Gut Microbiota Shifts Favorably with Delivery of Handwashing with Soap and Water Treatment Intervention in a Prospective Cohort (CHoBI7 Trial)

doi:10.21203/rs.3.rs-752679/v1

Cholera can result in the expulsion of important microbiota from the gut and result in death if left untreated. The disease transmits mainly via drinking water carrying Vibrio cholerae; and household contacts (HHC) of cholera patients are at elevated risk during the first week of infection. The gut microbiota profiles of HHC-children of cholera patients at Dhaka city slums were investigated before (day 0) and after (day 8) delivery of chlorinated water as part of the cholera-hospital-based intervention for 7 days (CHoBI7), a randomized controlled trial. Results of sequencing and analysis of bacterial community DNA revealed the predominance of two bacterial phyla: Bacteroidetes and Firmicutes at day 0 with a relative abundance of 62 ± 6 (mean ± SEM %) and 32 ± 7, respectively. The pattern reversed at day 8 with a decreased relative abundance of Bacteroidetes (39 ± 12) and an increased abundance of Firmicutes (49 ± 12). Of 65 bacterial families confirmed at day 0, six belonging to Proteobacteria including Vibrionaceae disappeared at day 8. Interestingly, the relative abundance of four Firmicutes families– Lachnospiraceae, Bifidobacteriaceae, Clostridiaceae, and Ruminococcaceae was increased in all five study children at day 8. The observed exclusion of pathogenic Proteobacteria and enhancement of beneficial Firmicutes in the gut of children delivered with chlorinated water as part of WASH intervention reflect a great promise of the CHoBI7 program in preventing cholera and improving child health.

Scientific Communication

Infectious Diseases

Cholera

Handwashing with Soap

CHoBI7 WASH Intervention

Chlorine-treated Drinking Water

Household-contact Children

Gut Microbiota

The intestinal tract (gut) of humans serves as an important habitat for diverse microbial commensals, also known as microbiota colonizing and adapting to the gut environment creating a state of homeostasis^1,2. Microbiota colonization of the gut begins soon after childbirth and continues throughout one’s lifetime depending on the immediate ecosystem they belong to and their overall lifestyle. The gut of a normal individual human can harbor microbiota accounting for more than 10¹⁴ microbial cells. These commensal microbiota regulate a number of host processes from nutrition and development to immune responses functionally regulating both health and disease ^3,4. Any qualitative and/ or quantitative change in the gut microbiota composition can alter the set equilibrium resulting in loss of microbiota balance, also known as dysbiosis ^5,6. With evidence of a causal link between microbiota composition and diverse immuno-compromised infectious and invasive diseases, the microbial repertoire has been a rapidly growing area of research of increasing public health importance in the host-microbe interactions.

Cholera is an acute dehydrating diarrheal disease that can result in death if not treated. Vibrio cholerae, the bacteria that causes cholera, is transmitted through contaminated drinking water and poor water, sanitation, and hygiene (WASH) infrastructure and practices in low-resource settings ⁷. Cholera remains a major public health problem in many parts of the world and can causes an estimated 4.0 million cases and 95,000 deaths annually ⁸. While reported from all around the globe, cholera infections are of primary importance in low -income countries where this disease is endemic due to the lack of safe drinking water and poor WASH infrastructure ⁹. Bangladesh continues to have a high disease burden of cholera ⁸ and related diarrheal diseases. Due to high population density and living in close proximity sharing food and drinking water, the household contacts of cholera patients are at a 100 times higher risk of developing the disease than the general population ^10–13.

In Dhaka, Bangladesh millions living in slum areas lack access to improved water sources ¹⁴; and they rely on a communal standpipe which is often an illegal connection to the municipal water supply and highly likely to be fecally contaminated ^15,16. Globally, unsafe drinking water and poor sanitation and hygiene are among the major contributors to diarrhea, resulting in over 829,000 deaths annually ¹⁷, with 19,464 of these deaths occurring in Bangladesh alone ¹⁸. Microbial safe drinking water remains key to save people from diarrhea and related deaths ¹⁹. Frequent use of unsafe water carrying multiple pathogens are responsible for recurrent diarrhea and environmental enteric dysfunction (EED), which is an acquired enteropathy of the small intestine characterized by enteric inflammation, villus blunting and decreased crypt-to-villus ratio that allows pathogenic bacteria to colonize and settle in the gut, as does good bacteria as commensals. EED has been associated with chronic malnutrition (stunting), wasting and reduced vaccine efficacy among children living in low-resource settings ²⁰. Frequent diarrhea (3–5 episodes/ year) and related microbiota dysbiosis ²¹ are responsible for growth faltering in children who reaches their adulthood as stunted and can have poor cognitive development ^22,23.

The household contacts of cholera patients are at potentially high risk of infection as V. cholerae can spread through drinking contaminated water and through person-to-person transmission. We conducted a randomized controlled trial (RCT) in Dhaka city slums of CHoBI7 household contact-children of index cholera patients and delivered a water, sanitation, and hygiene (WASH) intervention including chlorinated water (chlorine tablets) as part of the CHoBI7 to prevent cholera. As in the main RCT, we found drinking chlorine-treated water and handwashing by soapy water during key time points to be highly beneficial for the HHCs as overall V. cholerae infection was 47% less in intervention households ²⁴.

Chlorine is a WHO recommended disinfectant and is widely used for water treatment to prevent diarrhea and related infections ²⁵. In laboratory experiments, we found that the concentration of free chlorine required to inactivate 10⁵ colony-forming units (CFU)/mL of V. cholerae serogroups O1 and O139 were estimated to be 0.1 mg/L and 0.2 mg/L, respectively. The concentration of free chlorine generated by a single chlorine tablet (sodium dichloroisocyanurate [33 mg]) after a 30-minute reaction time in a 10-L sealed vessel containing Dhaka city municipal supply water was 1.8 mg/L; and the concentration declined to 0.26 mg/L after 24 hours ²⁶. Therefore, it was recommended that CHoBI7 program households use one chlorine tablet every 24 hours for household stored drinking water.

In this study, we investigated the gut microbiota composition of household children at baseline (Day 0) and one week after (Day 8) initiating delivery of the CHoBI7 program. The aim was to determine if delivery of the CHoBI7 WASH program including chlorine tablets would change microbial communities in the gut.

Study subjects

This study is part of the CHoBI7 trial ²⁴. Five children were included residing in the household of culture-confirmed cholera patients admitted in Dhaka icddr,b hospital between May to September 2014. All children resided in slum areas of Dhaka, Bangladesh. Three children were from Karail slum in Gulshan-1 ward of Dhaka, one child from Pirerbag, Mirpur and one child from Gabtoli (Fig. 1). All children in this analysis were 3–7 years of age. After enrolment of cholera patients at icddr,b hospital, the corresponding household contact of cholera patients were enrolled the same day or the following day. Household contacts were defined as those sharing the same cooking pot with the cholera patient for the past three days. CHoBI7 includes 1) a pictorial (“Chobi” in Bangla) module on how cholera can spread through the environment (e.g., contamination of household drinking water sources and stored water), how persons can spread cholera to each other by contaminating food and water in their home, and instructions on proper handwashing with soap and treatment of water; and 2) a cholera prevention package containing a 3-month supply of chlorine tablets (Aquatabs sodium dichloroisocyanurate; Medentech, Wexford, Ireland, UK) for water treatment, soapy water bottles (a low-cost alternative to bar soap made using detergent), a handwashing station, a sealed water vessel with cover to ensure safe water storage, and cue-to-action cards with instructions about promoted behaviors. A trained health promoter at Dhaka Hospital delivered this pictorial module and cholera prevention package to cholera patients and their accompanying family members during a consultation session in the hospital. These behavioral recommendations were reinforced through daily household visits by the health promoter for the 1-week intervention period. Fecal samples were collected from five household children of cholera patients at day 0 and at day 8. After collection of fecal samples, they were immediately preserved at − 20˚C freeze and stored until processing for DNA extraction. We collected a water sample from the household’s water source and drinking water stored in the home at each household visit to test for the presence of free chlorine, by using a digital colorimeter²⁶ as per manufacturer’s instruction (Hach, Loveland, CO, USA) which is a proxy measure of water treatment.

Extraction of total genomic DNA

DNA was extracted from fecal samples according to previously published methods ²⁷. 125 mg (wet weight) fecal sample was suspended in 625 µl breaking buffer [0.8 mol/L guanidinium isothiocyanate, 4% N-lauroyl sarcosine, 20 mmol/L Tris (pH 8.0), 80 mmol/L sodium phosphate buffer (pH 8.0)] and incubated for 1 hour at 70°C. Afterward, 750 µl glass beads 0.1mm in diameter (Sigma, St Louis, MO, USA) and 15 mg polyvinylpolypyrrolidone were added. Bacterial cells were lysed in a vortex mixer at high speed (10 cycles consisting of 1-minute vortexing and 1-minute storage in ice). The mixture was centrifuged at 20,000 g for 3 minutes at 4°C. After recovery of the supernatant, the pellet was washed 3 times with 200 µl TENP (50mm Tris-HCl [pH 8.0], 20mm EDTA [pH 8.0], 100mm NaCl, and 1% [w/v] polyvinylpolypyrrolidone). All the four supernatants obtained for a sample were pooled. Nucleic acids were extracted with one volume of phenol. The aqueous phase was washed twice by using chloroform-isoamyl-alcohol (24:1). DNA was precipitated using 100% isopropanol, and the pellet was washed with 70% v/v isopropanol, dried, and resuspended in 50 to 100 µl of sterile water and stored at -20°C. The amount and integrity of DNA were estimated by use of 1% (w/v) agarose gel electrophoresis containing ethidium bromide (1 mg/ml) in 1 X TBE (Tris Borate EDTA).

Universal primer PCR and Illumina MiSeq sequencing

Bacterial 16S ribosomal RNA gene targeted-sequencing was performed. The general bacterial 16S primers used were 341f (CCTACGGGNGGCWGCAG) and 805r (GACTACHVGGGTATCTAATCC), which amplified the V3-V4 region of the 16S rRNA gene. The sequencing library was prepared by following a published protocol ²⁸. The amplicon libraries were cleaned up with Zymo’s Select-a-Size DNA Clean & Concentrator™ (> 200 fragments were kept), quantified with Tape Station, normalized and pooled together. The final library was quantified with quantitative PCR and sequenced on Illumina MiSeq with v2 reagent kit (500 cycles). The sequencing was performed with > 10% PhiX mix and in paired-end mode.

Raw sequence reads were trimmed with Trimmomatic-0.33 ²⁹. The two paired end reads in each pair were assembled to construct a complete amplicon sequence with SeqPrep (https://github.com/jstjohn/SeqPrep). Chimeric amplicon sequences were identified and removed with Usearch (v. 6.1) ³⁰ in ref mode against a curated database (http://drive5.com/uchime/rdp_gold.fa). Amplicon sequences smaller than 320 bp were removed. For each sample, 50,000 sequences were randomly sampled to reduce potential bias caused by uneven sampling. These amplicon sequences were compiled, clustered and analyzed with QIIME 1.8.0 ³¹. Operational taxonomic units (OTUs) were picked by the workflow of pick_open_reference_otus.py script using GreenGene database (gg_13_8) as the reference database.

Statistical analysis

Data were analyzed using SPSS version 11.5 (LEAD Technologies Inc., Charlotte, NC, USA). Differences between time points were analysed for significance using t-tests. Data are presented as mean ± SEM.

In this study, the gut microbiota of children living in Dhaka city slums was investigated before (day 0) and after (day 8) chlorination of drinking water and handwashing with soap was promoted for seven days as part of a major RCT of cholera hospital-based WASH intervention (CHoBI7). A total of five household contact (HHC) children included in this study were aged between 3–7 years, and for this prior written consent was taken from parents or formal guardians of these children to collect fecal samples once before, and again after 7 days of drinking chlorine-treated water. The baseline characteristics of each of the children with the intervention package (materials) are listed in Table 1.

Table 1

Baseline characteristics of children included as household contacts of cholera patients.
Criteria	Children with cholera (n = 5)
Age (yr)	5.1 ± 1.9*
Male/Female	1/4
Use of chlorine treated drinking water during sample collection	Yes
Diarrhea during last 7 days	No
Antibiotic use in last 7 days	No
Socio-economic status	Moderate to low
mean ± SDmean ± SD

The average free chlorine in stored drinking water over the one-week surveillance period was found to be ≥ 0.66 mg/L in each household (Fig. 2). All above the cutoff value which is needed to deactivate cholera bacterium V. cholerae²⁶. None of the study children developed cholera or any other diarrhea infection during this one-week period, and they were in apparently healthy condition.

Illumina MiSeq sequencing of the microbial community DNA in stool samples, collected at day 0 (before drinking chlorine-treated water) found 90–95% of the sequences to be reflecting microbiota. The two most predominant bacterial phyla identified at day 0 include Bacteroidetes and Firmicutes, which showed the relative abundance (mean ± SEM %) of 62 ± 6 and 32 ± 7, respectively. The pattern observed at day 0 changed at day 8 when the relative abundance of bacteria belonging to phylum Bacteroidetes declined (39 ± 12), while those belonging to phylum Firmicutes rose (49 ± 12) and became predominant (p = 0.057) in all the study children. The Proteobacteria and Actinobacteria were the next predominant phyla showing the relative abundance of 4 ± 1 and 1 ± 0.7 at day 0; and 4.7 ± 1.9 and 2.3 ± 1.3 at day 8, respectively. The observed decrease in the number of bacteria belonging to the phylum Bacteroidetes after delivering the CHoBI7 WASH program was significant (p = 0.034). Bacteria belonging to phyla Euryarchaeota, Verrucomicrobia, Elusimicrobia, Fusobacteria, Spirochaetae, Synergistetes, Tenericutes, and Cyanobacteria did not significantly change their relative abundance as observed at day 0 and day 8.

When the top ten bacterial families were considered, substantial inter-individual variation in the dominant bacterial families was observed at day 0 (Fig. 3). The dominant bacteria belonging to the family Prevotellaceae were common for all the five individual children, and bacteria belonging to the family Ruminococcaceae, Streptococcaceae, Succinivibrionaceae, and Lactobacillaceae accounted for the second most predominant bacteria found in the gut of children at day 0.

Figure 3. Dominant bacterial groups in the gut of household children at day 0 and day 8 at the taxonomic level of family. Alphabets in the bar reflect bacterial families as indicated below: P, Prevotellaceae; E, Enterobacteriaceae; Le, Leuconostocaceae; R, Ruminococcaceae; La, Lachnospiraceae; St, Streptococcaceae; B, Bifidobacteriaceae; Pep, Peptostreptococcaceae; S, Succinivibrionaceae; Sp, Spirochaetaceae; V, Veillonellaceae; L, Lactobacillaceae.

Next, we considered families of bacteria belonging to the four major phyla Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria, and found significant differences in the gut microbiota profiles for all the five children at day 8. 65 bacterial families were confirmed at day 0. The bacterial families were reduced for all five children at day 8 and only 60 families were present with 6 families that were newly acquired. Total 54 families were common for all five children at both time points (day 0 and day 8) (Fig. 4).

Also, as the cohort of children represented household contacts of hospitalized cholera patients, bacteria belonging to the family Vibrionaceae were present in the gut of all five children at day 0, but not after the CHoBI7 WASH program delivery at day 8. Enterobacteriaceae, a family belonging to the phylum Proteobacteria, was common for all five children at the two-time points (day 0 and day 8). The six Proteobacteria families that were found at day 0, but not at day 8 included Vibrionaceae, Phyllobacteriaceae, Sphingomonadaceae, Erythrobacteraceae, Rhodocyclaceae, and Oxalobacteraceae.

Overall results revealed marked differences in the gut microbiota profile in children before and after the CHoBI7 WASH program delivery. When top three bacterial families belonging to the phyla Bacteroidetes, Firmicutes, and Actinobacteria were considered, they showed higher abundance at day 8 compared to that of the phylum Proteobacteria (Table 2).

Table 2

Effect of drinking chlorine-treated water on top three bacterial families among minor children in Bangladesh.
Child		Bacterial family
	Before intervention	Relative abundance (%)		After intervention	Relative abundance (%)
	Before intervention	Day 0	Day 08	After intervention	Day 08	Day 0
1	Prevotellaceae	44.3	2.8	Leuconostocaceae	28.4	12.4
	Ruminococcaceae	15.1	3.3	Streptococcaceae	20.0	0.9
	Leuconostocaceae	12.4	28.4	Lactobacillaceae	14.5	7.0
2	Prevotellaceae	54.8	11.8	Ruminococcaceae	32.0	6.3
	Streptococcaceae	11.5	1.6	Lachnospiraceae	24.3	9.5
	Lachnospiraceae	9.5	24.3	Prevotellaceae	11.8	54.8
	Prevotellaceae	84.7	67.9	Prevotellaceae	67.9	84.7
3	Succinivibrionaceae	4.4	1.4	Ruminococcaceae	10.8	2.8
	Ruminococcaceae	2.8	10.8	Lachnospiraceae	8.5	2.0
	Prevotellaceae	71.5	47.8	Prevotellaceae	47.8	71.5
4	Ruminococcaceae	12.3	11.3	Lachnospiraceae	17.4	7.5
	Lachnospiraceae	7.5	17.4	Spirochaetaceae	14.9	XX
	Prevotellaceae	54.1	62.3	Prevotellaceae	62.3	54.1
5	Lactobacillaceae	9.7	4.2	Veillonellaceae	12.7	4.0
	Streptococcaceae	8.4	0.9	Enterobacteriaceae	6.8	3.6
XX – Not presentXX – Not present

Families of anaerobic bacteria including Prevotellaceae, Bifidobacteriaceae, Lachnospiraceae, Clostridiaceae, and Veilloneleceae in the gut of children after receiving the CHoBI7 WASH program delivery. While these five families were widely distributed, showing high relative abundance at day 8 in all five children, the bacterial families– Lactobacillaceae and Ruminococcaceae remained unchanged; and members of the family Enterobacteriaceae increased in two of the five children receiving CHoBI7 WASH program delivery (Fig. 5).

Children receiving the CHoBI7 water treatment and handwashing with soap intervention in cholera patient households had significant reductions in harmful Proteobacteria and an increase in beneficial Firmicutes. The overall floral shift observed was uniform for all five of the children in the present study. These findings are likely attributed to pathogenic bacteria belonging to Proteobacteria being reduced with an increase of beneficial commensals occurring in healthy individuals. These promising findings further show the health benefits of the CHoBI7 program and build on our previous findings showing the CHoBI7 intervention significantly reduced cholera.²⁴

The chlorine concentration needed to deactivate pathogens in drinking water varies by setting and is based on source-water quality parameters such as turbidity³². In a study conducted among the present cohort, we found 0.1 mg/L of free available chlorine needed to deactivate V. cholerae and other gram-negative bacteria ²⁶. Consistent with the efficacy of this level of free chlorine concentration, which was promoted in our intervention, in households of intervention arm, no stored water samples had any detectable V. cholerae, and individuals had symptomatic cholera infections ²⁴. Likewise, our microbiota data showed no presence of bacteria belonging to the family Vibrionaceae in the cohort of children drinking chlorine-treated water at day 8. None of the study children developed cholera or any other diarrhea infection during this one-week period, and they were apparently in good health condition. In Dhaka city, most of the low-income groups collect drinking water from various sources, which are unreliable and highly likely to be contaminated with fecal bacteria, including V. cholerae, as reported by Rafique, et al., 2016 ³³. While disinfection of water by boiling is not an easy option for such marginal people, the low-cost point of use water treatment by chlorination appears to be an effective method to prevent them from cholera, as shown for other waterborne diseases in low-income countries ^34–37. Recent report showed possible contamination of drinking water in the household after collection as it presented much higher pathogen counts than that of supply water sources ³⁸. As per our study, chlorination could ensure post-collection safety of drinking water as the recommended residual chlorine will prevent recontamination of water during collection, transport, and storage at the household ^34,39. Nonetheless, chlorination of drinking water at the point of collection was shown to significantly reduce childhood diarrhea in low-income groups residing in urban settings with irregular water supply in Bangladesh ⁴⁰.

In a previous study, we have shown the multitude of multi-drug resistant pathogenic bacteria in the gut of young children in Bangladesh ⁴¹. The predominance of such pathogenic bacteria belonging to phyla Proteobacteria in the gut was associated with poor child growth ^42,43. Children in our cohort carried six families of bacteria belonging to phylum Proteobacteria, which included Vibrionaceae at day 0 in all five children, suggesting household level transmission of cholera. The observed dysbiosis of microbiota belonging to the six families at day 8, including Phyllobacteriaceae, Sphingomonadaceae, Erythrobacteraceae, Rhodocyclaceae, Oxalobacteraceae and Vibrionaceae resulted by drinking chlorine-treated water as part of handwashing with soap practices for seven days suggests the potential of WASH and safe water intervention to prevent cholera among the household members of the disease during the high-risk one-week period. Of 15 Proteobacteria families that were common in all five children before and after drinking chlorinated water, most (ten) showed very low relative abundance (< 0.001%), while the predominance of five families including Enterobacteriaceae, Pasteurellaceae, Campylobacteraceae, Helicobacteraceae, and Succinivibrionaceae reflected a high relative abundance in these children. This result appears consistent with the previously published report showing multiple pathogens in the gut of Bangladeshi children ⁴⁴, attributed presumably to contaminated drinking water and poor living condition in slums.

Bacteria belonging to the phylum Bacteroidetes are known to possess genes encoding enzymes for carbohydrate metabolism, which is known to help Bacteroides to colonize the human gut ⁴⁵. Conversely, the phylum Firmicutes includes bacteria that can influence the energy absorption from food; and it has been becoming increasingly evident that the members of the phyla Firmicutes are involved in energy resorption and obesity ^46,47. Bacteroidetes and Firmicutes are well known beneficial commensals. Firmicutes to Bacteroidetes ratio (F/B ratio) has been shown to be correlated with obesity and other diseases in human and amimal studies ⁴⁸.The observed increase of Firmicutes over Bacteroidetes at day 8 was positive in the present study is consistent with our previous study which showed a higher proportion of Firmicutes (43 ± 4; mean ± SEM %) over Bacteroidetes (33 ± 3) with healthy children in Bangladesh ⁴². A recent microbiota study carried out in Bangladesh showed tremendous success in improving child health by attracting beneficial gut microbiota via microbiota directed complementary foods (MDCF) ⁴⁹. Although the MDCF alone has succeeded in increasing weight for length Z score with positive changes in the level of some plasma proteins in study children, we would propose to combine MDCF study with the CHoBI7 WASH program to improve the gut microbiota composition of young children.

The major limitation of this study was the analysis of fecal samples from a small number of children. Nevertheless, the data presented in this study on the gut microbiota using next-generation sequencing is the first of its kind to provide a snapshot of the positive changes in gut bacterial community, which we observed in children receiving the CHoBI7 WASH program. Second, our study focused households with an index patient with cholera, and therefore may not be generalizable to other settings. Third, we cannot determine how much of the impact of intervention delivery was attributed to the water treatment versus handwashing with soap component of the intervention since both were combined. This should be explored in future studies.

The gut microbiota dysbiosis of harmful Proteobacteria and increase of beneficial Firmicutes in household children of an index cholera patient suggest that delivery of a WASH intervention promoting chlorination of drinking water and handwashing with soap can change microbial communities. Future studies are needed that investigate this association in our settings globally.

Ethics approval and informed consent

Informed consent was obtained from all study participants. All study procedures were approved by the research review and ethical review committee of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) and Institutional Review Board of the Johns Hopkins Bloomberg School of Public Health.

Acknowledgements:

This research was supported by the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, the Center for Global Health at Johns Hopkins University, USA, and the National Institutes of Infectious Diseases (NIID), Japan. We also pay our gratitude to Professor R Bradley Sack for his generous support for the study. icddr,b acknowledges the commitment of these collaborating institutes to its research efforts, and thanks the Governments of Bangladesh, Canada, Sweden, and the UK for providing core/unrestricted support. Authors acknowledge the support of hospital and laboratory staffs of icddr,b.

Author Contributions Statement:

MA, CMG, and SM conceived designed and supported the study. SM carried out the study involving SKIA, M-uR, KZR and MH. MA, SM, CMG, FZ, SIB, TP, TA, DS, IB, FTJ, AI, F-TJ, MS, KMSUR and HW were involved in the overall study in its implementation, data analysis, and manuscript writing. All authors read and approved the final manuscript.

Conflict of Interest Statement:

The authors declare that the research was conducted in the absence of any personal, professional or financial relationships that could be construed as a potential conflict of interest.

Ohland, C. L. & Jobin, C. Microbial activities and intestinal homeostasis: a delicate balance between health and disease. Cellular and molecular gastroenterology and hepatology, 1, 28–40 (2015).
Wu, H. J. & Wu, E. The role of gut microbiota in immune homeostasis and autoimmunity. Gut microbes, 3, 4–14 (2012).
Mai, V. & Draganov, P. V. Recent advances and remaining gaps in our knowledge of associations between gut microbiota and human health. World journal of gastroenterology: WJG, 15, 81 (2009).
Yan, F. & Polk, D. B. Commensal bacteria in the gut: learning who our friends are. Current opinion in gastroenterology, 20, 565–571 (2004).
Krogius-Kurikka, L. et al. Microbial community analysis reveals high level phylogenetic alterations in the overall gastrointestinal microbiota of diarrhoea-predominant irritable bowel syndrome sufferers. BMC gastroenterology, 9, 95 (2009).
Ley, R. E., Turnbaugh, P. J., Klein, S. & Gordon J. I. Human gut microbes associated with obesity. Nature, 444, 1022–1023 (2006).
Organization, W. H. Diarrhoeal disease Fact sheet.WHO library(2017).
Ali, M., Nelson, A. R., Lopez, A. L. & Sack, D. A. Updated global burden of cholera in endemic countries. PLoS neglected tropical diseases, 9, e0003832 (2015).
Lee, K. The global dimensions of cholera. Global change and human health, 2, 6–17 (2001).
Hughes, J. M. et al. Epidemiology of eltor cholera in rural Bangladesh: importance of surface water in transmission. Bulletin of the World Health Organization, 60, 395 (1982).
Weil, A. A. et al. Clinical outcomes in household contacts of patients with cholera in Bangladesh. Clinical infectious diseases, 49, 1473–1479 (2009).
Spira, W. M., Khan, M. U., Saeed, Y. A. & Sattar, M. A. Microbiological surveillance of intra-neighbourhood E1 Tor cholera transmission in rural Bangladesh. Bull World Health Organ, 58, 731–740 (1980).
Glass, R. I. et al. Seroepidemiological studies of EI Tor cholera in Bangladesh: association of serum antibody levels with protection. Journal of infectious diseases, 151, 236–242 (1985).
UNICEF. http://www.unicef.org/bangladesh/Urban_water_challenges_in_Bangladesh.pdf.. Urban Water Challenges in Bangladesh (2011).
Clasen, T., Saeed, T. F., Boisson, S., Edmondson, P. & Shipin, O. Household water treatment using sodium dichloroisocy anurate (NaDCC) tablets: a randomized, controlled trial to assess microbiological effectiveness in Bangladesh. The American journal of tropical medicine and hygiene, 76, 187–192 (2007).
Sirajul Islam, M. et al. Faecal contamination of drinking water sources of Dhaka city during the 2004 flood in Bangladesh and use of disinfectants for water treatment. Journal of applied microbiology, 103, 80–87 (2007).
WHO. Mortality and Burden of Disease from Water and Sanitation. Available at: http://www.who.int/gho/phe/water_sanitation/burden/en/.. (2016a).
WHO. Inadequate Water, Sanitation and Hygiene in Low- and Middle-Income Countries. http://apps.who.int/gho/data/view.main (2016b).
Esrey, S. A. Water, waste, and well-being: a multicountry study. Am J Epidemiol, 143, 608–623 https://doi.org/10.1093/oxfordjournals.aje.a008791 (1996).
Tickell, K. D., Atlas, H. E. & Walson, J. L. Environmental enteric dysfunction: a review of potential mechanisms, consequences and management strategies. BMC medicine, 17, 181 (2019).
Monira, S. et al. Metagenomic profile of gut microbiota in children during cholera and recovery. Gut Pathog, 5, 1 https://doi.org/10.1186/1757-4749-5-1 (2013).
Kar, B. R., Rao, S. L. & Chandramouli, B. Cognitive development in children with chronic protein energy malnutrition. Behavioral and Brain Functions, 4, 31 (2008).
Crookston, B. T. et al. Impact of early and concurrent stunting on cognition. Matern. Child Nutr, 7, 397–409 (2011).
George, C. M. et al. Randomized Controlled Trial of Hospital-Based Hygiene and Water Treatment Intervention (CHoBI7) to Reduce Cholera. Emerg Infect Dis, 22, 233–241 https://doi.org/10.3201/eid2202.151175 (2016).
WHO. Progress on Drinking Water and Sanitation: Special Focus on Sanitation. Available at:. http://www.who.int/water_sanitation_health/monitoring/jmp2008/en/ (2008).
Rashid, M. et al. Chlorination of Household Drinking Water Among Cholera Patients' Households to Prevent Transmission of Toxigenic Vibrio cholerae in Dhaka, Bangladesh: CHoBI7 Trial. The American journal of tropical medicine and hygiene, 95, 1299–1304 (2016).
Magne, F. et al. Low species diversity and high interindividual variability in faeces of preterm infants as revealed by sequences of 16S rRNA genes and PCR-temporal temperature gradient gel electrophoresis profiles. FEMS microbiology ecology, 57, 128–138 (2006).
Kozich, J. J., Westcott, S. L., Baxter, N. T., Highlander, S. K. & Schloss, P. D. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl. Environ. Microbiol, 79, 5112–5120 (2013).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data., 30, 2114–2120 (2014).
Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C. & Knight, R. UCHIME improves sensitivity and speed of chimera detection., 27, 2194–2200 (2011).
Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nature methods, 7, 335 (2010).
Lantagne, D. S. Sodium hypochlorite dosage for household and emergency water treatment. Journal-American Water Works Association, 100, 106–119 (2008).
Rafique, R. et al. Transmission of infectious Vibrio cholerae through drinking water among the household contacts of cholera patients (CHoBI7 trial). Frontiers in microbiology, 7, 1635 (2016).
Supply, W. U. J. W., Programme, S. M., Organization, W. H. & UNICEF. Progress on drinking water and sanitation: Special focus on sanitation (World Health Organization, 2008).
Arnold, B. F. & Colford, J. M. Jr Treating water with chlorine at point-of-use to improve water quality and reduce child diarrhea in developing countries: a systematic review and meta-analysis. The American journal of tropical medicine and hygiene, 76, 354–364 (2007).
Clasen, T. & Edmondson, P. Sodium dichloroisocyanurate (NaDCC) tablets as an alternative to sodium hypochlorite for the routine treatment of drinking water at the household level. International journal of hygiene and environmental health, 209, 173–181 (2006).
Clasen, T., Schmidt, W. P., Rabie, T., Roberts, I. & Cairncross, S. Interventions to improve water quality for preventing diarrhoea: systematic review and meta-analysis., 334, 782 (2007).
Mondal, D. et al. Diarrhoeal health risks attributable to water-borne-pathogens in arsenic-mitigated drinking water in West Bengal are largely independent of the microbiological quality of the supplied water., 6, 1100–1117 (2014).
Wright, J., Gundry, S. & Conroy, R. Household drinking water in developing countries: a systematic review of microbiological contamination between source and point-of‐use. Tropical medicine & international health, 9, 106–117 (2004).
Pickering, A. J. et al. Effect of in-line drinking water chlorination at the point of collection on child diarrhoea in urban Bangladesh: a double-blind, cluster-randomised controlled trial. The Lancet Global Health, 7, e1247–e1256 (2019).
Monira, S. et al. Multi-drug resistant pathogenic bacteria in the gut of young children in Bangladesh. Gut Pathogens, 9, 19 (2017).
Monira, S. et al. Gut microbiota of healthy and malnourished children in Bangladesh. Frontiers in microbiology, 2, 228 (2011).
Perin, J. et al. A Retrospective Case–Control Study of the Relationship between the Gut Microbiota, Enteropathy, and Child Growth.The American Journal of Tropical Medicine and Hygiene,tpmd190761(2020).
Subramanian, S. et al. Persistent gut microbiota immaturity in malnourished Bangladeshi children. Nature, 510, 417 (2014).
Larsbrink, J. et al. A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes. Nature, 506, 498 (2014).
Ley, R. E. et al. Obesity alters gut microbial ecology. Proceedings of the National Academy of Sciences 102, 11070–11075(2005).
Ley, R. E., Turnbaugh, P. J., Klein, S. & Gordon J. I. Microbial ecology: human gut microbes associated with obesity. Nature, 444, 1022 (2006).
Castaner, O. et al. The gut microbiome profile in obesity: a systematic review. International journal of endocrinology 2018 (2018).
Chen, R. Y. et al. A microbiota-directed food intervention for undernourished children. New England Journal of Medicine, 384, 1517–1528 (2021).

No competing interests reported.

Gut Microbiota Shifts Favorably with Delivery of Handwashing with Soap and Water Treatment Intervention in a Prospective Cohort (CHoBI7 Trial)

Status:

Version 1

Abstract

Figures

Introduction:

Materials And Methods:

Study subjects

Extraction of total genomic DNA

Universal primer PCR and Illumina MiSeq sequencing

Statistical analysis

Results:

Discussions:

Conclusion:

Declarations