 25 25 25 25 25 25

7 7 7 7 7 7 7

1.3 1.6* <1.6* <1.6* <1.6* <1.6* <1.6*

Wickramanayake et al. (1984) Langlais and Perrine (1986)

Chang (1978) Rubin et al. (1983)

Ercken et al. (2003)

Estimated from data in cited reference.

Table 7 Inactivation of Acanthamoeba cysts by different disinfectants.

*

Disinfectant

Acanthamoeba species (strain)

Temperature (◦ C)

pH

Ct 99% (mg min/L)

Reference

Chlorine

A. culbertsoni (A1) A. spp. 4A A. polyphaga

25 25 20–22

7.3–7.4 7.3–7.4 7.5–8

1260–6480* 5040* 1300

De Jonckheere and Van de Voorde (1976)

Chlorine dioxide

A. polyphaga

20–22

7.5–8

20

Loret et al. (2008b)

Ozone

A. polyphaga (1501/3a) A. spp. (MR4) A. culbertsoni (A1) A. royreba (OR) A. polyphaga

25 25 25 25 20–22

7 7 7 7 7.5–8

2.5* 1.6* <1.6* <1.6* 5

Langlais and Perrine (1986)

Estimated from data in cited reference.

Loret et al. (2008b)

Loret et al. (2008b)

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Legionella spp./mL, were found in the sediments from distribution systems. The pathogenic species L. pneumophila however represented only 8% of all Legionella spp. detected in this study. Similar results were obtained by Nakache-Danglot et al. from a survey of three French treatment plants supplied with ground and surface waters (Nakache-Danglot et al., 2006). Legionella spp. were detected by real-time PCR in treated and distributed waters from the three plants at maximum concentrations of 105 genome units/L. Again, L. pneumophila represented a minority, with only 2.4% of all Legionella detected in this study. Wullings and Van der Kooij also detected Legionella spp. by real-time PCR in all samples from 16 surface and 81 ground water treatment plants, representing 67% of the total drinking water production in The Netherlands (Wullings and Van der Kooij, 2006). Maximum concentrations in treated waters were in the order of 105 cells/L. They also observed on some occasions an increase in Legionella concentration after GAC filtration, and found that L. pneumophila represented less than 1% of all detected Legionella. The high prevalence of Legionella in drinking water was confirmed in a survey of 250 buildings in The Netherlands (Diederen et al., 2007). In this study, 87% of tap water samples were found positive for Legionella spp. by real-time PCR, with L. pneumophila representing only 3.9% of all detected Legionella. In all these studies, the presence of Legionella was mainly detected by using molecular methods, and rarely detected by culture-based methods.

and L. anisa (Thomas et al., 2006b). Thomas et al. and Corsaro et al. also applied systematically this technique to water samples collected at different steps of drinking water treatment plants (Corsaro et al., 2010; Thomas et al., 2008), and observed the presence of a large spectrum of ARB, including some new bacterial species (Corsaro et al., 2009; Thomas et al., 2006a, 2007). As an example, Table 5 summarizes the bacterial species recovered by amoebal co-culture from the raw and treated water of a French drinking water treatment plant supplied with water from the River Seine, as observed during a one-year monitoring. In these studies, the maximum biodiversity of ARB was observed in the raw water and in the biofilm from the sand filters. Although this biodiversity then decreased along the treatment lines, ARB were recovered from all the treatment steps, and an increased biodiversity was observed at distant points from the treatment plant in the distribution system, especially in biofilm and sediment samples. In the latter, mycobacteria represented about one third of all bacterial species recovered from the distribution system. A significant correlation between the presence of FLA and mycobacteria was observed in Spanish drinking water distribution systems (Corsaro et al., 2010), thus confirming the correlation observed by Thomas et al. (2006b), and supporting the role of FLA as a widespread reservoir for ARB. All these results tend to demonstrate that FLA favour the protection and dissemination of ARB in drinking water systems, and that controlling pathogenic bacteria that can survive or develop in biofilms would require a better control of FLA in these systems.

Mycobacteria Non-tuberculosis mycobacteria (NTM) constitute another interesting example of ARB demonstrating a behaviour comparable to that of Legionella spp., at least in drinking water systems. Thus, Le Dantec et al. observed on two treatment plants a decrease in mycobacteria concentrations along the treatment lines, from raw to ozonated water, an increase after GAC filtration, due to filter colonization, and again a decrease after chlorination (Le Dantec et al., 2002). In this study, NTM were present in 72% of tested samples obtained from the distribution system of Paris supplied by these two plants, with potentially pathogenic mycobacteria accounting for 16% of all positive samples. Hilborn et al. observed the persistence of NTM in a municipal drinking water distribution system despite the addition of ozonation and GAC filtration at the treatment plant serving a major metropolitan area in the USA (Hilborn et al., 2006), and Falkinham et al. recovered NTM from eight water distribution systems in the USA, supplied by ground or surface water treatment plants (Falkinham et al., 2001). They also observed in that case a decrease in prevalence just after the treatment, followed by an increase in the distribution system, with numbers up to 7 × 105 CFU/L, and frequently recovered mycobacteria in high numbers from biofilms. Other amoebae-resisting bacteria The use of axenically grown FLA has been recently proposed to recover amoebae-resisting micro-organisms from different clinical or environmental samples, and has since then proved to be an effective tool to isolate new bacterial species that cannot be otherwise cultured (Greub et al., 2004; Greub and Raoult, 2004). By using this technique, Pagnier et al. recovered a large number of ARB species from natural and artificial water systems, including a majority of environmental bacteria, and several human pathogens belonging essentially to the alpha, beta, and gamma-Proteobacteria (Pagnier et al., 2008). By applying the same technique to biofilm and water samples from a hospital water network, Thomas et al. also recovered a large diversity of ARB, composed of 30.5% of alpha-Proteo-bacteria, 20.5% of mycobacteria, and 5.5% of gamma-Proteobacteria, including Legionella pneumophila

Elimination of free-living amoebae in drinking water treatment Most studies conducted to date have been focused on the effectiveness of disinfectants against pathogenic FLA, and have targeted more especially Naegleria and Acanthamoeba species. Inactivation data for both amoebal genera for disinfectants applied in drinking water treatment are summarized in Tables 6 and 7. De Jonckheere and Van de Voorde observed that Naegleria was much more sensitive to chlorine than Acanthamoeba, with Ct (Concentration × time) values in the range of 20–60 mg min/L for a 4-log reduction of Naegleria cysts, whereas Acanthamoeba cysts required several thousands of mg min/L for the same reduction (De Jonckheere and Van de Voorde, 1976). This relative sensitivity of Naegleria to chlorine allows effective control by simple chlorination of drinking water supplies, and operational control and management of Naegleria based on the application of contact times greater than 30 mg min/L and maintenance of a free chlorine residual of 0.2 mg/L at the end of the distribution systems has proved to be effective in Australia (Trolio et al., 2008). On the other hand, control of Acanthamoeba cannot be achieved by current disinfection practices of drinking water. Loret et al. observed the persistence of FLA in a pilot scale domestic water system, despite the continuous application during three months of various disinfectants, including 2 mg/L chlorine, 0.5 mg/L chlorine dioxide or ozone (Loret et al., 2005). Among the natural FLA populations that colonized the system, an Acanthamoeba polyphaga strain was found to be able to resist all disinfectants tested in this study (Loret et al., 2004). Cysts of this strain were used for laboratory experiments and exposed to increasing concentrations of disinfectants, and were found to be able to survive a 2-h exposure to chlorine at 100 mg/L at 20 ◦ C. No inactivation of the cysts was observed until a threshold concentration of chlorine or chlorine dioxide was applied (25 and 2 mg/L, respectively). No threshold value was observed with ozone (Loret et al., 2008b). A similar threshold effect had been observed previously by Kilvington and Price with cysts of another A. polyphaga strain (Kilvington and Price, 1990). In that case, the lowest concentration of free chlorine that resulted in no excystation was found

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to be 75 mg/L for a contact time of 18 h at 25 ◦ C. Data for other FLA identified as potential hosts for ARB are scarce. Langlais and Perrine observed that Hartmannella vermiformis was highly sensitive to ozone and found that the necessary Ct for a 2-log reduction was <1.6 mg min/L at 25 ◦ C (Langlais and Perrine, 1986). Kuchta et al. observed that H. vermiformis survived chlorine concentrations up to 4 mg/L during 30 min at 20 ◦ C and pH 7.6–7.8, but did not survived after exposure of 30 min at 10 mg/L (Kuchta et al., 1993). Hijnen et al. reviewed inactivation credits of UV radiation for different protozoan cysts in water (Hijnen et al., 2006). Available data show that sensitivity of Acanthamoeba spp. is in the same order of magnitude as for adenovirus, with only 1-log reduction achieved with a fluence of 40 mJ/cm2 . An offset fluence of 30 mJ/cm2 before inactivation starts has been observed. It has also been demonstrated that photocatalytic disinfection of A. castellanii is ineffective (Sökmen et al., 2008). FLA cysts are also resistant enough to survive temperature levels applied to domestic hot water systems, which are generally below 60 ◦ C, and in many cases below 55 ◦ C. As for disinfectants, Naegleria appears to be more sensitive to heat, compared to Acanthamoeba. Chang observed that Naegleria spp. cysts were able to survive 120 min at 51 ◦ C, but only 2.5 min at 65 ◦ C (Chang, 1978). Loret et al. observed no significant inactivation of Acanthamoeba polyphaga cysts below 62 ◦ C (Loret et al., 2008a). At 62 and 65 ◦ C, a 5-log reduction was observed after a 2-h contact time, but viable A. polyphaga could still be recovered by culture in these conditions. Total elimination (>5 log) of A. polyphaga cysts required 1-h exposure to 70 ◦ C. Comparable results were obtained by Storey et al. who observed that A. castellanii and I4 Acanthamoeba cysts retained their viability for maximum values of 30 min at 70 ◦ C and 10 min at 80 ◦ C (Storey et al., 2004). Again, data for other FLA are scarce and limited to Hartmannella, for which Kuchta et al. observed a >3.3 log reduction of H. vermiformis cysts in 30 min at 55 ◦ C, and a total elimination (>5.4 log) in 30 min at 60 ◦ C (Kuchta et al., 1993). Although disinfection effectiveness on FLA cysts is limited, and consequently removal processes seem more appropriate to eliminate these micro-organisms, the effectiveness of physical processes applied in drinking water production (principally clarification and filtration) is poorly documented in the literature. The effect of clarification was studied by Loret et al. who performed Jar tests with river water spiked with Acanthamoeba polyphaga cysts, and compared two commercial coagulants (Loret et al., 2008a). They observed that ferric chloride performed better than aluminium chlorosulphate, with respective log reductions of 0.7 and 1.8 at the optimum dosage. A monitoring of FLA at seven full scale treatment plants, representing a total of nine different treatment lines in France and Spain, confirmed the effectiveness of coagulation, flocculation and sedimentation. A mean 1.5 log reduction of FLA was observed at the clarification step of these plants, and in that case no difference in performance was observed between the different clarification processes in place (aluminium or iron-based coagulants, static, sludge blanket or sludge re-circulation technologies). The filtration step at these treatment plants showed a limited effectiveness, with a mean reduction of only 0.5 log, and no difference in performance was observed between the different filtering media in place (sand, GAC or biolite). Consequently, the overall mean removal of FLA for coagulation, sedimentation and filtration at these treatment plants was 2.0 log (range: 0–3.5). Similar observations had been made by Hoffmann and Michel on six drinking water treatment plants in Germany, where overall reduction of FLA for flocculation, sedimentation and filtration was found to be in the range of 1–2 log (Hoffmann and Michel, 2001). Loret et al. hypothesized that the limited effect of filtration on FLA, in comparison with other protozoa such as Cryptosporidium or Giardia, was due to filter colonization (Loret et al., 2008a). This hypothesis was based on the observation of important FLA populations in filter media, and on the

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observation on several occasions of FLA concentrations at the outlet of the filters that were higher than those in the inlet water. This hypothesis was confirmed by a large survey of 15 drinking water treatment plants in Europe, North America and Asia, from which FLA levels were found to be all in the same range of 10–100/mL of medium, regardless of the raw water source, filtration stage, filter medium, or filter depth (Glucina et al., 2007). Additional pilot-scale and full-scale studies demonstrated that FLA colonization is rapid and takes place within the first weeks following the installation of a new filtration medium, and that filter backwashing is poorly effective in removing these micro-organisms, whatever the backwash mode or frequency (Baudin et al., 2008). As expected, ultra-filtration (UF) has proved to be an effective technology to remove FLA. Loret et al. treated water spiked with Acanthamoeba polyphaga cysts and observed a total removal of cysts (>5.5 log reduction in the experimental conditions) (Loret et al., 2008a). Taking into account the potential presence of damaged fibres at full-scale installations, they hypothesized a minimum achievable 4-log reduction under operational conditions with this technology. Conclusions and perspectives Free-living amoebae are widespread in environmental waters and consequently are present in both ground and surface waters used for drinking water production. Their prevalence and concentration is correlated with the level of organic matter present in the water, and their concentration is especially high in biofilms and sediments which constitute ecological niches where they can feed on bacteria. The high amount of biofilm fixed on filtration media can explain the important colonization of granular filters by FLA in drinking water production, as well as the limited effectiveness of this technique in removing these micro-organisms. Due to their capacity to encyst, amoebae are also highly resistant to disinfection, and consequently their presence can still be observed in treated waters, whatever the water source and treatment processes applied. Re-growth of FLA takes place in distribution systems, and to a larger extent in domestic water systems, where again sediments and biofilms may be present in large quantities. Temperatures encountered in hot water systems do not constitute an obstacle to their proliferation, except if they are above 60 ◦ C. Some FLA can act as hosts for amoebae-resisting bacteria and favour their protection and dissemination in water systems. Biofilms seem to be the major place where infection by ARB takes place. ARB recovered from drinking water systems include: (i) a large biodiversity of common environmental bacteria of unknown pathogenic role, (ii) non-pathogenic non-tuberculosis mycobacteria, as well as (iii) recognized and emerging pathogens able to cause respiratory diseases, and mainly belonging to the Legionellaceae and Parachlamydiaceae families. Amoebal co-culture or PCR methods may easily detect these ARB and may reveal a high prevalence and important concentrations for some of them (i.e. Mycobacterium spp. and Legionella spp.). The strong association between FLA and ARB demonstrates that preventing the risk associated with pathogenic ARB requires a better control of FLA in drinking water systems. With regard to water treatment, control of FLA cannot be based exclusively on disinfection. Removal processes including sedimentation, granular or membrane filtration constitute better options, ultrafiltration being the best available technology identified to date. Control strategies of FLA should also address the factors that favour their re-growth, i.e. organic matter, biofilms and sediments. Therefore, efficient elimination of organic matter by clarification, good management of sludge from clarifiers, adapted frequency of filter backwash, biofilm and sediment control in distribution systems are all essential measures to prevent FLA re-growth in drinking water distribution systems.

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Further research however is still required to better control FLA. The development of novel approaches for disinfection, based on the knowledge of the encystment/excystation mechanisms, would be useful to better eliminate the most resistant FLA such as Acanthamoeba from filter media and biofilms. Additional studies aiming at assessing the infectivity for humans of the environmental bacteria found to be resistant to FLA, and precisely defining the pathogenic role towards humans of the different ARB is also required. Such work may in the future allow the identification of new agents of pneumonia, a disease whose etiology still remains unknown in about 50% of cases.

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