Presence of enterotoxigenic Escherichia coli in biofilms formed in water containers in poor households coincides with epidemic seasons in Dhaka

Presence of enterotoxigenic Escherichia coli in biofilms formed in

water containers in poor households coincides with epidemic seasons

in Dhaka.

Dilruba Ahmed1*, Mohammad Shahidul Islam1*, Yasmin Ara Begum1,

Anders Janzon2, Firdausi Qadri1 and Åsa Sjöling2

1 International Centre for Diarrhoeal Disease Research, GPO Box 128, Dhaka, 1000,

Bangladesh

2 Department of Microbiology and Immunology, Institute of Biomedicine, the

Sahlgrenska Academy, University of Gothenburg, PO Box 435, SE-405 30, Göteborg,

Sweden

*shared first author

E-mail addresses: D Ahmed: [email protected], M S Islam: [email protected]

YA Begum: [email protected], A Janzon: [email protected], F Qadri:

[email protected], Å Sjöling: [email protected]

Corresponding author:

Åsa Sjöling

Department of Microbiology and Immunology, Institute of Biomedicine,

Sahlgrenska Academy, University of Gothenburg

Box 435, 405 30 Göteborg, Sweden

Tel +46-31-7866232

Fax +46-31-7866205

e-mail: [email protected]

Abstract

Background: Bacteria in aqueous conditions tend to form thin coatings of bacterial

communities, i.e biofilms, on abiotic or biotic surfaces and such biofilms may be

potential reservoirs for waterborne pathogens such as enterotoxigenic Escherichia coli

(ETEC).

Results:We show for the first time that enterotoxigenic Escherichia coli (ETEC), the

causative agent of acute watery diarrhea and travelers’ diarrhea is present in biofilms

formed on glass slides submerged into household water tanks in an urban area of

Dhaka, Bangladesh. Biofilms were detected every month over a two year period.

Multilayered mature biofilms were found significantly more often on glass slides

collected in the monsoon period between the two annual ETEC peaks i.e between

May and August. Sixty-four percent (49/76) of the biofilms were ETEC positive as

detected by quantitative real-time PCR. ETEC positive biofilms were found every

month of the year and followed a seasonal pattern with significantly more ETEC-PCR

positive biofilms during the epidemic peaks before and after the monsoon rains and

during flooding. Analyses of drinking water specimens from the same households

showed that planktonic ETEC was present in water during all seasons but no

correlation between presence or numbers of ETEC in water and the epidemic peaks

could be found.

Conclusions: We conclude that ETEC is continuously present in water and biofilms in

household water reservoirs in this area which has a high prevalence of ETEC diarrhea.

The frequency of biofilms with ETEC was significantly associated to seasonal

epidemic peaks of ETEC diarrhea.

Keywords: Enterotoxigenic Escherichia coli, ETEC, Drinking water, Biofilm

Background

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of acute

diarrheal disease in developing countries, causing up to 400 million diarrheal cases

annually and approximately 400, 000 deaths among children under the age of five

every year [1-3]. ETEC is also a cause of severe to mild disease in adults. ETEC

produce two main groups of plasmid-encoded virulence factors: colonization factors

(CFs), which are adhesion molecules on the bacterial surface that mediate

colonization of the small intestine by binding to the enterocytes [4, 5], and the heat-

stable (ST) and heat-labile (LT) enterotoxins. The enterotoxins ST and LT induce

diarrhea by binding to receptors on the human intestinal epithelium where both toxins

activate signaling pathways that ultimately result in the secretion of large amounts of

water and electrolytes into the intestinal lumen [6-8]. ETEC strains express either ST

only, LT only, or both toxins simultaneously. Two genotypes of ST are expressed by

strains causing disease in humans; STh and STp [9].

In Bangladesh, diarrheal diseases are a major health problem and ETEC accounts for

about 11-20% of all diarrheal cases and affects both the local population and travelers

to the region [2, 10, 11]. Bangladesh is a subtropical country characterised by heavy

rainfalls. Three seasons are generally recognized: hot, humid summer season from

March to May, a cooler rainy monsoon period from June to September and a cool, dry

winter from October to February. In Bangladesh where ETEC is endemic the infection

rates typically rises in the warm summer season before the monsoon rains with a peak

in April-May and a second peak when the rains abate in September-October every

year [2] but the reason for these epidemic peaks is still elusive. Bangladesh is

sometimes affected by flooding during the monsoon period and severe floods in recent

years occurred in 1998, 2004 and 2007 of which the latter occurred during the period

of this study. The incidence and severity of diarrheal disease increase during floods

[10, 12]. We have previously shown that ETEC is present in drinking and

environmental water in Dhaka, the capital of Bangladesh [13-15] and viable after long

term water incubation [16], suggesting that water may be a reservoir for ETEC and a

possible route of transmission.

However it is well known that bacteria in aqueous conditions prefer to form biofilms

[17]. Biofilms are surface associated bacterial communities surrounded by an

extracellular matrix that shelters the bacteria from outer stress such as antibiotic

treatment and dehydration. Biofilms form readily on solid surfaces in contact with

non-sterile water or in liquid/air interfaces [18]. In nature biofilms are a dominating

habitat for waterborne species and are often composed by multiple species including

bacteria, protozoa and virus and the composition varies with environmental factors

[19]. Bacterial biofilm formation is regulated by complex regulatory systems that

respond to environmental signals [20, 21]. The first steps in biofilm development is

formation of a reversible monolayer of bacteria attached to the surface, this step is

initially dependent on flagellar motility and on expression of surface adhesion

molecules [22, 23]. Later stages of microcolonies and subsequently more developed

biofilms are composed of complex multilayer structures with different bacterial

populations surrounded by exopolysaccharides, external DNA and proteins [20, 24].

In the present study we sought to determine if ETEC form biofilms in household

drinking water in Dhaka. We found that biofilms form on glass slides submerged in

household water all months of the year and that planktonic ETEC was present in the

drinking water samples during the whole year. However, a significant correlation

between presence of ETEC in biofilms and the epidemic peaks was found.

Results

Biofilm formation in drinking water tanks

A total of 101 glass slides from 75 households in Mirpur were collected every month

over a period of 2 years (August 2006-August 2008). Formation of biofilm on the

glass slides were analysed by gram staining and 85% of the samples were found to

have biofilms. Biofilms were found at all months of the year ranging from 60% to

100% positive samples per month (Figure 1A). There was no significant difference in

the number of slides positive for biofilm growth found during the three different

seasons; hot summer between March - May, hot rainy season in June - September and

the dry cold season October - February. The biofilm positive glass slides were scored

for intensity and thickness of the formed biofilm using a 3 graded scale (1 - 3) and the

mean value of the intensity was plotted against the month of collection (Figure 1B and

1C). The highest median scores of intensity were recorded in May and October but

there were no significant association of more developed biofilms scored as 3 with any

of the seasons, however biofilms scored as 3 were significantly more often isolated in

the months May to August when precipitation gradually increase and reach a

maximum than in the other months (p < 0.03). To determine if thicker multilayered

biofilms correlated with ETEC diarrheal peaks we defined the epidemic peaks to

April-May in 2007 and 2008 and July-September in 2007 during the period of

flooding in Bangladesh in 2007 [10] and September-October in 2006. We found a

trend but no significant correlation of presence with biofilms graded as 3 and the

epidemic periods (p < 0.063).

ETEC is present in biofilms

Seventy-six glass slides were further analysed for presence of ETEC using real-time

PCR quantification with a PCR product standard curve as described in detail

previously [15]. Biofilms positive for at least one of the ETEC toxin genes LT, STh

and STp were identified by real-time PCR in totally 49 (64%) of the analysed samples

and during all months. The percentage of ETEC positive glass slides ranged from 20%

in November to 100% in May. No association of ETEC positive biofilms to any of the

seasons were found but significantly more ETEC positive biofilms were collected

during the epidemic peak seasons in April-May and July-September 2007 and

September-October in 2006 relative to the rest of the year (p < 0.0082) (Table 1).

Real-time PCR quantification of the bacterial load expressed as number of ETEC

gene copies per biofilm present on one glass slide ranged from 40 to 65.000 (median

1015) and the PCR mainly detected the genes for LT and STh. However, no

significant differences were found in gene copy numbers per biofilm when comparing

the seasons or when comparing the peak months to the rest of the year (figure 2).

Identification and bacterial load of ETEC in drinking water does not follow the

epidemic peaks.

Drinking water samples were collected at the same time as the biofilm samples from

households in Mirpur during the course of the study. The water samples were also

tested for growth on MacConkey agar plates. A total of 70 water samples were tested

for ETEC growth by culture followed by toxin GM1-ELISA and 62 of these water

samples were also filtered and subjected to DNA extraction and subsequently tested

by real-time PCR. The frequency of ETEC positive samples detected by ELISA was

20% while 63% were real-time PCR positive for one or several of the toxin genes.

Neither real-time PCR nor ELISA results detected any association of ETEC positive

water samples to the epidemic peaks or season. Thirty-one samples that were positive

in real-time PCR were negative in ELISA. Real time PCR quantification revealed that

100 ml water contained a median of 3100 gene copies [15]. No significant difference

was found in gene copy numbers for LT and STh present in drinking water between

the different seasons or in the epidemic peak seasons (figure 2).

Discussion

Enterotoxigenic E. coli and other diarrheal bacterial pathogens are believed to spread

by the fecal-oral route of transmission presumably by contaminated food and water.

We and others have repeatedly reported presence of ETEC in environmental and

drinking water in endemic areas [13 - 15, 25] but since bacteria in aqueous

environments tend to exist mainly in the form of biofilms [17] the presence of ETEC

in biofilms in drinking water sources needed to be established. This is to our

knowledge the first study to confirm the presence of ETEC in biofilms formed in

drinking water sources in an endemic area with high rates of acute watery diarrhea.

Household drinking water biofilms were found during all months of the year.

Developed multilayered biofilms were found significantly more often on glass slides

collected in the period between the two annual ETEC peaks i.e between May and

August (figure 1B). These months have the highest average temperatures in Dhaka

and also this is the peak period for the rainy season. Since the households in this study

were mainly from low income groups and consisted mainly of sheds and one-roomed

over crowded homes the temperatures inside homes and in the drinking water was

presumably also higher during these months. We did however not measure

temperature during the time of the sampling. Biomass and changed biodiversity of

biofilms in estuarine waters has previously been found to be associated with higher

temperatures and rainfall in a subtropical area [26]. Other studies have reported a link

between higher temperature and earlier biofilm formation as well as higher bacterial

biofilm biomass [27]. Hence formation of biofilm in drinking water tanks probably

occurs faster during the warm and humid months in Bangladesh.

In 2007 Bangladesh was hit by massive flooding between July to September that

caused extensive flooding of several areas of Dhaka and contributed to an earlier

epidemic of ETEC cases than in other years with a peak in August [10]. Despite the

flood we could recover slides during this period and they were analysed as slides

collected during epidemic peaks. We found significantly more ETEC positive

biofilms detected by real-time PCR during the epidemic peaks of the entire study

period indicating that ETEC in biofilms are linked to the epidemic peaks. This could

indicate that more ETEC are circulating between the environment and community

during epidemics and hence present in environmental water during the epidemic peaks

and we have also shown this in a previous study [15]. However the presence of ETEC

in drinking water was not found to be higher during the epidemic peaks in this study

in contrast to previous evidence of significantly higher numbers of ETEC positive

drinking water samples during epidemics [15]. This previous report was however not

based on samples taken over the entire year and only covered the autumn peak and the

winter season.

Similar to earlier studies we detected culturable ETEC colonies in the water samples

[14, 15]. However, since we had to detach the biofilms using harsh methods we could

not establish the presence of viable ETEC from the biofilms with confidence which

made a comparison between viability in biofilms and water impossible. In addition,

we could not establish any significant link between presence of culturable ETEC in

water and the epidemic peak seasons or other seasons. The fact that the PCR method

detects more positive samples and higher numbers of bacteria further emphasizes that

conventional culture methods might underestimate the numbers of potentially

infectious bacteria in environmental samples, hence we choose to compare the results

obtained with real-time PCR in this study.

Quantification of the bacterial load in household biofilm and water showed that the

numbers varied over four order of magnitudes with up to ten thousand ETEC gene

copies per biofilm or 100 ml of drinking water. We hypothesizsed that higher loads of

bacteria would be present during the epidemic peaks but the numbers of ETEC gene

copies did not change significantly either in water or biofilms (Figure 2). Biofilms

may vary in biomass and composition and in the more developed stages of biofilms

planktonic bacteria may be released from the biofilms or large clumps of biofilms can

detach. Such clumps could contain high densities of pathogenic bacteria and might

therefore be sources of infection [17]. Further studies of drinking water is needed to

establish if shedding of large clumps of detached biofilm occurs in household waters.

The infection load of ETEC has been determined to be rather high requiring 107-109

bacteria and according to our results the biofilms in the households do not contain

such high numbers of ETEC and it does not seem possible that ingestion of a single

cup of water could cause disease. However, the infection load decreases by several

order of magnitudes in high-risk populations such as small children, elderly and

immuno-compromised individuals. The population in the study area suffers from

malnutrition and poverty and would probably be susceptible to lower levels of

bacteria. In addition, V. cholerae biofilms isolated from water sources have been

reported to be hyperinfectious and able to out-compete planktonic bacteria as well as

establish an infection with several order of magnitudes lower doses of inoculated

bacteria in a mouse model [28]. Hence it might be possible that a small dose of

thousands of ETEC bacteria from biofilms may be able to cause disease in high-risk

individuals. Since mature biofilms formed more often in the warm rainy periods there

might be a link between onset of epidemics and detachment of biofilms. This however

needs to be further investigated. In a previous study we were able to show that ETEC

of similar phenotypic characteristics were isolated both from water and patients with

diarrhea indicating water transmission [14]. On the other hand the constant presence

of low levels of ETEC in biofilms and drinking water found in this study may

contribute to the fact that ETEC are often recovered from the feces of asymptomatic

individuals. Although asymptomatic carriers of ETEC may be immune due to

repeated infections of the same type of ETEC strains [29], studies of ETEC numbers

in fecal specimens of symptomatic and asymptomatic individuals in endemic areas

would be interesting to perform.

Conclusions

In conclusion this is the first study to our knowledge that has established presence of

ETEC in household drinking water biofilms. ETEC is endemic in Bangladesh and the

results of this study indicate a constant presence of ETEC in the drinking water and

biofilms of households in poor areas. However the number of biofilms that contained

ETEC increased significantly during the epidemic periods which might suggest that

biofilms could be a reservoir for ETEC. .

Materials and Methods

Selection of sample sites

Households in Mirpur, which is a poor slum area in Dhaka, Bangladesh were selected

based on results from a previous birth cohort where children in Mirpur that suffered

from several incidences of ETEC diarrhoea 2002-2004 were identified [30].

Households with the highest incidence of ETEC infections in children in the previous

cohort study, i.e most likely to have ETEC within the household, and with household

water storage in water tanks, a requirement for this study, were selected for inclusion

in the present study. The water tanks are large or smaller water reservoirs kept outside

or inside the home that usually contain several hundred litres of water and members of

the household use the water within the tanks for daily consumption. The tanks were

drained every third to six month and a few tanks were drained every month. Totally 75

households were included in the study and samples were taken distributed over all

months of the year. The adults of each household were informed about the purpose of

the study and gave oral consent to collect water and to perform biofilm screening.

Bacterial samples were analysed mainly at the International Centre for diarrhoeal

disease research, Dhaka, Bangladesh icddr,b who has the premises for working with

pathogens.

Household sampling procedures

Biofilm samples were collected by submerging glass slides disinfected with 99,5%

ethanol and mounted into Petri dishes into the water tanks. Two holes were made in

the lid of the Petri dishes so that water could pass freely through the dish. The lid and

plate were tied with a rope and a piece of cork was attached at the other end to keep

the dish floating at a depth of 15-20 cm. The glass slides inside the Petri dishes were

placed into household water tanks of 6-8 households on each occasion. After 30 days

the Petri dishes with glass slides were collected and a water sample was taken from

the water tank.

Staining and DNA extraction

After collection the samples were brought to the laboratory at the international centre

for diarrhoeal disease Research, Bangladesh (iccdr,b) in Dhaka and the slides were

washed with 3x 10 ml of PBS (phosphate buffered saline) to remove the non adherent

cells and other debris. One slide was air dried and gram stained to analyse for the

formation of biofilms. The formation of biofilm was graded on a scale from 1 to 3 on

biofilm positive slides. Adherent cells were collected from the second slide by

applying 250 ul PrepMan®Ultra (Applied Biosystem) on top of the biofilm to

dissociate the attached bacterial cells. Bacterial cells were extracted by scraping off

the biofilms from the slides with the open end of a sterile 1000 µl pipette tip. The

cells were collected in a sterile 1.5 ml micro-centrifuge tube (Eppendorf). Extraction

of bacterial DNA was performed by using the DNeasy Blood and tissue kit (Qiagen,

Hilden, Germany) following the manufacturer’s protocol for extraction of Gram

negative bacterial DNA. Water samples were collected by filtration of 500 ml

drinking water through a 0.22 µm pore filter. Of each filter, one quarter was used for

DNA extraction and one quarter for E. coli culture on MacConkey agar plates as

described previously [15]. The rest of the filter was frozen at -20 ºC.

Culture, ELISA and PCR analyses of the samples

To determine the presence of culturable ETEC all water samples were cultured on

MacConkey agar followed by toxin ELISA or multiplex PCR analyses of lactose-

fermenting colonies as described previously [31]. The procedure for water samples

has been described earlier [15]. To determine and quantify the presence of ETEC

enterotoxin genes, STh, STp and LT, directly in the water and biofilm samples

extracted DNA was analysed by real-time PCR using a PCR product standard curve as

described previously [15]. Briefly, a single band PCR product of each gene was

purified using a PCR purification kit (Qiagen, Hilden, Germany). The concentration

(g / µl ) was measured and the number of double stranded PCR products per µl was

calculated using the length of the fragment multiplied with the mole weight of dsDNA

nucleotides (660 g / mole). Using Avogadro’s number 6.022 x 1023 molecules per

mole the number of PCR fragments per µl was calculated [32]. Serial ten-fold

dilutions were then used as a standard curve and compared to the Ct value of each

sample to subsequently calculate the number of gene copies per biofilm or water

sample.

Statistical analysis

Statistical significance was tested by GraphPad Prism version 5 using the 2 sided

Fishers exact test. A p-value < 0.05 was considered significant.

Competing interests

The authors declare that they have no competing interests

Author’s contributions

DA conceived of and participated in the design of the study, performed and analysed

biofilm straining and helped to draft the manuscript. MSI performed the house-hold

samplings, performed culture and biofilm staining experiments, analysed the samples

and helped to draft the manuscript. YAB performed house hold samplings, culture

experiments, analysed and organized data and revised the manuscript. AJ performed

molecular real-time PCR analyses and helped to revise the manuscript. FQ conceived

of and designed the study and revised the manuscript. ÅS conceived of and designed

the study, performed real-time PCR analysed all data and wrote the manuscript. All

authors read and approved the final manuscript

Acknowledgements

The study was funded by the joint Formas Sida/SAREC funded programme for

research on sustainable development in developing countries (209-2009-2018) and the

Signe and Olof Wallenius foundation to ÅS and the Swedish International

Development Cooperation Agency (Sida, Grant-INT-icddr,b-HNO1-AV) and the

International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) to FQ.

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Table 1. Presence of ETEC DNA detected by real-time PCR in biofilms is associated

with the epidemic seasons in Bangladesh.

Epidemic peak seasonsa n = 35Endemic seasons n = 41

ETEC positive biofilms n = 48 28 (80 %)* 20(49%)

ETEC negative biofilms n = 28 7 (20 %) 21(51 %)

aEpidemic months were defined as April-May in 2007 and 2008 and July-September

in 2007 (the flood epidemics) and September-October in 2006.

*The association between ETEC positive biofilms and the epidemic peak seasons

was considered to be statistically significant by the two-tailed Fishers exact test. (p <

0.0082).

Figure legends

Figure 1. Biofilm frequency and intensity over the year.

A The percentage of biofilm positive submerged glass slides recovered each month

over a period of two years, August 2006 to August 2008 (n = 4-15 slides per month).

B The intensity of formed biofilms was scored as 1 to 3. The mean and SEM is

plotted for all months of the year.

C Examples of stained glass slides with a monolayer biofilm graded as 1

D A multilayered biofilm graded as 3.

Figure 2. The number of gene copies encoding the two major virulence factors ST

and LT.

The number of gene copies encoding the two major virulence genes encoding the

toxins ST and LT recovered from one glass slide biofilm or from 100 ml of drinking

water collected at epidemic and endemic months during the study period. The

numbers of gene copies from each sample were quantified in duplicates using real-

time PCR and a DNA standard curve, the mean value of the two duplicates is shown

for each samples and the median value of all samples is indicated as a dash. No

significant differences were found between epidemic and endemic periods.

A

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Endem

ic S

T bio

film

epid

emic

LT w

ater

endem

ic L

T wat

er

Epid

emic

ST w

ater

Endem

ic S

T wat

er

10

Figure 2