biocidal efficacy of monochloramine against planktonic and biofilm-associated ...

ORIGINAL ARTICLE

Biocidal efficacy of monochloramine against planktonic andbiofilm-associated Naegleria fowleri cellsS. Goudot1,2,3*, P. Herbelin1, L. Mathieu3,4, S. Soreau1, S. Banas2,3 and F.P.A. Jorand2,3

1 EDF Recherche et D�eveloppement, Laboratoire National d’Hydraulique et Environnement, Chatou Cedex, France

2 Universit�e de Lorraine, LCPME, UMR 7564 CNRS – UL, Institut Jean Barriol, Villers-l�es-Nancy, France

3 CNRS, LCPME, UMR 7564 CNRS – UL, Villers-l�es-Nancy, France

4 Ecole Pratique des Hautes Etudes (EPHE), LCPME, UMR 7564 CNRS-UL, Vandoeuvre-l�es-Nancy, France

Keywords

Ct value, Free-living amoeba, freshwater

biofilm, monochloramine, Naegleria fowleri.

Correspondence

Laurence Mathieu, LCPME UMR 7564

CNRS-UL, 15 avenue du Charmois, 54500

Vandoeuvre Les Nancy, France.

E-mail: [email protected]

Pascaline Herbelin, EDF – R&D, Laboratoire

National d’Hydraulique et d’Environnement /

Groupe QEE, 6 quai Watier, 78401 CHATOU

Cedex, France.

E-mail: [email protected]

*Present address: EDF - DIN – CEIDRE, DETU -

Service CME - Groupe Source Froide, 2 rue

Amp�ere, F-93206, Saint-Denis Cedex, France

2013/1671: received 16 August 2013,

revised 17 December 2013 and accepted

30 December 2013

doi:10.1111/jam.12429

Abstract

Aims: Free-living amoebae (FLA) in aqueous systems are a problem for water

network managers and health authorities because some are pathogenic, such as

Naegleria fowleri, and they have also been reported to operate as reservoirs and

vectors of several pathogenic bacteria. Therefore, to better control the

occurrence of such amoebae, we evaluate the efficacy of monochloramine

against planktonic forms (trophozoites and cysts) and also biofilm-associated

cells of N. fowleri as FLA are often associated with biofilms.

Methods and Results: From a freshwater biofilm growing in a pilot reactor

and inoculated with N. fowleri, we obtained Ct values ranging from 4 to 17 mg

Cl2 min l�1 at 25°C and pH 8�2 on both planktonic and biofilm associated

cells. In addition, the inactivation pattern of biofilm associated was

intermediate between those of trophozo€ıtes and cysts.

Conclusions: The monochloramine efficiency varies with the life stage of

N. fowleri (trophozo€ıte, cyst, and biofilm-associated). The sensitivity to

disinfectant of amoeba, that is, trophozo€ıtes and cysts, in the biofilm life stage

is as high as that of their planktonic cyst form.

Significance and Impact of the Study: This study gives Ct values for cysts and

biofilm-associated N. fowleri. This may impact on water treatment strategies

against amoebae and should be considered when controlling N. fowleri in man-

made water systems such as cooling towers or hot water systems.

Introduction

Natural aquatic environments (rivers, lakes and springs)

and man-made water systems (drinking water networks or

poorly chlorinated swimming pools) are both common

habitats of free-living amoebae (FLA) (Sibille et al. 1998;

Thomas et al. 2004; Jamerson et al. 2009; Loret and Greub

2010; Marciano-Cabral et al. 2010; Buse et al. 2013; Garcia

et al. 2013; _Zbikowska et al. 2013). Some genera of these

FLA are opportunistic or nonopportunistic pathogens

capable of causing severe human diseases such as keratitis

or gastroenteritis. One of the most serious diseases caused

by FLA is primary amoebic meningoencephalitis, a fatal

central nervous system disease. Naegleria fowleri is the

causative agent of this infection, which results from

amoeba-contaminated water entering the nasal cavity

(Marciano-Cabral 1988; Visvesvara et al. 2007; Kaushal

et al. 2008). This infection is rare and, to date, less than

300 cases have been reported worldwide since 1965 (De

Jonckheere 2011; Moussa et al. 2013; Tung et al. 2013).

N. fowleri is ubiquitous in natural and man-made warm

aquatic environments, such as lakes, rivers, geothermal

water, swimming pools, spas and cooling systems (Jamer-

son et al. 2009; Huang and Hsu 2010; Stockman et al.

2011; Kao et al. 2012, 2013; Wang et al. 2012).

In addition to being causative agents of infectious dis-

eases, FLA have been reported to operate as reservoirs

and vectors by promoting the survival and multiplication

Journal of Applied Microbiology 116, 1055--1065 © 2014 The Society for Applied Microbiology 1055

Journal of Applied Microbiology ISSN 1364-5072

of infectious bacteria such as Legionellaceae, Mycobacteria-

ceae, Enterobacteriacaeae and Vibrionaceae, as well as of

some toxigenic cyanobacteria (Corsaro et al. 2010; Marci-

ano-Cabral et al. 2010; Garcia et al. 2013). Taking into

account both their pathogenic properties and their inter-

actions with pathogenic bacteria in aqueous environ-

ments, controlling amoebae in water is clearly a public

health concern (Thomas and Ashbolt 2010).

Disinfection is the main practice for controlling the wide

variety of pathogenic micro-organisms and reducing the

level of microbiological contaminants transmitted by

waters (recreational, drinking or thermal). Despite various

data on sensitivity and resistance of FLA to biocides (Tho-

mas 2012) and increasing health concerns over FLA, there

is still a lack of information on the mechanisms of action

and efficacy of biocides on amoebae in real systems. A few

studies have investigated the effects of chlorine on Acan-

thamoeba spp. trophozo€ıtes (Cursons et al. 1980; Critchley

and Bentham 2009) and Acanthamoeba cysts (De Jonckhe-

ere and Voorde 1977; Thomas et al. 2004). Low doses of

chlorine – representative of drinking water disinfection

practices – proved to be ineffective. For instance, while

trophozo€ıtes of Acanthamoeba castellanii exposed to 5 mg

Cl2 l�1 exhibited a size reduction, cellular damage and a

99�9% decrease in cultivability after 30 s of exposure at

25°C and pH 7 (Mogoa et al. 2010), hyperchlorination of

Acanthamoeba spp. cysts with chlorine concentrations as

high as 50 or 100 mg l�1, for 18 h or 10 min, respectively,

remained ineffective (Kilvington and Price 1990; Storey

et al. 2004). However, such concentrations are more effec-

tive against Hartmannella or Naegleria suggesting discrep-

ancies in the oxidant susceptibility according to the genus

and even to the species of amoeba (Coulon et al. 2010;

Thomas 2012; Wang et al. 2012; Dupuy et al. 2013).

Chloramine is another halogen compound widely used

for water disinfection. Although less reactive than chlo-

rine, it has the advantage of not forming regulated disin-

fection by-products such as trihalomethanes. It also

appears to diffuse better through the polymeric matrix of

biofilms than other chlorine disinfectants (LeChevallier

et al. 1988; Tachikawa et al. 2005). However, as in the

case of chlorine, few studies have explored the inhibitory

efficacy of monochloramine on amoebae. Moreover, all

such studies were performed in a buffered liquid medium

on a pure culture of amoeba species (Ercken et al. 2003;

Dupuy et al. 2011; Mogoa et al. 2011), not representative

of natural ecology of the protozoa.

As bacteria are their main nutrient source, FLA are

mainly found on or near surfaces, where they graze on

biofilm bacteria. It has been postulated that biofilm could

also provide physical and chemical protection for FLA

against predators and disinfectants (Barbeau and Buhler

2001; Thomas et al. 2004). Biofilms are therefore

considered a major reservoir of FLA (Parry 2004; Huws

et al. 2005; Pickup et al. 2007; Puzon et al. 2009; Goudot

et al. 2012). However, only very few studies have exam-

ined the disinfection efficacy of oxidants on amoebic

communities within biofilms (Thomas et al. 2004; Loret

et al. 2005). They both demonstrated that a continuous

monochloramine treatment of 0�5 mg l�1 over several

weeks was ineffective against the amoebic community (all

species) in both water and biofilm.

Since 1990, French power stations have been monitor-

ing their cooling systems for the presence of N. fowleri as

their cooling waters are released into rivers. To protect

river users, particularly during recreational activities, and

to reduce health risks downstream from power stations,

chemical or physical treatments are implemented in sev-

eral cooling water systems to control and inactivate

pathogens in the water. In France, several cooling water

systems are currently treated with monochloramine to

prevent microbiological risks.

In this context, the main objective of this study was to

evaluate the effects of monochloramine against N. fowleri

in its three different stages of life cycle: trophozo€ıtes, cysts

and biofilm-associated amoebae. To get the latter form, we

used a biofilm reactor which allowed freshwater biofilms

to develop from raw river waters and experimentally intro-

duced pathogenic amoebae to colonize (Goudot et al.

2012). Monochloramination treatments were performed to

(i) assess the efficacy of this oxidant both on planktonic

cysts and on trophozoites and (ii) compare the mono-

chloramine sensitivity of the N. fowleri planktonic forms

(trophozo€ıtes and cysts) with that of biofilm-associated

amoebae. While there is no standardized method available

for testing the efficacy of disinfectants on amoebae, we

used the model defined by Watson and Chick to determine

the Ct99% (monochloramine concentration 9 contact time

leading to 99% inactivation of the amoebic population)

(Chick 1908; Watson 1908). Ct99% values were assessed for

inactivation of the planktonic form of N. fowleri in both

life stages – cysts and trophozo€ıtes. Ct99% values were also

assessed for the inactivation of the biofilm-associated

N. fowleri. Monochloramine has a biocidal effect on plank-

tonic and biofilm-associated forms of N. fowleri, and its

efficacy appears to depend both on the intrinsic resistance

of the amoebae (cyst form) and the surrounding environ-

ment (water or biofilm).

Materials and methods

Naegleria fowleri strain, culture conditions and

inoculum preparation

The AMI005 strain of N. fowleri (EDF internal collection,

LNHE, Chatou, France) was isolated from the cooling

Journal of Applied Microbiology 116, 1055--1065 © 2014 The Society for Applied Microbiology1056

Monochloramination of N. fowleri S. Goudot et al.

water of a power station. It was grown (for 3–5 days) at

43°C on non-nutrient agar (NNA, Indicia Biotechnology,

Oullins, France) previously overlaid with an Escherichia

coli suspension and identified by an enzyme-linked

immunosorbent assay (Indicia Biotechnology) using

monoclonal antibody 5D12 (Pougnard et al. 2002).

N. fowleri trophozoite and cyst suspensions were pre-

pared separately. Trophozoites were harvested, under

microscope examination, after a 2-day culture on E. coli

mats against a 5-day culture for cysts (Figure S1). Sus-

pensions of N. fowleri trophozo€ıtes or cysts were prepared

by gently scraping the amoebic migration front or encyst-

ments, respectively, of ten plates and poured into 5 ml

phosphate buffer saline (PBS) for further use.

Naegleria fowleri and thermophilic FLA cell counting

Thermophilic FLA, including N. fowleri, were counted

using the most probable number (MPN) approach

described by Pougnard et al. (2002). The MPN approach

determines the concentration of viable N. fowleri – both

trophozoites and cysts – without allowing the two forms

to be discriminated. Briefly, immediately after sample col-

lection (river water or biofilm after desorption from the

support), five 1 ml replicate subsamples of each tenfold

serial dilutions were spread onto NNA plates previously

overlaid with E. coli. The plates were incubated at 43°C,and the presence of an amoebic migration front was

assessed daily for 5 days by microscopic examination.

Naegleria-positive samples were further analysed to deter-

mine the presence of flagella by incubating vegetative

forms in demineralized water at 37°C for 4 h. Finally,

N. fowleri were identified using an enzyme-linked immu-

nosorbent assay (Indicia Biotechnology) with monoclonal

antibody 5D12 as previously described by Pougnard et al.

(2002). Moreover, morphological examinations of the

amoebae under microscope allowed identification of ther-

mophilic FLA other than N. fowleri as recently recom-

mended by De Jonckheere et al. (2012).

Preparation of monochloramine

A monochloramine stock solution was prepared by mix-

ing under agitation a sodium hypochlorite solution

(152 g l�1, ACROS Organics) in an ammonia solution

(30%, ACROS Organics) at a Cl2/N mass ratio of 4�8 and

at a pH of 8�3. Under these stoichiometric conditions,

the theoretical concentration of monochloramine stock

solution was about 1000 mg Cl2 l�1. Monochloramine

solution was prepared daily and used extemporaneously.

Its concentration was determined by the DPD method

using Hach Methods 8167 on a DR/2500 spectrophotom-

eter (Hach Company, Loveland, CO) at 530 nm.

Disinfection assays on planktonic Naegleria fowleri

Monochloramine disinfection on planktonic N. fowleri

was performed separately on trophozoite or cyst suspen-

sions under batch conditions. Two sets of assays were

performed: three independent assays (named T1 to T3)

were dedicated to the disinfection of the trophozoite

form of N. fowleri whereas three other independent assays

(named C1 to C3) were assigned to the disinfection of

the cyst form. Each monochloramine treatment was

performed on 150 ml autoclaved freshwater (the same as

used to supply the reactor) (pH 8�2) inoculated with

N. fowleri suspension containing only trophozoites or

only cysts to achieve final concentrations of around

3 9 104 to 8 9 104 amoebae l�1 depending on the

assays. The freshwater had previously been autoclaved to

remove any naturally present amoebae and to also retain

the physicochemical characteristics of the freshwater close

to those in the reactor. A volume of the monochloramine

stock solution was then added to obtain a theoretical

final concentration of 1 mg Cl2 l�1. The concentration of

monochloramine and the survival of N. fowleri trophozo-

ites (assays T1 to T3) or cysts (assays C1 to C3) were reg-

ularly monitored for 60 min at 25°C under agitation

(magnetic stirrer). Sterile sodium thiosulfate 0�1 mol l�1

was added in excess in each treatment flask for neutral-

ization of monochloramine residuals. For each experi-

ment, control flasks without addition of monochloramine

were performed in parallel and sampled at the beginning

and end of the assay.

Disinfection assays on biofilm-associated Naegleria

fowleri

Freshwater biofilm formation and Naegleria fowleri

inoculation set-up

A flat-plate open channel reactor previously described by

Goudot et al. (2012) was operated in continuous flow

mode (Figure S2). The inlet flow and the recycle flow

rate were maintained at 1�9 and 810 ml min�1, respec-

tively. The hydraulic retention time was 24 h. The flow

presented a laminar velocity profile in the length direc-

tion characterized by a shear rate of 17 s�1.

The reactor was fed with freshwater (Loire River,

France), collected in June 2011 and stored in the dark in an

agitated, refrigerated (4°C) tank for the duration of the

experiments. Microbial and physico-chemical characteris-

tics of the inlet water are presented in Table 1. Biofilm col-

onization was carried out at 42°C on glass coupons

(~22 cm²) placed for at least 8–10 days in the freshwater

reactor. Averages of microbial and physico-chemical

characteristics of the biofilm are also presented in Table 1.

As previously described, a suspension of N. fowleri in


S. Goudot et al. Monochloramination of N. fowleri

trophozoite form (108–109 amoebic trophozo€ıtes l�1) was

prepared extemporaneously and was inoculated into the

reactor in a single injection 24 h after its startup to reach

105 trophozoites l�1 (final concentration). A previous

study had established that these conditions allowed viable

N. fowleri to colonize biofilms (Goudot et al. 2012).

Monochloramine treatment of biofilm-associated Naegleria

fowleri

Five disinfection assays were conducted on biofilm-asso-

ciated amoebae. Three of them (B1–B3) were performed

at a monochloramine concentration of 1 mg Cl2 l�1 with

increasing exposure time from 0 to 60 min. The two

remaining trials (B4 and B5) were achieved with a con-

tact time of 60 min and increasing concentrations of

monochloramine from 0 to 0�5 mg Cl2 l�1.

For each monochloramination assay, 14 glass slide cou-

pons colonized by an 8- to 10-day-old biofilm including

N. fowleri were sampled from the reactor and gently

rinsed with sterile PBS to remove cells and deposits not

strongly attached to the substrata (i.e., not considered as

part of the biofilm) and transferred to 700 ml of auto-

claved freshwater (pH 8�2). These biofilm samples were

incubated at 25°C with agitation and defined volumes of

monochloramine stock solution were added to reach the

theoretical final concentrations defined for the assays. The

concentration of monochloramine and the viability of the

indigenous thermophilic FLA and N. fowleri were moni-

tored over a 60-min period. Neutralization of residual

monochloramine was performed with sterile sodium thio-

sulfate in excess of 0�1 mol l�1. Controls without addition

of the oxidant were performed in parallel. For enumera-

tion of FLA and N. fowleri cells within the biofilm, two

glass slide coupons were scrapped with a sterile swab in

100 ml of bacteria-free PBS followed by ultrasound treat-

ment for 10 min (ultrasonic bath, 140 Watts, 50/60 Hz;

Thermo Fisher Scientific, Waltham, MA) (Goudot et al.

2012). In the biofilm trials, as the behaviour of the inocu-

lated N. fowleri (initially trophozoites) within the biofilm

could not be controlled, the MPN enumeration data of

biofilm-associated N. fowleri referred to a mixture of both

trophozoite and cyst forms as the method could not

differentiate between the two forms.

Calculation of the Ct99% and k disinfection rate

coefficient

To evaluate the effectiveness of a disinfectant, the micro-

bial inactivation (loss of cultivability) was recorded as a

function of Ct (concentration 9 time), which corre-

sponds to the disinfectant exposure (mg min l�1). The

Ct99% is the Ct necessary for 2-log inactivation.

Calculation of the Ct is based on the Chick–Watson

model (Chick 1908; Watson 1908) (Eqn 1), as follows:

InN

N0¼ �kCnt ð1Þ

where N is the concentration of micro-organisms after

exposure to the disinfectant (amoebae l�1 or amoebae

cm�2), N0 is the concentration of amoebae prior to expo-

sure to the disinfectant (amoebae l�1 or amoebae cm�2),

k is the disinfection rate coefficient (l mg�1 Cl2 min�1),

C is the disinfectant concentration (mg Cl2 l�1), t is the

time (min) and n is the disinfection dilution factor.

For planktonic disinfection assays, we assumed n = 1.

This assumption is supported by a previous planktonic dis-

infection study on N. fowleri (Leprince 2000). Conversely

for biofilms, in our experiments, n was estimated to be

equal to 0�94 (Data S1). Thus, the monochloramine inacti-

vation of N. fowleri in water and biofilm corresponds to a

first-order reaction that gives equal importance to the

monochloramine concentration factor and the time factor.

As a result, Eqn 1 was simplified by Eqn 2:

InN

N0¼ �kCt ð2Þ

Statistical analysis

To define statistical significance, we used the nonpara-

metric Mann–Whitney test using a 95% confidence level.

All analyses were performed with SigmaPlot Version 10

(Systat Software, Inc., Chicago, IL).

Table 1 Microbial and physico-chemical characteristics of the river

water (Loire, collected in June 2011) at the inlet of the reactor, and

of the biofilm growing within. For the biofilm, the value in brackets is

the standard deviation of three independent measures. FLA stands for

Free-Living Amoebae

Parameters Inlet water Biofilm

Thermophilic FLA

(cells l�1 or cells cm�2)

<105* 392 (150)

N. fowleri


<105* 295 (90)

Bacteria


4�2 9 108 7�2 9 105 (2�4 9 105)

pH 8�2 –

Conductivity (lS cm�1) 288 –

DO (mg l�1)† 5�6 –

TOC (mg l�1 or mg cm�2)‡ 3�2 0�05 (0�02)DOC (mg l�1)§ 2�9 –

DSS (mg l�1 or mg cm�2)¶ 7 0�70 (0�24)

*Detection limit.

†Dissolved oxygen.

‡Total organic carbon.

§Dissolved organic carbon.

¶Dried suspended solids.



Results

Inactivation of planktonic forms of Naegleria fowleri

To assess the disinfection efficiency of monochloramine

on planktonic forms of N. fowleri, suspensions of troph-

ozoites and cysts were exposed for 60 min to initial con-

centrations of monochloramine ranging from 1�04 to

1�12 mg Cl2 l�1. Monochloramine was quite stable dur-

ing the 60-min exposure as its consumption was less than

or equal to 14% at the end of the experiments (Table

S1). Results of inactivation are shown as semi-log curves

(log N/N0) as a function of the Ct values (mg Cl2 min

l�1) for three independent assays named T1–T3 and C1–C3 for trophozoites and cysts, respectively (Fig. 1). Com-

pared with the control samples (N. fowleri decrease <0�4log), extensive amoebic inactivation (>2 log) took place

during these experiments, indicating that monochlor-

amine was effective at eradicating both planktonic troph-

ozoites and cysts of N. fowleri. Its efficacy was 4�3 times

higher for the trophozoites than for the cyst forms and

Ct99% values were evaluated at 3�6 to 3�9 mg Cl2 min l�1

and 14�1 to 17�3 mg Cl2 min l�1, respectively (Fig. 1).

These Ct99% values corresponded to disinfection rate

coefficients (k) comprised ranging between 0�50 and

0�58 l mg�1 Cl2 min�1 for N. fowleri trophozoites and

0�12 and 0�14 l mg�1 Cl2 min�1 for N. fowleri cysts

(Fig. 2).

The statistical analysis (Mann–Whitney test) confirms

that the efficacy of monochloramine disinfection is signif-

icantly different (P < 0�05) between trophozoite and cyst

forms, as well as between tests and controls (without

monochloramine).

Inactivation of biofilm-associated Naegleria fowleri cells

Before the introduction of N. fowleri on day 1, no indige-

nous thermophilic FLA or N. fowleri were detected in the

freshwater biofilm (concentration below the detection

limit of 0�3 amoeba cm�2). On day 1, 2 h after its intro-

duction into the bulk water, around 10 N. fowleri cm�2

were found in the biofilm, indicating the transfer of

N. fowleri to the substratum (Fig. 3). Optical microscopy

allowed the observation of the amoebae both on the sup-

port surface and on the biofilms (Fig. 4). A significant

increase in N. fowleri density, up to 200–300 N. fowleri

cm�2, then occurred during the first 3 days. After this

point, the density remained quasi-stable until the 12th

day. Over these periods, N. fowleri was the main detect-

able thermophilic FLA. Some other indigenous thermo-

philic FLA (mainly Hartmannella), originating from the

freshwaters feeding the reactors were also detected and

identified by their morphological characteristics by use of

Page’s taxonomy keys for optical microscopy (Pages

1976). Their presence in the biofilm appeared constant

and systematically lower than the N. fowleri population

(<102 thermophilic amoebae/cm² other than N. fowleri).

Biocidal assays were performed on 8 to 10-day-old

biofilm-associated N. fowleri using a batch approach as

described in the materials and methods section. As in the

batch assays with planktonic trophozoites, biocide

consumption after 1 h at 25°C was low and represented

less than 18% at the end of the experiment (Table S1).

The monochloramine was thus assumed to be stable.

Ct (mg Cl2 min l–1)

0 10 20 30 40 50 60 70

Log

(N/N

0)

–3

–2

–1

0

Figure 1 Reduction in cultivability of planktonic Naegleria fowleri

cells as a function of Ct values (monochloramine initial concentration

of 1 mg Cl2 l�1 for 60 min at 25°C), on trophozoite forms (three

independent assays): (T1) ●, (T2) ■, (T3) ▲or cyst forms (C1) ○,(C2) □, (C3) M; controls without monochloramine for trophozoites

(♦) and cysts (◊). The grey tone points are those obtained from values

below the detection limit. Dotted lines are visual aids only.

Assays

T1 T2 T3 C1 C2 C3 B1 B2 B3 B4 B50·0

0·1

0·2

0·3

0·4

0·5

0·6

k (l

mg–

1 C

l 2 m

in–1

)

Figure 2 Disinfection rate coefficients (k) of Naegleria fowleri for

planktonic trophozoites (T1–T3), planktonic cysts (C1–C3) and

biofilm-associated cells (B1–B5).



Compared with control samples (N. fowleri decrease <0�5log), the amoebae exhibited a significant decrease in cul-

tivability (≥3 log), indicating that monochloramine was

effective in eradicating biofilm-associated N. fowleri cells

(Fig. 5a). As the MPN approach for counting N. fowleri

from biofilm samples cannot distinguish between the

trophozoite and cyst forms, the data on biofilm-associ-

ated N. fowleri probably refers to a mix of both.

The Ct99% values were evaluated at 8�6–16�1 mg

Cl2 min l�1, for disinfection rate coefficients (k) com-

prised between 0�11 and 0�24 l mg�1 Cl2 min�1 (Fig. 2).

In contrast, the decrease in the other biofilm-associated

thermophilic FLA cells did not exceed a maximum of 2

log for Ct values, up to 60 mg Cl2 min l�1 (Fig. 5b),

indicating that monochloramine was less effective in

eradicating indigenous thermophilic FLA associated with

biofilm than those experimentally injected.

Statistical analyses confirmed this discrepancy as a sig-

nificant difference in inhibition between biofilm-associ-

ated N. fowleri cells and other thermophilic FLA cells, as

well as between test and control samples (P < 0�05).

Discussion

Naegleria species are typical inhabitants of freshwater

microbial ecosystems (Gianinazzi et al. 2010; Kao et al.

2012, 2013; Painter et al. 2013). Among these protozoa,

N. fowleri have been traced to recreational water-related

activities (Marciano-Cabral 1988; Tyndall et al. 1989;

Lares-Villa and Hern�andez-Pe~na 2010), and only in rare

cases, they have been found in domestic water sources

(Cabanes et al. 2001; Marciano-Cabral et al. 2003; Blair

et al. 2008). The presence of such pathogenic amoebae in

disinfected waters represents a major threat to public

health. Numerous strategies for controlling FLA in artifi-

cial water systems focused on the effectiveness of disinfec-

tants against pathogenic FLA and have targeted more

especially Naegleria and Acanthamoeba species. Loret and

Greub (2010) recently reviewed the available information

on inactivation data for both amoebic genera following

treatments of drinking water with disinfectants. They

pointed out differences in the sensitivity of FLA in a

planktonic stage according to their genera – that is,

Naegleria cysts being more sensitive when exposed to

chlorine than Acanthamoeba cysts – and to the type of

oxidant used (Loret et al. 2005). Such discrepancy could

be related to their cyst wall compositions, as that of Nae-

gleria could be formed as a single thick fibrillar layer and

is thought to contain chitin (Fouque et al. 2012), while

that of Acanthamoeba cysts exhibits a double-layered wall,

composed of at least acid-insoluble proteins and cellulose,

which increases resistance to most chemical agents.

In the present study, we mainly investigated the sensi-

tivity of N. fowleri according to three life stages (plank-

tonic cysts, planktonic trophozoites and sessile forms)

when exposed to monochloramine. We evaluated the

disinfectant concentration 9 time (Ct) values leading to

2-log reduction of FLA, including N. fowleri, and we

compared monochloramine inactivation of trophozoites

versus cysts on the one hand and of planktonic versus

biofilm-associated N. fowleri cells on the other hand.

Trophozo€ıtes are considered relatively sensitive to most

chemicals, but cysts have been shown to be more resistant

(Thomas et al. 2004; Coulon et al. 2010). We first

Time (days)

0 2 4 6 8 10 12

Am

oeba

e (c

ells

cm

–2)

103

102

101

100

10–1

Figure 3 Changes in cell density of thermophilic FLA cells (●) and

Naegleria fowleri cells (○) in the biofilm over time, below 42°C. The

thermophilic FLA counts correspond to the sum of the indigenous

FLA from freshwater and the inoculated N. fowleri. The black arrow

indicates the N. fowleri spike. The dashed arrow indicates the time at

which the biofilm coupons were sampled for disinfection assays.

20 µm

Figure 4 Optical microscopy picture of the biofilm on a glass coupon

extracted from the reactor after Naegleria fowleri spike on the 10th

day. Circles indicate amoebae on the surface.



determined that, in their planktonic stage, N. fowleri cysts

were fivefold more resistant to monochloramine than

trophozoites, and Ct99% values ranged approximately from

15 to 4 mg Cl2 min l�1, respectively. This was in line with

the other few studies on Naegleria inactivation (De Jonc-

kheere and Voorde 1977; Chang 1978). Recent findings

have reported Ct values of 6 mg Cl2 min l�1 for N. fowleri

trophozoites and 31 mg Cl2 min l�1 for cysts, leading to

3-log inactivation of the pathogenic amoeba with chlorine

(Gerba et al. 2009; Sarkar and Gerba 2012). Operational

control and management of Naegleria based on the appli-

cation of contact times greater than 30 mg Cl2 min l�1

and maintenance of a free chlorine residual of 0�2 mg l�1

in the end section of water distribution systems was tried

in Australia and has proved to be effective (Trolio et al.

2008). The stronger resistance of cysts to oxidant disinfec-

tion is due to the structure and composition of the cyst

wall that is more resilient than that of the trophozo€ıtes

(Visvesvara et al. 2007; Johnston et al. 2009). In fact, Co-

ulon et al. (2012) demonstrated, using transmission elec-

tron microscopy and flow cytometry with calcofluor

staining, that the increased resistance of Acanthamoeba

cysts is likely due to cysts presenting thicker cell walls.

They also highlighted that laboratory encystment condi-

tions modified the resistance of the cysts. Trophozo€ıtes

grown using HEp-2 cells as a nutrient source produced

cysts that were significantly more resistant to the tested

biocides than cysts produced by nutrient starvation in

broth medium (axenic condition). Furthermore, Hughes

et al. (2003) and Johnston et al. (2009) showed that strain

age, the number of passages in axenic culture and the

method of encystment greatly affect the efficacy of thera-

peutic and biocide agents used to kill cysts.

Moreover, oxidant treatments were reported to have

different efficiency depending on the target strain (Cou-

lon et al. 2010; Dupuy et al., 2013), and to lead to mor-

phological modifications such as cell permeabilization

and size reduction, as well as to an increase in thiol

(Mogoa et al. 2011). These authors suggested that mono-

chloramine should have a different mode of action on

amoebic trophozo€ıtes as when compared to chlorine or

chlorine dioxide.

Biofilm-associated amoebae have never been explored

in terms of disinfectant sensitivity, while this life-style

stage appears to be of great importance in the survival

of amoebae in the environment. Sediments, deposits

and biofilms that may be present in large quantities in

both natural and artificial distribution systems, includ-

ing water treatment plants, and in which re-growth of

FLA takes place serve as reservoirs (Loret and Greub

2010).

While it is well known that biofilm micro-organisms

are less susceptible to the effects of antimicrobial treat-

ments (Behnke et al. 2011), the fate of amoebae within

microbial biofilms following biocidal treatment is under-

reported. Our results showed that monochloramine has

intermediate efficiency on the biofilm-associated N. fow-

leri cells compared to their planktonic forms. Ct values of

9 to 16 mg Cl2 min l�1 were necessary for the inactiva-

tion of biofilm-associated N. fowleri cells (Fig. 5), against

<4 mg Cl2 min l�1 and 14–17 mg Cl2 min l�1 for plank-

tonic trophozo€ıtes or planktonic cysts, respectively

(Fig. 1). This result suggests that N. fowleri associated

with the freshwater biofilm could be much less sensitive

to monochloramine treatment than planktonic trophozo-

ite forms, but much closer to the sensitivity of planktonic

cysts. We expected biofilm-associated amoebae to be less

0 10 20 30 40 50 60 70

0 10 20 30 40 50 60 70

Log

(N/N

0)

–4

–3

–2

–1

0

Log

(N/N

0)

–4

–3

–2

–1

0



(a)

(b)

Figure 5 Reduction in the number of biofilm-associated Naegleria

fowleri cells (a) and of biofilm-associated thermophilic FLA (b) as a

function of Ct values of monochloramine treatment. The thermophilic

FLA counts correspond to the sum of the indigenous FLA from fresh-

water and the inoculated N. fowleri. With B1 (●), B2 (■), B3 (▲),

B4 (♦), B5 (▼) and control (□). The grey tone points are those

obtained from values below the detection limit. Dotted lines are visual

aids only.



susceptible to oxidant than planktonic cysts because of

the protective role of biofilms, especially due to their exo-

polymeric matrix, widely described (Berry et al. 2010).

Beside the protective role of the extracellular polymeric

substances (EPS), the in-between position of the sessile

amoebae, as regards to disinfection sensitivity, is difficult

to explain at the present stage of knowledge and only a

few hypotheses can be put forward. It could first be

assumed that biofilm-associated amoebae are more resis-

tant than free-living amoebic trophozoites because they

partly consist of cysts, thus leading to an overestimation

of the monochloramine efficiency on biofilm-associated

amoeba. Indeed, when collected on biofilms, the amoebae

could be a mixture of trophozoites and cysts whose pro-

portions are unknown, as the MPN approach used can-

not distinguish between the two forms. Encystment is a

process which occurs during unfavourable conditions,

that is, depletion in the source of food, drying, pH or

temperature changes, osmolarity, etc. (Chang 1978;

Marciano-Cabral 1988). However, biofilms represent

favourable protective environment usually described as

the play-ground for numbers of micro-organisms, includ-

ing protozoa. Even if we cannot exclude the presence of

cysts, one can argue that our freshwater biofilms were

potentially highly productive environments to support

protozoan growth given the high density of bacteria

(>105 cells cm�2) and the likely presence of organic

deposits and EPS secreted within the biofilms that pro-

vide a suitable matrix for amoeba attachment, grazing

and growth (Wingender et al. 1999; Wey et al. 2012).

Even if it is unclear whether the biofilm matrix is a nutri-

ment source for protozoa, it is likely that some EPS

matrix will be ingested, even indirectly if embedded cells

are grazed on by protozoa (Parry 2004; Anderson 2013),

and more studies are required to explore this point of

view. Furthermore, Anderson (2010) questioned the com-

plex dynamics of the cycles of encystment and active

growth of amoeba in the environment, which is poorly

documented. He showed during laboratory trails that the

ratio of encysted to total naked amoebae of soil samples

reached a nearly constant value (43–46%) suggesting a

density-dependent equilibrium effect that maintains a rela-

tively steady-state balance between active and encysted

forms in the studied microcosms.

Additionally, when examining the growth response and

encystment of A. castellanii in laboratory culture when

fed with different bacteria, de Moraes and Alfieri (2008)

demonstrated that the presence of bacteria prey signifi-

cantly delayed the onset of encystment. This suggests

more strongly that biofilm-associated N. fowleri consists

of trophozo€ıtes able to grow and maintain at 42°C by

grazing the biofilm, as previously quantified by Goudot

et al. (2012).

The second hypothesis is that the freshwater biofilm

structure could influence the tolerance of the biofilm-asso-

ciated amoebae. This assumption is consistent with our

previous study (Goudot et al. 2012), which showed that

our 15-day-old biofilm was relatively thin and organized

into clusters heterogeneously distributed on the surface

material (>105 cells cm�2, surface coverage 5%) and that

its structure ranged from monolayers comprised of isolated

microbial cells to complex aggregates. The biofilm thick-

ness did not exceed a maximum of 100 lm (data not

shown), and cell clusters were ovoid and characterized by

an average size of 20 lm (Fig. 4). This biofilm structure

and the probable surface location of amoebae might have

facilitated the penetration of monochloramine, which is

also well recognized as a stable biocide with greater pene-

tration than chlorine (Berry et al. 2010), as well as its

accessibility to amoebae. This could explain the intermedi-

ate sensitivity of biofilm-associated amoebae to mono-

chloramine compared with cysts and trophozoites.

Moreover, Naegleria fowleri AAMI 005 was a laboratory-

grown strain experimentally inoculated in the reactor. As

for bacterial strains, a different behaviour could also be

expected between laboratory and environmental micro-

organisms, the latter being less sensitive to stress. This

could be related to the higher resistance to monochlor-

amine of the indigenous thermophilic biofilm-associated

FLA cells that displayed a different behaviour compared

with the biofilm-associated N. fowleri. A 2-log reduction of

the indigenous FLA trophozoites was never reached within

the biofilm even for Ct values >20 mg Cl2 min l�1, sug-

gesting that low disinfectant levels have only a limited

effect on FLA (Thomas et al. 2004). The higher resistance

of these indigenous thermophilic FLA could be explained

both by the weak sensitivity of these genera to the action of

oxidants and their location within the biofilm.

Whether it is the life stage of the amoeba (trophozoite

or cyst) rather than its ecological niche (biofilm-associ-

ated or planktonic) that determines the disinfection effi-

cacy of monochloramine remains under question and

further studies are needed to determine the fate of amoe-

bae within biofilms and their ability to encyst, and to

investigate the protective role of the biofilm against bio-

cides for these amoebic forms. Finally, our results provide

for the first time disinfectant exposure values for Naegle-

ria fowleri treatments in three life-style stages of the

amoebae – trophozoites, cysts and biofilm-associated –that might be used as references for disinfection of fresh-

water systems and also improve our understanding of the

persistence of N. fowleri in water systems.

In conclusion, our work has compared for the first time

the efficiency of monochloramine on N. fowleri in its

planktonic and sessile life stages and its trophozoite

and cyst forms. This oxidant was efficient on trophozoites,



cysts and biofilm-associated amoebae. However, its effi-

ciency appeared to vary with the different stages of its life-

style:

• Monochloramine was effective on both planktonic

and biofilm-associated N. fowleri cells with Ct values

ranging from 4 to 17 mg Cl2 min l�1 at 25°C and pH

8�2 in sterilized raw river water, corresponding to dis-

infection rate coefficients of 0�1–0�6 mg Cl2 min l�1.

• The inactivation pattern of biofilm-associated N. fow-

leri by monochloramine was intermediate between

those of trophozoites and cysts, but closer to that of

cysts and well below that of trophozoites.

• Compared to N. fowleri, other biofilm-associated FLA

cells expressed lower sensitivity to monochloramine.

This study could contribute to efforts to control

N. fowleri in water systems and help to adapt treatment

strategies against amoebae with the dual purpose of pro-

tecting health and the environment.

Acknowledgements

S. Morel, S. Barrouilhet and H. Salhi (EDF R&D) are

duly acknowledged for their excellent technical assistance.

This work was supported by EDF. S. Goudot is the reci-

pient of an industrial research-training contract (CIFRE)

between EDF and the ANRT (French national association

of research and technology).

Conflict of interest

We declare that we do not have any commercial or other

association that might pose a conflict of interest.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Figure S1 Pictures of the experimental setup.

Figure S2 Pictures of the experimental setup.

Data S1 Calculation of the disinfection dilution factor.

Table S1 Monochloramine consumption during the

different experiments.



biocidal efficacy of monochloramine against planktonic and biofilm-associated ...

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