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Micronutrient supplements with iron promote disruptive protozoan and fungal communities in the developing infant gut

06 Jul 2021-bioRxiv (Cold Spring Harbor Laboratory)-

Abstract: Supplementation with micronutrients, including vitamins, iron and zinc, is a key strategy to alleviate child malnutrition. However, adverse events resulting in gastrointestinal disorders, largely associated with iron, has resulted in ongoing debate over their administration. To better understand their impact on gut microbiota, we analysed the bacterial, protozoal, fungal and helminth communities of stool samples collected from children that had previously been recruited to a cluster randomized controlled trial of micronutrient supplementation in Pakistan. We show that while bacterial diversity was reduced in supplemented children, vitamins and iron may promote colonization with distinct protozoa and mucormycetes, whereas the addition of zinc ameliorates this effect. In addition to supplements, residence in a rural versus urban setting is an important determinant of eukaryotic composition. We suggest that the risks and benefits of such interventions may be mediated in part through eukaryotic communities, in a manner dependent on setting.
Topics: Micronutrient (56%), Malnutrition (53%)

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1
Title: Micronutrient supplements with iron promote disruptive protozoan and fungal
1
communities in the developing infant gut
2
3
Ana Popovic
1,2
, Celine Bourdon
3,4
, Pauline W. Wang
5,6
, David S. Guttman
5,6
, Sajid Soofi
7
,
4
Zulfiqar A. Bhutta
4,7
, Robert H. J. Bandsma
3,4
, John Parkinson
1,2,8,
* and Lisa G. Pell
4
5
6
1
Program in Molecular Medicine, Hospital for Sick Children Research Institute
7
2
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
8
3
Division of Gastroenterology, Hepatology and Nutrition, Hospital for Sick Children, Toronto,
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Ontario, Canada
10
4
Centre for Global Child Health, Hospital for Sick Children, Toronto, Ontario, Canada
11
5
Department of Cell & Systems Biology, University of Toronto, Toronto, Ontario, Canada
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6
Centre for the Analysis of Genome Evolution & Function, University of Toronto, Toronto,
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Ontario, Canada
14
7
Center of Excellence in Women and Child Health, the Aga Khan University, Karachi, Pakistan
15
8
Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
16
17
*To whom correspondence should be addressed:
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john.parkinson@utoronto.ca
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Keywords: Eukaryotic microbiota; Parasites; Malnutrition; Micronutrient Supplementation;
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Microbiome
22
.CC-BY 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 9, 2021. ; https://doi.org/10.1101/2021.07.06.451346doi: bioRxiv preprint

2
Abstract
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24
Supplementation with micronutrients, including vitamins, iron and zinc, is a key strategy to
25
alleviate child malnutrition. However, adverse events resulting in gastrointestinal disorders,
26
largely associated with iron, has resulted in ongoing debate over their administration. To better
27
understand their impact on gut microbiota, we analysed the bacterial, protozoal, fungal and
28
helminth communities of stool samples collected from children that had previously been recruited
29
to a cluster randomized controlled trial of micronutrient supplementation in Pakistan. We show
30
that while bacterial diversity was reduced in supplemented children, vitamins and iron may
31
promote colonization with distinct protozoa and mucormycetes, whereas the addition of zinc
32
ameliorates this effect. In addition to supplements, residence in a rural versus urban setting is an
33
important determinant of eukaryotic composition. We suggest that the risks and benefits of such
34
interventions may be mediated in part through eukaryotic communities, in a manner dependent on
35
setting.
36
37
38
.CC-BY 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 9, 2021. ; https://doi.org/10.1101/2021.07.06.451346doi: bioRxiv preprint

3
Introduction
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Malnutrition is a global health crisis with 149 million children stunted and 45 million children
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wasted under the age of five years
1,2
. With increased vulnerability to infection, undernourished
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children are at elevated risk of death, not least from diarrheal diseases
3,4
. Previous studies have
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demonstrated the role of gut microbiota in malnutrition, with microbiome immaturity (bacterial
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communities that are underdeveloped with respect to age) representing a key factor in disease
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development
5,6
. Beyond bacterial communities, parasites such as hookworm, Cryptosporidium and
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Entamoeba have also been associated with severe diarrheal disease and intestinal malabsorption
7,8
.
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However, much less is known regarding the role of other, potentially commensal, eukaryotic gut
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microbes in undernutrition. Of particular interest is their ability to interact with and alter bacterial
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communities. For example, indole-producing gut bacteria were found to confer protection against
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Cryptosporidium infection, while deworming treatments targeting helminth endemic communities
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reduced abundance of protective Clostridiales
9,10
. Mouse studies further showed that helminths
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and protozoa influence bacterial communities by modulating the host immune system
9,11,12
. While
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the number of published gut microbiome studies have increased rapidly over the last decade, few
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have explored the composition of eukaryotic gut communities and their potential interactions with
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bacteria. Previously, we applied 18S rRNA and internal transcribed spacer (ITS) sequence surveys
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to systematically characterize eukaryotic microbiota in severely malnourished Malawian children,
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and identified a high prevalence of protozoa, including commensals and pathobionts
13
. We
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furthermore associated Blastocystis colonization with increased gut bacterial diversity.
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Global health programs targeting vulnerable child populations include the use of micronutrient
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supplements, consisting of vitamins as well as essential minerals zinc and iron, that have been
61
.CC-BY 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 9, 2021. ; https://doi.org/10.1101/2021.07.06.451346doi: bioRxiv preprint

4
demonstrated to improve growth and reduce morbidity
14-16
. Such supplements are thought to
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address deficiencies that can impair immune responses to infectious pathogens and impact gut
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bacterial communities
17-20
. While beneficial, supplementation, especially with iron, may also
64
promote unintended pathogen growth, particularly where the host is unable to restrict
65
micronutrient bioavailability
21
. For example, it has been shown that surplus iron promotes the
66
growth of enteropathogens and induces intestinal inflammation in infants
22,23
. Furthermore, while
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known to reduce the duration of childhood diarrheal episodes, zinc supplementation has been
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associated with increased duration of Entamoeba histolytica infections
24,25
.
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In an attempt to understand the impact of micronutrient supplementation on the complex
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interactions between eukaryotic and bacterial microbiota in the maturing infant gut and health, we
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performed 18S rRNA and 16S rRNA amplicon surveys on stool samples obtained at 12 and 24
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months of age from 80 children, previously recruited as part of a cluster randomized trial conducted
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in Pakistan. The trial was designed to investigate the impact of micronutrient powders (MNP)
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containing vitamins and iron with or without zinc on growth and morbidity, and has shown an
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excess of significant diarrheal and dysenteric episodes among children receiving MNPs
26
.
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Microbial profiles were analysed in the context of supplementation, nutritional status, age and
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place of residence (i.e., urban or rural) to reveal a complex landscape of associations with microbial
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diversity, as well as specific taxa.
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Results
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Description of cohort
83
.CC-BY 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 9, 2021. ; https://doi.org/10.1101/2021.07.06.451346doi: bioRxiv preprint

5
A total of 80 children (160 paired stool samples at 12 and 24 months of age) from all three
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supplementation arms in the parent cRCT
26
(control (n=24), MNP (n=29), and MNP with zinc
85
(n=27)) conducted in Sindh, Pakistan were selected based on sample availability for inclusion in
86
this study (Supplementary Fig. 1). The cohort includes children from both urban (Bilal colony)
87
and rural (Matiari district) study sites (Fig. 1a). Children were stratified by weight-for-length z-
88
scores (WLZ) at 24 months into a reference WLZ (WLZ >-1) or undernourished (WLZ < -2) group.
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Subject characteristics are summarized in Table 1. The WLZ growth trajectories of the children
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selected as the reference WLZ group approximately tracked the upper 50th percentile of the
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original cohort, while the undernourished group started around the lower 50th percentile and
92
gradually dropped over time ending at the bottom 80
th
percentile of the cohort (Fig. 1b). This drop
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in the WLZ of the undernourished children was driven by poor weight gain (Supplementary Fig.
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2).
95
96
The developing infant gut is colonized by complex eukaryotic communities
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We applied 18S rRNA amplicon sequencing to profile the eukaryotic communities in all 160 stool
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samples. We generated a total of 11,639,233 paired 18S rRNA amplicon sequence reads (median
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70,642) of which 4,386,494 could be classified as a eukaryotic microbe (median 22,932;
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Supplementary Table 1). From these we identified a total of 859 eukaryotic OTUs (median 66;
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Supplementary Table 1), which included 438 protozoan, three helminth and 418 fungal OTUs (Fig.
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2a). Fungi, dominated by Mucoromycota and Ascomycota, accounted for 71% of all reads. The
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most abundant were species in the Candida-Lodderomyces clade, Saccharomyces, and taxa
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increasingly associated with rare but fatal infections known as mucormycoses: Rhizomucor,
105
Actinomucor and Lichtheimia. Alveolates accounted for 25% of reads, with
106
.CC-BY 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 9, 2021. ; https://doi.org/10.1101/2021.07.06.451346doi: bioRxiv preprint

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