- Extended genome report
- Open Access
Genomic insights into Mycobacterium simiae human colonization
Standards in Genomic Sciences volume 13, Article number: 1 (2018)
Mycobacterium simiae (Karassova V, Weissfeiler J, Kraszanay E, Acta Microbiol Acad Sci Hung 12:275-82, 1965) is a slow-growing nontuberculous Mycobacterium species found in environmental niches, and recently evidenced as an opportunistic Human pathogen. We report here the genome of a clinical isolate of M. simiae (MsiGto) obtained from a patient in Guanajuato, Mexico. With a size of 6,684,413 bp, the genomic sequence of strain MsiGto is the largest of the three M. simiae genomes reported to date. Gene prediction revealed 6409 CDSs in total, including 6354 protein-coding genes and 52 RNA genes. Comparative genomic analysis identified shared features between strain MsiGto and the other two reported M. simiae genomes, as well as unique genes. Our data reveals that M. simiae MsiGto harbors virulence-related genes, such as arcD, ESAT-6, and those belonging to the antigen 85 complex and mce clusters, which may explain its successful transition to the human host. We expect the genome information of strain MsiGto will provide a better understanding of infective mechanisms and virulence of this emergent pathogen.
Contrasting with the declining incidence of Mycobacterium tuberculosis -caused tuberculosis, the increasing number of nontuberculous mycobacteria infections is concerning. Amongst NTM, the Mycobacterium simiae complex contains 19 species , including M. simiae , which is considered the most important species in terms of its clinical relevance . M. simiae is a slow-growing saprophyte that has been isolated from several environments including water and soil [3, 4]. In addition to thriving in environmental niches, M. simiae has been associated to infections in both immunocompromised [4, 5] and immunocompetent patients , with clinical cases reported worldwide [5, 6]. Thus, M. simiae is considered an emergent pathogen [7,8,9]. Previous sequencing efforts have provided draft genome sequences for M. simiae , namely, strains MO323 (accession PRJNA276839) and DSM 44165 (accession PRJEB1560) , which were isolated in early years from the United States of America (1989) and India (1965), respectively. We expand here on this genomic data by reporting the genome sequence of a clinical isolate from a Mexican patient (bronchial lavage) obtained in 2011, designated as MsiGto, which is approximately 745 Kbp and 901 Kbp larger than the previously published M. simiae genomes, respectively. Additionally, we perform a comparative analysis between the genomes of strain MsiGto and the previously reported DSM 44165 and MO323 strains to provide insights that could unearth the transition from environmental bacteria to pathogenic NTM organisms.
Classification and features
To identify strain MsiGto as an M. simiae isolate, typical DNA markers for mycobacterial identification, such as the 16S rRNA, rpoB and hsp65 genes [11,12,13] were localized on the sequenced MsiGto genome, and compared to other publicly available sequences using BLASTN on its default settings. The MsiGto 16S rRNA gene exhibited 100% identity with previously deposited M. simiae sequences (strains DS39, DS34, DS31, DS24, DS19, DS3, DS2, MO323 and ATCC 25275). The MsiGto rpoB gene sequence showed 99% identity with M. simiae MO323. Similarly, there was 100% identity between the MsiGto and MO323 hsp65 genes. For comparison, a BLAST of these markers from DSM 44165 and MO323 evidenced a similar trend with 99% identity for the 16S rRNA gene and 100% identity for rpoB and hsp65. Figure 1 shows the phylogenetic position of M. simiae MsiGto within the M. simiae complex, based on a concatenated gene tree including the sequences of the 16S rRNA, rpoB and hsp65 genes (1507 bp in total).
Further analysis such as the Average Nucleotide Identity and Average Amino Acid Identity, preformed as previously reported , confirmed the high resemblance between M. simiae MsiGto and M simiae strains MO323 and DSN 44165 (Table 1). The ANI values were above 95% in all cases, indicating that the compared organisms belong to the same species. Similar results were found using Genome-to-Genome Distance Calculator [15, 16] with a calculated distance of 0.0292 and a DNA-DNA hybridization estimate of 77.40% using the formula for draft genomes. These results provide a taxonomic background in which the genome insights presented in the following sections can better accounted for, namely, large genotype differences of MsiGto, despite close taxonomic relationships with DSM 44165 and MO323 strains.
Genome sequencing information
Genome project history
Despite M. simiae being one of the most relevant NTM due to its emergence as a human pathogen, the genomic features and genetic potential of this species remain poorly described. In collaboration with the State Laboratory of Public Health of Guanajuat, located in central Mexico, we had access to a clinical isolate of M. simiae , termed MsiGto. The sample was isolated from a 90 years old female patient from León, the largest and most industrialized city of the State of Guanajuato, México, in 2011 (Table 2). This sample was selected for genomic sequencing due to the emerging clinical importance of strains from the M. simiae complex worldwide [8, 9, 17,18,19], combined with a lack of representative genomic sequences from Mexico to this date. The available genome sequences could allow comparative analyses in order to increase our understanding of this opportunistic bacterium.
Growth conditions and genomic DNA preparation
MsiGto was isolated from a sputum specimen. Briefly, 2 mL sample were transferred to a sterile tube and decontaminated by adding an equal volume of 4% NaOH with phenol red. The mix was immediately vortexed and incubated at 37 °C for 15 min. The liquefied sample was centrifuged at 3000 g at 4 °C for 15 min, the supernatant was discarded and pH was adjusted between the 6.5 and 7.2 range using 1 N HCL. Finally, 0.2 mL of sample were inoculated into Lowenstein-Jenssen slants (Difco) and incubated at 37 °C for three weeks.
Biomass was collected and suspended in phosphate buffered saline solution, pH 7.4, and inactivated by heating at 80 °C for 45 min. After centrifugation, genomic DNA was extracted from the pellet using the FastDNA SPIN Kit for Soil (MP Biomedicals) according to manufacturer’s instructions. Extracted gDNA was assessed for quantity and quality using a NanoDrop Spectrophotometer (Thermo Fisher Scientific).
Genome sequencing and assembly
The MsiGto genome was sequenced on a HiSeq 2000 platform with a 100 bp paired-end cycle according to standard Illumina protocols, at Macrogen facilities. Quality of the generated sequencing reads (13,676,836 reads, total read length: 1,381,360,436 bp) was checked with FastQC. Reads were filtered before assembly, such that both sequences in paired-end reads exhibited more than 90% bases of quality greater than or equal to Q20. Post-filtering Q20% was 97.69 and Q30% was 88.6 at the base level. The filtered reads were assembled using SOAP de novo aligner , yielding 50 contigs. Scaffolding was performed with SSPACE Standard  and resulted in 12 scaffolds, generating a genome size of 6.7 Mb with a coverage of 216X (Table 3).
Gene prediction and functional annotation were performed using the Rapid Annotation using Subsystem Technology platform . A prophage region prediction was completed using PHAST . CRISPRs were searched using the CRISPR finder, and antibiotic resistance genes were investigated using Resistance Gene Identifier from the Comprehensive Antibiotic Resistance Database . Genome-to-genome comparisons were performed using multiple approaches, including conservation analysis of protein families across genomes with the Protein Family Sorter from the PATRIC Platform , and comparing functionally related clusters using the function-based comparison of the RAST server. MsiGto and related genomes were also aligned using a genome-wide BLAST comparison, and visualized through the Artemis Comparative Tool  for manual inspection.
Genomic assembly yielded a total length of 6,684,413 bp fragmented in 50 contigs, the largest M. simiae genome reported to date. The MsiGto genome consists of a unique chromosome, as no plasmid DNA was found. The GC content of the genome was 66.08%, consistent with other reported M. simiae strains. Gene prediction analysis documented 3 rRNAs, 49 tRNAs and 6409 coding sequences. Out of these, 4462 genes (69.62%) were assigned to putative functions, and 3669 genes (approximately 62.57%) were assigned to clusters of orthologous groups functional categories. Sequence searches using RGI evidenced resistance genes to amynoglycosides, ethambutol and beta lactams, which is a concern as to date standard antibiotic regimes for this species include the use of ethambutol, the aminoglycoside amikacin, as well as the macrolides azithromycin and clarithromycin . The genome properties and statistics are summarized in Tables 4 and 5. Gene distribution among the COG functional categories is shown in Table 6. A circular map of MsiGto chromosome is provided in Fig. 2.
Insights from the genome sequence
We report here the third genome sequence of M. simiae . MsiGto was isolated from a clinical sample obtained from an elderly woman, and is the largest M. simiae genome reported to date with an approximate 745 Kbp additional to the next largest genome (strain MO323). As pathogenic mycobacteria tend to undergo genome decay [28, 29], the large genomic size of MsiGto speaks out of an organism capable of thriving in both environmental conditions and in the human host. Indeed, by comparison, the genome of M. tuberculosis , an obligate pathogen, is significantly smaller with 4.41 Mb . Meanwhile, with 6406 genes, the genome size of MsiGto is similar to that of the opportunistic pathogen Pseudomona aeruginosa (5570 predicted ORFs), which thrives in a variety of environments including soil, water, as well as the multiple human tissues it infects . Analysis of the P. aeruginosa genome evidenced its large size arose from genetic expansion to enhance functional diversity rather than from gene duplication . Interestingly, investigating protein families in MsiGto, in comparison with MO323 and DSM 44165, evidenced a larger proportion of proteins uniquely found in MsiGto (1.17 times more than DSM 44165 and 4.7 times more than MO323), suggestive of a relatively increased versatility in accordance with the multiple niches it can thrive in (Fig. 3).
The genome of MsiGto is rich in virulence factors, genes conferring infective mechanisms, and host immune response evasion systems. Some of these proteins are common to other pathogenic mycobacteria, and shared by the two previously sequenced M. simiae genomes, such as ESAT-6, known to modulate host immune responses by affecting human T-cell responses [33, 34]. Interestingly, all M. simiae strains including MsiGto have three of the four antigen 85 complex genes (fbpA, fbpC, and fbpD) responsible for cell wall synthesis. The enzyme products of these genes are responsible for the conversion of trehalose monomycolate (TMM) into the Cord Factor trehalose dimycolate (TDM) , which is considered one of the most important virulence factors of mycobacteria . Other genera belonging to the Mycobacteriaceae family that include both pathogenic and environmental species, such as Rhodococcus , Corynebacterium and Nocardia , also produce TDM .
Mammalian Cell Entry proteins are cell surface exposed proteins that play a crucial role in M. tuberculosis virulence by permitting the bacteria to enter mammalian cells and survive inside the macrophage, modulating the immune response [40, 41]. Mce clusters consist of 4 homologous operons in M. tuberculosis (mce1, mce2, mce3, mce4) with a similar arrangement: two genes encoding integral membrane proteins followed by six mce genes (A, B, C D, E and F) . Mce proteins are also involved in lipid metabolism, acting as transporters and allowing cholesterol degradation to free carbon and energy for use by M. tuberculosis . In the genome of MsiGto we found that the cluster mce3 is overrepresented. While the DSM 44165 and MO323 genomes present none and a single copy of mce3, respectively, two complete copies of the mce3 cluster were found in MsiGto. As the transition from an environmental organism to a pathogen has been associated with the acquisition of Mce genes in actinobacteria , and Mce3 proteins are expressed by M. tuberculosis during the infection phase , it is tempting to speculate the increased number of Mce3 copies found in MsiGto provided the strain with human infection potential.
The presence of multiple copies of these potential lipid transporters in mycobacterial genomes is consistent with the finding that pathogenic mycobacteria switch from carbohydrates to lipids as their main carbon and energy source inside cells . The evolution of this locus, through duplication and divergence, has almost certainly contributed to virulence in mycobacteria, and even in other distantly related actinobacteria, such as Streptomyces [42, 45]. Given that the ability to acquire cholesterol from the host is crucial to maintain a chronic infection, we postulate that cells having a large mce copy number, such as that found in MsiGto, may potentially evolve pathogenicity relatively faster when compared with other environmental mycobacteria.
A total of 493 protein families were found exclusively present in MsiGto when compared to the other two available M. simiae genomes (MO323 and DSM 44165). Consistent with findings in the Pseudomonas aeruginosa  genome , it seems that MsiGto has undergone genome expansion. Within the gene pool unique to MsiGto we found the arginine/ornithine antiporter gene arcD, which is involved in the persistence of the zoonotic pathogen Streptococcus suis  in host cells . Additional unique genes found in the MsiGto genome participate in aromatic amino acid metabolic pathways. For instance, 2-oxo-hepta-3-ene-1,7-dioic acid hydratase (hpcG gene, EC 184.108.40.206) participates in the degradation of tyrosine and the 2-keto-3-deoxy-D-arabino-heptulosonate-7-phosphate synthase I alpha participates in the synthesis of chorismate (aroG gene, EC 220.127.116.11). The latter is an interesting observation as tyrosine is a key nutrient source during infectious growth within macrophages of some pathogenic fungus [49, 50]. At this stage, however, it would be risky to rule out the involvement of these specific genes in important environmental functions that allow MsiGto to survive outside the host, or during both lifestyles.
Mycobacterium simiae is an organism of interest for genomic studies due to the scarce genomic data available, and its recent emergence as a human pathogen. Here we present the largest genome sequence of this species to date. The genome of M. simiae MsiGto presents characteristics in accordance with its adaptation to infect the Human host, with the presence of numerous virulence genes, plus some specific features that deserve further investigation. Additional M. simiae genomes, from both environmental and clinical isolates, should be sequenced to provide a wider evolutionary picture with functional implications. Indeed, our comparative analysis helps to better understand the evolution of host-pathogen interactions, and the molecular mechanisms of virulence, of this emergent human pathogen.
Artemis Comparative Tool
Comprehensive Antibiotic Resistance Database
State Laboratory of Public Health of Guanajuato
Mammalian Cell Entry
Rapid Annotation using Subsystem Technology
Resistance Gene Identifier
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We thank the Secretaría de Salud del Estado de Guanajuato and María Guadalupe Hurtado Torres from the LaESaP for their invaluable contribution to obtain the biological material, and Karina Gutiérrez-García for help with bioinformatic analysis.
The authors thank Fomix-Gto (grant number GTO-2011-C04–165962) for funding of this work, and Conacyt for a postdoctoral scholarship to EA (CVU No. 248685).
The authors declare that they have no competing interests.
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