specific detection of cultivable helicobacter pylori cells from wastewater treatment plants

Specific Detection of Cultivable Helicobacter pylori Cells fromWastewater Treatment PlantsYolanda Moreno* and Mª Antonıa Ferrus†

*Instituto Universitario de Ingenierıa del Agua y Medio Ambiente, Universitat Politecnica de Valencia, †Departamento de Biotecnologıa, Universitat

Politecnica, 46022, Valencia, Spain

Keywords

Helicobacter pylori, detection, culture, FISH,

PCR, viable but not culturable.

Reprint requests to: Mª Antonıa Ferrus,

Departamento de Biotecnologıa, Universidad

Politecnica, Camino de Vera 14, 46022 Valencia,

Spain. E-mail: [email protected]

Abstract

Background: Helicobacter pylori is present in surface water and wastewater,

and biofilms in drinking water systems have been reported as possible reser-

voirs of H. pylori. However, its ability to survive in an infectious state in the

environment is hindered because it rapidly loses its cultivability. The aim of

this study was to determine the presence of cultivable and therefore viable

H. pylori in wastewater treatment plants to understand the role of waste-

water in the pathogen’s transmission.

Materials and Methods: A modified filter technique was used to obtain a

positive H. pylori culture, and specific detection of this pathogen was

achieved with FISH and PCR techniques.

Results: A total of six positive H. pylori cultures were obtained from the

water samples, and molecular techniques positively identified H. pylori in 21

culture-negative samples.

Conclusions: The combination of a culturing procedure after sample filtra-

tion followed by the application of a molecular method, such as PCR or

FISH, provides a specific tool for the detection, identification, and direct

visualization of cultivable and therefore viable H. pylori cells from complex

mixed communities such as water samples.

Helicobacter pylori is an etiological agent of gastritis, and

peptic or duodenal ulcer disease. Infection with this

organism is also a recognized risk factor in the develop-

ment of gastric mucosa-associated lymphoid tissue lym-

phoma and adenocarcinoma. Despite the major public

health impact of H. pylori, the design of prevention

measures is difficult because of our limited knowledge

of its transmission pathways. Although the transmission

of H. pylori is not entirely clarified, human-to-human

spread through either the oral–oral or fecal–oral route

is thought to be the most plausible [1].

Helicobacter pylori is present in surface water and

wastewater, and biofilms in drinking water systems

have been reported as possible H. pylori reservoirs.

There is a growing consensus that considers the bacte-

rium to be a waterborne pathogen. However, its ability

to survive in an infectious state in the environment is

not easy to demonstrate because it is difficult to recover

from aquatic environments [1]. Helicobacter pylori can

survive in water but rapidly loses its cultivability.

Attempts to culture H. pylori cells from environmental

water samples have largely been unsuccessful. There-

fore, most methods used to detect H. pylori in environ-

mental samples are based on culture-independent

molecular techniques such as PCR or fluorescent in situ

hybridization, FISH [1]. PCR, however, detects viable

and nonviable cells. Recently, a DVC–FISH procedure

demonstrated the existence of viable H. pylori cells in

water samples [2], but the organism could not be

cultured from any sample.

As molecular detection of H. pylori in environmental

samples does not indicate that the bacteria are infective,

the concept of waterborne transmission is likely to

remain in question until this organism is cultured from

natural sources [3]. In many countries, treated sewage

effluents are increasingly being discharged into the

environment and used for irrigation. Moreover, in

industrialized countries, the use of treated wastewater

for domestic, industrial, and agricultural purposes is

currently the most common method of reusing waste-

water. This fact can lead to an increased risk of human

infection [4]. The presence of H. pylori infective cells in

© 2012 Blackwell Publishing Ltd, Helicobacter 1

Helicobacter ISSN 1523-5378

doi: 10.1111/j.1523-5378.2012.00961.x

reused water is a possible way by which the organisms

reenter the water chain, which is a public health con-

cern. Culturing of the bacteria from fecal-contaminated

water is needed to assess the infectivity of H. pylori cells

and thus the possibility of infection via a fecal–oral

route.

Materials and Methods

Wastewater Samples

A total of 45 wastewater samples were obtained from

two secondary wastewater treatment plants (A and B)

located in Valencia, Spain. Both plants receive urban

and industrial wastewater (40,000 m3/day) and apply

biological secondary and tertiary UV disinfection treat-

ment. Plant B also includes a sand filtration step before

disinfection. For both plants, the final effluent is dis-

charged into the sea or used for irrigation purposes.

Seven sample campaigns (H1 to H7) were taken from

plant A from three sites: influent (raw), after secondary

treatment, and after UV disinfection (effluent). A total

of 24 samples were collected from plant B through six

sampling campaigns (H8 to H13) from four sites: influ-

ent, after secondary treatment, after sand filter filtra-

tion, and effluent. All of the samples were placed into

sterile glass bottles, refrigerated, and processed within a

few hours.

A total of 300 mL of each water sample was centri-

fuged at 8000 g for 30 minutes. The supernatant was

discarded, and the pellet was resuspended in 3 mL of

PBS 1X (130 mmol/L sodium chloride, 10 mmol/L

sodium phosphate, pH 7.2). Aliquots of 1 mL were

obtained and used for culture, FISH, and PCR analysis.

For enrichment, another 300 mL of each sample was

filtered through 0.45-lm membrane filters (Whatman,

Maidstone, UK), which were transferred to flasks con-

taining 100 mL of Columbia broth (OXOID, SA, Spain)

supplemented with Dent selective supplement (Oxoid)

and incubated in microaerobic conditions at 37 °C for

48 hour. After incubation, aliquots of 1 mL of the

enrichment broths were used for culture, FISH and PCR

analysis.

Culture

To isolate H. pylori cells, different culture techniques

were used. First, aliquots of 0.1 mL of each water sam-

ple, before and after enrichment, were spread directly

on pylori agar (Biomerieux, Mercy L’Etoile, France)

and Columbia agar base supplemented with 10% defi-

brinated horse blood and Dent selective supplement

and incubated under microaerobic conditions at 37 °C

for 3–4 days. In addition, a modified filter technique

that was previously described by Steele and McDermott

[5] was also applied. Briefly, 0.65-lm cellulose acetate

membrane filters (Whatman) were placed onto both

types of H. pylori selective media plates, and then

100 lL portions of the samples were placed on the fil-

ters and incubated at room temperature for 30 minute.

Afterward, the membranes were removed from the

agar, and the plates were incubated at the optimal

conditions for H. pylori as described above.

The agar plates were examined for the presence of

characteristic colonies at 48 hour, and 3, 7, and

10 days. Presumptive H. pylori colonies were subcul-

tured on Dent agar and Gram-stained. For confirma-

tion, cultures were fixed for FISH analysis and

processed for PCR identification as described below.

Selective agar plates containing high amounts of

background wastewater microbiota were also analyzed

for the presence of H. pylori by collecting all of the

surface content and processing it for FISH and PCR

analysis.

FISH

For the FISH analysis, H pylori presumptive isolates

were resuspended in PBS buffer and immediately fixed

with three volumes of 4% paraformaldehyde for 2 hour

at 4 °C. The fixed samples were centrifuged, washed

with PBS buffer, and finally resuspended in 1 : 1 PBS/

ethanol (v/v) as previously described [6].

FISH analysis was performed with a 16S rRNA

LNA (locked nucleic acid) probe specific to H. pylori

(HPYCTGGAGAGACTAAGCCCTCC-) based on the

specific sequence designed by Moreno et al. [7] and

synthesized by EXIQON (Exiqon A/S Vedvaek,

Denmark). Although the specificity of the HPY probe

had been previously confirmed [6], a new gapped

BLAST search was performed to compare the probe

with all of the known rRNA sequences contained in

the database.

The EUB 338 universal probe, which is complemen-

tary to a region of 16S rRNA of the domain bacteria,

was used as a positive control to simultaneously visual-

ize the rest of the water microbiota [8]. The use of this

probe ensures that the hybridization procedure has

been performed properly and the oligonucleotides can

penetrate the cells and attach to rRNA. The samples

were checked for autofluorescence before hybridization,

and a fluorescent oligonucleotide sequence that was

not complementary to eubacterial rRNA (a non-EUB

probe) was used as a negative control to check for non-

specific binding of HPY probe to hydrophobic sample

components [9].

© 2012 Blackwell Publishing Ltd, Helicobacter2

Detection of Cultivable H. pylori Moreno and Ferrus

PCR Analysis and DNA Sequencing

DNA was purified from a 1-mL aliquot of each sample

using the Realpure Genomic DNA Isolation Kit (Durviz,

Valencia Spain) according to the manufacturer’s

instructions. H. pylori-specific VacA primers described

by Nilsson et al. [10] were used to amplify a 394-bp

fragment from the vacA gene. For the PCR, a final reac-

tion volume of 50 lL was used, and it included 5 lL of

each DNA template, 0.5 lmol/L of each primer,

0.2 mmol/L of each deoxynucleotide, 1.5 mmol/L

MgCl2, and 5 U of Taq polymerase (BIORON GmbH,

Ludwigshajen Germany). The amplification consisted of

an initial DNA denaturation step, at 95 °C for 5 min-

ute, followed by a 33-cycle reaction (94 °C for 1 min-

ute; 57 °C for 1 minute; 72 °C for 1 minute) and a

final extension step at 72 °C for 5 minute. PCR ampli-

cons were purified with a QIAquick PCR purification

kit (Qiagen Iberia, S.L., Madrid, Spain) according to the

manufacturer’s instructions and were used for nucleo-

tide sequencing. Both DNA strands were sequenced

commercially (Sistemas Genomicos S.L., Valencia,

Spain). The homology of the amplified sequences to the

corresponding H. pylori vacA gene fragment was deter-

mined by a BLAST alignment. For the presumptive

strains isolated from wastewater, additional sequencing

of the 16S rDNA gene was performed [11]. Sequences

were compared by alignment to the 16S rDNA Heli-

cobacter (http:www.ncbi.nem.nih.gov.blast) sequences

available in the gene bank by using BLAST software.

Results and Discussion

Culture Detection of Helicobacter pylori in

Wastewater

Although many studies have focused on improving the

recovery of H. pylori from water systems [12, 13], to

date, only one study has reported the isolation of

H. pylori from raw municipal wastewater after immuno-

magnetic capture [14]. Massive growth of competitive

biota in selective media is, together with viable but not

cultivable stages of the organism, one of the main chal-

lenges to isolation of H. pylori from the environment

[13, 15, 16]. In this work, we have used a modified fil-

ter method to isolate H. pylori from wastewater. The

technique seems to eliminate large amounts of competi-

tive microbiota, allowing H. pylori cells to pass through

the membrane and grow on the selective media. When

applied to our samples, H. pylori presumptive colonies

were easily observed in selective agar after incubation,

and plates presented much less contaminating micro-

biota. Observation of presumptive colonies on the same

selective agar plates without previous filtration was

unsuccessful because of the massive growth of other

bacterial species (Fig. 1). For this reason, these samples

were considered ‘‘negative’’ because characteristic colo-

nies could not be observed.

Thirteen H. pylori presumptive cultures were

obtained from eight different water samples (Table 1).

H. pylori cells could not be completely isolated because

of the growth of competitive biota in selective agar,

which demonstrates the inefficacy of presently available

culture media for the isolation of the bacteria from envi-

ronmental samples [3, 15]. When samples of the pre-

sumptive colonies were analyzed, Gram-negative cells

exhibiting typical H. pylori morphology were observed

but were mixed with other bacillary non-H. pylori

forms. Therefore, to confirm the presence of cultivable

H. pylori cells in plates, mixed cultures were analyzed

using the specific FISH assay, specific vacA PCR and 16S

rDNA sequencing. Six cultures were confirmed to con-

tain H. pylori cells. All of the water samples from which

A

B

Figure 1 Growth of bacteria from water samples on Dent agar plates:

(A) without and (B) with previous filtration.


Moreno and Ferrus Detection of Cultivable H. pylori

H. pylori was cultured were also determined to be posi-

tive by FISH (Fig. 2) or PCR techniques. In one case,

H. pylori was cultured after secondary treatment.

Molecular detection of Helicobacter pylori in

wastewater

When molecular methods were applied directly to

wastewater samples, FISH detection was positive in 26

of 45 samples: 10 raw, 8 after secondary treatment, 4

after the sand filtration step, and 4 after UV disinfection

(Table 1). Following enrichment, all samples were

H. pylori negative, confirming, as previously described

[7], the inadequacy of this step for FISH detection in

wastewater.

Although the PCR method is considered to be more

sensitive than either FISH or the culture methods, more

H. pylori-positive samples were obtained by the FISH

Table 1 Detection of Helicobacter pylori in non-inoculated wastewa-

ter samples. Only positive samples for any assay are shown. Results

were obtained prior to enrichment, unless indicated

Samples

Treatment

plant Origin Culturea FISH

DVC-

FISH mPCR

M1 A Raw � +F � �M3 A Raw � + � +F

M7 A Raw � + + +F

M8 A Raw � + � +F

M11 A Raw � + � +F

M13 A Raw � +F � �M17 A Raw +F + + +F

M18 A Raw +F + + +F

M20 A Raw +F + + +F

M21 A Raw +F + + +F

M23 A Raw + + + +F

M23 A After secondary

treatment

+ + + +F

M30 A Raw � +F � �M30 A Effluent � + � �M34 A Raw � +F � �M34 A After secondary

treatment

� +F � �

M40 A Raw +F +F � �M42 A Raw + + + �M42 A After secondary

treatment

� +F � �

M44 A Raw � + � +F

M44 A After secondary

treatment

� + � �

M44 A Effluent � � � +F

M48 B Raw +F + + +F

M49 B Raw +F + + �M51 B Raw +F + + +F

M52 B Raw � + � �M53 B Raw + + � +F

M53 B After secondary

treatment

� + + +F

M55 B Raw +F + + +F

M56 B Raw + + + +F


treatment

� +F � +F

M58 B Raw +F + + +F

M59 B Raw +F +F � +F


treatment

+ + + +F

M61 B Raw + +F � +F

M63 B Raw +F + � �M65 B Raw � + + �M66 B Raw +F + � �M67 B Raw � + � +


treatment

� + + +

M67 B Effluent � + + �M70 B Raw +F + + �M70 B After secondary

treatment

+ + + �

(Continued)

Table 1 (Continued)

Samples

Treatment

plant Origin Culturea FISH

DVC-

FISH mPCR

M73 B Raw +F + + �M76 B Raw � + � +F

M79 B Raw � + � +

M85 B Raw � + + �M85 B After secondary

treatment

� + � �

aIsolate identified as H. pylori; FPositive results obtained only after

enrichment.

Figure 2 FISH identification of H. pylori in water samples with the

specific LNA probe.



technique than by culture or PCR analysis, confirming

previous reports [2, 17]. PCR analysis allowed for the

direct detection of H. pylori DNA in only three raw sam-

ples. Following enrichment, four raw and one effluent

samples yielded positive results. Some authors have sug-

gested that inhibitory substances present in wastewater,

such as humic acids, can have a significant effect on the

activity of the Taq polymerase enzyme, yielding false-

negative results [18]. An enrichment step dilutes inhibi-

tors of the sample, thus improving detection rates [19].

In 21 culture-negative samples, H. pylori was

detected by PCR or FISH. According to other authors,

molecular methods appear to overestimate the H. pylori

presence in water while culture methods could under-

estimate it [20]. This could indicate the presence of via-

ble but nonculturable (VBNC) cells. This fact may be

important from a public health point of view; some

authors have suggested that pathogenic VBNC bacteria

can maintain their virulence, becoming a potential

reservoir of disease [3].

The presence of H. pylori was also detected by

molecular techniques after secondary treatment and

even in the final effluent after tertiary disinfection. This

is in agreement with some authors’ reports about the

fact that H. pylori could tolerate disinfection treatments

better than classical fecal indicators [12]. It was not

possible to culture H. pylori from samples after disinfec-

tion, but in six of them the organism was detected by

molecular methods. These results could be false-posi-

tives because of the detection of dead cells. However,

an enrichment step prior to PCR dilutes free DNA from

dead cells [21], while FISH allowed for the observation

of typical bacillary forms directly in the samples. Thus,

positive molecular results are more likely due to the

presence of VNBC cells. This indicates that the organism

could survive wastewater treatment and reenter the

human environment when treated water is reused.

In conclusion, the combination of a culture proce-

dure after filtration with a molecular method, such as

PCR or FISH, is a very specific tool for the detection,

identification, and direct visualization of cultivable

H. pylori cells from complex mixed communities such

as wastewater samples. This work demonstrates that

cultivable H. pylori cells are present in wastewater, con-

firming that fecal-contaminated water may act as a

transmission vehicle for the bacteria.

Acknowledgements and Disclosures

This work was supported by the grant AGL2008-05275-C03-02

from Ministerio de Ciencia e Innovacion, Spain.

Competing interests: the authors have no competing

interests.

References

1 Khean-Lee G, Wah-Kheong C, Shiota S, Yamaoka Y. Epidemi-

ology of Helicobacter pylori Infection and Public Health Implica-

tions. Helicobacter 2011;16(Suppl. 1):1–9.

2 Piqueres P, Moreno Y, Alonso JL, Ferrus MA. A combination of

direct viable count and fluorescent in situ hybridization for esti-

mating Helicobacter pylori cell viability. Res Microbiol

2006;157:345–9.

3 Bellack NR, Koehoorn MW, MacNab YC, Morshed MG. A con-

ceptual model of water’s role as a reservoir in Helicobacter pylori

transmission: a review of the evidence. Epidemiol Infect

2006;134(3):439–49.

4 Rubiano ME, Agullo-Barcelo M, Casas-Mangas R, Jofre J,

Lucena F. Assessing the effects of tertiary treated wastewater

reuse on a Mediterranean river (Llobregat, NE Spain):

pathogens and indicators. Environ Sci Pollut Res Int

2012;19:1026–32. Epub 2011 Jul 1.

5 Steele TW, McDermott SN. Technical note: the use of mem-

brane filters applied directly to the surface of agar plates for the

isolation of Campylobacter jejuni from faeces. Pathology

1984;6:263–5.

6 Amann RI, Ludwig W, Schleifer KH. Phylogenetic identification

and in situ detection of individual microbial cells without

cultivation. Microbiol Rev 1995;59:143–69.

7 Moreno Y, Ferrus MA, Alonso JL, Jimenez A, Hernandez J. Use

of fluorescent in situ hybridization to evidence the presence of

Helicobacter pylori in water. Wat Res 2003;37:2251–6.

8 Buswell CM, Herlihy YM, Lawrence LM, McGuiggan JTM,

Marsh PD, Keevil CW, Leach SA. Extended survival and persis-

tence of Campylobacter spp. in water and aquatic biofilms and

their detection by immunofluorescence-antibody and rRNA

staining. Appl Environ Microbiol 1998;64:733–41.

9 Amann RI, Ludwig K, Schleifer KH. Identification and in situ

detection of individual bacterial cells. FEMS Microbiol Lett

1992;59:143–69.

10 Nilsson H, Blom J, Al-Soud W, Ljungh A, Andersen L,

Eadstrom T. Effect of cold starvation, acid stress and nutrients

on metabolic activity of Helicobacter pylori. Appl Environ Microbiol

2002;68:11–9.

11 Engel AS, Porter ML, Stern LA, Quinlan S, Bennett PC. Bacte-

rial diversity, and ecosystem function of filamentous microbial

mats from aphotic (cave) sulfidic springs dominated by chemo-

lithoautotrophic “Epsilonproteobacteria”. FEMS Microbiol Ecol

2004;51:31–53.

12 Azevedo NF, Pacheco AP, Keevil CW, Vieira MJ. Nutrient

shock and incubation atmosphere influence recovery of cultur-

able Helicobacter pylori from water. Appl Env Microbiol

2004;70:490–3.

13 Degnan AJ, Sonzogni WC, Standridge JH. Development of a

plating medium for selection of Helicobacter pylori from water

samples. Appl Environ Microbiol 2003;69:2914–8.

14 Lu Y, Redlinger TE, Avitia R, Galindo A, Goodman K. Isolation

and genotyping of Helicobacter pylori from untreated municipal

wastewater. Appl Environ Microbiol 2002;68:1436–9.

15 Flanigan D, Rodgers M. A method to detect viable Helicobacter

pylori bacteria in groundwater. Acta Hydroch Hydrob 2003;31:

45–8.

16 Moreno Y, Piqueres P, Alonso JL, Jimenez A, Gonzalez A,

Ferrus MA. Survival and viability of Helicobacter pylori after

inoculation into chlorinated drinking water. Water Res

2007;41:3490–6.


Moreno and Ferrus Detection of Cultivable H. pylori

17 Lepeuple S, Delabre K, Gilouppe S, Intertaglia L, De Roubin

MR. Laser scanning detection of FISH-labelled Escherichia coli

from water samples. Water Sci Technol 2003;47:123–9.

18 Lemarchand K, Berthiaume F, Maynard C, Harel J, Payment P,

Bayardelle P, Masson L, Brousseau R. Optimization of microbial

DNA extraction and purification from raw wastewater samples

for downstream pathogen detection by microarrays. J Microbiol

Methods 2005;63:115–26.

19 Liu D. Preparation of Listeria monocytogenes specimens for

molecular detection and identification. Int J Food Microbiol

2008;122(3):229–42.

20 Queralt N, Araujo R. Analysis of the survival of H. pylori within

laboratory based aquatic model system using molecular and

classical techniques. Microbial Ecol 2007;54:771–7.

21 Josephson KL, Gerba CP, Pepper IL. Polymerase chain reaction

detection of non-viable bacterial pathogens. Appl Environ

Microbiol 1993;59:3513–5.



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