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Microbiology and Biotechnology Letters

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Molecular and Cellular Microbiology (MCM)  |  Microbial Genetics, Physiology and Metabolism

Microbiol. Biotechnol. Lett. 2024; 52(4): 428-440

https://doi.org/10.48022/mbl.2407.07019

Received: July 30, 2024; Revised: October 16, 2024; Accepted: November 12, 2024

Characterization of a Fast-Growing Mycobacteria, Isolated from a Healthy Individual from Kelantan, Malaysia

Anis Fadhilah Zulkipli1, Maria E. Sarmiento1*, Nyok-Sean Lau2, Kai Ling Chin3, Nur Hidayati MB1, Zulaikah Mohamed4, Fatin Syamimi Mohamad Zahidan1, Nik Zuraina Nik Mohd Nor5, Ezzeddin Kamil Mohamed Hashim1, Pannerchelvam S1, Siti Suraiya5, Armando Acosta1*, and Mohd Nor Norazmi1,6*

1School of Health Sciences, Universiti Sains Malaysia, Health Campus, Kubang Kerian, Kelantan, Malaysia
2Centre for Chemical Biology, Universiti Sains Malaysia, Bayan Lepas, Pulau Pinang, Malaysia
3Faculty of Medicine and Health Sciences, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia
4Johor Bahru Public Health Laboratory, Ministry of Health of Malaysia, Johor Bahru, Johor, Malaysia
5School of Medical Sciences, Universiti Sains Malaysia, Health Campus, Kubang Kerian, Kelantan, Malaysia
6Malaysia Genome and Vaccine Institute, National Institutes of Biotechnology Malaysia, Jalan Bangi, 43000 Kajang, Selangor, Malaysia

Correspondence to :
Armando Acosta,        ducmar13@gmail.com
Maria E. Sarmiento,     mariesarmientogarcia@gmail.com
Mohd Nor Norazmi,    norazmimn@usm.my

Abstract

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The human microbiome is critical to understanding health and disease. The presence of mycobacteria in the normal microbiome (mycobacteriome) holds significant interest for its potential role in autoimmunity, allergy, cancer, infections, and particularly in modulating immune responses to pathogenic mycobacteria and tuberculosis vaccines. In this regard, in the context of the study of the mycobacteriome in healthy individuals, the aim of this study was to characterize a Fast-Growing Mycobacteria (FGM) isolated from a healthy individual, which was characterized by: I) Genotypic studies: a) whole genome sequencing (WGS), b) phylogenetic study for 19 mycobacteria strains (general comparison) and 24 Mycolicibacterium fortuitum (Mfor) genomes (specific comparison), and c) homology study with Mfor strains and Mycobacterium tuberculosis (Mtb). II) Phenotypic studies: a) biochemical studies b) antimicrobial susceptibility testing (AST), c) temperature tolerance, d) tolerance to 5% NaCl, and e) photo-reactivity. III) Immunological study based on an antigenicity study using sera from pulmonary TB patients (PTB) and healthy individuals with positive tuberculin skin test (TSTp) or negative (TSTn). Based on the genotypic and phenotypic results, FGM-USM could be considered as a new strain, belonging to Mfor group, named Mycolicibacterium fortuitum strain kelantanesensis. The reactivity of PTB sera suggest the presence in the strain of homology to Mtb proteins expressed in vivo during infection. The homology and antigenicity results suggest the potential evaluation of the strain for the development of vaccine candidates against TB.

Keywords: Fast-growing mycobacteria, Mycolicibacterium fortuitum, Mycolycibacterium kelantanesensis, Mycobacterium tuberculosis, tuberculosis

Graphical Abstract


Introduction

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The human microbiome is a dynamic element, evolving since birth, changing in the context of several factors, such as age, body area, age, sex, diet, disease, and genetic background, among other factors, representing an important safeguard for health, although its imbalance is associated to multiple pathological manifestations [1]. The explosive development of genomics and informatics have helped to unveils the extreme diversity of the human microbiome, allowing their study in health and disease [1−3]. Mycobacteria are members of the microbiome (mycobacteriome), but it remains as one of its components less studied, despite the growing evidence of the importance of colonizing mycobacteria in health and disease, including their potential role on the protection and susceptibility to Tuberculosis (TB), the immune response to TB vaccination, and their potential use for development new vaccines, and immunotherapy for TB, other mycobacterial diseases, and non-communicable diseases, such as allergic, autoimmune and degenerative diseases [4, 5]. To contribute to the study of the mycobacteriome, the aim of this study was to characterize a Fast- Growing Mycobacteria (FGM) isolated from a healthy individual by genetic, biochemical, phenotypic, and immunological studies.

The genotypic and phenotypic results included the strain as part of the Mycolycibacterium fortuitum (Mfor) species, proposed to be named as M. fortuitum strain kelantanesensis. Bioinformatics and immunologic studies suggest the potential of the strain for prophylaxis and immunotherapy of TB.

Materials and Methods

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Sample

A fast-growing mycobacterium strain was isolated from the foreskin tissue of a healthy 10-year-old boy, obtained before an elective circumcision procedure performed at a local clinical facility in Kelantan, Malaysia. The swab was taken from the inner part of the foreskin before the tissue was sterilized for the surgical procedure. The swab was briefly dipped several times in sterile saline (500 μl) and 50 μl was seeded in Lowenstein Jensen agar and incubated (37℃/3−7 days). After 3 days of incubation, several colonies were analysed by Ziehl- Neelsen staining and a positive colony, of a presumptive fast-growing mycobacterium (FGM-USM), was inoculated in Vegitone Luria Bertani Broth (Merck, Germany) and incubated (37℃/200 rpm/3 days). and seeded in Vegitone Nutrient Agar and incubated (37℃/3 days). This study was approved by the Universiti Sains Malaysia (USM) Human Research Ethics Committee: USMKK/PPP/JEPEM (245.3 [16]). An informed consent was obtained and signed by the child’s father before the sample was obtained.

I. Genotypic studies:

DNA purification

Genomic DNA was extracted using the NucleoSpin® Tissue kit (Germany) according to the manufacturer’s instructions with some modifications. Briefly, a loop of the strain from the culture plate was added to a tube containing lysozyme solution and incubated (1 h/37℃). This solution was divided into two 1.5 ml microcentrifuge tubes (180 μl/tube), adding Proteinase K solution (25 μl) and incubated (overnight/56℃). The samples were vortexed and Buffer B3 (200 μl) was added, vortexed vigorously and incubated (70℃/10 min) followed by a brief vortex. Absolute ethanol (210 μl) was added, followed by vigorous vortex. Each sample was placed on a NucleoSpin® Tissue Column and centrifuged (1 min/ 11,000 ×g). The column was washed with BW buffer (500 μl), and B5 buffer (600 μl) was added and centrifuged (1 min/11,000 ×g). The residual ethanol was removed by centrifugation (1 min/11,000 ×g) and the DNA was eluted with BE buffer (300 μl) after incubation (70℃/5 min), followed by the addition of BE buffer (60 μl) and incubation (room temperature/2 min) followed by centrifugation (1 min/11,000 ×g).

Whole-Genome Sequencing (WGS)

The genome was sequenced using PacBio RS II system, which generated one SMRT cell of sequencing data from 20-kb insert library with size selection. 150,292 subreads totaling 552 Mb of data were produced from the sequencing. The PacBio sequencing reads were assembled by PacBio Hierarchical Genome Assembly Process 3.0 (HGAP 3.0) in SMRT Portal v2.3.0. The assembly was improved, and errors were corrected by Quiver in the SMRT Portal. The primary assembly by HGAP 3.0 consists of 2 contigs. The complete genome sequence of FGM-USM was deposited in GenBank database under the accession number of CP089608-CP089609.

Gene prediction and annotation

The gene prediction and annotation were carried out by CLgenomics (http://www.chunlab.com/).

Methylation analysis

Methylation analysis was carried out by RS Modification and Motif Analysis in SMRT Portal.

Comparative analysis of WGS

(a) General comparison (using 19 Mycobacterium genomes)

i) Pan-Genome SNP Identification. Complete WGS of the 19 Mycobacterium genomes were downloaded from NCBI. RefSeq (NCBI GenBank) sequence databases (Supplementary Table S1). The identification of pangenome SNP was done using the kSNP program (v3.0). The kSNP3 identifies SNPs by matching unique nucleotide sequences at k-mer length 19 bp. A total of 1,781,556 SNPs were found to be common in all 20 Mycobacterium genomes including our FGM-USM. The core SNPs detected were used to build a consensus parsimony tree with 100 bootstrap estimates.

ii) Average nucleotide identity (ANI) and correlation of tetranucleotide signatures (Tetra) analysis. ANI and TETRA analysis for our FGM-USM against the other Mycobacterium samples was performed using JSpecies, version 1.2.1 [6]. For calculation of ANI, the MUMmer version 3.1 [7] option was selected.

(b) Specific comparison (using 24 Mfor genomes)

Complete WGS of 24 Mfor and M. abscessus ATCC 19977 were downloaded from NCBI. RefSeq (NCBI GenBank) sequence databases (Supplementary Table S2). ANI and TETRA analysis was made as in (a).

(c) Study of homology

i) FGM-USM against 24 Mfor genomes. (Supplementary Table S2). The determination of the numbers of homologous (80% full sequence coverage and 80% identity) and non-homologous of the predicted CDS in the FGM-USM strain, in comparison to the CDS in 24 Mfor genomes obtained from NCBI (refer to Supplementary Table S3 for the complete list of Mfor genomes with their accession numbers and hyperlinks to their respective NCBI sites) was made using tBLASTn tool from NCBI.

ii) FGM-USM against Mtb. The determination of the numbers of homologous and non-homologous of the predicted proteins in the FGM-USM strain in comparison to the proteins in Mtb H37Rv strain obtained from NCBI (https://www.ncbi.nlm.nih.gov/genome/166?genome_assembly_id=159857 ) was made using tBLASTn, NCBI https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). Proteins from the strain with more than 80% full sequence coverage and 80% identity with Mtb proteins were considered as homologs and their cellular location was determined using PSORT protein (http://psort.hgc.jp/form.html) and CELLO v.2.5: subCELlular LOcalization predictor programmes (http://cello.life.nctu.edu.tw).

II. Phenotypic studies

Biochemical studies

(a) GEN III MicroPlate test (BIOLOG, USA)

A FGM-USM colony was inoculated on Vegitone Luria Bertani Broth (Merck), incubated (37℃/200 rpm/3 days), and cell suspensions were tested in a test reaction performed in a 96 well microplate. The results were compared against 2900+ phenotypic pattern (“metabolic fingerprint) using Biolog’s Identification Systems software (BIOLOG, USA). This system included 23 Mycobacterium reference phenotypic patterns. The cut-off of similarity index value (SIM) was set at 0.5, for strain identification.

(b) Analytical profile index (API Corine) Test: (bioMerioux, USA)

A FGM-USM colony was inoculated on Vegitone Luria Bertani Broth (Merck), incubated (37℃/200 rpm/3 days) and two APIs, bioMerieux, USA, were used:

i) API-20NE panel tests (substrates). adipate (ADI), arabinose (ARA), arginine (ADH), caprate (CAP), citrate (CIT), esculin (ESC), gelatine (GEL), gluconate (GNT), glucose (GLU), malate (MLT), maltose (MAL), mannitol (MAN), mannose (MNE), N-acetyl-glucosamine (NAG), oxidase test (OX), phenyl-acetate (PAC), p-nitrophenyl- β-Dgalactopyranoside (PNPG), Potassium nitrate (N03), tryptophane (TRP), and urea (URE).

ii) API-Coryne tests (reactions). Alkaline Phosphatase (PAL), Alpha GLUcosidase (∝GLU), Beta GALactosidase (βGAL), Beta GlucURonidase (βGUR), CATalase (ESC or GEL test) (CAT), ESCulin (βGlucosidase) (ESC), Fermentation: GLUcose (GLU), GELatine hydrolysis (GEL), GLYcoGen (GLYG), LACtose (LAC), MALtose (MAL), MANnitol (MAN), N-Acetyl-B Glucosaminidase (BNAG), NITrate reduction (NIT), Pyrazinamidase (PYZ), Pyrrolidonyl Arylamidase (PyrA), RIBose (RIB), Sucrose (SAC), UREase (URE), and XYLOSE (XYL).

Antimicrobial Susceptibility Testing (AST)

AST was performed by the Kirby Bauer Method [8] following the Clinical and Laboratory Standards Institute (CLSI) guidelines (https://clsi.org/) and as described previously [9]. The AST was performed against the following eighteen antimicrobials: Amikacin, Cefaperazone, Cefoperazone, Cefotaxime, Ceftazidime, Ceftriaxone, Cefuroxime, Chloramphenicol, Ciprofloxacin, Erythromycin, Gentamicin, Imipenem, Linezolid, Meropenem, Ofloxacin, Sulphamethoxazole, Tetracycline, and Vancomycin.

Temperature Tolerance Test (TTT)

A FGM-USM colony was inoculated on Vegitone Nutrient Agar and incubated for 3 days at 25℃, 37℃ and 45℃ respectively under aerobic conditions.

5% NaCl tolerance test

A FGM-USM colony was inoculated on Vegitone Nutrient Agar with 5% of sodium chloride (NaCl) and incubated for 3 days at 37℃ under aerobic conditions. Vegitone Nutrient Agar was used as control.

Photo-reactivity test

A FGM-USM colony was inoculated on Vegitone Nutrient Agar and incubated for 3 days at 37℃ under aerobic conditions with light/dark conditions.

III. Immunological study

Western blot was performed to analyze the human antigenicity by using pooled sera from microbiologically confirmed pulmonary TB patients (PTB), and healthy individuals with tuberculin skin test positive (TSTp) or negative (TSTn). This study was approved by the USMHuman Ethics Committee and a signed informed consent was obtained from each individual. SDS-PAGE was conducted as described by Laemmli with some modifications [10]. One ml of autoclaved bacterial culture (Vegitone Luria Bertani Broth/37℃/200 rpm/3 days) was centrifuged (14500 rpm/5 min) and the pellet was resuspended into 100 μl of lysozyme solution (10 mM Tris- HCL, pH 8; 10 mg/ml), vortexed and incubated (37℃/1 h). Ten ul of the sample was mixed with 10 μl of 2X buffer sample (1:1) and boiled for 10 min. The boiled sample was then centrifuged (10,000 rpm/1 min) and the supernatant was loaded into the wells of stacking gel. The electrophoresis was run at a constant current of 25 mA per gel for 50 min. The stacking gel was detached and removed from the resolving gel after the electrophoresis was completed. After SDS-PAGE, the polyacrylamide gel, nitrocellulose (NC) membrane and fiber pads were equilibrated in Towbin transfer buffer with 20% methanol for 15 min (~100 ml per gel) [11]. Subsequently, the gel, NC membrane and fiber pads were assembled according to the manufacturer’s protocol and transferred via semidry blot (Biorad, USA) for 90 min at 13 V. After the transfer, the NC membrane was rinsed with dH2O, and the profile of transferred proteins was checked using Ponceau-S stain. The NC membrane with the separated proteins was rinsed with copious dH2O to destain the Ponceau-S stain and was blocked with blocking reagent (Nacalai Tesque, Japan) for 1 h. After the blocking step, the NC membrane was rinsed 3 times with PBS and was cut into several strips according to the number of sera that were tested, later the strips were probed with human serum (5 μl in 500 μl PBS). The unbound primary antibodies were removed by washing (3 × 10 min) with 0.05% PBS-T. Subsequently, the NC membrane strips were incubated with the secondary antibody, monoclonal mouse anti-human IgG conjugated with HRP 1:300 (Sigma, USA). The unbounded secondary antibodies were removed by washing (3 × 10 min) with 0.05% PBS-T. Finally, the NC membrane strips were washed once with PBS and incubated in the same buffer prior development with 4-chloro-1-naphthol substrate solution for 15 min in the dark. The reaction was stopped by washing the NC membrane strips with dH2O. All incubations were carried out under constant shaking.

Results

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I. Genomic Studies

WGS analysis

Regarding the WGS analysis of the FGM-USM, the HGAP3 assembly followed by consensus polishing using Quiver, resulted in two contigs with high accuracy (>99.98% consensus) (Supplementary Table S4). The chromosome is 6,419,378 bp and the plasmid 220,108 bp, both of which are circular (Supplementary Table S4). The total length/size of the genome was 6,639,486 bp with a GC content of 66.06% (Supplementary Table S4).

Gene prediction and annotation

A total of 7,168 open reading frames (ORF) were predicted with the identification of 53 tRNAs and 6 rRNAs genes. Of these, 6,869 coding sequences (CDS) are found on chromosome and the remaining on plasmid. Most of the CDS on plasmid are annotated as hypothetical proteins of unknown function.

Functional categories based on Clusters of Orthologous Groups of proteins (COG)

The prediction of functional categories based on COG is shown in Supplementary Table S6. The most abundant known functions in this strain are transcriptions (9.15%), energy production and conversion (6.89%), and lipid transport and metabolism (6.61%).

Methylation analysis

The methylation analysis identified 7 types of motifs with the presence of 13,588 motifs in the genome, 6 belonged to m6A and one motif is m4c type (Supplementary Table S7).

Comparative analysis of WGS

General comparison (19 Mycobacterium Genomes)

The phylogenetic relationship of FGM-USM within the Mycobacterium genus was analyzed based on core SNPs of 19 mycobacterial species. FGM-USM clustered within the Mfor complex group and is sister to Mfor 852014-51789_SCH564012 (Fig. 1). Genomic metrics such as ANI and Tetra are frequently used for classification and identification of prokaryotic species, overcoming limitations of traditional techniques used for the same species. Then, to complement the kSNP study, ANI and TETRA analysis were carried out using the same mycobacterial genomes (Supplementary Table S8). The results of ANI and TETRA correspond to the kSNP results. The 6 Mfor genomes included in the general comparison had higher values than 98.36 ANI and 0.99942 TETRA compared to FGM-USM, which also support to consider the FGM-USM as a member of the Mfor species (Supplementary Table S8). To further explore the possibility that FGM-USM represent a new strain of the Mfor species, TETRA (Supplementary Table S9A) and ANI (Supplementary Table S9B) studies were carried out comparing the 6 Mfor included in the study and FGM-USM against each other. From these pairwise comparison data, can be seen that different Mfor strains (known strains) can have scores as high as 0.99991 (TETRA, Supplementary Table S9A) and 98.84 (ANIm, Supplementary Table S9B). The scores between FGM-USM with all other Mfor strains are below this, thus this result suggest the possibility that FGM-USM is a different Mfor strain (Supplementary Table S9, Fig. 1). However, not all known Mfor strains were included in the analysis, so with these results was not possible to confidently propose that FGM-USM is a new Mfor strain. To clarify this aspect, we compare FGM-USM with the 24 Mfor genomes available.

Figure 1.Cladogram SNP-based phylogenomics (kSNP) analysis of the Fast-Growing Mycobacteria (FGM-USM) using 19 Mycobacteria genomes. Cladogram of the phylogeny estimated from SNP positions found in 90% of the samples with branch support.

Specific comparison (24 Mfor genomes)

The pairwise ANI and TETRA values were plotted in a heat map using the corrplot R package, version 0.84 (Supplementary Fig. S1 and Fig. S2). All 24 Mfor sequences and M. abscessus as an inter-species reference have been included. Although the Tetra values range for intra-species comparisons is very narrow, Tetra and ANI both clustered FGM-USM with the 8520xx/SCHxxxxxxx strains while keeping it separate from the other strains (Supplementary Table S10, Fig. 2). FGM-USM exhibits ANI values of 98.97−99.15% to the 8520xx/SCHxxxxxxx strains. As FGM-USM and 8520xx/SCHxxxxxxx are different isolates from different strains, their ANI values are slightly lower that the same strain values observed between DSM 46621 strains (99.47−99.91%) (Supplementary Table S10, Fig. 3). While FGM-USM is most like the 8520xx/ SCHxxxxxxx strains, FGM-USM and 8520xx/SCHxxxxxxx are different strains of the same species.

Figure 2.ANI. Cluster dendrogram. Comparative analysis of the whole genome sequencing (WGS) of the Fast-Growing Mycobacteria (FGM-USM) with 24 Mycolicibacterium fortuitum (Mfor) genomes used for the specific comparative analysis.
Figure 3.TETRA. Cluster dendrogram. Comparative analysis of the whole genome sequencing (WGS) of the Fast-Growing Mycobacteria (FGM-USM) with 24 Mycolicibacterium fortuitum (Mfor) genomes used for the specific comparative analysis.

Study of homology

i) FGM-USM against 24 Mfor strains. The results of the study of homology of FGM-USM compared to 24 Mfor strains are shown in Supplementary Fig. S3, Supplementary Table S10. In average, 89% of CDS in FGMUSM strain are homologous to CDS inside 24 Mfor strains. Vice versa, 87% of CDS in Mfor strains are homologous to CDS in FGM-USM strain. High correlation of homologous CDS between them indicate that FGM-USM is a close relative to Mfor species (Supplementary Fig. S3). The phylogenetic study (Fig. 1) shows the estimated speciation of FGM-USM and Mfor species. The 25th organism i.e. Mycobacteroides abscessus, is a purposely selected outlier to demonstrate the difference in homologous percentages between close relatives and a distant species. The results obtained demonstrate differences in the protein coding sequences of FGM-USM compared with Mfor strains, which additionally support the notion that FGMUSM is a differentiated strain belonging to Mfor species.

ii) FGM-USM against Mtb. After the bioinformatics analysis, 497 proteins were identified with more than 80% identity with Mtb proteins, which demonstrated the presence of shared conserved antigens and epitopes between FGM-USM and Mtb. Regarding the cell location of the identified proteins, 7% belong to extracellular proteins, 15% to membrane proteins, and 78% are predicted to be located intracellularly (Supplementary Fig. S4).

II. Phenotypic studies

Biochemical study

i) GEN III MicroPlate test. The GEN III MicroPlate test panel provides a standardized micro method using 94 biochemical tests to profile and identify a broad range of Gram-negative and Gram-positive bacteria. Five databases are available for a broad spectrum of aerobic and anaerobic bacteria, yeasts, and filamentous fungi, including 23 Mycobacteria phenotypic patterns. All SIM (similarity index value) obtained with the FGM-USM were less than 0.5, which means that Biolog’s Identification Systems software could not identify the strain. Supplementary Table S11 shows the results of the study of FGM-USM compared to 23 Mycobacterium reference phenotypic patterns. Positive results were obtained for: D-Fructose-6-PO4, D-Galacturonic Acid, L Galactonic Acid Lactone, D-Glucuronic Acid, and Glucuronamide.

ii) API Panel tests. There were positive results with catalase and nitrate reduction. The negative results were obtained with PYZ, PyrA, PAL, βGUR, βGAL, ∝GLU, BNAG, ESC, URE, [GEL], O, GLU, RIB, XYL, MAN, MAL, LAC, SAC, GLYG, N03, TRP, GLU, ADH, URE, ESC, GEL, PNPG, [GLU], [ARA], [MNE], [MAN], [NAG], [MAL], [GNT], [CAP], [ADI], [MLT], [CIT], [PAC], OX.

Antimicrobial susceptibility testing (AST)

The FGM-USM was sensitive to: Amikacin (AN 30), Meropenem (MEM 10), Imipenem (IPM 10), Ciprofloxacin (CIP 5), Ofloxacin, Vancomycin (VA 30) and Linezolid (LZD 30) and resistant to: Gentamicin, Chloramphenicol, Cefoperazone, Cefotaxime, Ceftazidime (CAZ 30), Cefuroxime (CXM 30), Ceftriaxone (CRO 30), Cefaperazone (CFP 25), Erythromycin, Sulphamethoxazole (SXT 25) and Tetracycline.

Temperature tolerance test (TTT)

The FGM-USM grew at 25℃, 37℃ and did not grow at 45℃.

Tolerance to 5% NaCl test

The results of the Tolerance to 5% NaCl Test shows that the strain did not grow with 5% of NaCl.

Photo-reactivity test (PT)

The strain did not produce pigment in both light and dark conditions.

III. Immunological study

Western blot analysis showed the recognition of bands in the range of the 55 kDa−40 kDa, 35 kDa−25 kDa and 15 kDa−10 kDa (Fig. 4) in all the groups studied [pulmonary TB patients and healthy individuals (PPD positive and negative)], which could represent the immune response to FGM environmental strains. However, TB patients recognized bands in the range of 25 kDa−15 kDa which were not recognized by other groups.

Figure 4.Characterization of human antigenicity study by Western blot. NC membrane was cut into several strips according to the number of sera that were tested: 1: ladder, 2: Lysozyme probed with TB sera, 3: Sample probed with TB sera, 4: Lysozyme probed with PPD positive, 5: Sample probed with PPD positive, 6: Lysozyme probed with PPD negative, 6: Sample probed with PPD negative.

Discussion

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The importance of mycobacteriome in health and disease have been recognized, but its study have been hampered, among other factors by the difficulties associated to the biology of mycobacteria and their intrinsic characteristics, which make difficult the extraction of mycobacterial genomic DNA from different human samples using the current methods and commercial kits involved in the study of the human microbiome [4, 5]. The human mycobacteriome is highly diverse, including non-tuberculous mycobacteria (NTM), BCG vaccine strains, which persist for years after vaccination, and pathogenic mycobacteria, such as Mtb as in the case of latent tuberculosis infection (LTBI) [5]. This heterogeneous population, which vary regarding multiple factors such as age, body location, genetic background, geographical region, and environmental characteristics, among other factors, has potential important relevance in health and disease, which deserves a detailed study and characterization.

In the context of the study and characterization of the human mycobacteriome, the aim of the present study was to characterize, using multiple methodologies, a FGM isolated from a healthy individual.

Genome comparison is a process that compare genomic data to identify unique or shared features. Bacterial and viral genomes are characterized by multiple and massive insertion-deletions (indels), inversions, and transpositions of blocks of DNA from one part to another part of the genome. The term “pan-genome” has been coined to describe the collective content of the genomes of a bacterial species. The pan-genome consists of core genes, those genes that are present in all members of a species, and accessory genes, those that are present in some, but not all members of the species. Indeed, there is at least as much variation in the presence/absence of DNA sequences as there is variation in SNPs.

The genome size of this strain is like other RGM [12]. The GC content is typical of mycobacteria [12]. The sequencing study of M. fortuitum subsp. fortuitum Type strain DSM46621 reported similar results regarding to genome size, GC content and number of ORF [13]. Nowadays, WGS is becoming available in diagnostic microbiology and research laboratories for identification and characterization of the isolated organisms. To facilitate a more trustworthy genetic identification for FGM-USM we have characterized the WGS and the genome-wide DNA methylation profile for this strain. Regarding vaccine development, this tool is very important for the characterization of the vaccine strains as in the case of BCG vaccine [14, 15].

The genome size of Mycobacterium species varies from 3.2 million base pair (MB) to 7 MB with an average GC content of 64 to 69% [12]. A comparative genome size study has been conducted on 44 mycobacterial genomes and discovered that the non-pathogenic has a bigger genome size compared to those of opportunistic or totally pathogenic mycobacteria that can be ranged from 6,988,209 bp to 3,268,071 bp [16].

G+C (GC) content refers to the guanine and cytosine contained in a biological sequence [17]. Mycobacteria belong to the phylum of Actinobacteria which are characterized by their large size of genome and high GC content. Due to their varied lifestyles and environmental niches, the genome sizes are different [18]. It has been suggested that variations in GC content among prokaryotic genomes maybe largely due to the GC content of protein coding sequences, which commonly occupy most of the genome [19].

DNA methylation is a process where methyl groups are added to DNA bases catalyzed by DNA methyltransferases (MTases). Three functional classes of MTases have been acknowledged in bacteria and archaea. Two of MTases transfer a methyl group from S-adenosyl-Lmethionine (SAM) to the exocyclic amino groups of adenine (A) and cytosine (B) bases in duplex DNA to produce N6-methyladenine (m6A) and N4-methylcytosine (m4C). The third MTases transfers the methyl group of SAM to C5 of cytosine to yield 5-methylcytosine (m5C) [20].

In this study, methylation analysis was carried out by RS Modification and Motif Analysis in Single-molecule real-time (SMRT) Portal which is one of the technologies to study the methylation analysis. One study has applied similar technology for methylome analysis for Mtb complex strains suggesting that this technology can accomplish unbiased GC coverage with amazingly long reads (up to 20 kb), thus it is appropriate to be used in the genomes with high GC content and large numbers of repetitive regions [21].

The positive results obtained with catalase and nitrate and the negative results with beta galactosidase, mannitol and citrate are typical of Mfor, whereas the negative results obtained with urease and pirazynamidase are atypical for Mfor [22−24]. It is important to note that, in general, despite the shared traits in Mfor species, some variations can be found in different strains and subspecies [25−27].

For AST, other studies with Mfor reported similar results [9, 28−30]. The result of temperature tolerance is compatible with the behavior of Mfor strains [22, 25]. The results of tolerance to 5% NaCl differs of the general growing pattern of Mfor strains, which present tolerance to 5% NaCl [22−24], but other studies reported similar results to our study with some Mfor subspecies [25]. The results of the photo-reactivity test is compatible with Mfor strains and subspecies [22, 24, 25].

Tuberculosis (TB), caused by M. tuberculosis (Mtb), represent a global health challenge [31]. Approximately, 10 million new TB cases and 1.5 million deaths are reported annually, which is complicated by the rising appearance of multidrug-resistant strains (MDR), the presence of latent TB infection (LTBI) in 25% of the human population and the non-availability of a fully effective vaccine [31−33].

BCG, the vaccine in use, is effective in the prevention of the severe forms of TB in children but has suboptimal efficacy in the prevention of adult pulmonary TB and the transmission [34]. There is a high genetic and antigenic similarity between mycobacteria [26], which supports the evaluation of non-tuberculous mycobacteria (NTM) for the development of prophylactic and immunotherapeutic tools for TB control [26].

Formulations derived from NTM demonstrated protection against TB in animal models and the induction of reactivity against Mtb [35-38]. Experimental and clinical studies with live and inactivated NTM strains demonstrated to be safe with prophylactic and immunotherapeutic potential [39−50].

Considering these antecedents we decide, in addition to the genomic, biochemical, and phenotypic characterization, to also implement a preliminary evaluation of the isolated strain to have evidence about its potential to be evaluated in future studies aimed to explore immunotherapeutic and protective effect against Mtb infection.

The results of the study of homology with Mtb agrees with the reported high degree of antigenic homology among mycobacteria species [51]. The considerable number of antigens and epitopes with homology with Mtb in FGM-USM highlights its potential as vaccine candidate against TB. The presence of shared antigens with Mtb have the potential to elicit potent immune responses directed to Mtb upon immunization with this strain. It has been demonstrated the possibilities to induce cross reactive responses and protection against TB in animals immunized with experimental vaccines derived from other RGM [36−38, 52].

Based on the location of the identified homologous proteins, we can predict that different vaccine candidates derived from FGM-USM, containing proteins from different cellular compartments, will elicit immune responses against Mtb as there is a good representation of proteins shared with Mtb in different cellular compartments. In previous studies, the bioinformatics prediction of epitopes from Mtb in M. smegmatis proteoliposomes [53] was correlated with protection in a challenge study with Mtb in mice [37].

The recognition of differentiated bands in the Western blot study by PTB sera compared to healthy individuals could represent the expression of proteins/epitopes in vivo by Mtb which are shared with FGM-USM. This is an important finding that confirm the potential usefulness of the strain as vaccine candidate against TB as the proteins expressed in vivo by Mtb during infection represent important elements for the survival of bacteria and at the same time are the only targets available for the immune system during infection. The presence of these shared antigen epitopes in FGM-USM could elicit protective immune responses after immunization.

Conclusion

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Based on the genotypic and phenotypic results, we can conclude that FGM-USM could be considered as a new strain, belonging to Mfor species. Gupta et al., after a comprehensive phylogenomic and comparative genomic analyses propose a new division of the known mycobacterial species into an emended genus Mycobacterium including the major human pathogen and four novel genera (Mycolicibacterium gen. nov., corresponding to Foruitum- Vaccae clade; Mycolicibacter gen. nov., corresponding to Terrae clade; Mycolicibacillus gen. nov., corresponding to Triviale clade and Mycobacteroides gen. nov., corresponding to Abscessus-Chelonae clade [26].

Based on the division of mycobacterial species proposed by Gupta et al, we proposed that FGM-USM could be named as: M. fortuitum strain kelantanesensis, belonging to the Mycolicibacterium fortuitum group. Based on the presence of shared homologous proteins in M. fortuitum strain kelantanesensis with Mtb, and the results of the immunological study, it could be speculated that protective immune responses could be elicited after immunization with this strain, which need to be explored in the future.

Ethics Approval and Consent to Participate

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This study was approved by the Universiti Sains Malaysia (USM) Human Research Ethics Committee (USMKK/PPP/JEPEM (245.3[16])) and an informed consent was obtained and signed by the child’s father before the sample was obtained. The study was conducted in accordance with Helsinki declaration.

Author Contributions

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Conceptualization, M.E.S., A.A., and M.N.N.; methodology and investigation, A.F.Z., M.E.S., N.S.L., K.L.C., N.H.M., Z.M., F.S.M.Z., N.Z.N.M.N., E.K.M.H., S.S., A.A., and M.N.N.; formal analysis, A.F.Z., M.E.S., N.S.L., K.L.C., F.S.M.Z., E.K.M.H., P.S., S.S., A.A., and M.N.N.; funding acquisition, M.N.N. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

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All data generated or analyzed during this study are included in this published article [and its supplementary information files]. The complete genome sequence of FGM-USM was deposited in GenBank database under the accession number of CP089608-CP089609.

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