Manuscript Details

Manuscript number TUBE_2020_52

Title Microbial biodiversity in the throats of pulmonary tuberculosis patients and

tuberculosis skin test (TST) positive and negative healthy individuals in
Malaysia.

Article type Original Article

Abstract
The purpose of this study was to investigate the composition of throat microbiota in pulmonary tuberculosis patients
(PTB) in comparison to healthy tuberculin skin test positive (TSTp) and negative individuals (TSTn). Throat swabs
samples were collected, and the microbiota was characterized. Higher amount of unique operational taxonomic units
(OTUs) were present in PTB group, compared to TSTp and TSTn. Regarding alpha diversity analysis there was a
higher community diversity in TSTn compared to TSTp. Beta diversity analysis showed different species composition
in TSTp compared to TSTn and PTB. There was higher presence of Firmicutes in PTB and TSTn compared to TSTp
group at phylum level. At the genus level, Leuconostoc and Enterococcus were higher in TSTn compared to TSTp and
Pediococcus, Chryseobacterium, Bifidobacterium, Butyrivibrio, and Bulleidia were higher in PTB compared to TSTn.
Streptococcus was higher in TSTn compared to PTB and Lactobacillus in PTB compared to TSTp. At species level,
Streptococcus sobrinus and Bulleidia moorei were higher in PTB compared to TSTn individuals, while Lactobacillus
salivarius was higher in PTB compared to TSTp. The differences in the microbiome composition could influence the
resistance/susceptibility to Mtb infection.

Keywords Microbiome, pulmonary tuberculosis, latent tuberculosis, Tuberculin Skin Test,

metagenomic, throat swabs.

Manuscript category Mechanisms of Pathogenesis

Corresponding Author Armando Acosta

Corresponding Author's Universiti Sains Malaysia

Institution

Order of Authors Noreafifah Semail, Siti Suraiya, Romel Calero, Mayelin Mirabal, Humberto
Carrillo, Ezzeddin Kamil Amil Mohamed Hashim, Maria E. Sarmiento, Mohd Nor
Norazmi, Armando Acosta

Suggested reviewers Boon Huat Lim, Pere Joan Cardona, Nadine Alvarez, Rogelio Hernandez

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February 19, 2020

Cover letter

Dear Dr. Brian D. Robertson

Enclosed please find the manuscript entitled “Microbial biodiversity in the throats of
pulmonary tuberculosis patients and tuberculosis skin test (TST) positive and negative
healthy individuals in Malaysia.” to be considered for publication in “Tuberculosis” journal.

All the authors have read and approved the manuscript and agree with its submission.

Regards;

Prof. Armando Acosta. Corresponding author. Universiti Sains Malaysia.

Prof. Norazmi Mohd Nor. Corresponding author. Universiti Sains Malaysia.

Assoc. Prof. Dr. Siti Suraiya. Corresponding author. Universiti Sains Malaysia
 1 Title page

2 Title: Microbial biodiversity in the throats of pulmonary tuberculosis patients and tuberculosis skin test (TST)
3 positive and negative healthy individuals in Malaysia.

4 Authors: Noreafifah Semail1, Siti Suraiya1, Romel Calero2, Mayelin Mirabal2, Humberto Carrillo2,3, Ezzeddin
5 Kamil Mohamed Hashim4, Maria E. Sarmiento4, Armando Acosta4 and Mohd Nor Norazmi4
6

7 1School of Medical Sciences, Universiti Sains Malaysia (USM), 16150 Kubang Kerian, Kelantan, Malaysia.
8 2Center of Complexity Science, National Autonomous University of Mexico (UNAM), Mexico
9 3Faculty of Science, National Autonomous University of Mexico (UNAM), Mexico
10 4School of Health Sciences, Universiti Sains Malaysia (USM), Malaysia.
11

12 Corresponding authors: Mohd Nor Norazmi (norazmimn@usm.my), Siti Suraiya (ssuraiya@usm.my) and
13 Armando Acosta (armando@usm.my)
14
Order Authors email Institution

1 Noreafifah Semail noreafifah93@gmail.com School of Medical Sciences, Universiti Sains Malaysia

2 Siti Suraiya
3 Romel Calero
4 Mayelin Mirabal
5 Humberto Carrillo
6 Ezzeddin Kamil
Mohamed Hashim
7 Maria E. Sarmiento
8 Armando Acosta
9 Mohd Nor Norazmi
15
(USM), 16150 Kubang Kerian, Kelantan, Malaysia
ssuraiya@usm.my School of Medical Sciences, Universiti Sains Malaysia
(USM), 16150 Kubang Kerian, Kelantan, Malaysia
rcalero@gmail.com Center of Complexity Science, National Autonomous
University of Mexico (UNAM), Mexico
mayelin.mirabal@gmail.com Center of Complexity Science, National Autonomous
University of Mexico (UNAM), Mexico
hcarrillocalvet@gmail.com Center of Complexity Science, National Autonomous
University of Mexico (UNAM), Mexico and
Faculty of Science, National Autonomous University of
Mexico (UNAM), Mexico
Universiti Sains Malaysia School of Health Sciences, Universiti Sains Malaysia
(USM), 16150 Kubang Kerian, Kelantan, Malaysia
mari@usm.my School of Health Sciences, Universiti Sains Malaysia
(USM), 16150 Kubang Kerian, Kelantan, Malaysia
armando@usm.my School of Health Sciences, Universiti Sains Malaysia
(USM), 16150 Kubang Kerian, Kelantan, Malaysia
norazmimn@usm.my School of Health Sciences, Universiti Sains Malaysia
(USM), 16150 Kubang Kerian, Kelantan, Malaysia

1
16 Title: Microbial biodiversity in the throats of pulmonary tuberculosis patients and tuberculosis skin test (TST)
17 positive and negative healthy individuals in Malaysia.

18

19 Abstract
20 The purpose of this study was to investigate the composition of throat microbiota in pulmonary tuberculosis
21 patients (PTB) in comparison to healthy tuberculin skin test positive (TSTp) and negative individuals (TSTn).
22 Throat swabs samples were collected, and the microbiota was characterized. Higher amount of unique
23 operational taxonomic units (OTUs) were present in PTB group, compared to TSTp and TSTn. Regarding alpha
24 diversity analysis there was a higher community diversity in TSTn compared to TSTp. Beta diversity analysis
25 showed different species composition in TSTp compared to TSTn and PTB. There was higher presence of
26 Firmicutes in PTB and TSTn compared to TSTp group at phylum level. At the genus level, Leuconostoc and
27 Enterococcus were higher in TSTn compared to TSTp and Pediococcus, Chryseobacterium, Bifidobacterium,
28 Butyrivibrio, and Bulleidia were higher in PTB compared to TSTn. Streptococcus was higher in TSTn compared
29 to PTB and Lactobacillus in PTB compared to TSTp. At species level, Streptococcus sobrinus and Bulleidia
30 moorei were higher in PTB compared to TSTn individuals, while Lactobacillus salivarius was higher in PTB
31 compared to TSTp. The differences in the microbiome composition could influence the resistance/susceptibility
32 to Mtb infection.

33 Keywords:

34 Microbiome, pulmonary tuberculosis, latent tuberculosis, Tuberculin Skin Test, metagenomic, throat swabs.

35 List of abbreviations

Abbrev Abbrev
BCG Bacillus Calmette-Guerin TB tuberculosis
IFN Interferon TST Tuberculin skin test
Mtb Mycobacterium tuberculosis TSTn Healthy TST negative
OTU Operational taxonomic Unit TSTp Healthy TST positive
PCA Principal Component Analysis UPGMA Unweighted Pair-group Method with Arithmetic Means
PCoA Principal Coordinates Analysis WHO World Health Organization
PTB Pulmonary tuberculosis WPGMA Weighted Pair-group Method with Arithmetic Means

36

2
37 1.0. Introduction

38 Tuberculosis (TB) is a communicable disease caused by Mycobacterium tuberculosis (Mtb) that remains as one
39 of the major health problems throughout the world [1]. Despite the existence of numerous effective anti-TB drugs,
40 it continues to be a major public health challenge [1]. As reported by World Health Organization (WHO) in 2019,
41 approximately ten million new TB cases, which is equivalent to 130 cases per 100 000 populations worldwide,
42 has been estimated [1]. About 1.2 million people died with TB of which 250 000 deaths resulting from co-infection
43 with HIV [1]. Despite TB incidence had been declining about 2 % each year due to effective diagnosis and
44 treatment, the mortality rate is unacceptably high, and remains as one of the top 10 causes of death and the
45 main cause of mortality due to infectious diseases worldwide [1]. It is estimated that ¼ of humanity is latently
46 infected with Mtb (LTBI) [2], with a 10% lifetime risk to develop active TB, so, the identification of factors
47 associated with LTBI and progression to active TB is a priority [3].

48 Numerous factors conspire against the TB control, which includes the low performance of traditional diagnostics
49 methods, associations with other diseases such as HIV and diabetes mellitus, development of resistant strains
50 and the non-availability of an efficient vaccine to prevent the disease and its transmission [1,4]. Bacillus
51 Calmette-Guerin (BCG), the current vaccine, is protective against severe forms of TB in children but is not
52 effective against adult pulmonary TB (PTB). Therefore, many efforts are underway with different approaches to
53 develop new vaccines with the possibility to confer better protection against TB compared to BCG [5,6].

54 The composition and function of microbiota colonizing the human body differ depending on the body sites, age,
55 sex, race, diet, morbidities, genetic factors and lifestyle of the host [7-10]. Commensal bacteria colonize the
56 human host from the moment of birth and this bacterial community will eventually develops into a more varied
57 ecosystem and become a vital part of the host throughout the lifetime [11]. The host-bacterial relationship will
58 change into a beneficial over time in which the microbiota can act as a defense mechanism against colonization
59 by foreign pathogens as well as providing essential nutrients to the host [12]. Each component of the microbiota
60 has their own functions and ecosystem which depend on the microenvironment they live in [13].

61 Prior to the appearance of advanced sequencing technologies, the analysis of microbial communities was done
62 using traditional techniques which include staining and culture-based approaches. These techniques have been
63 used to distinguish many bacterial groups and has been a vital approach for microbial identification and
64 classification [14]. However, these techniques lacked taxonomic resolution and were limited to only a minority
65 of microorganisms, especially to those microbes that can be cultured in the laboratory [15].

66 Recently, high-throughput sequencing technologies have been applied to characterize the human microbiota in
67 various body habitats such as gut, oral cavity, and respiratory tract, among others [10,16,17]. This massive DNA-
68 based approach has been considered as a fast and cost-effective analysis and has provided valuable insights
69 and significant improvement to the studies that involve the human associated microbial communities [18,19].
3
70 The studies of the microbiota in association with TB have been described in several reports, which involved the
71 determination of the microbiota diversity in the lung, nasopharynx, and oropharynx as well as in the gut of PTB
72 [17,20-22]. Oropharynx or throat is one of the important parts of the respiratory tract that serve as an ecological
73 niche for microbiota where the colonizing microbial communities play an important role in human health and
74 disease [17]. The study of throat microbiota in PTB has become an area of great interest [17].

75 A study that investigated the microbiome in the throat and the sputum of PTB reported the resemblance of
76 microbial communities in these samples which suggested the similarity of the microbiome in the throat and lungs
77 [17]. The similarity between the microbiome of the throat and lower respiratory tract was also reported in a study
78 of healthy individuals. These studies suggested the possibility to use throat samples as surrogate of lower
79 respiratory tract samples for microbiome studies [16,17].

80 Although the difference of the throat microbiome between PTB and healthy individuals have been reported [23],
81 Tuberculin skin test positive (TSTp) and negative (TSTn) individuals have not been studied yet. Therefore, this
82 study was designed to investigate the biodiversity of the throat microbiota and to compare the abundance of the
83 microbiota in PTB, TSTp and TSTn. The study of the composition of the microbiota could give insights into the
84 influence of its composition in the resistance or susceptibility to TB and LTBI.

85

4
 86 2.0. Materials and methods
87 This study was approved by The Human Research Ethics Committee, Universiti Sains Malaysia (study protocol
88 code USM/JEPeM 17050275). All subjects participating in this study signed the informed consent.

89 For TST test, Tuberculin PPD RT 23 (Statens Serum Institute, Copenhagen, Denmark) was used and the
90 transverse diameter of the induration was determined. The procedure was carried out by trained professionals.

91 Twenty-four subjects divided in three groups (n: 8/group) were recruited based on strict inclusion/exclusion
92 criteria:

93 PTB: Patients with pulmonary TB microbiologically confirmed (smear and/or culture) before start the TB
94 treatment, without malignancies, acute diseases or immunosuppressive treatment, more than 20 years old. The
95 throat swab samples were collected at Chest Clinic, Hospital Universiti Sains Malaysia (HUSM), Kelantan,
96 Malaysia. Patient’s demographic data is shown in Table S1.

97 TSTp: Healthy Adults with TST more than 11mm with more than 20 years old, without acute or chronic diseases
98 with compromised immunity, Immunosuppressive, or antibiotic treatment and/or previous clinical TB. The throat
99 swab samples were collected at Chest Clinic, Hospital Universiti Sains Malaysia (HUSM), Kelantan, Malaysia.

100 TSTn: Healthy Adults with TST between 0-9mm older than 20 years old, without acute or chronic diseases with
101 compromised immunity, Immunosuppressive, or antibiotic treatment and/or previous clinical TB. The throat
102 samples were collected at Department of Medical Microbiology and Parasitology USM.

103 The throat samples were obtained using FLOQ Swabs, Copan Technologies, Italy and kept at -80°C in 3 ml of
104 Universal Transport Media (Copan Italia, Italy) until further analysis. Total genomic DNA was extracted from the
105 throat swabs using QIAamp® DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s
106 instructions. The genomic DNA were sent to First Base Laboratory (Malaysia) for sequencing processing.

107 Different 16S gene regions (V3-V4) were amplified using specific primers 515F (5’-
108 GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) with the barcode [24]. All
109 PCR reactions were carried out with Phusion®High-Fidelity PCR Master Mix (New England Biolabs). Same
110 volume of 1X loading buffer (containing SYB green) was mixed with PCR products and run on 2% agarose
111 electrophoresis gel for detection. Samples with a bright main band between 400-450bp were chosen for further
112 experiments. PCR products were mixed in equidensity ratios. Then, the mixture of PCR products was purified
113 with Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using TruSeq® DNA
114 PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer's recommendations, thus index
115 codes were added. The library quality was assessed with the Qubit@ 2.0 Fluorometer (Thermo Scientific) and
116 Agilent Bioanalyzer 2100 system. After that, the library was sequenced on an Illumina HiSeq 2500 platform and
117 250 bp paired-end reads were generated.
5
118 Paired-end reads were assigned to samples based on their unique barcode and truncated by cutting off the
119 barcode and primer sequence. Paired-end reads were merged using FLASH (V1.2.7,
120 http://ccb.jhu.edu/software/FLASH/) [25], a very fast and accurate analysis tool designed to merge paired-end
121 reads when at least some of the reads overlap the read generated from the opposite end of the same DNA
122 fragment. The resulting splicing sequences were called raw tags.

123 Specific filtering conditions were applied to raw tags to obtain high-quality clean tags according to the Qiime
124 quality-controlled process [26,27]. The tags were compared with the reference database (Gold database) using
125 UCHIME algorithm to detect and remove chimera sequences [28,29]. Thus, the effective tags were finally
126 obtained.

127 Sequence clustering was performed by Uparse software (Uparse v7.0.1001，http://drive5.com/uparse/) [30].

128 Sequences with similarity ≥97% where assigned to the same OTUs. For each OTU, representative sequences
129 was screened for further annotation using the Green Gene Database (http://greengenes.lbl.gov/cgi-bin/nph-
130 index.cgi) based on RDP classifier (Version 2.2, http://sourceforge.net/projects/rdp-classifier/) algorithm to
131 annotate taxonomic information [31,32]. In order to study the phylogenetic relationship of different OTUs, and
132 the difference of the dominant species in different samples (groups), multiple sequence alignment was
133 conducted using the PyNAST software (Version 1.2) against the "Core Set" dataset in the Green Gene database
134 [33]. OTUs abundance information were normalized using a standard of sequence number corresponding to the
135 sample with the least sequences. Subsequent analysis of alpha and beta diversity was all performed based on
136 this output normalized data.

137 Alpha diversity was performed to analyze complexity of species diversity for each sample through various alpha
138 indices: Observed-species, Chao1, Shannon, Simpson, ACE, which were calculated with QIIME (Version 1.7.0)
139 and displayed with R software (Version 2.15.3). Two indices were selected to estimate community richness,
140 Chao1 and ACE, and two indices were used to estimate community diversity: Shannon and Simpson.

141 Beta diversity analysis was used to evaluate differences of samples in species complexity. Beta diversity on
142 both weighted and unweighted unifrac were calculated by QIIME software (Version 1.7.0).

143 Cluster analysis was preceded by principal component analysis (PCA), which was applied to reduce the
144 dimension of the original variables using the FactoMineR package and ggplot2 package in R software (Version
145 2.15.3).

146 Principal Coordinate Analysis (PCoA) was performed to get principal coordinates and visualize from complex,
147 multidimensional data. A distance matrix of weighted or unweighted unifrac among samples obtained before
148 was transformed to a new set of orthogonal axes, by which the maximum variation factor is demonstrated by

6
149 first principal coordinate, and the second maximum one by the second principal coordinate, and so on. PCoA
150 analysis was displayed by WGCNA package, stat packages and ggplot2 package in R software (Version 2.15.3).

151 Unweighted Pair-group Method with Arithmetic Means (UPGMA) Clustering was performed as a type of
152 hierarchical clustering method to interpret the distance matrix using average linkage and was conducted by
153 QIIME software (Version 1.7.0).

154 To determine differences in species abundance, Metastat software was used.

155 The general workflow of this study is shown in Figure 1.

156

7
157 3.0. Results and Discussion

158 3.1. Sequencing data processing.

159 Out of twenty-four subjects enrolled in this study, one sample (TSTp08) was not further analyzed after library
160 construction and sequencing due to very poor effective tags quality, which might happen due to the presence of
161 chimeras, the hybrid products between multiple parent sequences that can be falsely interpreted as novel
162 organisms. The effective tags averages were: Individuals (70535), Taxon (70479), Unclassified (0), Unique (56),
163 and OTUs (28) (Figure S1).

164 3.2. OTU analysis and species annotation

165 From the analysis of the identified effective tags, 92 OTUs were assigned with a 97% similarity cut-off level.
166 After clustering a normalized OTU table was obtained and both the shared and unique OTUs of the sample
167 groups were displayed in a Venn diagram (Figure 2), 29 OTUs were shared among all the three groups, 5 OTUs
168 were shared between PTB and TSTp, 6 OTUs were shared between TSTp and TSTn and 32 OTUs were shared
169 between PTB and TSTn. There were 14 unique-OTUs for PTB and 3 unique OTUs for each TSTp and TSTn,
170 respectively.

171 Regarding the OTUs clustering, it was evident the presence of higher amount of unique OTUs in PTB group,
172 compared to TSTp and TSTn groups, which suggest that the active TB infection created a suitable growth
173 condition for selected species [20]. Surprisingly, there was more shared OTUs between PTB and TSTn groups
174 (n=32), which is an unexpected result considering that PTB and TSTp are Mycobacterium tuberculosis (Mtb)
175 infected individuals, which should be differentiated from non-infected individuals (TSTn). It is interesting also the
176 differentiation of TSTp from PTB and TSTn sharing few OTUs with these groups, which could suggest a very
177 unique niche created by the LTBI.

178 3.3. Alpha diversity

179 Alpha diversity analysis which measure the richness or evenness in a sample was performed. Rarefaction curve
180 tends to plateau at 60 000 sequences which indicated that the sequencing depth was enough to completely
181 capture the species present in the throats of all the studied subjects (Figure S2).

182 No significant differences were observed with Chao1, ACE and observed species indices which indicate that the
183 community’s richness was almost similar for all the groups.

184 Diversity was higher in TSTn compared to TSTp group, both, by Simpson (p=0.0420) (Figure 3A) and Shannon
185 indices (p=0.0445) (Figure 3B). Differences between PTB and TSTp were not significant (p>0.05).

186

187
8
188 3.4. Beta diversity
189 Beta diversity analysis was performed to compare the microbial composition between groups. UniFrac is a
190 phylogenetical measure of beta-diversity that can be used to compare microbial diversity. The weighted unifrac
191 measure takes the relative abundance of both groups into account, while the unweighted unifrac measure only
192 the distance between two communities by calculating the fraction of the branch length in a phylogenetic tree that
193 only leads to descendant’s in either of the compared groups. Using unweighted unifrac TSTp group showed
194 lower values compared to PTB (Tukey p=0.0000004 and Wilcoxon p=0) and TSTn (Tukey p=0.00037 and
195 Wilcoxon p=0) (Figure 4A). Regarding weighted unifrac, TSTp showed higher values compared to PTB (Tukey
196 p=0) and TSTn (Tukey p=0) (Figure 4B).

197 Based on the results of weighted and unweighted unifrac analysis, we conclude that the TSTp individuals
198 exhibits a significantly different species composition compared to PTB and TSTn.

199 A summary by groups are showed in Figure 5, based on heatmaps of unweighted and weighted unifrac. There
200 can be seen the composition similarity of PTB and TSTn and the dissimilarity of the groups PTB-TSTp and
201 TSTn-TSTp. These facts are revealed by the unweighted and weighted unifrac distances between PTB-TSTn,
202 TSTp-TSTn, PTB-TSTp, which were 0.206, 0.400, and 0.471 respectively for the unweighted index and 0.167,
203 0.334 and 0.350 for the weighted unifrac.

204 The results of PCA (Figure 6) and PCoA (Figures 7A and 7B), are consistent with the previous beta diversity
205 unifrac analysis results (Figures 4 and 5): Majority of derived variables from PTB located nearer to TSTn group
206 compared to TSTp group, which indicate the similarity of the microbiota inhabiting the throats of PTB and TSTn
207 compared to TSTp individuals.

208 The results of the UPGMA analysis, based on unweighted and weighted unifrac matrices, are shown in Figures
209 8A and 8B. In both cases, PTB and TSTn groups were clustered together, which could be explained as a result
210 of similarity not only in their composition (unweighted matrix) but also in the relative abundance (weighted matrix)
211 of phyla in their samples. The more predominant phyla in the three groups is Firmicutes, however, its presence
212 in PTB and TSTn is notably higher than in TSTp.

213 According to the previous results, using different criteria of analysis, it was demonstrated marked differences in
214 microbiota composition between TSTp compared to PTB and TSTn, which showed similar microbiota
215 composition. The meaning of this finding is difficult to interpret because it could be expected more similarity
216 between Mtb infected groups. In this case, non-infected (TSTn) and active TB patients (PTB) clustered together
217 with a marked difference compared to LTBI individuals (TSTp).

218 The change of microbiota composition in TSTp group compared to healthy TSTn and PTB could be a
219 consequence of the host immune response induced by the Mtb latent infection, which may kill or eliminate

9
220 microbiota components with cross reactive epitopes with Mtb after the Mtb infection, producing an adjustment
221 in the microbiota [20,34]. It should be noted that the interpretation of these results should be taken with caution
222 as the definition of LTBI is based on the immune response to Mtb antigens and not on the presence of Mtb, as
223 a test for the direct detection of Mtb infection in LTBI is currently unavailable [2].

224 In the present study, there was a clear distinction between TSTp and TSTn regarding TSTreactivity. The TST
225 reading in TSTn group were 0mm in all individuals except TSTn06 (4mm). All the individuals from TSTp group
226 were above of 12mm of induration (Supplementary information: Table S1)

227 3.5. Relative abundance of microbiota in the different groups at different taxonomic levels.
228 Firmicutes was the most represented phyla observed in the study. It showed higher abundance in PTB and
229 TSTn compared to TSTp in the analysis at the phylum level (Table 1). Firmicutes is a gram-positive bacterium
230 that was highly dominant in the healthy subjects in previous oral microbiome studies [17,22,35,36]. Another
231 study also suggested that Firmicutes are part of commensal microbiome due to their presence in more than 95%
232 of individuals in non-diseased populations. Bacteroidetes, Actinobacteria and Proteobacteria were also
233 abundant in TSTp in the present study. Regarding the differences in the presence of Firmicutes between TSTn
234 and PTB compared to TSTp, it could be due to the previously mentioned adjustment of the microbiota
235 composition as consequence of the latent infection. The study of the relative abundance at the class level
236 showed a higher presence of Bacilli in PTB and TSTn compared to TSTp (Table 1).

237 At the class level, the clustering of TSTn and PTB was present regarding the relative abundance of Bacilli.
238 Erysipelotrichi was more abundant in PTB compared to TSTn (Table 1), which could be explained by the
239 microbial environment created by TB, which could favor the grow of this class of microorganism or alternatively
240 its presence could predispose to active TB infection.

241 At the order level, Lactobacillales were more abundant in PTB and TSTn compared to TSTp, which evidence
242 that latent TB infection influences the relative abundance of some components of the microbiota.
243 Bifidobacteriales and Erycipelotrichales showed more abundance in PTB compared to TSTn; if this contribute
244 to the development of active TB or if it is an epiphenomenon of the disease need to be confirmed in future studies
245 (Table 1).

246 At the family level, the abundance of Enterococcaceae was higher in TSTn compared to TSTp. The presence
247 of Streptococcaceae was higher in TSTn compared to PTB. According to these results, it could be speculated
248 that the presence of these families could confer some resistance to Mtb infection. Bifidobacteriaceae,
249 Erysipelotrichaceae and Leuconostocaceae were more abundant in PTB compared to TSTn, which could be
250 associated to predisposition to the development of active TB. Leuconostocaceae and Lactobacillaceae were
251 more abundant in PTB compared to TSTp individuals, which could associate the presence of these families to
252 the progression from LTBI to active TB infection (Table 1).
10
253 The analysis of the relative abundance, at the genus level, showed that Streptococcus was more abundant in
254 TSTn compared to PTB, while Leuconostoc and Enterococcus were more abundant in TSTn compared to TSTp
255 (Table 1). These results could indicate that the presence of these genera may confer some degree of protection
256 against Mtb infection. This finding is consistent with previous studies that found Streptococcus as more prevalent
257 in control populations [22,37]. Pediococcus, Chryseobacterium, Bifidobacterium, Butyrivibrio and Bulleidia were
258 more represented in PTB compared to TSTn. This could indicate changes in the composition of the microbiota
259 due to active TB infection or an increase of the susceptibility to develop active infection due to the presence of
260 these genera. Regarding Lactobacillus, the abundance was increased in PTB compared to TSTp which could
261 be an indicative of predisposition to progress from latent to active TB (Table 1). This fact is in contradiction with
262 the demonstrated immunopotentiating and immunomodulatory effects of Lactobacillus. [38,39].

263 At species level, both Streptococcus sobrinus and Bulleidia moorei were significantly higher in the throats of
264 PTB compared to TSTn individuals suggesting a potential role of these species for the development of clinical
265 TB. Lactobacillus salivarius was significantly higher in PTB compared to TSTp individuals which could be
266 associated to the progression from LTBI to active TB.

267 The microbiota, which influence the immune response to Mtb, is modified by the living conditions and the
268 associated environmental infections [40,41]. In developed countries, due to higher socio-economic standards,
269 the microbiota is characterized by a less diverse composition, with a low presence of parasitic infections, in
270 contrast to developing countries, where a more diverse microbiota is present with high presence of
271 environmental mycobacteria and parasitic infections [40,41]. This fact, together with the characteristics of the
272 exposure to Mtb has a high influence on the immune response to Mtb infection [40,41]. In the developed world
273 the exposure to Mtb is characterized by a sporadic and low dose challenge, whereas in developing countries,
274 the Mtb exposure is produced in a sustained way with a high dose challenge [40,41]. All these factors could
275 determine the predominance of LTBI in developed countries and progressive disease in low resource areas,
276 where the presence of a predominant Th2 pattern of the immune response is associated with progressive
277 disease with lung damage and fibrosis and not with a Th1 pattern, which is associated with protection [40,41].

278 The influence of the living conditions and the microbiota in the developing world had been advocated to be linked
279 to the lack of protection associated with BCG vaccination compared with the higher protective capacity of the
280 vaccine reported in developed nations [40,41]. It had been reported that Helicobacter hepaticus infection in mice
281 can suppress the protection induced by a subunit TB vaccine [42], which reinforce the notion that microbiota
282 composition has influence in the immune response to TB vaccines.

283 There are several reports suggesting the influence of microbiota in the protection or susceptibility to TB infection.
284 The presence of Helycobacter pylori (H. pylori) infection in humans had been reported to protect against the
285 progression from LTBI to active TB [43], LTBI with H. pylori infection, has a predominant IFN gamma response

11
286 against Mtb antigens, with or without parasitic co-infection [44].

287 Regarding parasitic infections, trichuriasis and giardiasis have been associated to protection against Mtb
288 infection [45,46]. Helminth coinfection in TB patients had been associated with more severe clinical presentation,
289 as compared to helminth-free patients [46]. Several studies reported associations between parasitic infections
290 (e.g. Toxoplasma gondii, Trichuris trichiura, Giardia intestinalis, Ascaris lumbricoides, Strongyloides stercoralis,
291 and hookworms) and TB [45-49].

292 Experimental results suggest that Mtb infection could induce changes in microbiota composition, in this regard,
293 mice infected aerogenically with virulent strains of Mtb produced changes in the intestinal microbiota,
294 characterized by a transient decrease in the diversity of the microbiota, followed by changes in the composition
295 of the microbiota in advanced phases of the infection compared to non-infected animals, suggesting the
296 presence of immune cross-talk between the lungs and the gut [50].

297 According to the previous information it can be suggested that Mtb and the microbiota has a dynamic bi-
298 directional interaction influencing the microbiota composition, the evolution of the Mtb infection, determining
299 resistance or susceptibility to latent and active Mtb infection as well as with the capacity to modulate the immune
300 response to TB vaccines.

301 4.0. Conclusion

302 This is the first study investigating the throat microbiome of PTB and healthy individuals grouped according to
303 TST reactivity, demonstrating differences between these groups. The composition of the microbiome,
304 determined by multiple factors, could influence the susceptibility/ resistance to Mtb infection (active and latent),
305 and progression from latent to active TB. Another potential explanation of these differences could be the
306 microbiota adjustment by the presence of Mtb infection.

307 Funding

308 This research was funded by Long Research Grant Scheme (203.PPSK.67212001) and Research University
309 Grant (1001.PPSK.812200). The funding bodies did not participate in the design of the study and collection,
310 analysis, and interpretation of data and in writing the manuscript.

311 Conflict of interest statement

312 The authors declare no conflict of interests.

313 Acknowledgements:

314 We would like to thank Madam Norhayati for her help during sampling at the Chest Clinic and all the subjects
315 that participated in this project.
12
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15
429 Captions to illustrations
430

431 Figure 1. Workflow of the study

432 Figure 2. Venn diagram. OTUs among PTB, TSTp and TSTn groups.

433 Figure 3. Alpha diversity analysis (Wilcoxon test). A. Simpson. B. Shannon

434 Figure 4. Beta diversity analysis Tukey (T) and Wilcoxon(W) test. A. Unweighted Unifrac and B. Weighted Unifrac

435 Figure 5. Beta diversity index. Each grid represents pairwise dissimilarity coefficient between pairwise samples, in which

436 Weighted (above) and Unweighted Unifrac distance displayed

437 Figure 6. PCA plot. The percent variation explained 48.29%. Each dot represents one sample from each group.

438 Figure 7. PCoA of A. Weighted Unifrac and B. Unweighted Unifrac The PCoA combined explained 77.21% and 47.86% of

439 the variations respectively. Each dot represents one sample from each group.

440 Figure 8. A. Tree unweighted (UPGMA) and B. Tree weighted (WPGMA).

441 Table 1. Comparison of bacterial community between PTB group, TSTp and TSTn groups.
442

443

16
444 Figures and Table

445
DNA extraction
446

447
PCR amplification, quantification &
448 purification Species annotation
• Phylogenetic tree
449 • Abundance Heatmap
Advanced análisis: Metastat
Library preparation
450

451 Alpha diversity

Sequencing (Hiseq) • Rarefaction curve
452
Alpha diversity index:
• Chao1
453
Paired end reads merge. OTU Cluster • ACE
Raw data • Shannon
Quality, foltering, chimeras Venn diagram
454 • Simpson
• Observed species
455

456
Beta diversity
457 • (Un) weighted Unifrac distance
• Index Heatmap
458 • PCA/ (Un) weighted PCoA
• Clustering analysis of UPGMA &
WPGMA
459

460

461

462 Figure 1
463

17
464

465

466 Figure 2
467

468

469 A B

470 Figure 3

471

18
472

473 A B

474 Figure 4

475

476

477 Figure 5

478

19
479

480 Figure 6.
481

482

483 A B

484 Figure 7.
485

20
486

487

488 A

489

490

491 B

492 Figure 8

493

21
494 Table 1
Mean Relative Abundance TSTn > TSTp TSTn > PTB PTB > TSTn PTB > TSTp

TSTn TSTp PTB p q p q p q p q

Phylum
Firmicutes 0.9665 0.4404 0.9839 0.0038 0.0307 0.0004 0.0064
Class
Bacilli 0.9663 0.4404 0.9828 0.0018 0.0272 0.0004 0.006
Erysipelotrichi 0 0 0.00002 0.0009 0.015
Order
Lactobacillales 0.8469 0.2973 0.8475 0.0007 0.0151 0.0012 0.0272
Bifidobacteriales\ 0 0 0.00004 0.00002 0.001 0.01
Erysipelotrichales 0 0 0.00002 0.00001 0.001 0.01
Family
Streptococcaceae 0.7211 0.2847 0.1396 0.001 0.01
Lactobacillaceae 0.0696 0.0057 0.0538 0.002 0.05
Enterococcaceae 0.0019 0 0 0.0009 0.0348
Leuconostocaceae 0.0541 0.0054 0.6222 0.002 0.02 0 0
Bifidobacteriaceae 0 0 0.00004 0.001 0.01
Erysipelotrichaceae 0 0 0.00002 0.001 0.01
Genera
Streptococcus 0.7211 0.2847 0.1396 0.001 0.007
Leuconostoc 0.0022 0.0003 0.0595 0.002 0.04
Lactobacillus 0.0696 0.0057 0.0535 0.002 0.04
Enterococcus 0.00195 0 0.03174 0.0009 0.04
Pediococcus 0 0.00001 0.0026 0.001 0.007
Chryseobacterium 0 0.00003 0.00211 0.001 0.007
Bifidobacterium 0 0 0.00004 0.001 0.007
Butyrivibrio 0 0 0.00002 0.001 0.007
Bulleidia 0 0 0.00002 0.001 0.007
Species
Lactobacillus 0.0027 0.0007 0.0311 0.002 0.04
salivarius
Streptococcus 0 0 0.00001 0.001 0.01
sobrinus
Bulleidia moorei 0 0 0.00001 0.001 0.1

495
496

22
 1 Supplementary Information
2
3 Title: Microbial biodiversity in the throats of pulmonary tuberculosis patients and
4 tuberculosis skin test (TST) positive and negative healthy individuals in Malaysia.
5
6 Authors: Noreafifah Semail1, Siti Suraiya1, Romel Calero2, Mayelin Mirabal2, Humberto
7 Carrillo2,3, Ezzeddin Kamil Mohamed Hashim4, Maria E. Sarmiento4, Armando Acosta4 and
8 Mohd Nor Norazmi4
9
10 1School of Medical Sciences, Universiti Sains Malaysia (USM), Malaysia.
11 2Center of Complexity Science, National Autonomous University of Mexico (UNAM), Mexico
12 3Faculty of Science, National Autonomous University of Mexico (UNAM), Mexico

13 4School of Health Sciences, Universiti Sains Malaysia (USM), Malaysia.

14
15 Corresponding authors: Mohd Nor Norazmi (norazmimn@usm.my), Siti Suraiya
16 (ssuraiya@usm.my) and Armando Acosta (armando@usm.my)
17
18 Captions of Figures and Table
19
20
21 Table S1: Demographic data of participant in this study.

22 Figure S1: Statistical analysis of tags distribution and OTUs analysis of each
23 samples. The Y1-axis titled "Tags Number" means the numbers of tags: "Total tags" (Red
24 bars) means the numbers of effective tags; "Taxon Tags" (Blue bars) means the numbers
25 of annotated tags; "Unclassified Tags" (Green bars) means the numbers of unannotated
26 tags; "Unique Tags" (Orange bars) means the numbers of tags with a frequency of 1 and
27 only occurs in one sample. The Y2-axis titled "OTUs Numbers" means the numbers of
28 OTUs which displayed as "OTUs" (Purple bars) to identify the numbers of OTUs in different
29 samples and represent the plots for PTB (PTB01-08), TSTp (TSTP01-07) and TSTn
30 (TSTN01-08) groups respectively.

31 Figure S2: Rarefaction curves of microbiota communities based on observed OTUs

32 at 97% sequences.

33
34
35 Group Sample Age Gender TST (mm)
code
36 PTB01 65 M -
37 PTB02 60 M -
PTB03 49 M -
38 PTB04 34 M -
PTB PTB05 54 M -
39 PTB06 53 F -
PTB07 69 F -
40 PTB08 54 M -
41 TSTP01 58 M 16
TSTP02 23 F 18
42 TSTp TSTP03 19 F 13
TSTP04 51 M 16
43 TSTP05 60 M 15
TSTP06 34 F 15
44
TSTP07 54 F 14
45 TSTN01 25 F 0
TSTN02 27 F 0
46 TSTN03 26 F 0
TSTN04 26 F 0
47 TSTn TSTN05 26 F 0
TSTN06 35 F 4
48
TSTN07 24 M 0
49 TSTN08 24 M 0

50 Table S1:
51
Figure S1.
Figure S2