Gut bacteria influence Blastocystis sp. phenotypes and may trigger pathogenicity

Arutchelvan Rajamanikam; Mohd Noor Mat Isa; Chandramathi Samudi; Sridevi Devaraj; Suresh Kumar Govind

doi:10.1371/journal.pntd.0011170

. 2023 Mar 29;17(3):e0011170. doi: 10.1371/journal.pntd.0011170

Gut bacteria influence Blastocystis sp. phenotypes and may trigger pathogenicity

Arutchelvan Rajamanikam ^1,^*, Mohd Noor Mat Isa ², Chandramathi Samudi ^3,^*, Sridevi Devaraj ^4,^*, Suresh Kumar Govind ^1,^*

Editor: Matthew Brian Rogers⁵

PMCID: PMC10057785 PMID: 36989208

Abstract

Whilst the influence of intestinal microbiota has been shown in many diseases such as irritable bowel syndrome, colorectal cancer, and aging, investigations are still scarce on its role in altering the nature of other infective organisms. Here we studied the association and interaction of Blastocystis sp. and human intestinal microbiota. In this study, we investigated the gut microbiome of Blastocystis sp.-free and Blastocystis sp. ST3-infected individuals who are symptomatic and asymptomatic. We tested if the expression of phenotype and pathogenic characteristics of Blastocystis sp. ST3 was influenced by the alteration of its accompanying microbiota. Blastocystis sp. ST3 infection alters bacterial composition. Its presence in asymptomatic individuals showed a significant effect on microbial richness compared to symptomatic ones. Inferred metagenomic findings suggest that colonization of Blastocystis sp. ST3 could contribute to the alteration of microbial functions. For the first time, we demonstrate the influence of bacteria on Blastocystis sp. pathogenicity. When Blastocystis sp. isolated from a symptomatic individual was co-cultured with bacterial suspension of Blastocystis sp. from an asymptomatic individual, the parasite demonstrated increased growth and reduced potential pathogenic expressions. This study also reveals that Blastocystis sp. infection could influence microbial functions without much effect on the microbiota diversity itself. Our results also demonstrate evidence on the influential role of gut microbiota in altering the characteristics of the parasite, which becomes the basis for the contradictory findings on the parasite’s pathogenic role seen across different studies. Our study provides evidence that asymptomatic Blastocystis sp. in a human gut can be triggered to show pathogenic characteristics when influenced by the intestinal microbiota.

Author summary

Single-cell eukaryotes in the intestinal microbiota are increasingly gaining attention for their ability to influence microbiota composition. Blastocystis sp. is no exception. This study for the first time demonstrates gut microbiota alteration due to the colonization of Blastocystis sp. ST 3 in symptomatic and asymptomatic conditions. Colonization of Blastocystis sp. ST3, regardless of symptoms, significantly alters the diversity and microbiota composition. The abundance of Prevotella sp. was significantly elevated in symptomatic Blastocystis sp. carriage. Inferred metagenomic findings revealed that predicted metabolic functions were altered in Blastocystis sp. carriage in symptomatic and asymptomatic conditions. The current study demonstrates a bidirectional influence that seems to be crucial in Blastocystis sp.–microbiota interaction. Altering the accompanying microbiota of a symptomatic Blastocystis sp. with bacterial suspension from an asymptomatic condition resulted in the protozoan exhibiting asymptomatic characteristics. This implies, for the first time the effect of accompanying microbiota on Blastocystis sp. phenotypic characteristics. The findings lead to a postulation that a harmless protozoan parasite can be turned harmful by its accompanying microbiota in the gut.

Introduction

The gut microbiota is composed of not only prokaryotes; but also, certain eukaryotes, most notably, the intestinal protozoans that pose a serious health burden in developing countries. Recent studies suggest that although the gut microbiota is diverse in species, the temporal fluctuation of certain microbial species (denotes instability) commonly occurs [1,2]. Whether this fluctuation in gut microbiota exerts an influence on other eukaryotic inhabitants of the gut is not much explored.

Blastocystis sp. is an intestinal protozoan parasite that has been frequently associated with general gastrointestinal symptoms, colorectal cancer (CRC), and irritable bowel disease (IBS) [3]. Despite its high prevalence, Blastocystis sp. has unresolved controversies regarding its pathogenicity. It was considered as a harmless commensal with recent findings implying that there is a possibility of the existence of two variant forms i.e. disease (pathogenic) and non-disease (non-pathogenic) causing types in a single subtype. Blastocystis sp. has been reported to exhibit strong interaction with its accompanying microbiota [4]. Studies have reported on increased diversity of bacteria in Blastocystis sp. colonized gut [5,6] and strong association of specific subtype with gut microbial composition [7]. However, there is a paucity of studies on the influence of gut microbiota by a single and most prevalent subtype (ST3) of Blastocystis sp. isolated from symptomatic and asymptomatic individuals as well as the influence of varying accompanying microbiota on this intestinal protozoan cells.

Some of the common intestinal protozoan parasites that are pathogenic to human gut include Entamoeba histolytica and Giardia duodenalis. According to epidemiological data, colonization of these eukaryotic pathogens may not necessarily result in the manifestation of symptoms [8]. Some studies have posited that the association of certain bacteria resulted in increased pathogenicity and protective effect in the infection of Giardia sp. [9–11] and increased virulence in Entamoeba histolytica [12,13]. It is unknown if a similar mechanism occurs with Blastocystis sp. infection. Whether the microbial environment could influence shaping the parasite’s characteristics, thus altering the severity of infection is still a question.

In this study, we investigated the gut microbiota profiles in symptomatic and asymptomatic individuals with or without Blastocystis sp. ST3 infection. Subsequently, we altered the microbiome of Blastocystis sp. in in vitro culture to understand the response of the parasitic cell towards different microbiota.

Methods

Ethics statement

A verbal and written consent was obtained from all participants recruited. The study procedure was approved by University Malaya Medical Centre (UMMC) Medical Research Ethics Committee (MRECID: 201914–6975).

Stool sample collection and Blastocystis sp. carriage assignment

A total of 50 fecal samples were studied, with one fecal sample collected each from 50 individuals who participated. Twenty-eight individuals who did not experience any gastrointestinal illness were grouped as asymptomatic. These individuals were recruited from a voluntary stool survey. The balance of 22 was patients visiting the Gastroenterology Unit of University Malaya Medical Centre (UMMC) and Gastroenterology and Hepatology Specialist Clinic of Pantai Medical Centre, Kuala Lumpur, Malaysia. These patients, who experienced frequent non-specific gastrointestinal symptoms such as bloating, abdominal cramps, loose stool, and diarrhea at the time of recruitment were grouped as symptomatic. Both the symptomatic and asymptomatic individuals were sub-grouped into Blasotcystis sp.-infected and Blastocystis sp.-free groups. Only participants with Blastocystis sp. as the sole infective agent were recruited (for all Blastocystis sp.-infected individuals). For all the Blastocystis sp.-free individuals, the fecal specimens were screened to ensure no other parasitic infection was detected. Screening for other parasites was done using the formal-ether concentration technique as described previously [14]. Patients diagnosed with colorectal cancer (CRC), inflammatory bowel disease (IBD), or irritable bowel syndrome (IBS) and those who have consumed antibiotics within the last 30 days were excluded from participating. The clinicians obtained prior written consent before the recruitment of participants. The clinicians also confirmed the diagnosis of these individuals after a thorough examination. To maintain homogeneity in population and environment, only individuals from the most developed part of Malaysia (Kuala Lumpur) and who belonged to a high socio-economic group were included. This study was approved by the Medical Research Ethics Committee of UMMC (201914–6975). Stool samples collected in screw-capped containers were processed and stored at -20°C within 6 hours of collection.

Fecal DNA extraction

DNA extraction from fecal materials was carried out using MACHEREY-NAGEL NucleoSpin Soil kit (MACHEREY-NAGEL GmbH & Co. KG, Dü ren, Germany). The SL2 buffer was used with Enhancer SX in the lysis step of extraction. DNA was eluted in a final volume of 50 μl and stored at -80°C.

Patient and public involvement

Patients and the public were first involved during fecal sample collection and questionnaire administration. Recruited individuals were either identified by the healthcare professional or upon voluntary admission. All research questions and outcome measures were approved by the Medical Research Ethics Committee of UMMC and were explained to each participant in detail by the enumerators and the clinicians. There was no involvement of the patient or the public in the design of this study. The participants agreed verbally to have their results published.

Amplification of variable 3 (V3) and variable 4 (V4) region of 16S ribosomal RNA (rRNA) genes

The primer pair sequences that produced a single amplicon for V3 and V4 region of approximately 460 bp were used as described [15]. The library preparation was done according to the study. The amplification process was carried out using the 2X KAPA HiFi HotStart Ready Mix with microbial genomic DNA at a concentration of 5ng/μl in 10mM. The amplification was carried out with thermal cycling consisting of 95°C for 3 minutes, followed by 25 cycles of 95°C (30 seconds), 55°C (30 seconds), and 72°C (30 seconds) with a final extension of 72°C for 5 minutes. Subsequently, a dual index barcode Illumina sequencing adaptor was attached to the amplicon using Nextera XT Index Kit (Illumina). Prepared amplicons were cleaned up again using AMPure beads. The V3 and V4 regions were then sequenced on an Illumina MiSeq platform (Illumina, San Diego CA, USA) at the Texas Children’s Microbiome Center.

Quality filtering and analysis of filtered reads

The raw sequences were joined at the paired-end by trimming the low-quality bases. All sequences were imported and analyzed through Quantitative Insights Into Microbial Ecology 2 (QIIME 2 Version 2020.6) platform [16]. The paired-end sequences were joined, chimeric sequences filtered and low-quality reads were removed using DADA2 plug-in [17].

Analysis of filtered reads sequence diversity

Quality filtered reads were used as the sequence data. The Operational Taxonomic Units (OTU) abundance was identified and quantified with reference to the Greengenes reference database version 13_5 with a 97% homology cut-off (https://greengenes.secondgenome.com/). We generated a phylogenetic tree by first performing multiple sequence alignment of the sequence using the mafft [18]. Next, highly variable regions that add noise to the tree were removed and the phylogenetic tree was built using FastTree [19]. This pipeline was carried out in QIIME2.

The alpha diversity was measured using the Shannon index metrics, richness and observed OTUs. Alpha rarefaction curve was plotted after samples were rarefied to 4000 sequences per sample. Beta diversity was computed using Bray-Curtis distance matrix ordinated using non-metric multidimensional scaling (NMDS) and Principal Coordinate Analysis (PCoA).

Linear Discriminant analysis effect size (LEfSe) and predicting the functional composition of a metagenome

LEfSe algorithm from the Galaxy web application (https://huttenhower.sph.harvard.edu/galaxy/) was used to identify taxa/gene/pathways with differential abundance from different experimental classes. In this study, we have used the symptomatic and asymptomatic groups as main classes and Blastocystis sp. infection status as subclasses. LEfSe lists the taxa that are differential among the classes with statistical and biological significance and ranks them according to effect size [20].

Predicted functional metagenome was developed using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). An OTU table was built using closed-reference clustering method by comparing each OTU representative to the Greengenes database version 13_5 at 97% cutoff. The resulting OTU table was used for metagenome prediction in Galaxy web application using KEGG orthology classification schemes [21]. Subsequently LEfSe was used to compare differential predicted metabolic functions between the classes.

In vitro cultivation and genotyping of Blastocystis sp.

About 50 mg of fecal samples were inoculated into 3 ml Jones medium supplemented only with 10% horse serum as reported previously [22]. There were no antibiotics added to the medium to maintain the original bacterial composition. The cultures were incubated at 37°C and screened daily for 5 to 7 days. The presence of Blastocystis sp. vacuolar forms was regarded as a positive sample. The xenic parasite cell culture was maintained in vitro and passaged every 3 to 4 days. Basic aseptic techniques were maintained throughout the culture process.

DNA was extracted from Blastocystis sp. in vitro cultures using Macherey Nagel Soil DNA extraction kit. Extracted DNA was used as a template to amplify and sequence the 18S small subunit ribosomal RNA gene (18S SSU-rDNA) at the length of 600bp using the protocols and primers described previously [23]. Five isolates of Blastocystis sp. were selected randomly to represent the symptomatic and asymptomatic groups for subsequent analysis.

Alteration of microbiota surrounding Blastocystis sp.

Cells from three-day-old Blastocystis sp. (isolated from asymptomatic individuals) culture grown in 3 ml of Jones’ medium supplemented with only 10% horse serum were spun at 1000 rpm to sediment Blastocystis sp. cells. No antibiotics were added to the Jones’ medium. The supernatant containing mostly bacteria was isolated and washed with distilled water three times to lyse any remaining Blastocystis sp. cells. This bacterial suspension was then centrifuged at high speed and the pellet was re-suspended in 100 μl of Jones’ medium with 10% horse serum.

Three-day-old Blastocystis sp. cells from symptomatic individuals were washed three times in PBS and counted to a concentration of 1 x 10⁵ cells/ml in a final volume of 900 μl of Jone’s medium. The bacterial suspension extracted earlier from asymptomatic Blastocystis sp. cultures were added and allowed to incubate at 37°C. These steps were repeated every 3–4 days for 5 times before assessing the growth characteristics. The cells were then harvested and subjected to downstream processing. The experiment was repeated with asymptomatic Blastocystis sp. cell co-cultured with bacteria from symptomatic cultures. Four replicates were used in this experiment. Throughout this experiment, the xenic cultures that received bacterial suspension were regarded as co-cultured. Control experiments were those xenic cultures that were added with only sterilized Jones medium replacing bacterial suspension.

Phenotypic characterizations

Growth characterization

Three-days old cells from co-cultured and control experiments were counted using heamocytometer chamber and inoculated into 1 ml medium with a final concentration of 1 x 10⁵ cells/ml. The cells were counted every day for 10 days using trypan blue exclusion test to determine number of viable cells. The number of granular formation and amoebic formation per ml were counted.

Colorimetric protease quantification assay

Parasite isolates from co-cultured and control experiments were subjected to purification in order to recover parasites cells with minimal bacterial contamination. Purification was done through density-gradient centrifugation as described previously but with slight modification on the centrifugation speed [24]. The protease activity of the minimalized bacterial contamination was ensured to be at a negligible level as noted in the previous study [24]. The solubilized antigen of the purified Blastocystis sp. was extracted using the freeze-thaw technique. The purity and concentration were determined using Bradford Protein Assay (BioRad). The concentration of each sample was standardized to 0.1 mg/ml using a filter sterilized Jones medium before the assay. The specific protease activity was determined using azocasein colorimetric assay as reported previously [25,26].

Colon cell proliferation analysis

The antigen extracted previously were used to study colon cells proliferation. HCT 116 colonic cancer cells were obtained from American Type Cell Culture (ATCC) and maintained in RPMI medium supplemented with L-Glutamine, antibiotics and 10% fetal bovine serum (FBS). Colon cancer cells were maintained in T-25 vented culture flask at 37°C, 5% CO₂ and passaged every 4 to 5 days. To assess the proliferation, cells were standardized and seeded to 1000 cells/well in 100 μl using a 96-well plate as described by previous studies [27,28]. The cells were allowed to incubate at 37°C, 5% CO₂ for 24 hours. The antigens extracted earlier were added to the cells and incubated for another 48 hours. Cell proliferation was then determined using the MTT assay as described by the study cited above.

Statistical analysis

The difference between groups was evaluated by comparing the mean using statistical tests. A student’s t-test was conducted to compare the difference in alpha diversity and richness between groups using values derived from the diversity index. The student’s t-test was also used to compare means of protease levels and cell proliferation. A p-value less than 0.05 is considered significant.

Results

Microbial profile analysis

A mean of 57828 sequences per sample from 50 individuals who were symptomatic (22/50) and asymptomatic(28/50) was obtained. The sequences were deposited in the National Library of Medicine (NCBI) as a BioProject with accession number PRJNA881789. A total of 1725 unique features were identified. The core phyla across the samples were Firmicutes and Bacteroidetes with more than 50% of relative abundance followed by Actinobacteria and Proteobacteria. At the genus level, the core genera observed across all samples were Prevotella, Feacalibacterium, Bifidobacterium, Bacteroides, and Dialister (S1 and S2 Figs).

Variation of microbiota in symptomatic and asymptomatic individuals and effect of Blastocystis sp. colonization

Using the Shannon diversity index, significantly higher species diversity was observed in asymptomatic individuals (P = 0.028). The alpha rarefaction curve (at 5000 reads/sample) indicated that greater number of features seen in samples of asymptomatic individuals. Coverage index indicates the number of most abundant species occupying 50% of the community ecosystem. In this study, asymptomatic individuals showed significantly greater coverage compared to symptomatic individuals (P = 0.038). The evenness of the samples was measured using Pielou’s evenness index. There was lower evenness seen in symptomatic individuals (P = 0.017). Beta diversity analysis through ordination by Non-metric multidimensional scaling (NMDS) using Canberra distance matrix showed clustering of symptomatic and asymptomatic samples (Stress = 0.17). The difference in diversity between samples was significant using PERMANOVA (P = 0.026) (Fig 1).

Fig 1 — A Shannon diversity index showing higher diversity within gut flora of asymptomatic individual. B Pielou evenness and Coverage between the gut flora of symptomatic and asymptomatic individual. C Non-metric multidimensional scaling of gut microbial profile from symptomatic and asymptomatic individual ordinated based on Canberra distance matrix.

When the samples were generally classified into Blastocystis sp. infection status, we saw a significantly lower abundance of species in Blastocystis sp.-infected individuals using Chao1 richness estimator (P = 0.032) (Fig 2A). When the samples were grouped according to symptom, a significant difference in the abundance of species was only seen in asymptomatic individuals (P = 0.00026) while in the symptomatic group, there were no significant alterations to the species richness due to Blastocystis sp. infection (Fig 2B). Plotting of distance matrix using Canberra distance with the incorporation of abundance value revealed that infection of Blastocystis sp. regardless of symptoms has significant alterations in the gut microbiota (Fig 2C).

Fig 2 — A Chao1 diversity index showing higher diversity within gut flora of *Blastocystis* sp.-negative individual. B Chao1 comparing diversity in *Blastocystis* sp. infection in symptomatic and asymptomatic conditions. C NMDS plot including 95% confidence interval ellipses explaining 10% variation between groups (ADONIS: P<0.05, R² = 0.01). Each point represents samples ordained with the incorporation of abundance data of taxa up to species level using the Canberra distance matrix. The sample size of the groups is as following: BlastoNegative_Asymptomatic = 12;BlastoNegative_Symptomatic = 13; BlastoPositive_Asymptomatic = 16;BlastoPositive_Symptomatic = 9.

Differential association of bacterial taxa to Blastocystis sp. infection in symptomatic and asymptomatic individual

LEfSe was deployed to determine the bacterial taxa that was differentially present in symptomatic and asymptomatic conditions and in Blastocystis sp.-positive and Blastocystis sp.-negative subjects. In this study, logarithmic LDA score of 3.0 was used as cut-off for the detection of important taxonomic differences. We found that Prevotella sp. was differentially abundant in symptomatic subjects while in asymptomatic group, bacterial phyla belonging to Firmicutes, Bacteroidotes, Verrucomicrobiota and Desulfobacteriota was abundant with LDA score beyond the fixed cut-off value (Fig 3A).

Fig 3 — Differentially abundant bacteria taxa in A Gut microbiota of symptomatic and asymptomatic individuals. B Gut microbiota of symptomatic individuals with and without *Blastocystis* sp. C Gut microbiota of asymptomatic individuals with and without *Blastocystis* sp.

Within the symptomatic group, subjects that were Blastocystis sp.-positive showed that the family Prevotellaceae and Ruminococcceae were abundant whereas Blastocystis sp.-negative subjects showed an abundance of Akkermansia sp. and Bacteroides sp. (Figs 3B and S3). However, in the asymptomatic group, taxa from the phylum Firmicutes, specifically Megasphaera sp. and Butyricicoccaceae were differentially abundant in Blastocystis sp.-positive subjects while in Blastocystis sp. negative subjects, the increased abundance was in taxa belonging to the phyla Verrucomicrobiota, Firmicutes, and Bacteroidota (Fig 3C). The findings implicate that Blastocystis sp. in symptomatic and asymptomatic infection could be associated with different bacterial taxa.

Blastocystis sp. colonization, and alteration in gut microbial functions

Microbial functions of the microbiota in the subjects were determined by using the inferred metagenomics obtained from PICRUSt. LEfSe was used to identify a differently abundant pathway in the samples. Microbial function in symptomatic individuals with Blastocystis sp. colonization generally showed an abundance of pathways involved in translation, nucleotide metabolism, metabolism of cofactors and vitamins, digestive systems, and also pathways involved in metabolic diseases compared to the microbiota without Blastocystis sp. which had pathways involved in transcription, signal transduction and lipid metabolism (Fig 4A). However, microbiota colonized by Blastocystis sp. in asymptomatic individuals had functional pathways abundant in metabolism of cofactors, vitamins and amino acids (Fig 4B). In general, pathways involved in replication and repair, nucleotide metabolism, translation, metabolic diseases and digestive system are found in Blastocystis sp.-positive subjects. The findings demonstrate that, it was not just the microbes that were differently abundant but also the metabolic functions that seemed to be different in Blastocystis sp.-colonized microbiota isolated from symptomatic and asymptomatic individuals.

Fig 4 — Influence of *Blastocystis* sp. on the gut microbial function in A symptomatic and B asymptomatic individuals.

Genotyping and phenotypic characteristics of Blastocystis sp.

As reported in past studies [29], here we observed that Blastocystis sp. was found colonizing both symptomatic and asymptomatic individuals. All Blastocystis sp. isolated from this study belong to ST 3. The analysis of 18S partial length rDNA of Blastocystis sp. sequence analysis suggests that the ST 3 isolates belonged to allele 34. The 18S partial rRNA sequences of Blastocystis sp. ST3 isolated from symptomatic and asymptomatic individuals suggest close genotypic similarity and this means that any difference seen phenotypically would be solely due to external pressures. In this study, we assessed phenotypic expressions of the parasite from symptomatic and asymptomatic conditions. The phenotype was studied in terms of in vitro growth profile, specific protease activity, and ability to proliferate cancer cells. High peak cell count was observed specifically in Blastocystis sp. isolated from asymptomatic individuals than parasites isolated from the symptomatic individual (Fig 5A and 5C). Significantly greater total protease activity was seen in Blastocystis sp. isolated from symptomatic individuals. This was noticed in the control experiments in Fig 6. We also observed that the Blastocystis sp. obtained from symptomatic individuals had a predominance of cysteine protease whereas the parasite cells isolated from asymptomatic individuals possessed serine protease predominantly (Fig 6A).

Fig 5 — A Growth profile of parasites obtained from asymptomatic individuals. B Growth profile of parasites obtained from asymptomatic individuals co-cultured with bacterial suspension of symptomatic parasite culture. C Growth profile of parasites obtained from symptomatic individuals. D Growth profile of parasites obtained from symptomatic individuals co-cultured with bacterial suspension of asymptomatic parasite culture.

Fig 6 — A Changes in specific protease activity in *Blastocystis* sp. isolated from symptomatic and asymptomatic individuals after the introduction of bacterial suspension from asymptomatic and symptomatic parasite culture. Values are expressed as mean±SD from 4 replicates. *P<0.05 in Student’s t-test for comparison with control. Note: E64 = cysteine protease inhibitor; PMSF = serine protease inhibitor; PA (Pepstatin A) = aspartic protease inhibitor; EDTA = metalloprotease inhibitors. Ctrl: Protease activity without addition of inhibitors. B Cell proliferation by solubilized antigen of *Blastocystis* sp. isolated from symptomatic and asymptomatic individuals after the introduction of bacterial suspension from asymptomatic and symptomatic parasite cultures. Values are expressed as mean±SD from 3 replicates. *P<0.05 in Student’s t-test for comparison with control. Co-cultured: Experiments with introduction of bacterial suspension. Control: Experiments with introduction of sterile Jones medium instead of bacterial suspension.

Influence of bacteria on Blastocystis sp.

Blastocystis sp. from symptomatic individuals co-cultured with bacterial suspension from asymptomatic individuals showed increased growth of parasite numbers compared to the parasite isolates without introducing bacterial suspension from asymptomatic isolates. The average peak cell count of 2.46 x 10⁶ cells/ml increased about 3-fold to 6.54 x 10⁶ cells/ml. Whereas Blastocystis sp. obtained from asymptomatic individuals, which had high cell numbers showed a reduction in cell count upon introducing bacterial suspension from symptomatic individual. The average peak cell count decreased 4-folds from 6.17 x 10⁶ cells/ml to 1.45 x 10⁶ cells/ml (Fig 5).

Protease activity in Blastocystis sp. isolated from symptomatic individuals co-cultured with bacterial suspension extracted from asymptomatic individual showed only a slight increase, which was insignificant. However, isolate obtained from asymptomatic individuals when cultured with bacterial suspension isolated from symptomatic individuals showed significant increase in the protease activity (from 0.085 to 0.2789). The increase was statistically significant using Student’s t-test when compared to the control (P = 0.029). Blastocystis sp. isolated from asymptomatic individuals initially possessed significant predominance of serine protease. When bacterial suspension from symptomatic individuals was introduced to Blastocystis sp. obtained from asymptomatic individual there was an increase in the cysteine protease. This increase was found to be significant using the Student’s t-test (P<0.05) (Fig 6A).

Regarding the ability of Blastocystis sp. antigens to promote colonic cell proliferation, antigens isolated from symptomatic individuals that were cultured with bacteria from asymptomatic Blastocystis sp. culture showed insignificant proliferation compared to the control. Nonetheless, antigens from Blastocystis sp. isolated from asymptomatic individuals that were co-cultured with bacteria from symptomatic individuals produced significantly greater colonic cell proliferation than antigens from Blastocysits sp. isolated from asymptomatic individuals in autochthonous culture. There was about 3-fold increase from 19.8% proliferation to 65.8% (Fig 6B).

Discussion

Blastocystis sp. has been reported to have an intricate relationship with its surrounding bacteria [30]. A previous study had orally inoculated axenic, monoxenic and xenic Blastocystis sp. from symptomatic individuals into germ-free guinea pigs. It was found that about half of the rats inoculated with xenic parasite developed infections with watery diarrhea for more than a week duration and increased cellularity at the lamina propria region. In contrast, the rats inoculated with monoxenic had an infection and none was infected in rats inoculated with axenic Blastocystis sp.[31]. This finding was one of the earliest to highlight the importance of accompanying gut bacteria in Blastocystis sp. infection.

In this study, the phyla Firmicutes and Bacteroidetes were most predominant among all the subjects. This is consistent with a previous study on a similar Malaysian population [32,33]. In general, regardless of Blastocystis sp. infection, we found a significant difference in alpha and beta diversity, confirming that bacterial composition in symptomatic and asymptomatic samples is distinct. This confirms that gastrointestinal symptoms are associated with low species richness. LEfSe and relative abundance analysis further confirms alteration in Firmicutes/Bacteroidetes (F/B) ratio. Decreased F/B ratio seen in symptomatic individuals suggests dysbiosis, commonly also seen in inflammatory bowel disease (IBD) patients [34].

Our findings demonstrated that Blastocystis sp.-infected individuals, regardless of symptoms, had decreased alpha diversity and Pielou’s evenness. As observed in our recent study, greater amoebic forms and surface fuzzy coat commonly seen in symptomatic isolates [35] suggest a greater interaction with bacteria in these isolates which could contribute to the alteration of microbiota. On the other hand, lower peak cell numbers in the growth profile of symptomatic isolates implicate potential inhibition from accompanying microbiota. These observations suggest a bidirectional interaction between Blastocystis sp. and gut microbiota. However, more data on Blastocystis sp.-gut microbiota across multiple populations is required to corroborate this interaction. A similar study done on pooled symptomatic and asymptomatic populations showed contrasting results where no difference in alpha diversity was detected [36]. This discrepancy could have been potentially contributed by differences in environment and the studied population. Since subtype-influenced associations to gut microbiota have been demonstrated by Tito et al. [7] it is highly likely that the discrepancy is due to analyses being carried out on multiple Blastocystis sp. subtypes (ST 1–7). In this study, the association seen is unique and specific to only Blastocystis sp. ST3. While most other studies have compared Blastocystis sp.-gut microbiota association in diseased or healthy group [5,6,37,38], our study for the first-time reported association of a single subtype (ST 3) of Blastocystis sp. to symptomatic and healthy individuals.

A study by Nagel et al. on Blastocystis sp. from irritable bowel syndrome patients revealed insignificant influence on the gut microbiota [38]. Similarly, in this study, the presence or absence of Blastocystis sp. in symptomatic group did not significantly influence bacterial diversity but changed the abundance of certain bacterial taxa suggesting alterations in bacterial composition. However, in asymptomatic individuals, we saw a significant alteration in gut microbial diversity and composition in Blastocystis sp. infection. Interestingly, different composition of bacteria was seen to be associated with Blastocystis sp. in symptomatic and asymptomatic infections. In symptomatic individuals, bacteria from the family of Prevotellaceae and Rumunicoccaceae were predominant in Blastocystis sp. colonization. Our study is similar to previous reports where Prevotellaceae were positively associated with Blastocystis sp. colonization [5]. Studies have associated bacteria from Prevotellaceae with inflammatory disorders [39] and symptomatic Entamoeba histolytica infection [40]. However, its role especially in symptomatic Blastocystis sp. infection needs further exploration as the parasite often presents features such as amoebic forms and a sticky surface coat [41,42] implicating enhanced interaction with bacteria.

Asymptomatic individuals with Blastocystis sp. colonization were associated with a predominance of bacteria belonging to mainly Firmicutes with reduced diversity. A recent study on the Iranian population has reported similar findings where harmful bacteria were elevated in asymptomatic Blastocystis sp. infection [43]. The findings by Nieves-Ramirez et al [6] showed increased diversity in asymptomatic Blastocystis sp. infection, although there was a similar increase in Firmicutes. Population heterogeneity could be a reason as the study was conducted in the Mexican rural population while the current study was done on the urban population in Malaysia. A significant difference in bacterial composition between Malaysian and western populations [33] suggests the contrasting findings between this study and other studies on gut microbiota-Blastocystis sp. association [5,37]. However, the reduction in richness in asymptomatic Blastocystis sp. infection could be best explained ecologically by an alternative stable state [44], whereby perturbation in gut microflora results in the establishment of a different stable state with associated dynamics such as population fluctuations. This stable state may contribute to specific immunological adjustments as previous reports have noticed reduced fecal calprotectin, IgA level [6], and neutrophil levels [45] in asymptomatic Blastocystis sp. infection. Whether Blastocystis sp. instigates an anti-inflammatory environment for persistent asymptomatic colonization by altering gut bacterial composition warrants more study.

PICRUSt algorithm is commonly used for the functional prediction of the intestinal microbiota [33,46]. In adjunct to microbial diversity and composition, we used the PICRUSt algorithm to further add dimension to metabolic functions in gut microbiota and its alteration after Blastocystis sp. infection in studied subjects. The outcome, for the first time, implies that Blastocystis sp. could be related to modifications of resulting microbial functional pathways. This is likely due to the alteration of microbial composition in Blastocystis sp. infection. We postulated that asymptomatic Blastocystis sp. infection could be associated with an alternative stable state. In symptomatic individuals, Blastocystis sp. altered microbial composition despite the diversity not being significantly affected. Studies suggested that altered bacterial composition can influence how metabolites are processed, resulting in the metabolic pathway and profile changes [2]. Evidence from this study suggests the same, as significant change in metabolic processes are observed in Blastocystis sp. infection. However, the predicted microbial functional pathway only offers preliminary access to understanding microbiota function. Greater depth and details provided by the metabolomic approach and whole genome sequencing would be essential in identifying genes involved in the specific metabolic pathway and metabolite interactions during Blastocystis sp. infection.

To date, no studies have reported the influence of accompanying bacteria on Blastocystis sp. cells. Several studies have suggested important roles of accompanying bacteria in the pathogenesis of intestinal protozoan parasite [9,12]. These studies, however, do not mimic the natural condition of the gut as the parasite cells used were axenic. Hence, the changes seen in the parasites may not translate to the real-time scenario. Here we report the effects of altering the autochthonous bacterial composition in Blastocystis sp. culture. We found that parasite cells isolated from asymptomatic individuals resembled the cells from symptomatic ones upon consistent introduction of bacterial suspension from symptomatic individual and vice versa. These findings demonstrate the role of bacteria in influencing Blastocystis sp. up to protein expression levels where the solubilized protease levels and ability of antigens to proliferate colon cancer cells in vitro were also altered. Although the role of proteases is inconclusive in Blastocystis sp. infection, evidence of degradation of secretory immunoglobulin A [47] and activation of IL-8 gene expression [48] and well-studied pathogenic roles in other intestinal parasites [49,50] potentially implicate it as a virulent factor. Evidence on bacteria engulfing amoebic forms and increased protease activity [24] in Blastocystis sp. as well as lipopolysaccharide (LPS)-induced toll-like receptor activation [51] supports the obligatory role of bacteria in pathogenic characteristics in Blastocystis sp.

Earlier studies demonstrated the presence of physical features such as sticky surfaces, fuzzy coats, and amoebic morphologies indicating interactions with bacteria [52]. With current findings, we are certain that this interaction contributes to shaping the phenotypic feature of Blastocystis sp. cells. Several studies have used phenotypic features to ascribe pathogenic potentials in Blastocystis sp. ST3 when isolated from diseased and healthy groups [41,53,54]. The resulting phenotype reflects a specific microbiota composition, either due to various host-related factors or possibly influenced by Blastocystis sp. itself. The latter is highly likely, as we have seen here and in other recent findings, that Blastocystis sp. modifies the bacterial composition [6,55], although its influence on diversity is contradictory. Even so, nothing is conclusive until the mechanism of specific phyla in cross-talk with Blastocystis sp. is elucidated. Studies on the pathogenicity of Blastocystis sp. have been contradicting for a long time. However, case studies [56] reporting improvement of symptoms upon the extermination of this organism via drug treatment suggest a pathogenic role that could be restricted to only some individuals or certain gut microbial environments. Studies thus far have reported that pathogenic potentials are being assessed in terms of variations in the growth profile, cysteine protease activity, and ability to proliferate cancer cells [57]. Our finding showed that these factors, disparate in Blastocystis sp. isolated from symptomatic and asymptomatic individuals, are largely dependent and altered by the parasite’s microbial surroundings. We propose that the pathogenic characteristics may not be wholly exerted by the parasite itself but influenced by factors such as the gut microbiota as well.

Increasing number of studies is beginning to show differences in gut microbiota due to various factors [58]. An individual may undergo alterations in gut microbial environment as a consequence of changing dietary intakes, life stresses, medications, travel and migrations, which are rampant in recent years. A diverse and balanced microbiota profile provide protection to the mucosa [59] and secrete metabolic products such as short chain fatty acids (SCFA) that promote health [60]. Parasitic cells colonizing in such environment may remain asymptomatic [59]. When there is a change in the environment, especially when triggered by certain diet, antibiotic consumption or stress, the microbiota may be altered [1]. This influences the colonizing organism initially harmless to be pathogenic (Fig 7). Although this postulation was derived from the correlation of data without a causal relationship, we believe this is the way forward in understanding the role of Blastocystis sp. in disease and health.

Our study is limited in terms of sample size. This is due to difficulty in obtaining a single subtype of Blastocystis sp. and maintaining the parasite cells in vitro. A similar experimental design, applied to larger sample size, yields more conclusive evidence. This study is also limited in terms of the use of molecular diagnostics for the detections of Blastocystis sp. colonization. Therefore, it is possible that Blastocystis sp.-free group may have individuals who were infected with this organism but was not captured by in-vitro cultivation technique. Also, the quantitative burden of Blastocystis sp. could not be compared between the groups. However, this study, for the first time, has demonstrated gut variation associated with a single Blastocystis sp. subtype. Our study has also shown for the first time the influence of autochthonous bacterial alteration on the phenotype of Blastocystis sp. ST3 cells. In the future, it is essential to characterize the bacterial taxa in close interaction with Blastocystis sp. ST3 and its role in symptomatic and asymptomatic infections.

Conclusion

Many recent studies focused on the effect of Blastocystis sp. in altering the gut microbiota [5,61], however, this is the first study to demonstrate the influence of microbial environment on this prevalent intestinal protozoon. The findings open new vistas in understanding parasite-bacteria interaction, which could help us understand better the pathogenicity of Blastocystis sp. We postulate that the interactions seen between specific intestinal microbiota and Blastocystis sp. influence whether the protozoa will function in a commensal or parasitic role. This study also provides preliminary evidence of a typical intestinal protozoan reverting from a harmless organism to a harmful one.

Supporting information

S1 Table. The demographic profile of participants (n = 50) recruited into the study.

(XLSX)

Click here for additional data file.^{(11.6KB, xlsx)}

S1 Fig

Breakdown of core microbiota at (A) phylum and (B) genus level in symptomatic and asymptomatic individuals.

(TIFF)

Click here for additional data file.^{(4.1MB, tiff)}

S2 Fig

Breakdown of rare microbiota at (A) phylum and (B) genus level in symptomatic and asymptomatic individuals.

(TIFF)

Click here for additional data file.^{(4.1MB, tiff)}

S3 Fig

Breakdown of core microbiota at (A) phylum and (B) genus level in symptomatic and asymptomatic individuals with and without Blastocystis sp. colonization.

(TIFF)

Click here for additional data file.^{(4.1MB, tiff)}

Acknowledgments

We would like to thank the staff of Pantai Medical Hospital Specialist Clinic and Department of Parasitology, Faculty of Medicine, University Malaya.

Data Availability

All the data generated are included within the manuscript. The demographic data of the participants have been included in the Supporting Information files. The raw sequencing microbiome data have been added to a public repository, the National Library of Medicine (NCBI) as a BioProject with accession number PRJNA88178.

Funding Statement

This study was supported by Trans-disciplinary Research Grant Scheme, Ministry of Higher Education (TRGS) (TRGS/1/2018/UM/01/7/1) obtained by SKG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011170.r001

Decision Letter 0

Shan Lv, Matthew Brian Rogers

10 Aug 2022

Dear Dr Rajamanikam,

Thank you very much for submitting your manuscript "A harmless organism in the gut can be triggered to be harmful" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Apologies for delays in returning this manuscript. Two reviewers have carefully reviewed your manuscript, and both have determined that your manuscript could be accepted with major revisions. Some clarifications in the text and aesthetic modifications of the figures are required, but some methodological weaknesses need to be acknowledged as well. Finally please ensure that sequence data is deposited on a public repository and accession numbers are included in the text.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Matthew Brian Rogers, Ph.D.

Academic Editor

PLOS Neglected Tropical Diseases

Shan Lv

Section Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? Yes

-Is the study design appropriate to address the stated objectives? Yes

-Is the population clearly described and appropriate for the hypothesis being tested? No

A) please present the demographic characterizations of both groups in a table; B) please indicate whether healthy individual or infected groups had underlying diseases (e.g., diabetes, autoimmune disorders, etc.) or other gut infections; C) please indicate B. hominis group had single infection and did not have co-infection with other gut pathogens or parasites.

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? Yes

-Were correct statistical analysis used to support conclusions? Please add a statistical analysis section at the end of material and methods.

-Are there concerns about ethical or regulatory requirements being met? No

Reviewer #2: - the authors need to articulate in the methods the definition what is meant by symptomatic and asymptomatic. Presumptively, this means intestinal symptoms (? diarrhea). Were these definitions made a prior or only after participant recruitment and questionnaire review. Given the potential range of 'subclinical' associations between a disease state and presence of Blastocystis described in the introduction it is critical that the report be specific about what disease condition is being studied.

- it appears that there were 50 individuals and each provided one sample. Please indicate this clearly.

- 'maintaining utmost anonymity' is vague. Please elaborate on why written consent was not required and the procedures for de-identification of samples

- The recruitment procedures and any inclusion/exclusion criteria are absent. Coupled with a lack of definition for symptomatic vs asymptomatic, it makes it difficult to appraise the assignment of different groups.

- Please include a section for assignment of Blastocystis carriage status. What assignment based on culture recovery of parasites? qPCR data for Blastocystis would be more sensitive and allow for analyses based on parasite burden.

- Line 187 implies that there was a screen for pathogens in the stool samples, yet a list of what pathogens and by what method is not provided. If Blastocystis is a common co-pathogen, this should be considered in the interpretation of the 16S sequencing data. What is meant in line 188-189, eg. in Line 189, which groups?

-Please clarify if antibiotics are present in the Jones media. Though a reference for the media is provided, given the emphasis of this paper on parasite-bacteria interactions it is important to explicitly indicate what if any antibiotics were present in the media.

- Line 192. delete 'asymptomatic' as an adjective modifying Blastocystis, the parasite. This allows the authors to delete the paranethetical (ie. 3-days-old Blastocystis sp isolated from asymptomatic individuals...)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: -Does the analysis presented match the analysis plan? Yes

-Are the results clearly and completely presented? Yes

-Are the figures (Tables, Images) of sufficient quality for clarity? Yes

Please add accession numbers if you deposited your sequences in GenBank.

Reviewer #2: -Figures. It is customary to show the letter designation for figure panels in the upper left, not the upper right.

- Line 238. If separating the symptomatic from asymptomatic, groups, then report the mean of all 50 participants and separately the mean in symptomatic vs asymptomatic for each group. Likewise, a break down comparison of phyla and genera between symptomatic and asymptomatic should be performed.

- by 'features' do the authors mean 'assigned sequence variants'? What is a 'feature'?

-Figure 2: Please include a sentence about the 'n' of Blastocysts positive vs neg (A) and including by clinical status (B). C, What determined the circle sizes? Where are the individual samples? Is this absolute range of dots (where are the dots?), 95% CI?, some other measure of range?

-Lines 318-324: please reference to a figure or data to substantiate the claims made in the text. In addition, please comment on what methods were used to validate that the phenotypic differences between Blastocystis isolated from symptomatic versus asymptomatic participants were due to Blastocystis specifically and not the co-cultured bacteria in these xenic cultures. Can the authors axenize Blastocystis?

- Lines 327-334: does filtered or heat-killed bacterial suspension have a similar result?

- Line 336: please indicate how the authors know that the protease activity is a function of Blastocystis rather than the bacteria present in symptomatic xenic cultures. protease activity could be a composite of Blastocystis and bacteria/other microbes present in the culture.

- Line 348: How was the blastocysts antigen prepared and what measurements were done to confirm its purity compared with other proteins, including bacterial antigens, that may be present in the extract. Also, numerically, the difference in proliferation between Blastocystis antigen from asymptomatic (20%) and symptomatic (60%) stool is similar (and with less variability) to asymptomatic (20%) and co-culture with bacteria (~65%). Please clarify. Also, both figures in 6B read 'bacterial suspension from asymptomatic parasite culture'. This seems inconsistent with the text. Finally, is 'control' bacteria-free (axenic) or is 'control' with bacteria in the autochthonous xenic culture used to recover Blastocystis?

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: -Are the conclusions supported by the data presented? Yes

-Are the limitations of analysis clearly described? No

Please say your strengths, limitations, and suggestions for future researchers as a concise paragraph before conclusion section.

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study? somewhat

-Is public health relevance addressed? Yes

Reviewer #2: - The authors have performed multiple experiments to derive observations of potential Blastocystis - bacteria interactions. These questions are complex and the experiments are by extension challenging.

- That Blastocystis is accompanied by decreased alpha diversity and Pielou's evenness does not distinguish causality from association. In addition to potential strain-dependent influences the authors reference in lines 380-385, there are potentially other factors (environmental, individual patient, population, and methodological) that could explain a discrepancy between findings in this study and other manuscripts. Another interpretation is that the lower diversity stool selected for assignment of Blastocystis status (and therefore a molecular method to assign Blastocystis status should be pursued). It is harder to culture Blastocystis ST3 from some stool (ie. symptomatic as shown in Figure 5) than other stool and perhaps not surprisingly stool with less microbial diversity is less likely to have bacteria that inhibit Blastocystis growth.

-The conclusion overstates what was observed. Line 506 extends beyond the data observed in this manuscript. There is no measure of immune barrier and immune response in this study and such a conclusion is only conjecture. Also, the designation of Blastocystis as either commensal or parasite based on the data presented is not possible. There is no evidence that a) Blastocystis is contributing to a disease condition in the participants or that b) the in vitro readouts are sufficient to designate a 'parasitic' phenotype. Rather than stating 'here we present', the authors could raise their new hypothesis that interactions between specific intestinal microbiota and Blastocystis influence whether the protozoan is functioning in a commensal or parasitic role. Finally, Line 510, I think the authors would agree that the evidence presented is not strong. The study may be suggestive of the statement, but the presented data is not a complete mechanism.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The study is interesting and well designed. However, I have some comments and suggestions:

1. Regarding stool sample collection from case and control groups: A) please present the demographic characterizations of both groups in a table; B) please indicate whether healthy individual or infected groups had underlying diseases (e.g., diabetes, autoimmune disorders, etc.) or other gut infections; C) please indicate B. hominis group had single infection and did not have co-infection with other gut pathogens or parasites.

2. Please add a statistical analysis section at the end of material and methods.

3. Please add accession numbers if you deposited your sequences in GenBank.

4. Please read and use the recent paper regarding asymptomatic B. hominis and gut bacteria composition: Behboud, et al. "Alteration of gut bacteria composition among individuals with asymptomatic Blastocystis infection: A case-control study." Microbial Pathogenesis (2022): 105639.

5. Please say your strengths, limitations, and suggestions for future researchers as a concise paragraph before conclusion section.

Reviewer #2: Overall the authors present an intriguing concept that microbiota-protozoa interactions are critical for understanding intestinal protozoan behavior (in this case Blastocystis) and disease potential. These are difficult hypotheses to test and prove, and the authors used an in vitro system of mixed stool culture derived Blastocystis with bacteria present in the same stool or from stool from participants with a different clinical status. The strength of the study is the attempt to disentangle these complex microbe-microbe interactions. Weaknesses, however, are several including: incomplete descriptions of the participant cohort (especially the definition of symptomatic or asymptomatic), apparent lack of a molecular assay to assign Blastocystis status (and therefore the designation of Blastocystis status is contingent upon culture viability which that authors demonstrate may be influenced by autochthonous bacteria in the stool), apparent lack of axenic Blastocystis controls, and lack of reporting what measurements or manipulations were performed to indicate that the phenotypic changes seen from different mixes of microbial communities were derived from changes in Blastocystis rather than derived from the other microbes in the xenic assay system. Additional experiments are therefore needed to reach the authors' final conclusions.

Additional specific comments:

--Abstract: multiple changes are necessary 'microbiome' to 'intestinal microbiota'. Reserve the use of the term 'microbiome' in reference to descriptions of the intestinal microbial community genomic structure.

--"healthy" could easily be a misclassification of individual status and should be avoided. Rather these 'healthy' individuals are Blastocystis ST3 negative controls.

--change line 44. This reads as an overstatement and 'symptomatic' or 'asymptomatic' refer to host syndromes, not characteristics of a parasite. It would be better to just state the observation without assigning presumed consequences on the host:eg. ".... demonstrated diminished growth and diminished expression of putative virulence genes"..

--line 47 'microbial diversity' appears to refer to the genomic (16S amplicon?) diversity. This should be more clearly stated.

Introduction

-The introductory paragraph is difficult to read, in part because of the sentence structure, and in part because of the complexity of the concepts. First, there are plenty of studies to indicate that plasticity in response to dietary and environmental influences is a fundamental characteristic of intestinal microbiota (and therefore not unexpected). The first sentence also inappropriately lumps direct influences with established influences on intestinal microbial community structure (ie. diet) with those that are indirect, or require several steps to prove causality (ie. climate change, to this reviewer's knowledge had not been proven to directly alter intestinal microbiota). It is important when introducing this concept that the authors take care to delineate what is known and verifiable from what is speculation (even if plausible). In Line 60 and line 62 it is also not clear what specific influences on the host are relevant for the submitted manuscript. Susceptibility to what? It would be better for this paragraph to do more to introduce the 'holes' in our understanding of the intestinal microbiota as not only prokaryotes but also eukaryotes (as introduced in lines 66-67).

-Line 76 would be more accurate to state "increased diversity of bacteria in the presence of intestinal Blastocystis carriage." The prior sentences appropriately explain the questionable pathogenicity of this parasite, so the designation of 'infected gut' is therefore controversial.

-Please do not use the terms 'flora' and 'microbiota' and 'microbiome'. Suggest changing 'flora' to 'microbiota' for consistency. Otherwise it is not clear what the difference is between flora and microbiota and whether these terms are synonymous or meant to designate something different to the reader.

-The references to Giardia in line 86 are too restricted. Please include references to other studies including Bartelt et al PLoSPathogens, 2017; work by Andre Buret laboratory and work by Scott Dawson/Steven Singer group. For E histolytica see papers by Stacey Burgess.

--------------------

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Reviewer #1: Yes: Amir Abdoli

Reviewer #2: No

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PLoS Negl Trop Dis. 2023 Mar 29;17(3):e0011170. doi: 10.1371/journal.pntd.0011170.r002

Author response to Decision Letter 0

8 Oct 2022

Attachment

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011170.r003

Decision Letter 1

Shan Lv, Matthew Brian Rogers

29 Nov 2022

Dear Dr Rajamanikam,

Thank you very much for submitting your manuscript "A harmless organism in the gut can be triggered to be harmful" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Thank-you for the resubmission of your manuscript to PLOS Neglected Tropical Diseases. Two reviewers have carefully evaluated your manuscript, and reviewer 2 has noted some language that requires clarification surrounding the nature of your controls, and various other places to make the manuscript more easily understandable to a reader.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Matthew Brian Rogers, Ph.D.

Academic Editor

PLOS Neglected Tropical Diseases

Shan Lv

Section Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: Important clarifications have been made. There are still some apparent inconsistencies to resolve, for example "control" in Figure 6A and 6B don't appear to refer to the same thing, and that is confusing and requires significant work by the reader to comprehend (these concepts are complex).

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: Regarding comment #4/6. To clarify, these questions addressed how the authors knew that the protease activity (figure 6A) was due to Blastocystis proteases and not bacterial-derived proteases that are different between different bacterial communities. Part of the confusion for this reviewer was that the terms "control" and "bacterial co-culture" in Figure 6 are not well defined elsewhere in the manuscripts. Based on the methods (new line 239), it is now clearer that 'control' means 'minimalized bacteria' where as 'bacterial co-culture' means the the non-bacterial minimalzied/standard xenic culture. From a different perspective, the experimental group is therefore the minimalized bacterial group if blastocysts culture normally includes the autochthonous xenic bacteria (conversely, in Figure 6B it appears that control indeed does refer to autochthonous bacterial culture). Line 239 helps to alleviate concerns that the bacterial proteases account for differences in Figure 6A. I'd suggest re-labeling Figure 6A as following "Bacterial co-cultured" should read "autochthonous culture" and "control" should read "minimalized bacteria culture". This makes more clear what was done and what is being compared.

Line 383: This paragraph needs some editing for grammar and clarity. It is still hard to tell what is going on. First, include the term colonic cell proliferation so it is clear what is being measured. For example, change the opening clause to read "Regarding the ability of Blastocystis antigens to promote colonic cell proliferation,..." instead of "When tested on the ability to proliferate cells".

Line 384, should 'individual' be 'individuals'? Also, should the comma be removed? The authors are meaning antigens from Blastocystis isolated from symptomatic individuals that were later cultured with the bacteria from asymptomatic individuals. Correct?

Line 384, 'bacteria from asymptomatic INDIVIDUALS' not bacteria from asymptomatic isolates

Line 385-386 should this read "Nonetheless, [?ANTIGENS from?] Blastocystis sp isolated from asymptomatic individuals that were cultured with bacteria from symptomatic individuals produced greater proliferation than antigens from Blastocysits isolated from asymptomatic individuals in autochonthonus culture?" As written in line 387 it isn't explicit what comparison was made. Greater than what?

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: The toned down conclusion is much better and appropriate to the data presented.

However, new paragraph, Lines 542-550 needs revision. In Line 545-546, again the findings are associative and not causal. Instead of 'due to' the authors should state "associated with" ... a single subtype. Also, the only other pathogens screened were other parasites (not other bacteria or viruses) and the parasite diagnostics used were low sensitivity non-molecular based methods. So it is overstating to say that Blastocystis was a single infective agent. The entire clause "which is also a single infective agent" should be removed. Please add the limitation of not use molecular diagnostics and that therefore a) Blastocystis-free group may have had individuals with Blastocystis that wasn't cultivatable by the authors methods and that b) Blastocystis quantitative burden in stool cannot be compared between the two symptomatic groups.

--------------------

Editorial and Data Presentation Modifications?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

--------------------

Summary and General Comments

Reviewer #1: (No Response)

Reviewer #2: The authors have provided a detailed response to most of the critiques. Some responses were misinterpreted, but they are minor and need not be further addressed. (eg. by Blastocystis status I did not mean differentiating symptomatic from asymptomatic, but rather presence or absence of Blastocystis. In this paper they used culture-recovery methods rather than molecular methods to assign presence or absence of Blastocystis. Future studies should incorporate qPCR if possible--https://journals.asm.org/doi/10.1128/JCM.01392-10). Overall I think the findings are of interest, appropriate for PLoSNTDs, but a few more revisions are needed before this is publication-ready.

In addition to specific section comments above, please pay careful attention to semantics in the paper. As in line 209, Line 217 should read "Three-day-old Blastocystis sp. cells from symptomatic individuals". The Blastocystis cells are not inherently symptomatic or asymptomatic. This same problem is present in Figure 6 legend where there is reference to 'symptomatic' and "asymptomatic" parasite culture. It happens again in lines 357-359 where line 357 reads "symptomatic individuals' whereas line 358 reads "asymptomatic isolates". Line 376 should read "Isolates from asymptomatic individuals".Line 378 should read "bacterial suspension from symptomatic individuals". Please correct throughout the manuscript. For this reason I recommended additional copyediting below.

Finally, and now that clarifications have allowed me to better comprehend this study, I suggest a change in the title. The authors don't show 'harm' from Blastocystis in the in vitro models and likewise it is not possible from the data presented to assign the organism as 'harmless'. A more appropriate title would be "Gut bacteria influence Blastocystis phenotypes and may trigger pathogenicity"

--------------------

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Reviewer #1: No

Reviewer #2: No

Figure Files:

Data Requirements:

Reproducibility:

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

PLoS Negl Trop Dis. 2023 Mar 29;17(3):e0011170. doi: 10.1371/journal.pntd.0011170.r004

Author response to Decision Letter 1

27 Dec 2022

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011170.r005

Decision Letter 2

Charles L Jaffe, Matthew Brian Rogers

14 Feb 2023

Dear Dr. Rajamanikam,

We are pleased to inform you that your manuscript 'Gut bacteria influence Blastocystis sp. phenotypes and may trigger pathogenicity' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Matthew Brian Rogers, Ph.D.

Academic Editor

PLOS Neglected Tropical Diseases

Shan Lv

%CORR_ED_EDITOR_ROLE%

PLOS Neglected Tropical Diseases

***********************************************************

Thank-you for the re-submission of your manuscript, and apologies for the delay in returning this decision. After consideration by two reviewers we have made the decision that your manuscript is acceptable in it's current form for publication.

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011170.r006

Acceptance letter

Charles L Jaffe, Matthew Brian Rogers

13 Mar 2023

Dear Dr. Rajamanikam,

We are delighted to inform you that your manuscript, "Gut bacteria influence Blastocystis sp. phenotypes and may trigger pathogenicity," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. The demographic profile of participants (n = 50) recruited into the study.

(XLSX)

Click here for additional data file.^{(11.6KB, xlsx)}

S1 Fig

Breakdown of core microbiota at (A) phylum and (B) genus level in symptomatic and asymptomatic individuals.

(TIFF)

Click here for additional data file.^{(4.1MB, tiff)}

S2 Fig

Breakdown of rare microbiota at (A) phylum and (B) genus level in symptomatic and asymptomatic individuals.

(TIFF)

Click here for additional data file.^{(4.1MB, tiff)}

S3 Fig

Breakdown of core microbiota at (A) phylum and (B) genus level in symptomatic and asymptomatic individuals with and without Blastocystis sp. colonization.

(TIFF)

Click here for additional data file.^{(4.1MB, tiff)}

Attachment

Submitted filename: Response to Reviewers.docx

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Attachment

Submitted filename: Response to Reviewer 2.docx

Click here for additional data file.^{(19.6KB, docx)}

Data Availability Statement

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PERMALINK

Gut bacteria influence Blastocystis sp. phenotypes and may trigger pathogenicity

Arutchelvan Rajamanikam

Mohd Noor Mat Isa

Chandramathi Samudi

Sridevi Devaraj

Suresh Kumar Govind

Roles

Abstract

Author summary

Introduction

Methods

Ethics statement

Stool sample collection and Blastocystis sp. carriage assignment

Fecal DNA extraction

Patient and public involvement

Amplification of variable 3 (V3) and variable 4 (V4) region of 16S ribosomal RNA (rRNA) genes

Quality filtering and analysis of filtered reads

Analysis of filtered reads sequence diversity

Linear Discriminant analysis effect size (LEfSe) and predicting the functional composition of a metagenome

In vitro cultivation and genotyping of Blastocystis sp.

Alteration of microbiota surrounding Blastocystis sp.

Phenotypic characterizations

Growth characterization

Colorimetric protease quantification assay

Colon cell proliferation analysis

Statistical analysis

Results

Microbial profile analysis

Variation of microbiota in symptomatic and asymptomatic individuals and effect of Blastocystis sp. colonization

Fig 1. Analysis of alpha diversity and beta diversity of gut flora isolated from symptomatic and asymptomatic individuals.

Fig 2. Analysis of gut microbial diversity in Blastocystis sp. infection.

Differential association of bacterial taxa to Blastocystis sp. infection in symptomatic and asymptomatic individual

Fig 3.

Blastocystis sp. colonization, and alteration in gut microbial functions

Fig 4.

Genotyping and phenotypic characteristics of Blastocystis sp.

Fig 5. Growth profile of Blastocystis sp. upon introduction of bacterial suspension from symptomatic and asymptomatic culture.

Fig 6. Influence of bacterial alteration resulting in variation of protein expression.

Influence of bacteria on Blastocystis sp.

Discussion

Fig 7. Schematic diagram demonstrating the effect of microbiota in alternating the characteristic of Blastocystis sp.

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Shan Lv

Matthew Brian Rogers

Roles

Author response to Decision Letter 0

Decision Letter 1

Shan Lv

Matthew Brian Rogers

Roles

Author response to Decision Letter 1

Decision Letter 2

Charles L Jaffe

Matthew Brian Rogers

Roles

Acceptance letter

Charles L Jaffe

Matthew Brian Rogers

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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