ARTICLE IN PRESS

Available at www.sciencedirect.com

WAT E R R E S E A R C H 4 1 ( 2 0 0 7 ) 3 4 9 0 – 3 4 9 6

0043-1354/$ - see frodoi:10.1016/j.watres

�Corresponding autE-mail address: m

journal homepage: www.elsevier.com/locate/watres

Survival and viability of Helicobacter pylori after inoculationinto chlorinated drinking water

Yolanda Morenoa, Patricia Piqueresa, Jose L. Alonsob, Ana Jimeneza, Ana Gonzaleza,Marıa A. Ferrusa,�

aDepartamento de Biotecnologıa, Universidad Politecnica, Camino de Vera 14, 46022 Valencia, SpainbInstituto de Ingenierıa del Agua y Medio Ambiente, Universidad Politecnica, Camino de Vera 14, 46022 Valencia, Spain

a r t i c l e i n f o

Article history:

Received 22 January 2007

Received in revised form

30 April 2007

Accepted 4 May 2007

Available online 18 May 2007

Keywords:

H. pylori

Water

Chlorine

Culturability

Viability

DVC–FISH

nt matter & 2007 Elsevie.2007.05.020

hor. Tel.: +34 963877423; fferrus@btc.upv.es (M.A.

a b s t r a c t

The aim of this work was to assess the effect of chlorine water treatment on Helicobacter

pylori and to study the succession of cellular alterations in response to chlorine exposure. H.

pylori NCTC 11637 reference strain was used for inoculating water samples. The

culturability, substrate responsiveness combined with fluorescent in situ hybridization

detection (DVC–FISH assay), RNA content, DNA content, and mRNA changes of H. pylori

cells were analyzed. Culturability was lost at 5 min in water with 0.96 mg/l of free chlorine.

Viable cells were detected by DVC–FISH after 3 h of exposure to chlorine but not after 24 h.

The percentage of coccoid forms was higher than spiral forms after 40 s of chlorine

exposure, but even after 24 h, FISH detection revealed the presence of spiral cells. After 24 h,

amplification of the specific H. pylori 16S rDNA gene was achieved. Expression of the vacA

gene was detected with the same intensity at all time points tested, demonstrating that

these genes are expressed in non-culturable H. pylori cells. Levels of 16S rRNA were

constant during the chlorine treatment, so killing of bacteria with chlorine probably does

not involve ribosome degradation. According to our results, H. pylori could survive to

disinfection practices normally used in drinking water treatment in the viable but non-

culturable form, which would allow them to reach final consumption points and, at the

same time, enable them to be undetectable by culture methods.

& 2007 Elsevier Ltd. All rights reserved.

1. Introduction

Helicobacter pylori is one of the most common infective agents

worldwide. It is an etiological agent of gastritis, peptic, and

duodenal ulcer disease, and infection with this organism is a

recognized risk factor in the development of gastric mucosa-

associated lymphoid tissue lymphoma and adenocarcinoma

(Magalhaes Queiroz and Luzza, 2006). The public health

relevance of this infection is high. Although the prevalence

of H. pylori infection in the world is decreasing, it is assumed

to be near 50%, with higher prevalence in developing

countries than in developed countries (Brown, 2000).

r Ltd. All rights reserved.

ax: +34 963877429.Ferrus).

Design of prevention measures is difficult due to our

limited knowledge of transmission pathways. Several studies

have shown an association between the prevalence of

H. pylori and the source of drinking water (Percival et al.,

2004). It has been reported that the organism is able to

remain culturable in natural cold waters for 2–3 days (Adams

et al., 2003), and has been detected in domestic and

school drinking water distribution systems, both in

biofilms and in water samples (Park et al., 2000; Watson

et al., 2004).

Chlorination is the most frequent disinfection method for

drinking water. Some studies have analyzed the resistance of

ARTICLE IN PRESS

WAT E R R E S E A R C H 41 (2007) 3490– 3496 3491

H. pylori to usual levels of chlorine in water distribution

systems (Baker et al., 2002; Johnson et al., 1997), showing that

this bacteria is more resistant than Escherichia coli (Baker et al.,

2002) and can be detected in municipal distribution waters

after chlorination (Mazari-Hiriart et al., 2003). H. pylori has

shown to be able to survive disinfection practices with 2%

glutaraldehide too (Nurnberg et al., 2003). All these studies

strongly support the possibility that, after gaining access to

water distribution systems, H. pylori may remain viable in

drinking water for enough time to reach population (Bellack

et al., 2006).

Bacterial viability has been characterized for a long time in

terms of culturable versus non-culturable states of cells.

However, the use of direct methods, which allow checking for

the physiological status of cells, has led to the suggestion of

existence of a ‘viable but non-culturable’ (VBNC) state (Joux

et al., 1997). VBNC forms of H. pylori cells have been previously

described, frequently associated with morphological changes,

from rod to coccoid cellular forms (Nilsson et al., 2002). It has

been suggested that changing to these VBNC stages could be a

survival strategy under environmental stress conditions, and

that these cells could be responsible for transmission in the

environment (Citterio et al., 2004), but as in this form H. pylori

is non-culturable by ordinary techniques, its ability to survive,

viability and virulence is still a matter of controversy. In some

studies, in vitro reversion of coccoid forms to their original

spiral shapes, and subsequent growth in culture media, has

been reported (Andersen et al., 1997; Ren et al., 1999). Some

authors (Cellini et al., 1994; Wang et al., 1997) have been able

to induce H. pylori infection in mice inoculated with a

concentrated suspension of non-culturable coccoid forms.

Thus, the VBNC stage of H. pylori in water could be a problem

of great public health concern, since VBNC cells cannot be

detected by traditional methods.

Rapid bacterial detection and viability measurements have

been greatly enhanced by recent advances in the use of

fluorescent stains. A number of assays using fluorescent

probes of different cellular functions have been introduced

for the evaluation of viability of bacterial single cells

(McFeters et al., 1995). Many of the staining techniques are

indirect analysis, monitoring variously membrane potential,

and DNA and RNA persistence. More direct indicators of

viability are detection of respiratory activity and environ-

mental or substrate responsiveness (Keer and Birch, 2003).

The SYTOs RNASelectTM green fluorescent stain is a cell-

permeant nucleic acid stain that is selective for RNA.

Although virtually non-fluorescent in the absence of nucleic

acids, the SYTO RNASelect stain exhibits bright green

fluorescence when bound to RNA (absorption/emission max-

ima �490/530 nm) and only a weak fluorescent signal when

bound to DNA (Haugland, 2005).

Detection of mRNA has been proposed as a good viability

marker, as mRNA is turned over rapidly in living cells, with a

short half-life time (Sheridan et al., 1998). However, there is

increasing evidence that in many cases, mRNA persistence

depends on the targeted gene or the cells’ inactivating

conditions (Sheridan et al., 1998; Takayama and Kjelleberg,

2000). Supporting this fact, expression of VacA and urease

genes mRNA has been found in H. pylori non-culturable cold-

starved cells (Nilsson et al., 2002).

Direct viable count method, DVC (Kogure et al., 1979),

discriminates by direct microscopy between viable and non-

viable cells. This method is based on the incubation of

samples in the presence of nutrients and a single gyrase

inhibitor. The antibiotic inhibits DNA synthesis and prevents

cell division, but cells can continue metabolizing nutrients

and become elongated and/or fattened after incubation

(Besnard et al., 2000; Yokomaku et al., 2000). Viable cells can

then easily be discriminated from non-viable cells, due to

differences in their respective sizes. Ribosomal RNA probe

hybridization without cultivation (fluorescent ‘‘in situ’’ hybri-

dization, FISH) has become widely adopted for detection of

specific bacterial groups in mixed populations (Amann et al.,

1995). FISH has been used previously to specifically detect

H. pylori in aquatic systems, demonstrating the presence of

these bacteria in water and wastewater (Moreno et al., 2003).

The combination of DVC, which increases intracellular rRNA

levels, with the FISH technique, applied to the rapid

identification of a specific bacterial sequence, has been

proposed as a very accurate and specific method to determine

viable cells in aquatic environments (Garcia-Armisen and

Servais, 2004). Recently, a modified DVC–FISH assay for

H. pylori detection in water samples has been developed by

our research group (Piqueres et al., 2006).

The aim of this work was to determine whether H. pylori can

survive to chlorine water treatment and to study its cellular

alterations in response to chlorine exposure, in order to better

understand the transmission pathways of this organism.

The RNA content was evaluated by measurement of

fluorescent intensity after hybridization with an H. pylori-

specific oligonucleotide probe and SYTO RNASelect stain.

Viable cells were detected with DVC combined with FISH. The

integrity of DNA was measured by means of PCR of the 16S

rRNA gene and the integrity of mRNA was measured by

means of RT-PCR of the vacA gene.

2. Materials and methods

2.1. Bacterial strain and culture conditions

H. pylori NCTC 11637 reference strain was used for inoculating

water samples. H. pylori strain was grown on Columbia blood

agar (Difco) with 10% horse blood under microaerobic

conditions (5% O2, 10% CO2, and 85% N2) at 37 1C for 48 h.

2.2. Survival assay

Experiments were conducted in flasks containing 50 ml of

chlorinated water filtered through a 0.2mm pore size mem-

brane. Chlorine levels (1.16 mg/l total chlorine and 0.96 mg/l

free chlorine) were measured by an amperometric titrator

(mod. 716 DMS Tritino, Methrom, Switzerland). All glassware

was carefully cleaned to remove chlorine demand (Baker

et al., 2002). Cells of an exponential growth culture (48 h)

of H. pylori were harvested, washed twice with sterile PBS

(130 mM sodium chloride, 10 mM sodium phosphate

(pH 7.2)) to eliminate extracellular debries, and suspended

in flasks containing chlorinated drinking water. Samples were

removed aseptically immediately after inoculation (T0) and

ARTICLE IN PRESS

WAT E R R E S E A R C H 4 1 ( 2 0 0 7 ) 3 4 9 0 – 3 4 9 63492

after 10 s, 40 s, 1 min, 5 min, 3 h and 24 h. Prior to the analysis

of samples, chlorine was neutralized by the addition of

sodium thiosulfate (1% w/v). All assays were performed by

duplicate.

In order to determine the part of the chlorine in the

monitored effects, a control assay in which H. pylori cells were

suspended in non-chlorinated water was performed and

samples were removed at T0, 5 min, 3 h and 24 h.

2.3. Culturable cell counts

Samples were serially diluted in PBS buffer. Aliquots of 100mL

from each dilution were plated onto Columbia blood agar

(Difco) supplemented with Dent Selective Supplement (Oxoid)

and 10% of horse blood, and incubated for 72 h under

microaerobic conditions at 37 1C. Presumptive colonies were

confirmed to be H. pylori by Gram staining and examination of

urease production.

2.4. FISH analysis

Samples were fixed with paraformaldehyde (PFA) 4% as

described by Amann et al. (1995), and subsequently hybri-

dized with an H. pylori-specific probe (HPY-CTGGAGAGAC-

TAAGCCCTCC-) according to Moreno et al. (2003). Hybridized

samples were examined with an Olympus microscope BX50

equipped with a 100 W mercury high-pressure bulb and set

filters U-MWB, U-MWIB, and U-MWIG. Color micrographs

were taken with a digital camera Olympus DP 10 (Olympus

Optical Co., Hamburg, Germany). The fluorescent green

intensity signal was measured with the Olympus DP Softs

program.

For total cells counts, samples were also stained with

0.5mg/ml of DAPI (4,6-diamino-2-phenylindole, Sigma, St.

Louis, MO). All cell counts were measured with two replicate

determinations.

2.5. RNA selective stain

A concentration of 250 nM of SYTOs RNASelectTM green

fluorescent cell stain solution in phosphate-buffered saline

(PBS) was pre-warmed at 37 1C prior to application and used

immediately following the manufacturer’s instructions (Mo-

lecular Probes Inc.). Bacterial cells were resuspended in PBS

and centrifuged for 3 min at 9000 rpm. The cell pellet was

stained with the 250 nM labelling solution and incubated for

20 min at 37 1C. The staining solution was removed and cells

were washed two times in PBS. Stained cells were immobi-

lized on glass surfaces. All green cells were considered RNA

stained (Alonso et al., 2002). For each experiment, all cell

counts were measured with two replicate determinations.

2.6. DVC–FISH

The DVC–FISH method used was described in Piqueres et al.

(2006). Briefly, an aliquot of 1 ml of each drinking water

sample was taken from each flask and inoculated into 50 ml

of Brucella broth supplemented with 5% newborn calf serum

(PAA Laboratories, Austria) and 0.5mg/ml Novobiocin (Sigma

Chemical Co., St. Louis, MO) at 37 1C in microaerobic condi-

tions for 24 h. After incubation, cells from 1 ml of each sample

were harvested by centrifugation and fixed for FISH analysis.

According to previous works (Piqueres et al., 2006), we

estimated as viable (replicate capacity) the cells elongated at

least two times their original size.

2.7. DNA analysis

A volume of 1 ml of each inoculated drinking water sample

was used for DNA extraction, using the CTAB method

(Wilson, 1987). A 392 bp 16S rRNA fragment was amplified

using HP1 (50-CCT AAC CAA TTG AGC CAA GAA G-30) and HP2

(50-CTT TCT AAC ACT AAC GCG CTC A-30) primers. Five

microliters of each sample was added to a 50ml PCR reaction

mixture, consisting of 10� PCR buffer, 0.2 mM of each

deoxynucleotide, 1.5 mM MgCl2, 2 U of Taq polymerase (New

England Biolabs, UK), and 0.4mM of each primer.

Amplification was performed in a thermal cycler with an

initial cycle of 95 1C for 5 min, followed by 33 cycles of

94 1C for 1 min, 58 1C for 1 min, and 72 1C for 1 min. An

additional extension step at 72 1C for 5 min completed the

PCR.

For DNA visualization, electrophoresis of PCR products was

performed through 1.5% agarose gels containing 0.5 mg/ml

ethidium bromide and gels were photographed under UV

light. A 100 bp DNA ladder was used as a molecular weight

marker.

2.8. RT-PCR

Reverse transcription assays were carried out by using a one-

step RT-PCR System (Roche Molecular Biochemicals) accord-

ing to the manufacturer’s instructions, with 400 mM concen-

tration of each deoxynucleoside triphosphate and 0.3 mM of

each primer. Primers for vacA gene were used. The forward

primer, Vac1 (50-GGC ACA CTG GAT TTG TGG CA-30), and the

reverse primer, Vac2 (50-CGC TCG CTT GAT TGG ACA GA-30),

amplified a 372 bp product of H. pylori. The cDNA was

synthesized for 50 min at 70 1C and after incubation at 94 1C

for 2 min, the templates were subjected to 10 cycles of

denaturation at 94 1C for 30 s, annealing at 60 1C for 30 s and

extension at 70 1C for 1 min. After that, 25 more cycles were

performed, denaturation at 94 1C for 1 min, annealing at 60 1C

1 min and extension at 70 1C for 1 min. After a final extension

of 7 min at 72 1C, the PCR products were detected by 1.5%

agarose gel electrophoresis.

3. Results

3.1. Culturability

Culturability of H. pylori was progressively decreased until

1 min of exposition to chlorine. At 5 min, cells were not able to

form colonies on the agar (Table 1).

In non-chlorinated water, the number of culturable

cells was maintained at 5 min and 3 h. It decreased one log

unit during the assay, from 4.5�106 at T0 to 2.3�105

at 24 h.

ARTICLE IN PRESS

Table 1 – Results of H. pylori viability markers at different times in chlorinated drinking watera (1.16 mg/l total chlorine and0.96 mg/l free chlorine)

Technique Time

0 h 10 s 40 s 1 min 5 min 3 h 24 h

Culturabilityb 6.54 5.41 4.68 3.25 – – –

Total countsc 7.24 7.34 7.44 7.45 7.40 7.41 7.43

DVC-FISHd 7.14 6.00 5.40 5.35 5.27 4.88 –

Percentage of coccoid forms 20–30 30–40 60–70 80–90 490 490 490

rRNA contente 150 143 148 150 154 150 149

DNAf ++ ++ ++ ++ ++ ++ ++

mRNAg ++ ++ ++ ++ ++ ++ ++

a Data are means of duplicated assays.b Log of colony forming units/ml in Columbia Blood Agar.c Log of total cell counts/ml with DAPI stain.d Log of elongated (viable) cells/ml.e In situ hybridization fluorescence intensity measured with Olympus DP Softs program.f,g ++, strong band intensity; +, weak band intensity.

Fig. 1 – FISH detection of rods and coccoid H. pylori cells after

24 h exposition to chlorinated drinking water.

Fig. 2 – FISH identification of viable (elongated) H. pylori cells

in chlorinated drinking water after DVC treatment. Time

5 min sample.

WAT E R R E S E A R C H 41 (2007) 3490– 3496 3493

3.2. FISH analysis

At the beginning of the study, the number of total cells,

determined by FISH, was 2.7�107 cells/ml in chlorinated

water and 1.65�107 cells/ml in non-chlorinated water.

Microscopic examination of samples at T0 indicated that

most cells (70–80%) were present in the spiral form. Due to the

presence of multiple aggregates of cells, it was not possible to

determine exactly the percentage of coccoid forms present in

the samples. These data were consequently estimated after

observation of, at least, 25 fields from each sample prepara-

tion. The percentage of coccoid forms increased after 40 s of

chlorine exposition until 60–70%. When the culturable state

was lost, coccoid populations were predominant (more than

90%), but rods were also present. Spiral forms were also

detected in chlorinated water at 24 h (Fig. 1). After 5 min and

3 h, the percentage of coccoid forms in non-chlorinated

water was the same as that at T0 (20–30%). It raised until

30–40% after 24 h.

When FISH was performed on chlorinated and non-

chlorinated water, cells gave high fluorescence after probe

hybridization even at 24 h. The fluorescent intensity ranged

from 143 to 154 all along the assays, indicating that 16S rRNA

levels were constant during the chlorine treatment.

3.3. DVC–FISH

After DVC incubation of cells treated with chlorine, an

important reduction of cells with active or reactivable

metabolic activity was observed. Levels of viable cells were

diminished 10 times after 10 s of chlorine contact, and after

5 min we observed 1.8�105 viable elongated cells per ml

ARTICLE IN PRESS

Fig. 3 – mRNA expression of H. pylori vacA gene in

chlorinated drinking water samples: lane 1—time 0

sample; lane 2—time 10 s sample; lane 3—time 40 s sample;

lane 4—time 1 min sample; lane 5—time 5 min; lane 6—time

3 h sample and lane 7—time 24 h sample.

WAT E R R E S E A R C H 4 1 ( 2 0 0 7 ) 3 4 9 0 – 3 4 9 63494

(Fig. 2). The mean number of viable (non-culturable) cells after

3 h of chlorine exposition was 7.5�104 cells/ml, while the

number of total cells was approximately the same as at the

beginning of the assay, showing little cellular lysis. Treatment

with chlorine for 24 h led to negative detection of viable

elongated cells.

In non-chlorinated water, the number of viable cell level

was maintained at 5 min and 3 h. It decreased one log unit

during the assay, from 1.2�107 at T0 to 2.4�106 at 24 h.

3.4. DNA and RNA analysis

The specific amplicon for the 16S rRNA H. pylori gene (392bp) was

detected at all analyzed times for chlorinated and non-chlori-

nated water samples, even after 24h of chlorine exposure. The

intensity of DNA bands in agarose gel was constant too.

The fluorescent intensity of the SYTO RNASelect stained

cells remained constant in all analyzed time samples and for

all the morphologies observed.

3.5. mRNA analysis

Expression of the vacA gene was detected at all time points in

all the samples, demonstrating that these genes were

expressed in non-culturable H. pylori cells. Although after

24 h in chlorinated water no viable cells were observed, mRNA

continued to be detected, and there was no difference in band

intensity of the RT-PCR products at any tested time (Fig. 3).

4. Discussion

When several viability markers are simultaneously analyzed,

the temporal evolution of each positive or negative sub-

population, corresponding to the maintenance or loss of a

physiological function, allows for the identification of suc-

cessive physiological states during the disinfection process

(Lisle et al., 1999).

Our results clearly indicate that under chlorinated condi-

tions, H. pylori cells exhibit a succession of different cellular

alterations, which occur at different rates. In this study,

physiological indices of H. pylori cells submitted to chlorine

treatment were affected in the following order: viable

counts4substrate responsiveness4mRNA expression, rRNA,

and DNA content. The observed succession of changes in

physiological markers for H. pylori is consistent with our

knowledge of cell physiology (Joux et al., 1997): loss of

synthesis activities (CFU, DVC), decrease of metabolic activity,

and, finally, alteration of cell structure and components

(membrane permeability, DNA).

rRNA levels remained constant all along the experiment,

showing that killing of bacteria with chlorine did not involve

ribosome degradation. Results also showed that DNA is

detectable at least for 24 h in chlorine drinking water,

although cells were damaged and the culturability of the

cells was lost. Similar findings have been described for

Arcobacter butzleri cells in chlorinated drinking water (Moreno

et al., 2004).

Even after 24 h in chlorinated water, when no viable cells

were detected, mRNA continued to be detected with the same

intensity than the earlier assays. Although this was unex-

pected, the fact that different mRNAs have different half-life

periods have been previously reported, and, in some cases,

detection of mRNA 30 h after bacterial death has been found

(Birch et al., 2001).

Problems in the definition of a VBNC state are in part due to

the lack of a clear definition of bacterial death. Villarino et al.

(2000) consider death as an irreversible state where no

growth, cell elongation, or protein synthesis may occur. In a

study of different viability markers for UV-killed E. coli cells,

the authors concluded that only a combination of FISH with

DVC assay had the ability of clearly differentiating between

live and dead cells. Our results agree with these considera-

tions, and with those of Yu and McFeters (1994), who reported

that, in biofilms’ mixed bacterial populations, DVC response,

which depends on integrity of cellular physiology, was more

susceptible to biocides’ action than other more discrete

physiological processes. Furthermore, the DVC–FISH combi-

nation has been proved to provide a rapid method to

simultaneously detect and specifically identify viable cells of

H. pylori in drinking water samples (Piqueres et al., 2006).

Previous works about H. pylori resistance to chlorination,

together with evidence that the organism is present in

distribution systems, strongly suggest that drinking water

could be a potential infection source for H. pylori. Chlorine

mean level of 1.1 mg/l is a typical concentration for water

distribution systems at the point of first use, and residual

levels of 0.1–0.3 mg/l are expected along the distribution

system (Baker et al., 2002). According to our results, disinfec-

tion practices normally used in drinking water treatment

would be not totally adequate for controlling this organism,

as viable cells were detected by DVC–FISH after 3 h of

exposition. This period of survival, determined for a single

reference strain, cannot be generalized, as different H. pylori

strains have shown significant variations in their resistance

ARTICLE IN PRESS

WAT E R R E S E A R C H 41 (2007) 3490– 3496 3495

to environmental stress (Owen et al., 2006; Ohkusa et al.,

2004) and H. pylori NCTC 11637 strain has been subcultured

many times over the past 20 years. In this sense, it would be

interesting to have data on more recently isolated strains

from local patients. Nevertheless, our results are consistent

with previous findings of the organism in drinking water

distribution systems. Moreover, in biofilms, resistance of

H. pylori to chlorine increases significantly (Watson et al.,

2004; Yu and McFeters, 1994). It has also been demonstrated

that water-stressed H. pylori cells form large aggregates

adhered to the surface of pipe materials (Azevedo et al.,

2006). So, it is possible that if the organism enters a

distribution system, it may survive to disinfection treatment

within the biofilm matrix.

5. Conclusions

In this work, we have studied the response of H. pylori cells to

chlorination, at mean levels found in water distribution

systems. According to results, some considerations can be

summarized:

�

Nucleic acids content of stressed cells are not well related

to viability and, when detection of a pathogen in environ-

mental samples relies only on these markers, it cannot be

assured that detected cells are potentially infective.

�

We consider that substrate responsiveness (DVC) would be

adequate for monitoring H. pylori cellular viability in water.

Moreover, the DVC–FISH technique can provide a rapid and

specific method to detect and identify viable cells of

H. pylori in drinking water samples.

�

Our work confirms the ability of H. pylori cells to survive for

short periods of time in chlorinated drinking water in the

VBNC form, which would allow them to reach final

consumption points and, at the same time, enable them

to be undetectable by culture methods.

Acknowledgments

This work was supported by Grants no. AGL2002-04480-C03-

03 and AGL2005-07776-C03-01 from Ministerio de Ciencia y

Tecnologıa, Spain (national and FEDER fundings). P.P. had a

graduate student fellowship from Universidad Politecnica de

Valencia.

R E F E R E N C E S

Adams, B.L., Bates, T.C., Oliver, J.D., 2003. Survival of Helicobacterpylori in a natural freshwater environment. Appl. Environ.Microbiol. 69, 7462–7466.

Alonso, J.L., Mascellaro, S., Moreno, Y., Ferrus, M.A., Hernandez, J.,2002. Double-staining method for differentiation of morpho-logical changes and membrane integrity of Campylobacter colicells. Appl. Environ. Microbiol. 68, 5151–5154.

Amann, R.I., Ludwing, W., Schleifer, K.H., 1995. Phylogeneticidentification and in situ detection of individual microbial cellswithout cultivation. Microbiol. Rev. 59, 143–169.

Andersen, A.P., Elliot, D.A., Lawson, M., Barland, P., Haatcher, V.B.,Puszkin, E.G., 1997. Growth and morphological transforma-tions of Helicobacter pylori in broth media. J. Clin. Microbiol. 35,2918–2922.

Azevedo, N.F., Pacheco, A.P., Keevil, C.W., Vieira, M.J., 2006.Adhesion of water stressed Helicobacter pylori to abioticsurfaces. J. Appl . Microbiol. 101 (3), 718–724.

Baker, K.H., Hegarty, J.P., Redmond, B., Reed, N.A., Herson, D.S.,2002. Effect of oxidizing disinfectants (chlorine, monochlor-amine, and ozone) on Helicobacter pylori. Appl. Environ.Microbiol. 68, 981–984.

Bellack, N.R., Koehoorn, M.W., MacNab, Y.C., Morshed, M.C., 2006.A conceptual model of water’s role as a reservoir in Helicobacterpylori transmission: a review of the evidence. Epidemiol.Infect. 134 (3), 439–449.

Besnard, V., Federighi, M., Cappelier, J.M., 2000. Development of adirect viable count procedure for the investigation of VBNCstate in Listeria monocytogenes. Lett. Appl. Microbiol. 31 (1),77–81.

Birch, L., Dawson, C.E., Cornett, J.H., Keer, J.T., 2001. A comparisonof nucleic acid amplification techniques for the assessment ofbacterial viability. Lett. Appl. Microbiol. 33 (4), 296–301.

Brown, L.M., 2000. Helicobacter pylori: epidemiology and routes oftransmission. Epidemiol. Rev. 22 (2), 283–297.

Cellini, L., Allocati, N., Angelucci, D., Iezzi, T., Di Campli, E.,Marzio, L., Danielli, B., 1994. Coccoid Helicobacter pylori notculturable in vitro reverts in mice. Microbiol. Immunol. 38,843–850.

Citterio, B., Casaroli, A., Pierfelici, L., Battistelli, M., Falcieri, E.,Baffone, Y., 2004. Morphological changes and outer membraneprotein patterns in Helicobacter pylori during conversion frombacillary to coccoid form. New Microbiol. 27, 353–360.

Garcia-Armisen, T., Servais, P., 2004. Enumeration of viable E. coliin rivers and wastewaters by fluorescent in situ hybridization.J. Microbiol. Methods 58, 269–279.

Haugland, R.P., 2005. Assays for cells viability, proliferation andfunction. In: Spence, M.T.Z. (Ed.), Handbook of FluorescentProbes and Research Chemicals, 10th ed. Molecular ProbesInc., Oregon, pp. 699–717.

Johnson, C., Rice, E.W., Reasoner, D.J., 1997. Inactivation ofHelicobacter pylori by chlorination. Appl. Environ. Microbiol.63, 4969–4970.

Joux, F., Lebaron, P., Trousellier, M., 1997. Succession of cellularstates in a Salmonella typhimurium population during starva-tion in artificial seawater microcosms. FEMS Microbiol. Ecol.22, 65–76.

Keer, J.T., Birch, L., 2003. Molecular methods for the assessment ofbacterial viability. J. Microbiol. Methods 57, 175–183.

Kogure, K., Simidu, U., Taga, N., 1979. A tentative direct micro-scopic method for counting living marine bacteria. Can. J.Microbiol. 25, 415–420.

Lisle, J.T., Pyle, B.H., McFeters, G.A., 1999. The use of multipleindices of physiological activity to access viability in chlorinedisinfected Escherichia coli 0157:H7. Lett. Appl. Microbiol. 29,42–47.

Magalhaes Queiroz, D.M., Luzza, F., 2006. Epidemiology ofHelicobacter pylori infection. Helicobacter 11 (Sl), 1–5.

Mazari-Hiriart, M., Lopez-Vidal, Y., Ponce de Leon, S., Castillo-Rojas, G., Hernandez-Eugenio, C., Rojo, F., 2003. Bacteria anddisinfection byproducts in water from southern Mexico City.Arch. Environ. Health 58, 233–237.

McFeters, G.A., Yu, F.P., Pyle, B.H., Stewart, P.S., 1995. Physiologicalassessment of bacteria using fluorochromes. J. Microbiol.Methods 21, 1–13.

Moreno, Y., Ferrus, M.A., Alonso, J.L., Jimenez, A., Hernandez, J.,2003. Use of fluorescent in situ hybridization to evidence thepresence of Helicobacter pylori in water. Water Res. 37,2251–2256.

ARTICLE IN PRESS

WAT E R R E S E A R C H 4 1 ( 2 0 0 7 ) 3 4 9 0 – 3 4 9 63496

Moreno, Y., Alonso, J.L., Botella, M.S., Ferrus, M.A., Hernandez, J.,2004. Survival and injury of Arcobacter after artificial inocula-tion into drinking water. Res. Microbiol. 155, 726–730.

Nilsson, H.O., Blom, J., Al-Soud, W.A., Ljungh, A., Andersen, L.P.,Wadstrom, T., 2002. Effect of cold starvation, acid stress, andnutrients on metabolic activity of Helicobacter pylori. Appl.Environ. Microbiol. 68, 11–19.

Nurnberg, M., Schulz, H.J., Ruden, H., Vogt, K., 2003. Do conventionalcleaning and disinfection techniques avoid the risk of endoscopicHelicobacter pylori transmission? Endoscopy 35, 295–299.

Ohkusa, T., Miwa, H., Endo, S., Okayasu, I., Sato, N., 2004.Helicobacter pylori is a fragile bacteria when stored at low andultra-low temperatures. J. Gastroenterol. Hepatol. 19, 200–204.

Owen, R.J., Chisholm, S.A., Brick, G., Lee, J.V., Surnam-Lee, S., Lai,S., Said, B., Nichols, G., 2006. Culture of Helicobacter pylori fromdomestic water samples—the impact of strain variation ongrowth on solid and in liquid media. Water Sci. Technol. 54 (3),147–152.

Park, S.R., Mackay, W.G., Read, D.C., 2000. Helicobacter sp.recovered from drinking water biofilm sampled from a waterdistribution system. Water Res. 35, 1624–1626.

Percival, S.L., Chalmers, R.M., Embrey, M., Hunter, P.R., Sellwood,J., Wyn-Jones (Eds.), 2004. Microbiology of Waterborne Patho-gens. Elsevier, London.

Piqueres, P., Moreno, Y., Alonso, J.L., Ferrus, M.A., 2006. Acombination of direct viable count (DVC) and fluorescent insitu hybridization (FISH) techniques for estimation of Helico-bacter pylori cells viability. Res. Microbiol. 157 (4), 345–349.

Ren, Z., Pang, G., Musicka, M., Dunkley, M., Batey, R., Beagley, K.,Clancy, R., 1999. Coccoid forms of Helicobacter pylori can beviable. Microbios 97, 153–163.

Sheridan, G.E.C., Masters, C.I., Shallcross, J.A., Mackey, B.M., 1998.Detection of mRNA by reverse transcription-PCR as anindicator of viability in E. coli cells. Appl. Environ. Microbiol. 64,1313–1318.

Takayama, K., Kjelleberg, S., 2000. The role of RNA stability duringbacterias stress response and starvation. Environ. Microbiol. 2,355–365.

Villarino, A., Bouvet, O.M., Regnault, B., Martin-Delautre, S.,Grimont, P.A.D., 2000. Exploring the frontier between life anddeath in E. coli: evaluation of different viability markersin live and heat- or UV-killed cells. Res. Microbiol. 51,755–768.

Wang, X., Sturegard, E., Rupar, R., Nilsson, H.O., Aleljung, P.,Carlen, B., Willen, R., Wadstrom, T., 1997. Infection of BALB/cAmice by spiral and coccoid forms of Helicobacter pylori. J. Med.Microbiol. 46, 657–663.

Watson, C.L., Owen, R.J., Said, B., Lai, S., Lee, J.V., Surman-Lee, S.,Nichols, G., 2004. Detection of Helicobacter pylori by PCRbut not culture in water and biofilm samples from drinkingwater distribution systems in England. J. Appl. Microbiol. 97,690–698.

Wilson, K., 1987. Preparation of genomic DNA from bacteria. In:Ausubel, F.M., Brent, R., Kingston, R.E., et al. (Eds.), CurrentProtocols in Molecular Biology, Unit 2.4.1. Wiley, New York.

Yokomaku, D., Yamaguchi, N., Masao, N., 2000. Improved directviable count procedure for quantitative estimation of bacterialviability in freshwater environments. Appl. Environ. Microbiol.66, 5544–5548.

Yu, F.P., McFeters, G.A., 1994. Physiological responses of bacteriain biofilms to disinfection. Appl. Environ. Microbiol. 60,2462–2466.