Impact of Intestinal Parasites on Microbiota and Metagenomic Influences on Gene Encoding Cobalamin Pathway

Background: Approximately 30% of children worldwide are infected with gastrointestinal parasites. Depending on the species, parasites can disrupt intestinal bacterial flora affecting nutritional status. We implemented a multi-parallel quantitative real-time PCR and whole-genome sequencing analysis for bacterial microbiota and Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Trichuris trichiura , Cryptosporidium species , Entamoeba histolytica, and Giardia lamblia. Stool samples were collected from 37 asymptomatic children from rural Argentina. We focused this study on Giardia screening. Separate analyses were done for uninfected, Giardia only, Giardia /helminth coinfections, and helminth only groups. Results: For Giardia only infected children, DNA sequencing data showed a decrease in microbiota biodiversity compared to those uninfected that correlated with increasing Giardia burden. Clustering was statistically significant using Shannon alpha diversity. A non-significant increase in diversity was observed for helminth only infections with a compensatory decrease in Giardia /helminth coinfections. In Giardia only infections, microbiome taxonomy shifted from Firmicutes towards increasing proportions of Prevotella , with the degree of shift related to the intensity of infection compared to uninfected. An abundance of Prevotella bacteria was decreased in the helminths only group but increased for Giardia /helminth coinfections. Metagenomic analysis of the bacterial microbiota showed that the proportion of non-vitamin B12 producing bacteria ( Prevotella ) was higher in the Giardia group. Total cobalamin synthesis was decreased in the Giardia -infected only group compared to the control and helminth-infected group. Conclusion: Our data provide evidence for an effect of parasitic infections allowing permissive growth of anaerobic bacteria such as Prevotella , suggesting an altered capacity of vitamin B12 biosynthesis and potential impact on growth and development in children.

3]. Symptoms include chronic diarrhea, severe anemia, and can lead to intestinal obstruction.
Economically disadvantaged children have recurrent infections leading to growth and cognitive delays due to malnutrition [4]. These children have more difficulties in school and, subsequently, in the job market [4]. The cycle continues when they remain in poverty and have their children [4]. The link between intestinal helminths and malnutrition leading to growth stunting and anemia has been found by others [5][6][7][8][9][10][11][12], and a new Global Burden of Disease Study points to evidence that hookworm is a leading cause of anemia in resource-poor settings [13]. Valuable information from the Global Enteric Multicenter Study (GEMS) also reveals an unexpected global health impact caused by protozoa [14,15]. There are few studies attributing giardiasis to growth delays and no published studies showing the impact on the human intestinal microbiome using multi-parallel real-time quantitative (qPCR) detect the presence of Giardia and quantitating the burden of infection [16]. To date, most studies examining intestinal parasitism have not been able to study intestinal worms and protozoa simultaneously and to successfully dissect the relative contribution of each of the significant intestinal helminth or protozoan pathogens to specific diseases. The current state of diagnosing GI parasites uses the subjective method of microscopy. Depending on the parasite, single stool microscopy exam sensitivity ranges from 50-80% [17]. As a result, large numbers of infected children are not being diagnosed and treated.
With advances in Next Generation DNA sequencing, we are now able to determine a broader range of intestinal microbiota. The microbiome is associated with digestion, nutrition, and health, but There is increasing evidence that specific GI parasites (Ascaris) may increase the biodiversity of intestinal microbiota [21]. Since the burden of parasitic infection is directly correlated to morbidity and disease, the load may also impact the intestinal microbiota [21]. Our qPCR quantitates the burden of helminths and protozoa, determining the correlation of burden to changes in intestinal microbiota biodiversity.
Alterations in intestinal microbiota alter bacterial metabolisms, such as vitamin B12, reducing the capacity for human use. Unique to only bacteria is the synthesis of vitamin B12 (cobalamin) [22][23][24].
Vitamin B12 is a crucial coenzyme for humans cannot produce it, and it must be derived exogenously [25].

Bacterial overgrowth
In Giardia infections, microbiome analysis data shows a decrease in biodiversity in the infected parasite group compared to those non-infected, a bias toward increased Prevotella, with the degree of change related to the intensity of infection ( Figure 4). Giardia-infected children had significantly higher proportions of the genus Prevotella bacteria directly correlating to above 1 fg/µl Giardia DNA versus No Parasite group (p = 0.037). Comparing all the cohorts revealed the helminth-infected group with decreased Prevotella proportions to Giardia-infected (p = 0.024), but similar to the control group.
All four groups had different bacteria genera as their most abundant microbiota. Giardia infected children, including Giardia/helminth coinfection, had higher Bacteroidales, including Prevotella species ( Figure 3).

Metagenomics of Cobalamin Biosynthesis
Vitamin B12 InterPro identifier IPR002751 biosynthesis CbiM family was used in the analysis. High Giardia infection had fewer quantities of cobalamin DNA sequences than the No Parasite group (p = 0.002) ( Figure 4A). Children with Giardia and Giardia/Ascaris infections have decreased the proportion of vitamin B12 pathway DNA sequences, compared to children with no parasites (p = 0.021) ( Figure   4B).

Discussion
The impact of Giardia on diversity was seen in this study using whole-genome sequencing. While diarrhea caused by giardiasis can reduce the brush border layer in small intestinal cells, the subjects in this cohort were all asymptomatic at the time of stool collection. Giardia likely has a more prominent role, since its primary site of infection and replication is in the small intestine.

Conclusions
In this study, we propose a possible link on why Giardia and other parasites may cause growth and developmental delays in infected children. Evidence of Giardia's impact on diversity and available micronutrients is observed with the Giardia/helminth coinfection group, where alterations of the microbiome are seen in the helminth only group.
While the helminth-infected group did not have changes in diversity or decreased cobalamin synthesis genes, a possible explanation is that most of these helminths reside in the colon and do not alter the microenvironments as does Giardia.
We are currently extending these results to additional populations where Giardia and other intestinal parasite infections are high and are performing, confirming the biochemical analysis of the vitamin B12 pathway products in affected children.

Methods
This study aimed to determine the impact of intestinal parasites on bacterial microbiota and subsequent cobalamin metagenomics.

Study population
Samples were selected from a previously published study using qPCR in peri-urban Argentina[26].
Samples were randomized and selected as representatives of four cohorts, (1) a control group with no parasites detected by qPCR, (2) a Giardia only infected group, (3) a helminth, and Giardia coinfection group, and (4) a helminth only infected group. Helminths included in this study were either Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, and Strongyloides stercoralis (Table 1).

Multi-parallel real-time quantitative PCR
All stools were collected and immediately stored in ice and sealed in air-tight containers, frozen within . STAMP software was used for statistical analysis pertaining to taxonomic and metagenomic differences [54]. Microbial attributes were derived from Livermore Metagenomics Analysis Toolkit (LMAT) taxonomic output using Megan program ( Figure 5).

Statistics
qPCR results were recorded for each patient as positive or negative for each parasite and concentration of parasite DNA. All statistics were performed using Prism v. 7.0b (GraphPad, La Jolla, CA). Kruskal-Wallis test was used to compare multiple groups. Spearman rank to correlate parasite concentrations to Shannon alpha diversity. The Shannon alpha diversity is a commonly reported diversity metric that weights the numbers of species by their relative evenness data [55]. All statistical models used p values less than 0.05 as significant.    Flow chart for bioinformatics and data processing.