Metagenomic

De
et al. Gut Pathog (2020) 12:32

https://doi.org/10.1186/s13099-020-00371-8 Gut Pathogens
RESEARCH Open Access
Metagenomic analysis of gut microbiome

and resistome of diarrheal fecal samples
from Kolkata, India, reveals the core and variable
microbiota including signatures of microbial
dark matter
Rituparna De* , Asish Kumar Mukhopadhyay and Shanta Dutta
Abstract
Background: Metagenomic analysis of the gut microbiome and resistome is instrumental for understanding the
dynamics of diarrheal pathogenesis and antimicrobial resistance transmission (AMR). Metagenomic sequencing of 20
diarrheal fecal samples from Kolkata was conducted to understand the core and variable gut microbiota. Five of these
samples were used for resistome analysis. The pilot study was conducted to determine a microbiota signature and the
source of antimicrobial resistance genes (ARGs) in the diarrheal gut.
Results: 16S rRNA amplicon sequencing was performed using Illumina MiSeq platform and analysed using the
MGnify pipeline. The Genome Taxonomy Database (GTDB-Tk) was used for bacterial taxonomic identification. Diar-
rheal etiology was determined by culture method. Phylum Firmicutes, Bacteroidetes, Proteobacteria and Actinobac-
teria were consistently present in 20 samples. Firmicutes was the most abundant phylum in 11 samples. The Bacte-
roidetes/Firmicutes ratio was less than 1 in 18 samples. 584 genera were observed. 18 of these were present in all the
20 samples. Proteobacteria was the dominant phylum in 6 samples associated with Vibrio cholerae infection. Conser-
vation of operational taxonomic units (OTUs) among all the samples indicated the existence of a core microbiome.
Asymptomatic carriage of pathogens like Vibrio cholerae and Helicobacter pylori was found. Signature of Candidate
phyla or “microbial dark matter” occurred. Significant correlation of relative abundance of bacterial families of com-
mensals and pathogens were found. Whole-genome sequencing (WGS) on Illumina MiSeq system and assembly of
raw reads using metaSPAdes v3.9.1 was performed to study the resistome of 5 samples. ABRicate was used to assign
ARG function. 491 resistance determinants were identified. In 80% of the samples tetracycline resistance was the most
abundant resistance determinant. High abundance of ARGs against β-lactams, aminoglycosides, quinolones and
macrolides was found. Eschericia sp. was the major contributor of ARGs.
Conclusions: This is the first comparative study of the gut microbiome associated with different diarrheal patho-
gens. It presents the first catalogue of different bacterial taxa representing the core and variable microbiome in acute
diarrheal patients. The study helped to define a trend in the gut microbiota signature associated with diarrhea and
revealed which ARGs are abundantly present and the metagenome-assembled genomes (MAGs) contributing to
AMR.
*Correspondence: [email protected]
Division of Bacteriology, National Institute of Cholera and Enteric
Diseases, Kolkata, India
© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
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the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material
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De et al. Gut Pathog (2020) 12:32 Page 2 of 48
Background by various parameters like genetic make-up, ethnicity,

Diarrhea is a leading cause of mortality accounting for altitude, geographical location, mode of delivery and diet
more than 1.6 million deaths worldwide [1]. It causes among others and also changes with age, travel, exposure
nearly 5,25,000 deaths among children under 5 years of to antibiotics and infections [14, 15, 19–22] and onset of
age and leads to malnutrition, stunted growth and ane- diseases [23].
mia [2–7]. It is particularly prevalent in the low and The microbiota comprises archae, bacteria, viruses and
middle-income countries owing to poor hygiene and san- unicellular eukaryotes. These carry out essential func-
itation. India is the second most populous country in the tions which are indispensible for maintaining a healthy
world and is one of the top five countries with the high- state of the body and includes homeostasis, metabo-
est burden of diarrhea and high rates of mortality and lism, immunity. This symbiotic association between the
morbidity [8–10]. Recently, India has recorded the high- host and the microbiota is highly vulnerable as the frag-
est number of deaths among under five age group [11]. ile structure of the microbiota is prone to dysbiosis in
The Eastern region recorded the third highest mortal- the event of diseases. In the disease state the commensal
ity rate among under five age and diarrhea is one of the flora is subdued by pathobionts (opportunistic pathogens
leading causes of death in this region [11]. In India the and asymptomatically carried pathogens) [24]. The most
most common causes of diarrhea are Rotavirus, Crypto- common observation is Bacteroidetes/Firmicutes ratio
sporidium sp. Shigella sp., Enterotoxigenic Eschericia coli which is high in the healthy state is reversed in the dis-
[12, 16]. Antibiotic therapy is administered to diarrheal ease state with few exceptions [25]. Dysbiosis has been
patients along with ORS (oral rehydration solution) to frequently studied in metabolic disorders [26], cancer
assuage severity of symptoms. AMR (antimicrobial resist- [27], inflammatory diseases [28]. Specific microbes and
ance) has rendered antibiotic therapy in diarrhea partially specific signature of gut microbiota termed as entero-
or completely ineffective. The genetic determinants of types have been found to be associated with each of the
AMR reside in the gut and in the environmental micro- diseases [29]. Only few studies have addressed gut micro-
biota from where they spread and enter into diarrheal biota dysbiosis in diarrhea. Most of these studies have
pathogens by lateral gene transfer (LGT). Most of the been directed towards understanding dysbiosis in the
diarrheal pathogens like E.coli, Klebsiella pneumoniae, event of infection by individual pathogens [30–33] or in
Campylobacter sp., Shigella sp. have emerged as mul- hospital acquired infections (HAIs) [34] or in Traveler’s
tidrug-resistant (MDR) and extensively drug-resistant diarrhea (TD) [25].
(XDR) and fail to respond to empirical drugs like ami- The current study is an unbiased pilot study conducted
noglycosides and cephalosporin [13]. AMR is a global for characterizing the gut microbiota and the resistome
challenge which needs to be urgently addressed using from diarrheal stool and to see if we could find a statisti-
a multi-disciplinary approach. Surveillance of AMR in cally significant association of microbiota structure with
diarrheal patients based on next-generation sequencing diarrhea. We present the first comparative analysis of
(NGS) is a novel way of addressing the AMR threat [13]. gut microbiota from twenty fecal samples collected from
The structural and functional components of the micro- patients with symptoms of diarrhea. The stool samples
biota can be studied and mapped completely with the aid were collected at the Infectious Diseases Beliaghata Gen-
of culture-free techniques which have been possible due eral Hospital (IDH) and Dr. B.C. Roy Memorial Hospital
to the advent of NGS. Big data derived from sequencing for Children (BCH), both in Kolkata, in Eastern India.
metagenomes will help to understand the importance of These were subject to diagnostic test by classical micro-
the structural and functional components of the microbiological method and were found to be associated with
biota in the development and dissemination of AMR [13] either distinct diarrheal etiology or with mixed infec-
by detection, analysis of distribution and abundance of tions and for some the etiology could not be determined
AMR determinants and their source organisms. A large by culture method currently deployed in our laboratory.
number of studies have been undertaken over the last Eastern India is endemic for diarrhea. Kolkata is a cos-
decades to understand the human microbiome and its mopolitan city with a population of 5.8 million. It is the
association with disease [14, 15]. A lot of emphasis has capital of the state of West Bengal (Fig. 1) and a major
been put on defining a healthy microbiome signature and commercial hub of India where people of high, mid-
core microbiome culminating in the Human Microbi- dle and low-income groups throng for job and business
ome Project for cataloguing the microbial communities opportunities from across the country contributing to the
in different body sites. These projects have revealed that remarkable cultural and ethnic diversity of the city. The
the gut microbiome is one of the most diverse and com- Infectious Diseases and B.C. Roy Memorial Hospital in
plex [14, 16–18]. Although a core microbiome may exist Kolkata has specialized facility for the treatment of diar-
every individual has a unique microbiota which is shaped rheal patients. It is the apex referral centre and sentinel
Fig. 1 Showing West Bengal in Eastern India (Courtesy: thymapguide.in)
surveillance centre for infectious diseases in West Ben- diarrheal gut microbiota that may be contributing to
gal and Eastern India. Regular diarrheal stool collection diarrheal pathogenesis, AMR and identify organisms that
takes place from the outpatient ward and from hospital- may be exploited to counterfeit the effect of diarrhea. The
ized patients. Therefore, NGS applied to study diversity study helped to establish a catalogue of taxonomic units
of bacterial composition of the gut microbiome is antici- present in the gut microbiome of diarrheal subjects and
pated to reveal striking biodiversity. The results could be to understand the superiority of WGS over 16S amplicon
a valuable resource for understanding the gut microbiota sequencing in studying the structure of the microbiota.
composition and resistome in the region. In our study we
present the profile of the gut microbiota using 16S rRNA Results
amplicon sequencing and resistome using whole genome Demographic details and diagnosis of fecal specimen
shotgun (WGS) sequencing in diarrheal patients who Out of 20 diarrheal fecal samples 13 were from male
were not subjected to any selective bias. They were ran- patients and 7 were from females. The cohort included
domly selected to represent the heterogeneity in a real subjects from the age of 8 months to 56 years which
community to catalogue the diversity of bacterial species were divided into three, age groups namely, 0–5 years,
present in the gut microbiota of the local community and 6–15 years and above 15 years. Accordingly, 5 sam-
in spite of observed inter-individual differences in ente- ples could be assigned to 0–5 years group, 2 samples
rotypes to define a shared microbiome. The study helped were assigned to 6–15 years group and 13 samples were
to understand the importance of the composition of the assigned to above 15 years group. S1, S2, S4, S16 and S17
were from the outpatient ward while the remaining sam- The samples uniformly showed the presence of Superk-
ples were collected from hospitalized diarrheal patients. ingdom (SK) Bacteria as the major constituent of the
Diagnosis of diarrheal pathogen by culture-based diarrheal microbiota in every sample. SK Chloroplast
methods showed that S1, S2, S5, S11, S12, S14, S18, S20 was also found but in minute proportion compared to
were associated with Vibrio cholerae (VC) O1; S14 with Bacteria. SKs Archae, Mitochondria and Eukaryota also
VC O139; S4, S7 with VC non O1 non O139; S19 with appeared in minute proportion in many of the samples
Vibrio fluvialis; S15 and S16 with Aeromonas sp.; S3, S6, but not all.
S8, S9 suffered mixed infections; S17 with Shigella flexen- Histograms representing the relative abundance of dif-
eri; the diarrheal pathogen associated with S10 could not ferent phyla, class, order, family, genera and species were
be determined with culture method established in our constructed with 0% threshold and occur in Fig. 3. A
laboratory. total of 46 bacterial phyla were found by DNA sequence
Diarrheal study subjects’ demographic details and cul- homology to reference genomes on the GTDB-Tk data-
ture results have been presented in Table 1. base. Bacterial phyla that were present in all the twenty
samples were Firmicutes, Bacteroidetes, Actinobacteria
16S rDNA V3‑V4 amplicon sequencing and Proteobacteria. Firmicutes was the most dominant
Gut microbiota of diarrheal patients phylum in S3 (58.01%), S5 (44.97%), S7 (77.26%), S10
16S rDNA sequencing was carried out to study structural (51.81%), S11 (41.21%), S12 (37.64%), S14 (67.07%), S16
composition of diarrheal microbiome and the relative (75.69%), S17 (54.03%), S19 (40.16%), S20 (62.89%) irre-
abundance of various components of the microbiota. 16S spective of the diarrheal pathogen that was isolated from
rDNAV3-V4 sequencing of the diarrheal samples (Fig. 2) it followed by Proteobacteria which was the most domi-
yielded > 150 K raw reads per sample. Of these 88%–91% nant phylum in S1 (46.27%), S2 (25.01%), S4 (35.54%), S6
passed quality control. These processed reads ranged in (38.91%), S8 (14.1%), S18 (63.09%). Actinobacteria was
size from 100 to 478 bp with an average sequence size of the most dominant in S9 with 49.58% abundance rate fol-
200–300 bp for each sample. lowed by 47.82% of Firmicutes. Actinobacteria was the
most abundant in S15 with 42.76% followed by 31.82%
Table 1 Demographic details of the donors of diarrheal stool, the pathogen isolated, the most abundant phylum
and Bacteroidetes/Firmicutes (B/F) ratio
Sample ID Sex Age (Years Hospitalized District/State Pathogen isolated by culture Most abundant phylum B/F ratio
(y)/Months (H)/OPD(O)
(m))
S1 Male 29y O 24 Parganas VC O1 Inaba Proteobacteria 0.256637168

S2 Female 52y O 24 Parganas VC O1 Ogawa Proteobacteria 0.788744975
S3 Male 36y H (3 days) Kolkata EAEC,VC O1 Ogawa Firmicutes 0.00844682
S4 Male 2y O 24 Parganas VC Non-O1 nonO139 Proteobacteria 0.042666667
S5 Male 11y H (2 days) 24 Parganas VC O1 Ogawa Firmicutes 0.659106071
S6 Female 25y H (1 day) Kolkata VC O1 Ogawa + E.coli (ETEC LTST) Proteobacteria 0.105140187
S7 Female 43y H (1 day) Kolkata VC Non-O1 nonO139 Firmicutes 0.02601605
S8 Male 22y H (3 days) Burdwan VC O1 Inaba + Campylobacter sp. Proteobacteria 0.901763224
S9 Male 16y H (2 days) Kolkata VC O1 Ogawa + C.jejuni Actinobacteria 0.02446675
S10 Male 55y H (1 day) Kolkata UNRESOLVED Firmicutes 0.119475005
S11 Female 56y H (1 day) Kolkata VC O1 Ogawa Firmicutes 0.01601553
S12 Male 12y H (1 day) Kolkata VC O1 Ogawa Firmicutes 0.681455898
S13 Male 40y H (3 days) Kolkata VC O139 Bacteroidetes 1.157114228
S14 Male 50y H (1 day) Hooghly VC O1 Ogawa Firmicutes 0.023855673
S15 Male 1y H (1 day) Kolkata Aeromonas sp. Actinobacteria 1.536455818
S16 Female 8 m O Kolkata Aeromonas sp. Firmicutes 0.001056943
S17 Female 2y O Kolkata S.flexeneri (UT) Firmicutes 0.152137701
S18 Male 35y H (2 days) Bihar VC O1 Ogawa Proteobacteria 0.864882507
S19 Female 4y H (1 day) Kolkata V.fluvialis Firmicutes 0.745517928
S20 Male 42y H (1 day) Kolkata VC O1 Ogawa Firmicutes 0.043091111
Fig. 2 Flow-chart for 16S rDNA V3-V4 region amplicon sequencing and analysis of metagenomic data
of Bacteroidetes and 20.71% of Firmicutes. Bacteroidetes Different bacterial classes were found in variable pro-
was the most dominant phylum only in S13 (28.87%). S13 portion in the 20 samples. Actinobacteria, Bacilli, Bac-
was associated with VC O139. Table 1 shows the most teroidia, Coriobacteria, Clostridia, γ-Proteobacteria
abundant phylum present in each sample S1–S20. Fig- and Verrucomicrobiae were the most prominent classes
ure 4 shows the relative abundance (in percentage) of the observed. Bacilli was the most dominant class in seven
major phyla in each sample from S1 to S20. A large pro- samples (S3, S7,S8, S10, S11, S14, S16). S1 was mainly
portion of reads in every sample could not be assigned composed of unclassified bacteria and γ-Proteobacteria
any taxonomic rank below domain and was labeled as is the only annotated class that is present in high propor-
unassigned bacteria (Fig. 4). The mean of abundance of tion but < 50%. In this sample all other classes are present
various phyla in 20 samples was 38% Firmicutes, 10% in lower proportion. In S18 γ-Proteobacteria was found
Bacteroidetes, 12% Actinobacteria and 19% Proteobac- in relative abundance of > 50%. Other samples where
teria. Verrucomicrobia, Fusobacteria, Tenericutes, Spi- γ-Proteobacteria was present prominently but at < 50%
rochaetes, Lentisphaerae, Elusimicrobiae, Cyanobacteria, relative abundance were S2, S3, S4, S6, S8, S10, S11, S12,
Synergistetes, Deferribacteres, Acidobacteria, Armati- S13, S14, S16, S17, S19, S20.
monadetes, Caldotrichaeota, Chloroflexi, Deinococcus, S7, S9, S17 showed the presence of ~ 25% Erysipelotri-
Fibrobacteres, Gemmatomonadetes, Ignavibacteriae, cha while S2, S10, S15 and S19 have < 25%. Classes that
Nitrospinae, Kiritimatiellaeota, Planctomycetes, Candi- were found in > 0%– < 1% abundance in many of the sam-
date Phyla Radiation (CPR) also appeared in many samples were Acidimicrobia, Rubrobacteria, Armatimonadia,
ples. Candidatus Saccharibacteria or TM7 phylum, was Cytophagia, Flavobacteria, Calditrichae, Anaerolineae,
detected in many samples like S2 (0.04%), S3 (0.03%), S7 Deinococci, Negativicutes, Tissierellia, Fusobacteria, α,
(0.01%), S8 (0.03%), S11 (0.01%), S13 (0.01%), S14 (0.02%). β, δ, ε, ζ- Proteobacteria, Fimbrimonadia, Nitrilorup-
Verrucomicrobiae formed 19.91% of S5, 13.4% of S13 and toria, Ktedonobacteria, Sphingobacteria, Fibrobacte-
11.94% of S20. From all the three samples VC was iso- ria, Gemmatimonadetes, Ignavibacteria, Lentisphaeria,
lated as the diarrheal agent. In S1 and S2 which were also Phycisphaerae, Opitutae, Endomicrobia, Spiritrichae,
associated with VC Verrucomicrobia was present at an Saprospiria, Oligoflexia, Oligosphaeria, Spirochaetia,
abundance of > 0%–< 1%. Table 2 presents a catalogue of Synergistia, Mollicutes, Chloroflexia, Elusimicrobia, Aci-
the different phyla found in the study cohort. dithiobacillia, Solibacteres, Chitiniphagia, Chlamydiia,
Fig. 3 Histogram showing relative abundance of a Phylum, b Class, c Order, d Family, e Genus, f Species
Fig. 3 continued
Fig. 3 continued
90
80
70
60
50
40
30
20
10
0
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20
Unassigned Bacteria Proteobacteria Firmicutes Actinobacteria

Bacteroidetes Verrucomicrobiae Fusobacteria Tenericutes
Lentispharae Euryarchaeota Elusimicrobiae Cyanobacteria
Synergistetes Deferribacteres
Fig. 4 Relative abundance of the major bacterial phyla in the diarrheal gut microbiome. Bar-diagram showing relative abundance of the major
bacterial phyla in each diarrheal sample
Kiritimatiella, Halobacteria, Caldilineae, Dehalococ- between 0% and 1%. The abundance of Micrococcales
coidea, Thermomicrobia, Limnochordia, Planctomy- was as high as 3.3% in S8, 12.5% in S11 and 4.6% in S14.
cetia, Hydrogenophilalia, Balneolia, Spartobacteria, Clostridiales was a dominant order in S5, S9, S10, S12,
Holophagae, Thermideophilia, Longimicrobia. S13, S15, S17, S18, S19, S20 where its abundance rate was
Table 2 presents a list of all the different orders 8%–40% while in S2, S6, S7, S8, S14 they were present at
reported from this study. Order Actinomycetales, Bacte- 1%–2% abundance. Abundance of 3%–44% Coriobacteri-
roidales, Enterobacterales, Bifidobacteriales, Corynebac- ales was observed in S9, S12, S15, S17, S18, S19, S20 while
teriales, Micrococcales, Clostridiales, Coribacteriales, in all other samples its abundance rate was < 1%. Propor-
Erysipelotrichales, Lactobacillales, Pseudomonadales, tion ranging from 1% to 21.5% of Erysipelotrichales was
Tissierellales, Verrucomicrobiales, Vibrionales, Strepto- present in S2, S7, S8, S9, S10, S12, S15, S17, S18, S19 and
mycetales, Flavobacteriales, Bacillales, Selenomonadales, S20. In the remaining samples it occurred at < 1% abun-
Fusobacteriales, Rhizobiales, Rhodobacterales, Burk- dance. A proportion of the diarrheal microbiota com-
holderiales, Neisseriales, Desulfovibrionales, Myxococca- prised Lactobacillales in a majority of the samples. These
les, Campylobacterales, Aeromonadales, Cellvibrionales, were S3 (> 50%), S4 (> 5.5%), S6 (> 5.3%), S7 (> 46.5%),
Chromatiales, Pasteurellales, were found in variable pro- S8 (> 42%), S10 (31.4%), S11 (> 39%), S13 (> 1.4%), S14
portion in all the twenty samples. In S7, S8, S14 and S20 (> 55.7%), S15 (> 2.8%), S16 (> 71.7%), S17 (> 7.6%), S20
Actinomycetales was found at 2%–6% abundance. In S5, (> 10.1%). In the remaining samples presence of Lacto-
S13, S15, S19 Bacteroidales were observed at > 25% abun- bacillales was found at an abundance rate of < 1% Pseu-
dance. Abundance of Enterobacterales in S5, S9, S17, S18, domonadales was conspicuous in S12 (17.1%), S14 (1.5%),
S19 was > 0%–1%. In all others it was between 1 and 25%. S19 (3.2%) and Tissierellales in S20 (21.1%). In S5, S13
Bifidobacteriales was present at > 5% abundance in S2, and S20 order Verrucomicrobiales was present at 19.9%,
S7, S9, S10, S14, S15, S16, S17, S18, S19, S20 with > 25% 13.4% and 11.9% respectively. Vibrionales were conspic-
abundance in S15. In all other samples its abundance was uously abundant in S13, S14 and S18 and were found
Table 2 Catalogue of phyla, orders, families found in the study cohort

Phylum present in all subjects Phylum not present in all subjects
Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria Verrucomicrobia, Fusobacteria, Tenericutes, Spirochaetes, Lentisphaerae, Elusimicrobiae,
Cyanobacteria, Synergistetes, Deferribacteres, Acidobacteria, Armatimonadetes, Cal-
dotrichaeota, Chloroflexi, Deinococcus-Thermus, Candidatus, Fibrobacteres, Gemmato-
monadetes, Ignavibacteriae, Nitrospinae, Kiritimatiellaeota, Planctomycetes, Balneolaeota,
Chlamydiae,
Candidatus Falkowbacteria, Candidatus Moranbacteria, Candidatus Saccharibacteria, Candi-
datus Latescibacteria,
Candidatus Melainabacteria,Candidatus Peregrinibacteria, Thermodesulfobacteria, Can-
didatus Shapirobacteria, Candidatus Jorgensenbacteria, Candidatus Raymondbacteria,
Candidatus Schekmanbacteria, Candidatus Doudnabacteria, Candidatus Gracilibacteria,
Candidatus Portnoybacteria, Candidatus Yanofskybacteria,Candidatus Parcubacteria,
Candidatus Wolfebacteria,Candidatus Lindowbacteria,
Candidatus Pacebacteria
Orders present in all subjects Orders not present in all subjects
Actinomycetales, Bacteroidales, Enterobacterales, Bifidobacteriales, Veillonellales, Propionibacteriales, Eggerthellales, Pseudonocardiales,

Corynebacteriales, Micrococcales, Clostridiales, Coribacteriales, Erysipel- Gaiellales, Armatimonadales, Oceanospirillales, Cytophagales, Acid-
otrichales, Lactobacillales, Pseudomonadales, Tissierellales, Verrucomicro- aminococcales Rhodospirillales, Rickettsiales, Sphingomonadales,
biales, Vibrionales, Streptomycetales, Flavobacteriales, Bacillales, Selenom- Xanthomonadales, Oligoflexales, Sphaerobacterales, Kallotenuales,
onadales, Fusobacteriales, Rhizobiales, Rhodobacterales, Burkholderiales, Chroococcales, Calditrichales, Anaerolineales, Deinococcales, Caulo-
Neisseriales, Desulfovibrionales, Myxococcales, Campylobacterales, bacterales, Nitrosomonadales, Bradymonadales, Desulfobacterales,
Aeromonadales, Cellvibrionales, Chromatiales, Pasteurellales Alteromonadales, Arenicellales, Cardiobacteriales, Legionellales,
Immundisolibacterales, Thiotrichales, Mariprofundales, Spirochaetales,
Synergistales, Mycoplasmatales, Acidimicrobiales, Acidothermales,
Frankiales, Kineosporiales, Euzybyales, Gaiellales, Marinilabiliales,
Deferribacterales, Fibrobacterales, Gemmatimonadales, Victivallales,
Acidithiobacillales, Holosporales, Rhodocyclales, Desulfuromonadales,
Acholeplasmatales, Methylococcales, Bacteriovoracales, Bdellovibrion-
ales, Brachyspirales, Anaeroplasmatales, Opitutales, Puniceicoccales,
Saprospirales, Halanaerobiales, Ignavibacteriales, Planctomycetales,
Magnetococcales, Haloplasmatales, Acidobacterales, Solibacterales,
Rubrobacterales, Chitinophagales, Sneathellales, Micromonosporales,
Parachlamydiales, Caldilineales, Thermomicrobiales, Limnochordales,
Chthoniobacterales, Jiangellales, Streptosporangialles, Thermoanaero-
bacterales, Kordiimonadales, Acidiferrobacterales, Nakamurellales, Soli-
rubrobacterales, Dehalococcoidales, Endomicrobiales, Holophagales,
Ardenticatenales, Nostocales, Gloebacterales, Parvularculales, Pelagi-
bacteriales, Natranaerobiales, Desulfarculales, Syntrophobacterales,
Synechococcales, Geodermatophilales, Methylacidophilales, Kiloniella-
les, Hydrogenophilales, Nitriliruptorales, Balneolales, Ktedonobacterales,
Salinisphaerales, Chloroflexales, Ferrovales, Orbales, Nitriruptorales,
Fimbrimonadales, Oligosphaerales, Nitrospirales, Neviskiales, Entomo-
plasmatales, Acanthopleuribacterales, Pleurocapsales Longimicrobiales.
Table 2 (continued)
Families present in all subjects Families not present in all subjects
Actinomycetaceae, Bifidobacteriaceae, Corynebacteriaceae, Micro- Solibacteraceae, Gordoniaceae, Dietziaceae, Nocardiaceae, Brevibac-

bacteriaceae, Micrococcaceae, Streptomycetaceae, Atopobiaceae, teriaceae, Dermacoccaceae, Dermatophilaceae, Intrasporangiaceae,
Coriobacteriaceae, Bacteroidaceae, Prevotellaceae, Rickenellaceae, Nocardioidaceae, Propionibacteriaceae, Pseudonocardiaceae, Egg-
Flavobacteriaceae, Bacillaceae, Staphylococcaceae, Aerococcaceae, Car- erthellaceae, Dysgomonadaceae, Lentimicrobiaceae, Muribaculaceae,
nobacteriaceae, Enterococcaceae, Lactobacillaceae, Streptococcaceae, Odoribacteraceae, Paludibacteraceae, Prolixibacteraceae, Porphy-
Christencenellaceae, Clostridiaceae, Clostridiales Family XIII Incertae romonadaceae, Tannerellaceae, Cytophagaceae, Microscillaceae,
Sedis, Lachnospiraceae, Peptococcaceae, Peptostreptococcaceae, Rumi- Calditrichaceae, anaerolineaceae, Deinococcaceae, Saprospiraceae,
nococcaceae, Erysipelotrichaceae, Selenomonadaceae, Veillonellaceae, Alicyclobacilaceae, Planococcaceae, Leuconostocaceae, Caldicopro-
Peptoniphilaceae, Fusobacteriaceae, Methylobacteriaceae, Rhodobac- bacteraceae, Eubacteriaceae, Oscillosporaceae, Syntrophomonadaceae,
teraceae, Neisseriaceae, Campylobacteraceae, Succinivibrionaceae, Acidaminococcaceae, Leptotrichiaceae, Caulobacteraceae, Hyphomona-
Enterobacteriaceae, Pasteurellaceae, Vibrionaceae, Akkermansiaceae daceae, Erythrobacteraceae, Sphingomonadaceae, Burkholderiaceae,
Comamonadaceae, Suttarellaceae, Nitrosomonadaceae, Bradymona-
daceae, Desulfobulbaceae, Desulfovibrionaceae, Helicobacteraceae,
aeromonadaceae, Shewanellaceae, Arenicellaceae, Cardiobacteriaceae,
Helieaceae, Spongibacteraceae, Chromatiaceae, Erwiniaceae, Morga-
nellaceae, Pectobacteriaceae, Yersiniaceae, Immundisolibacteraceae,
Coxiellaceae, Alcanivoraceae, Endozoicomonaceae, Halomonadaceae,
Oceanospirillaceae, Thiotrichaceae, Xanthomonadaceae, Oligoflexaceae,
Mariprofundaceae, Spirochaetaceae, Synergiataceae, Mycoplasmataceae,
Fabaceae, Acidimicrobiaceae, Mycobacteriaceae, Sphingobacteriaceae,
Sporichthyaceae, Kineosporiaceae, Bogoriellaceae, Dermabacteraceae,
Microcystaceae, Deferribacteraceae, Elusimicrobiaceae, Fibrobacteraceae,
Defluvitaleaceae, Thermoactinomycetaceae, Sporomusaceae, Gemma-
timonadaceae, Acidithiobacillaceae, Candidatus Paracaedibacteraceae,
Hyphomicrobiaceae, Acetobacteraceae, Geminicoccaceae, Rickettsiaceae,
Oxalobacteraceae, Thiobacillaceae, Azonexaceae, Rhodocyclaceae, Zoog-
loeaceae, Desulfomicrobiaceae, Desulfuromonadaceae, Geobacteraceae,
Alteromonadaceae, Moritellaceae, Porticoccaceae, Halothiobacillaceae,
Hafniaceae, Legionellaceae, Methylococcaceae, Bactriovoracaceae,
Bdellovibrionaceae, Brachyspiraceae, Anaeroplasmataceae, Opitutaceae,
Puniceicoccaceae, Frankiaceae, Promicromonosporaceae, Listeriaceae,
Paenibacillaceae, Sporolactobacillaceae, Halanaerobiaceae, Halobacte-
roidaceae, Ignavibacteriaceae, Magnetococcaceae, Chromobacteriaceae,
Desulfobacteriaceae, Sandaracinaceae, Williamsiaceae, Gemmataceae,
Rhodospirillaceae, Kofleriaceae, Pseudoalteromonadaceae, Microbul-
biferaceae, Hanellaceae, Saccharospirillaceae, Rhodoanobacteraceae,
Bryobacteraceae, Beijerinckiaceae, Sneathiellaceae, Haloplasmataceae,Tsu
kamurellaceae, Micromonosporaceae, Barnesiellaceae, Cyclobacteraceae,
Parachlamydiaceae, Caldiliniaceae, Aurantimonadaceae, Rhizobiaceae,
Ectothiorhodospiraceae, Jiangellaceae, Cellumonadaceae, Rubrobacte-
raceae, Chitinophagaceae,
Bradyrhizobiaceae, Brucellaceae, Cellvibrionaceae, Nakamurellaceae, Seg-
niliparaceae, Amoebophilaceae, Cryomorphaceae, Endomicrobiaceae,
Pasteuriaceae, Clostridiales Family XVIII Incertae Sedis, Gracilibacteraceae,
Thermodesulfobiaceae, Isophaeraceae, Planctomycetaceae, Anaplas-
mataceae, Candidatus Midichloriaceae, Methylophilaceae, Polyangiaceae,
Oleiphilaceae, Leptospiraceae, Verrucomicrobiaceae, Holophagaceae,
Crocinitomicaceae, Gottschalkiaceae, Victivallaceae, Parvularculaceae,
Alcaligenaceae, Competibacteraceae, Psychromonadaceae, Woeseiaceae,
Acholeplasmataceae, Sanguibacteraceae, Thermoanaerobacterales Fam-
ily III Incertae Sedis, Desulfarculaceae, Geodermatophilaceae, Natramae-
robiaceae, Liminochordaceae,Anaeromyxobacteraceae, Hymenobacte-
raceae, Trueperaceae, Archangiaceae, Rubritaleaceae, Idiomarinaceae,
Hydrogenophilaceae, Nitriliruptoraceae, Flammeovirgaceae, Ichthyo-
bacteriaceae, Proteinivoraceae, Rhodobiaceae, Xanthobacteraceae,
Chlamydiaceae, Orbaceae, Blattabacteriaceae, Nitrospiraceae, Chthonio-
bacteraceae, Cohaesibacteraceae, Acanthopleuribacteraceae, Parviter-
ribacteraceae, Xenococcaceae, Longimicrobiaceae, Galiionellaceae,
Syntrophaceae, Thorselliaceae, Beutenbergiaceae, Thermoanaerobac-
terales, Family IV Incertae Sedis, Phyllobacteriaceae, Sterolibacteriaceae,
Ferrimonadaceae.
at 16.7%, 11% and 56.9% respectively. Bacillales were Selenomonadales was present at 6.2% in S2. Burkholde-
present above 1% in S6 (4.3%), S8 (6.7%), S14 (7.78%). riales and Neisseriales were present at 1% and 1.9%
respectively in S19. 2% Campylobacterales was present in at < 1% in all the samples except S5, S13 and S20. The
S12. Aeromonadales were present at 1.3% in S6 and 5.5% relative abundance of Akkermansia sp. in S5 was 19.2%,
in S19. Pasteurellales were present at 6% in S8, 1.5% in in S13 was 13.1% and in S20 was 11.6%. Clostridium sp.
S14 and 1.8% in S17. was found at < 1% abundance in all the 20 samples. Bifido-
Veillonellales were completely absent in S1. It was bacter sp. was present at 32% in S15. In all other samples
found in the remaining samples. Its proportion in its abundance was below 10%. A complete hierarchical
some of the samples were as follows: S2 (2%), S3 (3%), classification of the different microbial units found in the
S7 (1.2%), S8 (4.8%), S9 (1.2%), S10 (0.7%), S12 (3.7%), study by 16S rDNA sequence homology has been pre-
S13 (1.4%), S14 (0.25%), S17 (4.5%), S18 (0.63%), S19 sented in Additional file 1.
(1.8%), S20 (5.7%). Propionibacteriales and Eggerthel-
lales were absent in S4, Pseudonocardiales were absent Correlation of commensal and pathogen abundance
in S3, S5, S9, S10, S12, S18, S19, Gaiellales were absent in diarrhea
in S1, S3, S4, S8, S14 and S20. Armatimonadales were Differences in relative abundance of four different fami-
absent in S1, S2, S3, S4, S7, S8, S12, S18, S19. Oceano- lies namely Bifidobacteriacea, Enterobacteriaceae, Bac-
spirillales were absent in S16. Cytophagales were found teroidaceae and Vibrionaceae in diarrheal samples
to be absent in S4, S11 and S18. Acidaminococcales and S1 to S20 were observed and graphically presented in
Rhodospirillales were absent in S3. Rickettsiales were Fig. 6A(a). Families Bifidobacteriaceae and Enterobacte-
absent in S1 and S7. Sphingomonadales was absent in riaceae were negatively correlated with rs − 0.40695 and
S5. Xanthomonadales and Oligoflexales were absent 2-tailed p value of 0.07495. The association was non-sig-
in S5 and S9. Sphaerobacterales were present only in nificant. Negative correlation between Bifidobacteriaceae
S10 and Kallotenuales in S18 only. Chroococcales were and Vibrionaceae was found at rs − 0.03073 and the
found in S14, S17 and S18. Other orders that appear association was non-significant with 2-tailed p value
in Table 2 were observed in some samples in minute of 0.8977, Streptococcaceae and Enterobacteriaceae were
proportion. found to be positively correlated with rs = 0.29959 and
Table 2 shows the different bacterial families that were the association was found to be non-significant with a
found in the study. Streptococcaceae was the dominant 2-tailed p value of 0.19941.
family in 35% of samples, in 10% samples Coriobacte- A significant positive correlation with rs 0.4751 and a
riaceae was dominant and 5% samples Vibrionaceae was two-tailed p-value of 0.0343 between Bifidobacteriaceae
dominant. S1 consisted of predominantly Enterobacte- and Lachnospiraceae, significant negative correlation
riaceae and Unclassified Bacteria. Actinomycetaceae, Bifi- with rs − 0.6338 and − 0.6882 and two-tailed p-values
dobacteriaceae, Corynebacteriaceae, Coriobacteriaceae, of 0.0027 and 0.0008 respectively between Enterobac-
Bacteroidaceae, Prevotellaceae, Streptococcaceae, teriaceae and Lachnospiraceae and Enterobacteriaceae
Ruminococcaceae, Erysipelotrichaceae, Veillonellaceae, and Ruminococcaceae, significant positive correlation
Enterobacteriaceae, Pasteurellaceae, Vibrionaceae, between Lachnospiraceae and Ruminococcaceae with
Lachnospiraceae were found at greater than 1% in many rs 0.7111 and two-tailed p-value of 0.0004 and signifi-
samples (Fig. 5). In all other samples all these families cant negative correlation between Lachnospiraceae and
occurred at a relative abundance of less than 1%. Streptococcaceae with rs − 0.5215 and two-tailed p-value
Table 3 presents the genera and species under Kingdom of 0.0184, significant negative correlation was found
Bacteria that were found in the study cohort. A total of between Ruminococcaceae and Streptococcaceae with rs
584 genera were observed. 136 of these could be further − 0.6847 and two-tailed p-value of 0.0009. The Spear-
classified till the species level while the remaining 448 man’s rank correlation coefficient and two-tailed p-values
could not be classified further with 16S rDNA amplicon have been represented graphically in Fig. 6a.
sequencing. Akkermansia sp., Alloprevotella sp., Bacte-
roides sp., Bifidobacterium sp., Catenibacterium sp., Col- Difference in abundance of commensals and pathogens
linsella sp., Holdmanella sp., Streptococcus sp., Vibrio sp. in diarrhea
occurred at 1% and greater relative abundance. Genera Kruskal–Wallis test performed to compare difference in
present in all the 20 diarrheal samples were Actinomyces abundance among families of commensals namely, Bifido-
sp., Bifidobacterium sp, Corynebacterium sp., Bacteroides bacteriaceae, Ruminococcaceae and Lachnospiraceae in
sp., Alloprevotella sp., Lactobacillus sp., Streptococcus diarrhea showed a positive trend with H statistic 1.5543
sp., Clostridium sp., Blautia sp., Peptostreptococcus sp., (2, N = 60) and with a p-value of 0.4597. Kruskal–Wallis
Faecalibacterium sp., Holdemanella sp., Dialister sp., test performed to compare differences among families of
Methylobacterium sp., Neisseria sp., Acinetobacter sp., pathogens namely, Bacteroidaceae, Enterobacteriaceae
Vibrio sp., Akkermansia sp. Akkermansia sp. was found
a b c
f
h
g
Fig. 5 Sample-wise distribution of relative abundance of different bacterial families. Pie-chart showing families of commensals and pathogens in
diarrheal samples in which these were found at >1% relative abundance a Actinomycetaceae, b Bacteroidaceae, c Vellionellaceae d Vibrionaceae
e Bifidobacteriaceae f Streptococcaceae, g Enterobacteriaceae, h Coriobacteriaceae, i Erysipelotrichaceae, j Pasteurellaceae, k Prevotellaceae, l
Lachnospiraceae
j
i
l
Fig. 5 continued
and Vibrionaceae showed a significant difference with H and Enterobacteriaceae, Bifidobacteriaceae and
statistic 21.574 (2, N = 60) with p-value of 0.00002. Vibrionaceae, Lachnospiraceae and Vibrionaceae,
Unpaired t-test with Wilcoxon matched-pairs signed Lachnospiraceae and Enterobacteriaceae, Enterobac-
rank test was used to calculate and compare the differ- teriaceae and Vibrionaceae and Aeromonadaceae and
ence of relative abundance of family Bifidobacteriaceae Enterobacteriaceae. Figure 6B shows the differences in
Table 3 Genus and species catalogue

Genus Species under the corresponding genus Samples carrying these species
Blastocatella sp.
Actinomyces sp.*
A. odontolyticus S13, S7, S8
A. graevenitzii S7, S8, S14
A. oris S8
Arcanobacterium sp.
Bifidobacterium sp.*
B. longum S16, S9, S17, S15, S10
B. ramosum S16, S15
B. secularae S7
B. bifidum S17
B. pullorum S17
B. sp. MRM 8. 19 S2
B. aerophilum S15
B. angulatum S2, S9, S10, S15, S19, S17
B. animalis S15, S10
B. avesanii S15
B. biavatii S15
B. sp. MC10 S15, S17
Corynebacterium sp.*
C. falsenii S3
C. bovis S16
C. accolens S8, S10, S13, S14
C. kroppenstedtii S9, S20, S10, S13
C. cystitidis S20
C. sp. M72 S10
C. doosarense S1
C. simulans S14
C. sp. NML98-0116 S16
C. glaucum S7
C. sp. 1938BRRJ S7, S11, S13, S14
C. sp3210O2 S7, S8
C. sp. NML080024 S7
C. durum S20
Turicells sp.
T. otidis S1, S3, S4, S7, S14, S15, S20
Dietzia sp.
Rhodococcus sp.
Brevibacterium sp.
B. luteolum S17
Kytococcus sp.
Candidatus Planktoluna sp.
Microbacterium sp.
Micrococcus sp.
M. terreus S14
Rothia sp.
R. dentocariosa S7, S8, S11, S2, S13, S14
R. aeria S11
R. sp. HMSC065C03 S14
Friedmanniella sp.
Mamoricola sp.
Cutibacterium sp.
Streptomyces sp.
S. sp. Q1 S13, S14
Libanicoccus sp.
L. massiliensis S18, S19, S9, S17, S12, S13
Enterorhabdus sp.
Slckia sp.
S. hellotrinireducens S15, S17, S20, S12, S10, S13
Bacteroides sp.*
B. plebius S7, S10,
B. fragilis S15, S19
B. propionicifaciens S10, S15
B. sp. CAG:1060_57_27 S5
B. vulgatus S10, S13
B. acidifaciens S13
B. paurosaccharolyticus S13
Butyricimonas sp.
Odoribacter sp.
Porphyromonas sp.
P. sp. oral taxon 278 K S3
Alloprevotella sp.*
A. tannerae S15, S20
A. rava S2, S5, S19, S13
Prevotella sp.
p. enoeca S6, S11
P. micans S8
P. sp. AN5135 S15, S2
P. copri S19, S12, S2, S5, S13
P. sp. oral clone ASCG10 S19, S12
P. sp. HJM029 S12
P. sp. 310-5 S13
P. marshii S18, S7, S15, S19, S12, S2
P. nigrescens S8, S20, S11
P. stercorea S15, S19, S12, S5
P. sp. 152R-1a S19
P. intermedia S2
P. sp. HUN102 S2
P. sp. oral cloneKWO35 S2
Parabacteroides sp.
Flectobacillus sp.
Bergeyella sp.
B. sp. AF14 S8
Capnocytophaga sp.
C. sputigena S8
C. ochracea S11
C. sp. oral clone ID062-W S2
Chryseobacterium sp.
Deinococcus sp.
D. geothermalis S6
Exiguobacterterium sp.
Gemelia sp.
G. sanguinis S3, S8, S2, S14
G. morbillorum S8, S20
Effusibacillus sp.
Bacillus sp.
Planococcus sp.
Staphylococcus sp.
S. haemolyticus S14
S. simulans S14
Aerococcus sp.
A. vaginalis S13, S20
Dolosigranulum sp.
Granulicatella sp.
G. sp. BB-11 S8, S20, S11, S14
G. sp. canine oral taxon O95 S14
Enterococcus sp.
E. faecium S16
E. italicus S11, S14
E. xinjiangensis S16
E. casseliflavus S10
E. hermanniensis S10
E. durans S10
Lactobacillus sp.*
L. coryniformis S11
L. satsumensis S14
Lactococcus sp.
Streptococcus sp.*
S. agalactiae S2, S6, S3, S7, S8, S11, S10, S14
S. pneumoniae S8, S17, S11, S2, S10, S1, S14
S. parasanguinis S2, S5, S6, S3, S7, S8, S11, S10, S13, S14, S17, S20
S. anginosus S18, S3, S4, S7, S8, S17, S20, S2, S10, S13, S14
S. dannielliae S18, S3, S4, S16, S20, S11, S12, S2, S10
S. ferus S18
S. pantholopis S3
S. sp. oral clone ASCB12 S3, S7, S8, S17, S20, S11, S10, S14
S. sp. oral taxon GS9 S3, S11, S14
S. didelphis S16
S. equinus S4
S. parasuis S16
S. sanguinis S7, S8, S17, S14
S. sp. DD04(2016) S7, S11, S14
S. sinensis S7
S. sp. XJ149-N32 S7, S8, S11, S2, S14
S. vestibularis S7, S8, S17, S11
S. mutans S20
S. cristatus S8, S20, S14
S. equi S10, S14

S. gordonii S14
S. mitis S14
Clostridium sp.*
C. magnum S1, S2, S3, S4, S15, S17, S18, S19, S20
C. sp. Marseille S15, S19, S19, S12
C. sp. CE6 S19, S5, S10, S13
C. sp. CAG:288 S12
C. sp. Culture-Jar-56 S5
Lutispora sp.
Fusibacter sp.
Mogibacterium sp.
Alkalibacter sp.
Agathobacter sp.
Anaerosporobacter sp.
Anaerostipes sp.
Blautia sp.*
B. massiliensis S18, S19, S9, S20, S2, S5, S10, S13
B. hydrogenotrophica S10, S13
B. stercosis S13
Catonella sp.
Dorea sp.
D. formicigenerans S7, S19, S9, S17, S20, S12, S5, S10
D. longicatena S2, S19
Lachnoclostridium sp.
[Clostridium]polysaccharolyticum S15
Roseburia sp.
R. hominis S6, S17, S2, S5
R. sp. 1120 S9, S10
Shuttleworthia sp.
Stomatobaculum sp.
Oscilibacter sp.
Desulfotomaculum sp.
Peptostreptococcus sp.*
Faecalibacterium sp.*
F. prausnitzii S2, S12, S18, S19, S20
Fastidiosipila sp.
Ruminiclostridium sp.
Eubacterium siraeum S18, S19, S20, S12, S13
Clostridium leptum S19, S20, S12, S5
Ruminococcus sp.
R. sp. CE2 S16, S15, S19, S2, S10, S13
R. sp. YE58 S15, S9, S10
R. sp. RLB3 S9, S12
R. sp. W22 S9, S17, S10, S13
R. sp. 653 S17
R. bromii S12, S5
R. sp. YE281 S12
R. sp. ID1 S2
R. gauvreaucii S5
R. sp. Marseille-P328 S13
Subdoligranulum sp.
Dethiobacter sp.
Catenibacterium sp.
C. mitsuokai S18, S7, S15, S19, S9, S5, S17
Holdemanella sp.*
Solobacterium sp.
Turicibacter sp.
Phascolarctobacterium sp.
P. sp. 377 S17
P. sp. canine oral taxon 212 S17, S5
Megamonas sp.
Dialister sp.*
D. sp. 67 S18, S8, S15, S19, S9, S17, S20, S12, S2, S10, S13
D. succinatiphilus S17
Veillonella sp.
V. sp. oral clone OH1A S3, S8, S20, S10, S13.S14
V. sp. oral taxon 780 S8, S2, S13
V. atypica S17, S13
V. sp. 2011-11eoVSA-F2 S17
V. magna S20
Anaerococcus sp.
A. sp. S138 S4
A. prevotii S16
A. sp. S194 S16
A. octavius S7
Peptoniphilus sp.
Fusobacterium sp.
F. nucleatum S11
F. russii S2
Leptotricha sp.
L. sp. oral clone FP036 S11
L. sp. oral taxon 212 S11, S2, S14
L. sp. oral taxon 847 S11, S2
L. buccalis S14
Streptobacillus sp.
S. hongkonggensis S6, S20
Brevundimonas sp.
Methylobacterium sp.*
Paracoccus sp.
Actererythrobacter sp.
Novosphingobium sp.
Sphingomonas sp.
Lautropia sp.
L. mirabilis S2, S6, S11, S13, S14
Ralstonia sp.
Parasutterella sp.
P. excrementihominis S10
Sutterella sp.
S. sp. YIT-12072 S7, S5
Neisseria sp.*
N. meningitidis S18, S19, S20
N. shayeganii S18, S19, S20
N. elongata S11, S14
N. flavescens S11
N. lactamica S2
Simonsiella sp.
Campylobacter sp.
C. faecalis S18
C. concisus S11
Helicobacter sp.
H. pylori S2, S6, S7, S11, S14,
Shewanella sp.
Rheinheimera sp.
Buttiauxella sp.
Candidatus Benitsuchiphilus sp.
Candidatus Blochmannia sp.
Citrobacter sp.
C. amalonaticus S10
Eschericia sp.
E. coli S1, S2, S3, S4, S6, S8, S10, S11, S13, 16, S17, S19, S20
E. albertii S1, S2, S4, S6, S10, S11, S13, S20
E. sp. S1, S11
Klebsiella sp.
K. sp. A4 S1, S2, S6, S13, S18
K. pneumoniae S4, S7, S10
K. aerogenes S7
Shigella sp.
S. dysenteriae S6, S11
S. sonnei S6, S10, S16
Shimwella sp.
Xenorhabdus sp.
Brenneria sp.
B. sp. DAF NE_Bnig-1 S4
B. populi S10
Dickeya
Serratia sp
S. marcescens S3, S11
Alcanivorax sp.
Halomonas sp.
Aggregatibacter sp.
Haemophilus sp.
H. haemolyticus S4, S6, S8
H. parainfluenzae S4, S8, S14
H. pittmaniae S8
H. sp. oral clone BJ021 S17
H. sputorum S11, S17
H. influenzae S1
Acinetobacter sp.*
A. baumanii S19
A. sp. S19
A. calcoaceticus S12
Moraxella sp.
M. osloensis S6, S7
Pseudomonas sp.
P. putida S1
Vibrio sp.*
V. cholerae S1, S2, S5, S6, S8, S9, S11, S12, S15, S17, S18, S19, S20
V. metoecus S18, S13, S14
V. neptunius S18
V. proteolyticus S18
V. parahaemolyticus S13
V. sp. NJ-2 S13
Treponema sp.
T. pectinovorum S20
T. berlinense S5
Fretibacterium sp.
Mycoplasma
M. muris S10
Akkermansia sp.*
A. muciniphila S5, S6, S7, S8, S13, S15, S16, S18, S19, S20
A. glycaniphila S5
Mobiluncus sp.
Gardnerella sp.
Mycobacterium sp.
Brachybacterium sp.
Serinicoccus sp.
Glutamibacter sp.
Kocuria sp.
Nesterenkonia sp.
Propioniciclava sp.
P. sp. SCSIO_13291 S18
Atopobium sp.
Olsenella sp.
Collinsella sp.
C. aerofaciens S18, S4, S16, S8, S15, S19, S9, S17, S20, S11, S12, S2,
S5, S10, S1
C. bouchesdurhonensis S15, S18, S19, S9, S17, S20, S12, S14
C. ihuae S18, S15, S19, S9, S17, S20, S12, S14
C. sp. Marseille-P3740 S15, S9, S17, S20
C. phocaeensis S17
Senegalimassilea sp.
Gaiella sp.
Paludibacter sp.
P. jiangxiensis S11, S16, S18
Macellibacteroides sp.
Massiiprevotella sp.
M. massiliensis S1, S2, S5, S9, S12, S18, S19, S20

Prevotellamassilia sp
P. timonensis S18, S19, S17, S20, S12, S2, S10
Alistipes sp.
A. finegoldii S15
Empedobacter sp.
Arcticibacter sp.
Sphingobacterium sp.
S. sp. JAS3 S19, S20
Microcystis sp.
M. sp. SAG43. 90 S18
Elusimicrobium sp.
E. minutum S12
Fibrobacter sp.
Lactobacillalis bacterium HY-36-1)
Weissella sp.
Howardella sp.
H. ureilytica S2
Intestimonas sp.
Christensenella sp.
C. massiliensis S5, S19
Butyricoccus sp.
B. faecihominis S9, S17, S20, S13
Oxobacter sp.
Acidaminobacter sp.
Anaerofustis sp.
Eubacterium sp.
E. coprostanoligenes S18, S19, S20
E. eligens S2, S5, S10, S12, S17, S18, S19, S20
E. sp. oral clone DO 016 S3, S7, S8
E. sp. oral clone FX028 S7, S14
E. pyruvativorans S12
E. sp. oral clone El074 S14
Butyrivibrio
B. crossotus S12, S18, S19, S20
Eisenbergiella sp.
Fusicatenibacter sp.
Lachnospira sp.
Marvinbryantia sp.
M. formatexigens S20, S13
Moryella sp.
Oribacterium sp.
Tyzzerella sp.
Peptococcus sp.
Romboutsia sp.
Candidatus soleaferrea
Fournierella sp.
Negativibacillus sp.
Papillibacter sp.
Phocea sp.
Saccharofermentans sp.
S. acetigenes S20
Sporobacter sp.
Dielma sp.
Erysipelothrix sp.
E. larvae S7
Anaerovibrio sp.
Mitsuokella sp.
Selemonas sp.
S. sputigena S20
S. noxia S11
Sporomusa sp.
Allisonella sp.
A. histaminiformans S19, S9, S17, S12, S2
Megasphaera sp.
M. micronuciformis S13
Finegoldia sp.
Parvimonas sp.
Cetobacterium sp.
Craurococcus sp.
Roseomonas sp.
Candidatus Alysiosphaera
Sphaerotilus sp.
S. natans S9
Comamonas sp.
C. aquatica S12
C. testosteroni S12
Diaphorobacter sp.
Oxalobacter sp.
O. formigenes S18, S19, S10
Eikenella sp.
Kingella sp.
K. genomosp. P1 oral clone MB2_C20 S11
Thiobacillus sp.
Azonexus sp.
Zoogloea sp.
Desulfomicrobium sp.
D. baculatum S18, S3, S4, S15, S19, S17, S20, S2, S5, S1, S14
Bilophila sp.
Desulfovibrio sp.
D. putealis S3, S4, S15, S17, S2, S1, S14
D. sp. enrichment clone JdgSrb011 S11
Mailhella sp.
M. massiliensis S18, S19, S20, S12
Pelobacter sp.
Geobacter sp.
Arcobacter sp.
Sulfurospirillum sp.
S. deleyianum S2, S17, S20

Aeromonas sp.
Anaerobiospirillum sp.
Ruminobacter sp.
Succinivibrio sp
S. dextrinosolvens S18, S19
Plesiomonas sp.
Cronobacter sp.
Buchnera sp.
B. aphidicola S18
Edwardsiella sp.
Morganella sp.
Methyloparacoccus sp.
Actinobacillus sp.
Aliivibrio sp.
Lysobacter sp.
Stenotrophomonas sp.
Bacteriovorax sp.
Peredibacter sp.
Brachyspira sp.
Asteroleplasma sp.
Gordonia sp.
Tessaracoccus sp.
Candidatus Saccharimonas
Listeria sp.
L. floridensis S16
Abiotrophia sp.
A. defectiva S1, S2, S3, S8, S17, S20, S11, S13, S14
Facklamia sp.
Ignavigranum sp.
Melissococcus sp.
Pilibacter sp.
Leuconostoc sp.
Epulopiscium sp.
Anaerovorax sp.
Johnsonella sp.
J. ignava S14
Clostridioides
C. difficile S3, S8
Halanaerobium sp.
Anaeroglobus sp.
Murdochiella sp.
Ignavibacterium sp.
Magnetococcus sp.
Vogesella sp.
Marinobacter sp.
Franconibacter sp.
F. pulveris S1, S3, S11
Pantoea sp.
P. agglomerans S4
P. conspicua S16
P. sp. R21 S14
Coxiella sp.
Ureaplasma sp.
Varibaculum sp.
Lawsonella sp.
L. clevelandensis S7
Williamsia sp.
Arthrobacter sp.
Saccharopolyspora sp.
Flavobacterium sp.
F. caeni S2
Moheibacter sp.
Alkaliphilus sp.
A. sp. LacT S11
Acetobacterium sp.
A. woodii S11
Desulfosporosinus sp.
Anaerotruncus sp.
Negativicoccus sp.
Fimbriiglobus sp.
Haliangium sp.
Enterobacter sp.
E. cloacae S10
E. hormaechei S1
Salmonella sp.
S. enterica S11
Erwinia sp.
E. teleogrylli S16
Proteus sp.
P. mirabilis S14
Sodalis sp.
Hahella sp.
H. ganghwensis S4
Phocoenobacter sp.
Perlucidibaca sp.
Luteimonas sp.
Arcella sp.
A. hemisphaerica S1, S4
Bryobacter sp.
Aurantimicrobium sp.
Huakuichenia sp.
Propionimicrobium sp.
Emticicia sp.
Cloacibacterium sp.
Salinicoccus sp.
Proteiniclasticum sp.
Ethanoligenens sp.
Ezakiella sp.
Gallicola sp.
Helcococcus sp.
H. sueciensis S7
Limnobacter sp.
Dechloromonas sp.
Propionivibrio sp.
Acidibacter sp.
Nocardia sp.
Pseudopropionibacterium sp.
Barnesiella sp.
Culturomica sp.
Hydrogenispora sp.
Atopococcus sp.
Catellicoccus sp.
Fenollaria sp.
F. timonensis S7
Neofamilia sp.
N. massiliensis S7
Sarcina sp.
Acetoanaerobium sp.
Bulleidia sp.
B. extructa S8
Candidatus Stoquefichens
Coprobacillus sp.
Erysipelatoclostridium sp.
Sedimentibacter sp.
Aureimonas sp.
Aquabacterium sp.
Paraburkholderia sp.
Massilia sp.
Candidatus Babela
Cardiobacterium sp.
Legionella sp.
Bdellovibrio sp.
B. sp. SRP1 S9
Illumatobacter sp.
Acidothermus sp.
Haloactinopolyspora sp.
Quadrisphaera sp.
Georgenia sp.
Cellulomonas sp.
Dermabacter sp.
Pseudoclavibacter sp.
Nocardioides sp.
N. sp. N37 S15
Eggerthella sp.
Roultibacter sp.
R. timonensis S8, S20
Rubrobacter sp.
Dysgonomonas sp.
Tannerella sp.
T. forsythia S8, S2, S1
Asinibacterium sp.
Sediminibacterium sp.
S. sp. LT21-MRL S8
Marinoscilum sp.
Neochlamydia sp.
Paenibacillus sp.
Eremococcus sp.
Lacticigenium sp.
L. naphtae S8, S17, S20, S11, S10, S14
Tetragenococcus sp.
Vagococcus sp.
Lachnoanaerobaculum sp.
Filibacter sp.
F. alocis S14
Eggerthia sp.
E. catenaformis S8, S14
Hyphomicrobium sp.
Candidatus Ovatusbacter sp
Cellvibrio sp.
Yersinia sp.
Psychrobacter sp.
Pseudoxanthomonas sp.
Thermomonas sp.
Candidatus Ancillula sp.
Aeriscardovia sp.
Pseudoscardovia sp.
Candidatus Aquiluna sp.
Candidatus Limnoluna sp.
Haematomicrobium sp.
Nakamurella sp.
Dinghuibacter sp.
Pasteuria sp.
Alloiococcus sp.
Flavonifractor sp.
F. plautii S15, S17
Caloranaerobacter sp.
Caminicella sp.
Syntrophococcus sp.
Coprothermobacter sp.
Singulisphaera sp.
Pirellula sp.
Plantoptrus sp.
Devosia sp.
Amaricoccus sp.
Reyranella sp.
Thiomonas sp.
Undibacterium sp.
Duodenibacillus sp.
D. massiliensis S12
Desulfobulbus sp.
Pajaroellobacter sp.
Mariprofundus sp.
Luteolibacter sp.
Verrucomicrobium sp.
Holophaga sp.
H. foetida S19
Acidimicrobium sp.
Cellulosimicrobium sp.
Rikenella sp.
Marinifilum sp.
Candidatus Latescibacter sp.
Trichococcus sp.
Colidextribacter sp.
C. massiliensis S19, S20
Proteiniborus sp.
Caloramator sp.
Hungatella sp.
Acetitomaculum sp.
A. ruminis S10, S14
Cellulosilyticum sp.
Herbinix sp.
Robinsoniella sp.
Sellimonas sp.
Peptoclostridium sp.
Acetivibrio sp.
Anaerofilum sp.
Hydrogenoanaerobacterium sp.
Oscillospira sp.
Catenisphaera sp.
Dubosiella sp.
D. newyorkensis S19
Schwartzia sp.
Gottschalkia sp.
Victivallis sp.
Candidatus Paracaedibacter sp.
Enhydrobacter sp.
Komagataeibacter sp.
Ideonella sp.
Acidovorax sp.
Alicycliphilus sp.
Delftia sp.
Alysiella sp.
Vitreoscilla sp.
V. stercoraria S5
Dechlorobacter sp.
Azoarcus sp.
Thauera sp.
Geothermobacter sp.
Succinatimonas sp.
Pseodoalteromonas sp.
Psychromonas sp.
Woeseia sp.
Candidatus Rosenkranzia
Marinomonas sp.
Alkanindiges sp.
Candidatus Parabeggiatoa sp.
Dokdonella sp.
Piscicoccus sp.
Agromyces sp.
Sanguibacter sp.
Sanguibacteroides sp.
Enorma sp.
Paraeggerthella sp.
Nubsella sp.
Pedobacter sp.
P. terricola S9
Caldicoprobacter sp.
Lactonifactor sp.
Coprococcus sp.
C. catus S9, S5
Pseudobutyrivibrio sp.
P. sp. CA38 S9
Faecalibaculum sp.
Faecalitalea sp.
Merdibacter sp.
Gemmatirosa sp.
Microvirga sp.
Candidatus Thiosymbion sp.
Oblitimonas sp.
Alloscardovia sp.
Leucobacter sp.
Longispora sp.
Acetatifactor sp.
Cryptanaerobacter sp.
Breznakia sp.
Succiniclasticum sp.
Sneathia sp.
Kaistia sp.
K. sp. TBD058 S17
Hellea sp.
Pseudoruegeria sp.
P. marunistellae S17
Erythrobacter sp.
Candidatus Accumulibacter sp.

Rhodoferax sp.
Anaeromyxobacter sp.
Mannheimia sp.
M. haemolytica S17
Mesocricetibacter sp.
Fluviicoccus sp.
Photobacterium sp.
Dermacoccus sp.
Dactylosporangium sp.
Propionibacterium sp.
Coriobacterium sp.
Cryptobacterium sp.
Gordonibacter sp.
Phocoeicola sp.
Rufibacter sp.
Truepera sp.
Desulfuribacillus sp.
Oceanobacillus sp.
Sporosarcina sp.
Marinilactibacillus sp.
M. sp. G13. 51 S13
Candidatus Arthromitus sp.
Guggenheimella sp.
Proteocatella sp.
Terrisporobacter sp.
Quinella sp.
Acidaminococcus sp.
Anaerospora sp.
Dethiosulfatibacter sp.
Tissierella sp.
Asticcacaulis sp.
Sphingobium sp.
tepidomonas sp.
Halioglobus sp.
Candidatus Stammerula sp.
Izhakiella sp.
Pectobacterium sp.
Aquicella sp.
Scardovia sp.
S. inopinata S20
Luedemannella sp.
Rosemarinus sp.
Maritimimonas sp.
Bavariicoccus sp.
B. seileri S11, S14
Bradyrhizobium sp.
Rhizobium sp.
Burkholderia sp.
Candidatus Glomeribacter sp.

Polynucleobacter sp.
Curvibacter sp.
Conchiformibius sp.
Snodgrassella sp.
Stenoxybacter sp.
Tolumonas sp.
Idiomarina sp.
Photorhabdus sp.
Chromohalobacter sp.
Balneatrix sp.
Oleispira sp.
Galeibacterium sp.
Pasteurella sp.
P. muttocida S11
Anaerocella sp.
Myroides sp.
Jeotgalicoccus sp.
Pseudoflavonifractor sp.
Thermobrachium sp.
Mobilitalea sp.
Marseilibacter sp.
M. massiliensis S5, S12
Paeniclostridium sp.
Anaerobacterium sp.
Holdemania sp.
Pedomicrobium sp.
Rubellimicrobium sp.
Rivibacter sp.
Alcaligenes sp.
Sulfurimonas sp.
Pseudohongiella sp.
Zobellella sp.
Thiorhodovibrio sp.
Providencia sp.
neptunomonas sp.
Azotobacter sp.
Thiopseudomonas sp.
Beggiatoa sp.
Arenimonas sp.
Anaeroplasma sp.
Chlamydia sp.
Pectinatus sp.
Candidatus Gullanella sp.
Orbus sp.
achromatium sp.
Fermentimonas sp.
Nitribacter sp.
Natranaerovirga sp.
Parasporobacterium sp.
P. paucivorans S5
Acetanaerobacterium sp.
Angelakisella sp.
A. massiliensis S5
Ercella sp.
Pygmaiobacter sp.
Thermodesulfovibrio sp.
Caulobacter sp.
Candidatus hamiltonella sp.
Pyramidobacter sp.
Luteipulveratus sp.
Aestuarlimicrobium sp.
A. kwangyangense S10
Spirosoma sp.
Acidibacillus sp.
Sinibacillus sp.
Solibacillus sp.
Carnobacterium sp.
Constrictibacter sp.
Endozoicomonas sp.
Kistimonas sp.
Oleiphilus sp.
Synergistes sp.
Ornithinimicrobium sp.
Pseudonocardia sp.
Phoenicibacter sp.
Cytophaga sp.
Alsobacter sp.
Rhodobacter sp.
Magnetospira sp.
Paraglaciecola sp.
Candidatus Purcelliella sp.
P. pentastirinorum S1
Kosakonia sp.
Candidatus Schmidhempelia sp.
Parviterribacter sp.
Haoranjiania sp.
Hathewaya sp.
Acidiphilum sp.
Sulfurirhabdus sp.
Candidatus Regiella sp.
Ammoniibacillus sp.
Macrococcus sp.
Thermoactinomyces sp.
Jeotgalibaca sp.
Phenylobacterium sp.
Cupriavidus sp.
Methyloversatalis sp.
Desulfuromonas sp.
Ferrimonas sp.
Vulcaniibacterium sp.
Genus marked with an * were found in all the twenty diarrheal samples
mean abundance between the different families in diar- which Aeromonas sp. was isolated as the etiologic agent
rheal samples. Mean abundance of Bifidobacteriaceae of diarrhea (Table 1). Mean abundance of Aeromona-
was found to be lower than that of Enterobacteriaceae daceae was found to be lower than that of Bifidobacte-
and Vibrionaceae, however the two-tailed p-values riaceae, Lachnospiraceae and Enterobacteriaceae, but the
were non significant at 0.2571 and 0.3683 and median difference was non-significant with two-tailed p-values
values of 1.1 and − 0.1750 respectively. Mean abun- of 0.3476, 0.4938, 0.4298 respectively. These results have
dance of Lachnospiraceae was found to be lower than been represented in Fig. 6d.
that of Enterobacteriaceae and the two-tailed p-value
was non significant at 0.5412 and median of − 0.9.
Statistical analysis of Bacteroidetes/Firmicutes ratio
Mean abundance of Lachnospiraceae was significantly
The Bacteroidetes/Firmicutes (B/F) ratio was calculated
lower than that of Vibrionaceae with two-tailed p-value
to predict dysbiosis related to diarrhea. B/F ratio obtained
of 0.0233 and median was 1.240. Mean abundance of
was in the range of 0.001056943 to 1.536455818 (Table 1
Enterobacteriaceae was found to be significantly higher
and Fig. 7) with a median ratio of 0.11 and a mean ratio of
than that of Aeromonadaceae with a two-tailed p-value
0.407702313. The standard deviation was ± 0.454603761.
of
< 0.0001 and median of − 3.199 but non-signifi-
The normal Distribution Curve showed 68% of diar-
cantly higher than that of Vibrionaceae with two-tailed
rheal population has a B/F ratio of 0.86 to 0.05, 95% has
p-value of 0.0711 and median of − 2.640.
a ratio of 1.32 to 0.50 and 99.7% has a ratio of 1.77 to
0.96. The z-score ranged between − 0.33 and 2.48 stand-
Difference in abundance of commensals and pathogens
ard deviations of the mean value. In all samples except
in diarrheal samples confirmed with Vibrio sp. infection
S13 and S15, abundance of Firmicutes exceeded that of
Wilcoxon matched-pairs signed rank test was used to
Bacteroidetes.
compare the difference in mean abundance of Vibrion-
Samples were grouped according to various param-
aceae and Bifidobacteriaceae, Vibrionaceae and Lach-
eters like age, sex, diarrheal etiology and residential
nospiraceae, Vibrionaceae and Enterobacteriaceae in
location (urban or suburban). Difference in B/F ratio
samples from which Vibrio sp. was isolated as the etio-
between these groups was compared and significance of
logic agent of diarrhea. Mean abundance of Vibrionaceae
the difference determined by unpaired t-test. Difference
was found to be higher than that of Bifidobacteriaceae
of B/F ratio between male and female of age ≥2 years
and Lachnospiraceae but the difference was non-signif-
was found. Mean B/F ratio of male was 0.4 and that
icant with two-tailed p-values of 0.5186 and 0.5703 and
of female was 0.3. However, this difference was found
median of 0.07000 and − 0.05500 respectively. Mean
to be non-significant, Difference of mean B/F ratio
abundance of Vibrionaceae was found to be lower than
between samples with single diarrheal pathogen and
that of Enterobacteriaceae but the difference was non-
two pathogens was non-significant. Mean B/F ratio of
significant with two-tailed p-values of 0.5693 and median
samples from urban areas and samples from suburban
of − 1.405. These results have been depicted in Fig. 6c.
areas were 0.4 and 0.5 respectively and the difference
was non-significant. Analysis of variance (ANOVA)
Difference in abundance of commensals and pathogens was performed to determine the significance of B/F
in diarrheal samples confirmed with Aeromonas sp. ratio among samples of three age groups, 0–5 years,
infection 5–15 years and > 15 years and was found non-signifi-
Unpaired t-test with Welch’s correction was used to cant. One sample t and Wilcoxon test was performed
compare the difference in mean abundance of Aeromon- on B/F ratios of samples associated with V. cholerae
adaceae with that of Bifidobacteriaceae, Lachnospiraceae, (VC) infection and non-VC infections. The first group
and Enterobacteriaceae in samples S15 and S16 from was significant with two-tailed p-value of < 0.0001.
r=0.4751
40 a 60 b 80 c 40 d 25 e p=0.0343
20
30 60 30
40
r=0.2996 15
20 r=-0.03073 40 p=0.1994 20 r=-0.4069
p=0.8977 p=0.0750 10
20
Vibrionaceae
De et al. Gut Pathog
10 20 10
Lachnospiraceae
Streptococcaceae
5
Enterobacteriaceae
Relative abundance
0 0 0 0 0
0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40
a e a e a e ae ae ae
c e c e a c e a ce a ce a ce Bifidobacteriaceae
i a ia r Bifidobacteriaceae Enterobacteriaceae Bifidobacteriaceae
c ter ter rion occ spi occ
c b c o c
o ba oba Vi pto hn ino
d te r e c
ifi St
r La um
R
B En
(2020) 12:32
Family
25 40 40 25
f g h i 40 j
20 20
30 30
r=-0.6882 r=-0.5215 30
r=-0.6338
15 p=0.0008 r=0.7111 15 p=0.0184
p=0.0027
20 20 p=0.0004 20 r=-0.6847
10 10
p=0.0009
10 10
Lachnospiraceae
Lachnospiraceae
5 5 10
Ruminococcaceae
Ruminococcaceae
Ruminococcaceae
0 0 0 0
0
0 10 20 30 40 0 10 20 30 40 0 5 10 15 20 25 0 20 40 60 80
0 20 40 60 80
Enterobacteriaceae Enterobacteriaceae Lachnospiraceae Streptococcaceae
Streptococcaceae
80 60 40
60 30
40
40 20
20
20 10
Mean Abundance
Mean Abundance
Mean Abundance
0 0 0
e
ae ae e a e ae a e ae
ce
ae eae eae eae eae eae eae eae
c c ea
ce ce ea ce ce ce ce
ac rac ac ac ac ri i ac
ra ria n ac ia a ra
er ia ida ria
e e ri a r ad p i
on pi occ occ nad te sp ct io te te
a ct tero act ibri os c c o ac no br ac on ac os
b c b V n no to m ba Vi hn
d ob ch ro ob rom r ob c
do Ba tero ch mi rep ero i a t e i d e te a
ifi St A if L if A L
B En
La Ru B En B En
Family Family Family
B C D
Fig. 6 Comparison of abundance of bacterial families. A (a–j) Spearman’s correlation rank coefficient and p-vlues of different families of commensals and pathogens : Correlation of
relative abundance of families Bifidobacteriaceae, Enterobacteriaceae, Vibrionaceae and Bacteroidaceae in all the 20 samples B t-test to compare relative abundance of different bacterial
families in diarrhea C t-test to compare relative abundance of different bacterial families with Vibrionaceae in diarrheal samples diagnosed with Vibrio sp. D t-test to compare difference in
relative abundance in Aeromonas sp. infection
Page 34 of 48
Fig. 7 B/F ratio in diarrheal samples. Bacteroidetes/Firmicutes ratio in diarrheal samples shows a ratio of <1 in all the samples except S13 and S15.
The diarrheal agent isolated from the sample has been indicated in parenthesis beside each sample
Alpha and beta diversity Figure 9 represents the β-diversity among the sam-
Alpha diversity (α-diversity) is used to study the rich- ples. The PCA (Principal Component Analysis) shows
ness and evenness of species diversity within a sample that each sample is unique and has variable OTU diver-
while beta-diversity (β-diversity) is used to calculate sity and OTU abundance compared to one another even
the species diversity between two samples. Therefore, if the stool samples were associated with the same diar-
α-diversity was calculated to understand OTU diver- rheal pathogen. The axis PC1 was more informative than
sity, richness and evenness within each of the 20 diar- PC2 about the β-diversity. The samples were divided into
rheal samples and represented by Shannon-index while six groups based on etiologic agent of diarrhea isolated
β-diversity was used to compare OTU diversity among from the stool by culture method and in the Fig. 9 sam-
these twenty samples. Figure 8 shows the α-diversity ples were represented with a different colour to indicate
observed among the diarrheal samples. The samples the group it belongs to. These six groups are co-infec-
could be sequenced to a variable range of depth of tion (CI), V.cholerae (VC), other Vibrio (O_V) and VC
2e + 05 to 5e + 05 and showed variable evenness and nonO1/non O139 (VCN). Samples S3, S6, S8 and S9
richness of microbial diversity among them even if two associated with CI did not cluster together indicating
samples were associated with the same diarrheal patho- they have different β-diversity, similar trend was found
gen. S20 had the highest α-diversity while S1 had the in S4 and S7, associated with VCN. S12 associated with
lowest although VC O1 was isolated from both. From VC and S19 associated with other Vibrio sp.were closer
S8, S13, S14 different diarrheal pathogens were isolated although they were associated with different etiologic
but they showed the same Shannon index indicating agents of diarrhea.
the same level of richness and evenness of OTUs. The heat map in Fig. 10 was constructed to represent
the relative abundance of various bacterial families in
3
Alpha diversity (Shannon index)
2 Depth
5e+05
4e+05
3e+05
2e+05
S20 S19 S17 S12 S10 S2 S9 S14 S8 S13 S5 S15 S7 S18 S11 S3 S6 S16 S4 S1
Fig. 8 α-Diversity of twenty diarrheal samples. The individual samples show variable richness and evenness of microbial diversity on the basis of
Shannon index
Fig. 9 Principal component analysis of the diarrheal samples. Samples with the same diarrheal pathogen did not cluster together
Fig. 10 Heat-map showing the proportion of different bacterial families in diarrheal samples
the 20 samples. It shows that most of the families occur streptothricin, pleuromutilin. Resistance was high against
at low abundance. The samples were found to have 18 tetracycline, β-lactams, quinolones, aminoglycosides and
families in common and these occurred in variable pro- macrolides. Figure 11 shows all the antimicrobials against
portion even among samples associated with the same which resistance determinants were found and the rela-
diarrheal pathogen. S1 and S20, both were associ- tive proportion of these in each of the 5 samples. In S1
ated with V.cholerae O1 however, in S1 family Strepto- highest abundance of ARGs occurred against aminogly-
coccaceae occurred in low proportion while in S20 it cosides in S2 against tetracycline, both less than 25%, in
occurred in higher proportion while S17 associated with S8 against tetracycline and it was found to be more than
S. flexneri and S20 had comparable proportion of family 60%. ARGs against other classes in S8 were found to be
Streptococcaceae. within 5%. In S9 highest abundance of resistance determi-
nants occurred against tetracycline at greater than 30%.
Resistome mapping with WGS In S10 equal abundance of greater than 20% resistance
WGS analysis of five samples S1, S2, S8, S9 and S10 was determinants was found for tetracycline and quinolone.
performed and 491 resistance determinant against the Among 5 samples, tetracycline resistance was found to be
major classes of antibiotics were found by using the tool the highest in 4 samples. Therefore, 80% samples (based
ABRicate [51]. Identities of the antimicrobial resistance on calculations using 5 metagenomes) could be predicted
genes were determined using default parameters of ABRi- to carry tetracycline resistance determinants. All of the
cate, namely 75% nucleotide identity. Accordingly genetic samples showed resistance determinants against tetracy-
determinants were annotated to encoding resistance cline, β-lactams, macrolide, aminoglycoside, phenicol and
against tetracycline, aminoglycosides, β-lactams, qui- sulphonamide. Biosynthetic gene clusters (BGCs) associ-
nolone, macrolide, phenicol, glycopeptide, fosfomycin, tri- ated with secondary metabolites involved in antimicrobial
methoprim, sulfonamide, lincosinamide, metronidazole, resistance were also recovered and annotated with the
Fig. 11 Resistome of diarrheal samples. Whole genome shot-gun sequencing was used to study the resistome in five diarrheal samples. The
histogram presents the relative abundance of antimicrobial resistance determinants and secondary metabolites predicted to be present in the gut
microbiome of diarrheal subjects in the study
help of antiSMASH algorithm (Fig. 11). Highest number Discussion

of genes were annotated to bacteriocin in S8 and S10. S8 16S rRNA amplicon sequencing was used to study the gut
showed the highest diversity of BCGs as 11 BCGs could microbiota associated with diarrhea in twenty diarrheal
be assembled followed by 9 in S10. Nonribosomal peptide samples collected from two hospitals in Kolkata to define
synthetase (NRPS) was the only BCG that was present in the core and variable microbiota in this part of India.
all the five samples. Bacterial taxonomic identification was performed by
Genomes recovered from the 5 fecal samples were matching DNA sequence homology of the metagenomic
aligned using metaWRAP. It revealed the different bac- reads generated from 20 diarrheal samples to 1,45906
terial species present in each fecal sample. Phylogenetic reference genomes available on the Genome Taxonomy
tree in Fig. 12 shows the different OTUs predicted to be Database [50]. We have been able to identify taxonomic
the source of the antimicrobial resistance genes in each units at different taxonomic levels namely, phyla, class,
sample and the clonal relationship among these OTUs. order, family, genera and species which were found in
The highest number of OTUs occurred in S9. Origin of all the twenty diarrheal samples. Therefore, it may be
ARGs in S10 could be traced to Klebsiella pneumoniae, inferred that these constituents may be present as part of
Bacteroides B vulgatus, Bifidobacterium sp., Eggerthella the core microbiome in diarrheal patients. However, we
lenta, Collinsella sp. and CAG-83 sp., Catenibacterium cannot assert to what extent the proportion of these con-
sp., Holdemanella sp., Enterococcus B faecium, Strep- stituents has been altered or undergone dysbiosis com-
tococcus infantarius and Streptococcus pasteurianus. pared to the normal or non-diarrheal microbiota, since,
Signature of Eschericia coli D occurred at highest per- comparison of diarrheal and non-diarrheal stool sam-
centage in S1 (55.84%), S2 (50.07%) and S8 (32.19%) and ples was beyond the scope of our work. Next-generation
Streptococcus infantarius signature occurred at 15.3% in sequencing is not easily accessible due to the constraints
S10. Figure 13 shows the 41 MAGs and their contribu- of expenses incurred in the sequencing and analysis pro-
tion towards AMR in each sample. E.coli is the highest cess [55]. Therefore, we conducted a pilot study with a
contributor being the major MAG detected in 3 of the 5 small sample size of 20 diarrheal fecal samples to deter-
samples. MGYG-HGUT-2778 was the major contribu- mine microbiota composition during diarrhea and to
tor in S9 while Streptococcus pasteurianus was the high- define a bacterial signature for diarrhea, irrespective of
est contributor in S10. In S1, the ARGs originated from the pathogen causing diarrhea.
Eschericia sp. We aimed to see differences in microbiota structure
based on the diarrheal pathogen that was isolated by
classical microbiological method. We found in diarrheal
Fig. 12 Forty-one metagenomically-assembled genomes (MAGs) recovered from five samples by Whole-genome shot-gun sequencing
samples the dominant phylum is Firmicutes. In 11 out dominant phylum was Bacteroidetes and in S15 obtained
of 20 samples phylum Firmicutes was the most abun- from a male child of 1 year old, Actinobacteria was the
dant phylum (Fig. 4). The Bacteroidetes/Firmicutes is most abundant phylum. However, in this sample also
an important indicator of bacterial dysbiosis [25]. The Firmicutes was in higher abundance than Bacteroidetes.
healthy gut has been found to have higher proportion The gut microbiota primarily comprise Firmicutes, Bac-
of Bacteroidetes than Firmicutes [25]. 18 out of 20 diar- teroidetes, Actinobacteria, Proteobacteria, Tenericutes
rheal fecal samples showed higher abundance of Firmi- and Fusobacteria [56]. The adult Microbiota is dominated
cutes than Bacteroidetes. From our study we conclude by phyla Firmicutes, Bacteroidetes and Actinobacte-
that the diarrheal gut has a higher abundance of Firmi- ria and depends on a host of intrinsic and extrinsic fac-
cutes than Bacteroidetes. Two samples S13 associated tors while the infant gut is usually dominated by phylum
with V.cholerae O139 infection and S15 associated with Actinobacteria [20, 56–58]. In sample S16, which came
Aeromonas sp. were found to have a higher proportion from a female infant of 8 months old and Aeromonas sp.
of Bacteroidetes compared to Firmicutes. In sample S13, was the etiologic agent as confirmed by culture method,
which was obtained from an adult male of 40 years the the most dominant phylum was found to be Firmicutes.
Fig. 13 Metagenomically assembled genomes (MAGs) contributing to AMR in the diarrheal gut microbiome. WGS of gut microbiome yielded 41
MAGs. The resistance determinants could be traced to these 41 MAGs out of which 22 OTUs could be identified till the species level. The percentage
of occurrence of these 22 OTUs in the five samples has been presented here
Proteobacteria was the most abundant phylum in six with Aeromonas sp. infection. Under the superphylum
samples. From all these samples V.cholerae was isolated PVC [59] all phyla except Omnitrophica occurred in one
by classical culture method (Table 1, Fig. 4). B/F ratio of or the other sample. These were Planctomycetes, Verru-
diarrheal patients associated with V.cholerae infection comicrobiae, Chlamydiae and Lentisphaerae. Verrucomi-
was found to be statistically significant on the basis of crobiae associated with primarily beneficial bacteria and
one sample t and Wilcoxon test. This provided us with an of environmental origin [59] was found to be in low abun-
insight into the B/F ratio that might be associated with dance in most of the samples in which it occurred. Core
cholera. The study provided us with a trend in microbiota Microbiota varies with geographic location, nationality
structural composition in the diarrheal gut that could and diet among other factors [20, 60]. Previous reports by
also be indicative of dysbiosis. However, comparison of other researchers in other parts of the world have shown
the profile with that of non-diarrheal subjects will help the presence of Actinobacteria and Verrucomicrobia as
in establishing the baseline of Bacteroidetes/Firmicutes dominant phyla in healthy subjects [20, 60]. A suppres-
ratio. This could be assertively used as an indicator for sion in the proportion of these phyla in the diarrheal sub-
diarrhea. jects in our study indicate either a characteristic of the
Predominance of Proteobacteria and Firmicutes are Indian gut microbiota or dysbiosis associated with diar-
indicators of a disturbed gut microflora [55]. 42 other rhea. A study by Das et al. showed the healthy Indian gut
phyla including Tenericutes, Fusobacteria and Candi- consistently harbours 62% Firmicutes, 24% Bacteroidetes,
date phylum radiation (CPR) were found in some of the 5.2% Actinobacteria and 4.2% Proteobacteria and a low
samples. Their proportion was found to be very minute. abundance of Verrucomicrobia, Tenericutes and Fuso-
Although Tenericutes and Fusobacteria have been shown bacteria were found in most of the individuals participat-
to be a part of the core Microbiota [56], in our study these ing in the study [61]. In the present study we found 38%
were absent in samples like S16, which was associated Firmicutes, 10% Bacteroidetes, 12% Actinobacteria and
19% Proteobacteria. The difference in the abundance of and Oscillospira and were depleted in Bacteroides [68].
these phyla as observed in the present study could be due Our study is the first attempt to present a core micro-
to diarrhea and diet, ethnicity, geographical location and biota signature in diarrheal subjects from Eastern India.
other environmental factors influencing the proportion We found 18 genera that were present in all the 20 sam-
of these constituents. A study conducted by Monira et al. ples (Table 3). Prevotella sp. was absent in S10. The diar-
addressing the gut microbiota composition in healthy rheal etiology of this sample could not be successfully
and malnourished children in Bangladesh showed that determined by culture method. This sample was from a
in healthy children Proteobacteria and Bacteroidetes 55 year old male from Kolkata who was hospitalized for
accounted for 5% and 44% respectively [62]. As Eastern 1 day at ID Hospital in Kolkata. Prevotella sp. has been
India and Bangladesh are comparable demographies we found to be associated with the core human gut microbi-
may assume that the lower abundance of Proteobacte- ome [61]. It is a pathobiont of clinical significance. A pos-
ria in the healthy gut observed in Bangladeshi children itive correlation between the upsurge of Prevotella copri
has been altered in diarrheal subjects resulting in higher and diarrhea has been estimated by previous studies [69].
abundance of Proteobacteria in the diarrheal subjects of The study showed the presence of commensals, patho-
the present study. bionts and pathogenic bacteria in the diarrheal gut micro-
Candidate Phyla Radiation (CPR) like Candidatus biome. Pathobionts may cause inflammatory disorders or
Falkowbacteria, Candidatus Moranbacteria and oth- may cause infections in the event of compromised immu-
ers, belonging to the Parcubacteria group (Table 2) were nity [70, 71]. The presence of pathogens like V. cholerae,
found in our study. These are uncultured bacteria of envi- Helicobacter pylori etc. in addition to the etiologic agent
ronmental origin and involved in important ecological isolated by culture was found in many samples. This is a
activity like sulfur-reduction and other biogeochemical matter of grave concern as asymptomatic carriers act as
cycles like carbon and hydrogen cycles [63, 64]. These reservoirs of infections and expedite the transmission of
are of ancient lineage, mostly symbionts or episymbionts, infections. Commensals like Bifidobacterium sp., Rumi-
lack biosynthetic pathways and have not been cultured nococcus sp., Fecalibacterium sp., Lactobacillus sp., Lac-
due to their stringent metabolism [64]. Our metagen- tococcus sp., were found in the present study and are
omic data showed the presence of Candidatus Saccha- intrinsic colonizers of the human gut [72]. Commensals
ribacteria or TM7 phylum which has been found to be play a protective role by mediating colonization resistance
a potential pathogen with a parasitic lifestyle, associated and preventing colonization by pathogens and oppor-
with human inflammatory mucosal diseases and often tunistic pathogens, prevent intestinal barrier impair-
recovered from wastewater and clinical environments ment and suppresses pro-inflammatory factors thereby
[65, 66]. preventing diarrhea [73, 74]. Earlier studies showed that
The uncultivated Candidate phyla is referred to as abundance of certain commensals remained unchanged
“microbial dark matter” [66]. Its presence in diarrheal before, during and after recovery from acute diarrhea in
samples from patients in and around Kolkata is a matter children while others like Eubacterium sp., Fecalibacte-
of concern about environmental pollution and intestinal rium sp., Prevotella sp., Bacteroides sp., showed marked
colonization of organisms with pathogenic potential. It differences during acute diarrhea and after recovery [75].
will be interesting to investigate how they have adapted It will be interesting to investigate whether the reduction
to the intestinal habitat and about the transmission of in proportion of commensals prior to diarrheal onset lay
these organisms into the host from the environment. In the ground for diarrheal pathogenesis. Previous studies
the future it will be interesting to look for these metagen- on diarrhea associated microbiota had found a positive
omes in healthy/non diarrheal microbiome. correlation between diarrhea and pathogenic bacteria like
In the recent years a large number of published reports Eschericia sp., Shigella sp., Granulicatella sp, Streptococ-
attempting to define the core gut microbiome of Indians cus sp. [76], We found the existence of these pathogenic
are available [61, 67]. Bacterial composition at the genus genera in our study subjects. Eschericia sp. was not found
level has been found to be influenced by location and in S5, S7, S9, S12, S14, S18 and all of these samples were
diet [61]. Kulkarni et al. showed the presence of Prevo- associated with V. cholerae infection. These findings led us
tella sp., Bacteroides sp., Megasphaera sp., Roseburia to test if there is a correlation in the relative abundance of
sp., from fecal samples of 43 Indians. Das et al., showed various families of pathogens and among pathogens and
the presence of a core microbiota comprising 54 genera commensals which could be of significance for diarrheal
from fecal samples of individuals from rural, urban and etiology or bear implications for diarrheal treatment.
high-altitude dwellers in India [61]. Another study con- We found an association among the relative abun-
ducted by Lin et al. showed that healthy Bangladeshi dance of families of commensals and pathogens
chidren harboured more of Prevotella, Butyrivibrio, (Fig. 6a). Although some of the associations were not
statistically significant it succeeded to present a trend agonistic relationship among these and significant nega-
which may be useful for understanding the agonis- tive correlation among families of commensals and patho-
tic and antagonistic relationship among these families gens like Enterobacteriaceae and Lachnospiraceae and
and could show direction in preventive and therapeu- Enterobacteriaceae and Ruminococcaceae were observed.
tic modules of diarrheal diseases. These correlation All these observations indicate antagonistic relationship
could become statistically significant if performed on bearing promise of future exploitation of these tendencies
a larger sample size. We found that the commensals for development of probiotics.
Bifidobacteriaceae and Lachnospiraceae were nega- The samples had a variable range of α-diversity. S1 had
tively correlated with pathogens Enterobacteriaceae the least while S20 had the maximum diversity. Samples
and Vibrionaceae. Among the pathogenic groups, fam- like S1 and S20 associated with the same diarrheal etio-
ily Enterobacteriaceae was higher than both Vibrion- logic agent, VC, had stark differences in Shannon-indices
aceae and Aeromonadaceae thereby shedding light on indicating that other parameters are crucial for micro-
the trend observed in gut microbiota during diarrhea. biota structural composition. For analysis of β-diversity
Streptococcaceae and Enterobacteriaceae were posi- samples were grouped according to diarrheal agent iso-
tively correlated indicating that these two pathogenic lated from it by culture method. The samples did not
groups show the same trend in gut microbiota struc- group into clusters based on the etiologic agent. We
tural composition in diarrhea. Enterobacteriaceae are anticipate this was due to the small sample size and also
a family of potential pathogens and our study showed factors other than the etiologic agent of diarrhea deter-
that these outnumber other families of potential path- mining the bacterial composition in the gut.
ogens like Vibrionaceae and Aeromonadaceae in diar- The gut of diarrheal patients carries a high abundance
rhea implying the obvious trend in diarrheal dysbiosis. of antimicrobial resistance genes (ARGs) and the mem-
We observed differences in relative abundance among bers of the microbiota have been found to carry these
various families of bacteria in samples found to be asso- genes in their genomes and act as reservoirs of AMR in
ciated with V. cholerae or Aeromonas sp. as etiologic the gut [55, 77]. We used WGS to sequence five diar-
agents. These are two common diarrheal pathogens and rheal samples to study the resistome and understand the
we wanted to examine if we could derive any significant origin of ARGs in the gut microbiome. We selected the
association of any pathogenic or commensal family with fecal samples to see if variation in these aspects existed
these specific diarrheal etiology. We noted a trend in based on demography, etiology and α-diversity. In spite
the difference in abundance of Vibrionaceae with Bifi- of the differences in demography, etiology and α-diversity
dobacteriaceae and Lachnospiraceae and Enterobacte- all the samples showed the presence of the four classes
riaceae. Vibrionaceae was higher than the commensals of ARGS namely, tetracyclines, β-lactams, aminoglyco-
while lower than Enterobacteriaceae. Aeromonadaceae sides and macrolides. Even though samples like S1 and S2
abundance was lower than those of the commensals and were associated with the same diarrheal etiology V. chol-
Enterobacteriaceae but the difference was non-significant. erae and were from the same district, 24 Parganas, their
These findings suggest that in diarrhea commensals are resistome analysis revealed difference in relative abun-
suppressed by pathogens belonging to these families and dance of the same ARGs like tetracyclines, quinolones,
could bear implications for probiotic therapy in diar- β-lactams, aminoglycosides and macrolides. Although
rhea with commensal gut pathogens. This is also sugges- S1 had the lowest α-diversity, it did not have the lowest
tive of the pattern of dysbiosis occurring in diarrhea. The diversity of ARGs although had the lowest number of
same comparative analysis if performed in a healthy study total ARGs compared to the others. S9 and S10 were both
cohort may help to determine if the observed differences from Kolkata but S10 had the highest number of ARGs
in our analysis is due to dysbiosis associated with diarrhea. while S9 had much lower number of ARGs and S10 had
Mean abundance of Enterobacteriaceae was signifi- much higher relative abundance of each class of ARGs
cantly higher than that of Aeromonadaceae in the diar- compared to S9. Moreover, quinolones were absent in
rheal study cohort. Significant difference in mean S9. We conclude that in this region Eschericia sp. is the
abundance among Bacteroidaceae, Enterobacteriaceae major contributor of ARGs in the gut. This is of grave
and Vibrionaceae was observed. These findings suggest concern. Eschericia sp. includes both commensals and
that in diarrhea certain families of pathogens overpower pathogens. They are involved in metabolism and defense
others and this may lead to co-infections, co-morbidities mechanisms [78]. Eschericia sp. are resident microbes
leading to complications in diarrheal treatment. of the gut. These will act as reservoirs for dissemination
Statistically significant positive correlation was observed of ARGs into other bacteria in close proximity. Moreo-
among the families of commensals like Bifidobacteriaceae, ver, from the five fecal samples genomes of E. coli D, E.
Lachnospiraceae and Ruminococcaceae, indicating marmotae, E. albertii, E. fergusonii were reconstructed in
addition to others (Fig. 13). Many of these pathogens are Enterobacteriaceae, on the basis of Spearman’s correla-
MDR as confirmed by previous studies [79]. tion coefficient test. Bacteria with probiotic capability
We found a high abundance of resistance against tet- can be identified and these can be developed as probi-
racyclines, macrolides, aminoglycosides, quinolones and tocs for alternative therapy to replace or supplement
β-lactams. This presents a menacing picture of the AMR antibiotic therapy in diarrhea. The existence of “micro-
crisis in countries like India. These are last resort drugs bial dark matter” in diarrheal gut evident from our
against enteric pathogens like E.coli, K. pneumoniae, V. study is indicative of contamination of the gut micro-
cholerae which are common diarrheal pathogens in India. biota with rare and dangerous bacteria. This would
Our study revealed that resistance determinants against help in epidemiological analysis to trace the origin and
the most important classes of antimicrobials are present understand the route of transmission of members of
in the gut of people residing in this region. This will con- Candidate phyla into the diarrheal gut microbiome.
tribute to transmission and spread to the community and Consequently, it will be useful to reduce the occur-
the environment and lead to the emergence of MDR and rence of such organisms in the environment and the
XDR (Extensively Drug Resistant) strains. gut. Overall, the study on metagenomic sequencing of
Diarrhea is associated with dysbiosis of microbiota diarrheal microbiome is the first of its kind, from East-
[75]. The dynamics of gut microbiota has been well- ern India revealing the core and variable microbiota
studied in case of invading pathogens like V.cholerae associated with diarrhea and has immense implica-
[80]. We used NGS to study the gut microbiota in tions for understanding diarrheal etiology.
acute diarrheal patients in the present study. The
results showed that a core microbiota exists in diar- Methods
rheal patients. Specific signature of microbiota com- Collection of fecal samples
position corresponding to distinct diarrheal etiology Twenty diarrheal stool samples S1–S20 were collected at
could not be established. We anticipate it is due to the the IDH amd BCH, Kolkata. The donors of the fecal sam-
small sample size. The trend that we observed can be ples were patients suffering from acute diarrhea. They
confirmed by expanding the sample size in the future. were passing liquid stool more than three times a day and
The study helped to reveal the critically high abun- were suffering from dehydration. Five of these (S1, S2, S4,
dance of AMR determinants against the most crucial S16, S17) were collected from day patients at the outpa-
drugs administered for diarrheal treatment and con- tient ward at BCH and the remaining fifteen were from
firmed the existence of these determinants in the gut patients admitted to the IDH for 1–3 days for receiving
of diarrheal patients and originating from genomes of treatment for diarrhea. The samples were from both male
pathogens residing in the gut. From these ecological and female patients of age 8 months to 56 years. Nineteen
cross-talk future threat of infections by MDR bacteria of the donors were from Kolkata and the adjacent dis-
would emanate. The study highlights the presence of tricts in West Bengal in Eastern India while one was from
asymptomatic carriers of pathogens who are serving as the adjacent state of Bihar. The samples were brought
reservoirs of important infectious agents and expedit- to the Bacteriology laboratory at the adjoining National
ing community transmission of diarrheal pathogens. Institute of Cholera and Enteric Diseases (NICED) within
In the study two NGS techniques were used simul- few hours of collection. The samples were assigned labo-
taneously. 16S rRNA amplicon sequencing helps to ratory identification code and immediately aliquoted into
identify bacterial taxa but not function. WGS provides sterile 2 ml cryovials (catalogue number SCT-200-SS-C-
comprehensive information about both the structure S, Corning, USA) and stored at − 80 °C for isolation of
and function of the microbiota. It also helps to identify microbial DNA. A part of the sample was used for routine
the genomes contributing to those functions. Therefore diagnosis of the diarrheal pathogen by culture method.
we conclude that if molecular epidemiological labora- A list of the samples and their demographic details are
tories can overcome financial constraints, WGS would shown in Table 1. The samples were randomly selected
be the preferred technique for investigating the con- and were not subject to any selective bias regarding any
stituent genomes of the microbiome and annotate their demographic and clinical parameter. Figure 1 shows the
functional role. location of West Bengal on the map of India and the state
of West Bengal with its districts.
Conclusion
The pilot study revealed significant antagonistic Isolation of microbial DNA
correlation of families of commensals like Lachno- Microbial DNA was extracted by the Guanidinium thio-
spiraceae and Ruminococcaceae with pathogens like cyanate (GITC) method according to the THSTI protocol
described by Bag et al. [35] with minor modification. This and 5′-CAAGCAGAAGACGGCATACGAGAT[i7] GTC
method employs a combination of enzymatic, chemical TCGTGGGCTCGG where [i5, i7] are unique dual index
and mechanical lysis for the complete breakdown of the sequences to identify sample-specific sequencing data.
bacterial cell wall, cell membrane and removal of nucle- Round 2 PCR amplicons (sequencing libraries) were
Prep™ clean up system and quality checked. The library

ases. Accordingly, 200 µl stool sample was resuspended in analyzed on 1.2 percent agarose gel, cleaned using High-
Tris–EDTA buffer (pH 8.0) and homogenized using ster-
ile glass beads (2.5 mm) and the clear suspension collected was diluted to 4 nM using 10 mM Tris (pH 8.5) and 5 µl
after centrifugation was subject to enzymatic lysis at 37 °C of each library was aliquotted and mixed to pool the
for 1 h by a mixture of bacterial cell-wall lysis enzymes libraries. The pooled library was denatured by addition
containing lysozyme (10 mg/ml) (catalogue number of NaOH followed by heat denaturation and the DNA
L6876, Merck, Germany), lysostaphin (4 KU/ml) (cata- samples were diluted and finally loaded onto the Illumina
logue number L7386, Merck, Germany) and mutanolysin MiSeq system and sequencing was performed to generate
(25 KU/ml) (catalogue number M9901, Merck, Germany). (300*2) V3–V4 paired-end reads.
250 µl of 4 M GITCwas added to the suspension followed The Illumina paired end V3–V4 raw reads (300*2) were
by 300 µl of 10% N-Lauryl sarcosine and incubated at submitted to the European Nucleotide Archive (ENA) for
37 °C for 10 min. Mechanical disruption by 0.1 mm zirco- validation and further analysis using the MGnify pipeline
nia beads (BioSpec Products Inc., USA) ensued in a mini provided by the EMBL server. The study was assigned the
beadbeater (catalogue number 607EUR, BioSpec Products number MGYS00005131. The raw reads were processed
Inc., USA) using a 2 min cycle comprising 30 s beating using MGnify v4.1. SeqPrep [36] was used to merge the
and 30 s rest and followed by washing in PolyVinylPo- overlapping raw reads into a single longer read. Trim-
lyPyrollidone (PVPP) (catalogue number 77627, Merck, momatic [37] and Biopython [38] were used to trim
Germany). Removal of RNA was done using RNase A and filter these initial reads by removing > 10% undeter-
(10 mg/ml) (catalogue number R6513, Merck, Germany) mined nucleotides and adapter sequences and filtering
and incubating the suspension for 30 min at 37 °C. DNA out < 100 bp long sequences to generate processed reads
was finally extracted by adding 96% chilled ethanol and which were annotated using MAPseq [39] framework
spinning at 14,000 rpm for 10 min at 4 °C. The pellet was for taxonomic classification and Operational Taxonomic
with NanoDrop spectrophotometer and Qubit® dsDNA

air-dried followed by estimation of DNA concentration Unit (OTU) mapping. For classification of OTUs, paired-
end reads with > 97% sequence similarity were consid-
HS Assay Kit (catalogue number Q32854, Invitrogen, ered. The sequences of raw and processed reads can be
USA). The DNA concentration was in the optimal range accessed through the EMBL server with the accession
and estimated at 1 ng/µl–400 ng/µl. The 20 DNA samples number MGYS00005131.For multivariate analysis and
were used for library preparation for 16S V3–V4 ampli- graphical representation of the metadata tools Codaseq
con sequencing and 5 of the 20 DNA samples were used [40], Vegan [41] and Ape [42] on the Phyloseq [42] pack-
for WGS sequencing for resistome analysis. age, ggplot2 [43] on R Studio (R studio Inc, Boston, MA,
USA) were used. Biom files generated by MAPseq in the
16S rDNA sequencing and metagenomic analysis MGnify pipeline were imported into R package. Princi-
16S V3–V4 metagenome libraries were prepared using pal component Analysis (PCA) was performed using the
region-specific primers. DNA samples were loaded on Phyloseq package for analysis of abundance of OTUs and
clean up using HighPrep™ clean up system (catalogue

gel to examine the bands followed by 0.7 × Hiprep bead entitities within different taxonomic ranks namely, phy-
lum, class, order, family, genera and species. Abundance
numberAC-60050, MagBio genomics Inc., USA) to avoid was expressed as percentage. Relative abundance of differ-
impurities and amplified for 26 cycles of round 1 PCR ent entities within a taxonomic level was represented as
using KAPA HiFi Hot-Start PCR Kit (catalogue number histogram to show taxonomic diversity and abundance.
KM2602, KAPA Biosystems Inc., Boston, MA, USA). The 0–5 percent was used as the threshold. The top fifteen
forward and reverse primer concentration was kept at 5 to twenty-five OTUs within each taxon were plotted for
µM each. The amplicons were analyzed on 1.2% agarose each sample. α-diversity was calculated to estimate spe-
gel. 1 µl of diluted round 1 PCR amplicons were used cies richness and evenness of each sample. Accordingly,
for Indexing PCR (Round 2). Round 1 PCR amplicons OTUs were rarefied at even depth and Shannon index was
were amplified for 10 cycles to add Illumina sequencing calculated. To calculate β-diversity between the samples
barcoded adaptors (Nextera XT v2 Index Kit, catalogue ordination was performed and principal coordinates plots
number FC-131-1002 Illumina Inc., CA, USA). Illumina were generated based on pairwise weighted UniFrac dis-
Adapter Sequences used were: 5′-AATGATACGGCG tances. Pie, bar, stacked and interactive krona charts were
ACC ACCGAGATCTACAC[i5]TCGTCGG CAG CGTC generated by the taxonomic analysis steps of the MGnify
v 4.1 pipeline. Bacteroidetes/Firmicutes ratio was calcu- Amplicon Tagment Mix from the Nextera XT Kit. Twelve
lated and compared among the 20 samples. For bivariate cycles of Indexing-PCR (72 °C for 3 min followed by
analysis normal distribution, z-score, unpaired t-tests, denaturation at 95 °C for 30 s, cycling (95 °C for 10 s,
ANOVA (Analysis of Variance) and one sample t and Wil- 55 °C for 30 s, 72 °C for 30 s) and 72 °C for 5 min) were
coxon tests were calculated to represent the statistical sig- performed on the adapter tagged DNA to enrich the
nificance of the taxonomic composition and abundance adapter-tagged fragments. The PCR product was purified
data. Correlation coefficient using Spearman’s rank co- using JetSeq Magnetic Beads (Bio, 68031). Quantifica-
efficient test was used to study correlation among abun- tion of the prepared library was performed using Qubit
dance of families Bifidobacteriaceae, Lachnospiraceae, fluorometer according to the manufacturer’s instruc-
Ruminococcaceae, Enterobacteriaceae, Vibrionaceae, tions. The universal adapter sequence was 5′AATGAT
Streptococcaceae to derive if any significant association ACG G CGACC ACCGAGATC TAC ACTC TT TCCC TA
existed among them in diarrhea. Kruskal–wallis test was CACGACGCTCTTCCGATCT and adapter index was
used to compare abundance of three families of commen- 5′GAT C GG A AG A GC A CA C GT C TG A AC TCC A GT
sal bacteria namely Bifidobacteriaceea, Lachnospiraceae CAC[INDEX]ATCTCGTATGCCGTCTTCTGCTTG.
and Ruminococcaceae and three families of pathogenic The libraries were pooled and these were sequenced
bacteria namely Enterobacteriaceae, Bacteroidaceae and in the Illumina MiSeq System (Illumina Inc., CA, USA)
Vibrionaceae to see if an association could be established to generate paired-end raw reads. The raw reads were
among the relative abundance of these families which passed through the metaSPAdes v 3.9.1 [45] assembler
could have a significance for diarrheal etiology. pipeline after initial quality check with FastQC [46]
Unpaired t-test was used to compare difference in followed by removal of adapters and low quality bases
relative abundance of Bifidobacteriaceae with Entero- towards 3′-end by the program TrimGalore [47] and
bacteriaceae and Vibrionaceae, Lachnospiraceae with BWA (Burrows-Wheeler Aligner) [48] that removes
Enterobacteriaceae and Vibrionaceae, also between host contaminants. Binning was done using the soft-
Enterobacteriaceae and Vibrionaceae and between ware metaWRAP [49] and taxonomic annotation and
Enterobacteriaceae and Aeromonadaceae in diarrhea mapping was done using the GTDB Toolkit (GTDB-Tk)
and calculations were based on 20 samples. Wilcoxon [50]. The contigs generated by the metaSPAdes pipe-
matched-pairs signed rank test was used to compare dif- line was used for screening for acquired antimicrobial
ference in relative abundance of Vibrionaceae with Bifi- resistance genes using the tool ABRicate [51] and the
dobacteriaceae, Enterobacteriaceae, Lachnospiraceae and program antiSMASH [52] was used for screening and
Ruminococcaceae in samples diagnosed with Vibrio sp. annotation of secondary metabolite biosynthesis gene
by culture method. Unpaired t-test was used to compare clusters (BGCs). Multivariate analysis and graphical
differences in relative abundance of Aeromonadaceae representation of the metagenomic datasets were per-
with Bifidobacteriaceae, Enterobacteriaceae, Lachno- formed with ggplot2 on R Studio (R studio Inc, Boston,
spiraceae in samples diagnosed with Aeromonas sp. by MA, USA).
culture method.
Figure 2 presents the workflow of library preparation,
sequencing and metagenomic analysis. Culture of diarrheal pathogens from fecal samples
The fecal samples S1–S20 were streaked onto selec-
WGS sequencing and resistome analysis tive and differential media plates for the isolation of
De novo sequencing of DNA from five diarrheal sam- suspected diarrheal pathogens, Vibrio sp., E. coli, Sal-
ples S1, S2, S8, S9 and S10 was performed for resistome monella sp., Shigella sp., Aeromonas sp., Campylo-
profiling and to understand the presence of secondary bacter sp. in the Bacteriology Laboratory at NICED.
metabolites associated with AMR present in the diar- Accordingly, bacterial culture plates TCBS (Thiosul-
rheal metagenomes. The samples were from three differ- fate-citrate-bile salts-sucrose), HEA (Hektoen enteric
ent districts of West Bengal, suffering from diarrhea due agar), XLD (Xylose Lysine Deoxycholate), Mac Conkey,
to single infection or polymicrobial infections or unre- Blood agar were used for each fecal specimen. Culture
plates were incubated overnight at 37 °C (3–5 days for
Nextera® XT Library Preparation Kit (catalogue num-
solved etiology (Table 1) and with different α-diversity.
Campylobacter sp.) and single colonies from the cul-
ber FC-131-1024, Illumina Inc., CA, USA) was used to ture positive plates were used for phenotypic confirma-
prepare paired-end libraries according to the protocol tion of diarrheal pathogens with biochemical tests [53,
documented by Illumina (Illumina Inc., CA, USA) [44]. 54]. The confirmed strains were stored in nutrient agar.
Accordingly, 1 ng of Qubit quantified genomic DNA
was tagmented (fragmented and adaptor tagged) using
Supplementary information 7. Semba RD, de Pee S, Ricks MO, Sari M, Bloem MW. Diarrhea and fever
as risk factors for anemia among children under age five living in urban
Supplementary information accompanies this paper at https://doi.
slum areas of Indonesia. Int J Infect Dis. 2008;12(1):62–70.
org/10.1186/s13099-020-00371-8.
8. Kamath NA, Shetty K, Unnikrishnan B, Kaushik S, Rai SN. Prevalence,
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from metagenomic sequencing in the study. s12889-018-6213-z.
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on 3rd Dec 2019.
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We are grateful to Dr. G. Balakrish Nair for his contribution to the study. We are nationalmorbidity, mortality, and aetiologies of diarrhoealdiseases: a
thankful to Dr. Robert D. Finn and Dr. Alexandre Almeida at EMBL-EBI for their systematic analysis for the Global Burden of Disease Study 2015. Lancet
immense help with the analysis of the metagenomics data. Infect Dis. 2017;17(9):909–48.
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Authors’ contributions regional, and state-level all-cause and cause-specific under-5 mortality in
RD designed the study, performed the experiments, analyzed the data and India in 2000-15: a systematic analysis with implications for the Sustain-
wrote the manuscript; AKM did experiments and wrote the manuscript; SD able Development Goals. Lancet Glob Health. 2017;7(6):e721–34.
planned experiments and analyzed the data. All authors read and approved 12. Raju B, Parikh RP, Vetter VV, Kolhapure S. Epidemiology of rotavirus gastro-
the final manuscript. enteritis and need of high rotavirus vaccine coverage with early comple-
tion of vaccination schedule for protection against rotavirus diarrhea in
Funding India: a narrative review. Indian J Public Health. 2019;63:243–50.
We thank the Department of Health Research, Ministry of Health and Family 13. De R. Metagenomics: aid to combat antimicrobial resistance in diarrhea.
Welfare, India, for the financial support we received for the study via grant Gut Pathog. 2019. https://doi.org/10.1186/s13099-019-0331-8.
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et al. Gut Pathog (2020) 12:32

RESEARCH Open Access

Metagenomic analysis of gut microbiome

Background by various parameters like genetic make-up, ethnicity,

Fig. 1 Showing West Bengal in Eastern India (Courtesy: thymapguide.in)

S1 Male 29y O 24 Parganas VC O1 Inaba Proteobacteria 0.256637168

Unassigned Bacteria Proteobacteria Firmicutes Actinobacteria

Table 2 Catalogue of phyla, orders, families found in the study cohort

Actinomycetales, Bacteroidales, Enterobacterales, Bifidobacteriales, Veillonellales, Propionibacteriales, Eggerthellales, Pseudonocardiales,

Actinomycetaceae, Bifidobacteriaceae, Corynebacteriaceae, Micro- Solibacteraceae, Gordoniaceae, Dietziaceae, Nocardiaceae, Brevibac-

Table 3 Genus and species catalogue

S. equi S10, S14

M. massiliensis S1, S2, S5, S9, S12, S18, S19, S20

S. deleyianum S2, S17, S20

Candidatus Accumulibacter sp.

Candidatus Glomeribacter sp.

help of antiSMASH algorithm (Fig. 11). Highest number Discussion

Prep™ clean up system and quality checked. The library

with NanoDrop spectrophotometer and ­Qubit® dsDNA

clean up using HighPrep™ clean up system (catalogue

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Fig. 1 Showing West Bengal in Eastern India (Courtesy: thymapguide.in)

Table 2 Catalogue of phyla, orders, families found in the study cohort

Table 3 Genus and species catalogue

with NanoDrop spectrophotometer and Qubit® dsDNA