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BACKGROUND
Mycobacterium ulcerans (MU) is the causative agent of Buruli ulcer (BU) in immunocompetent hosts, which ranks third among human mycobacteriosis worldwide after tuberculosis and leprosy.1 This has been reported from 33 countries with tropical, subtropical, as well as temperate climates.2 The mode of transmission of this extracellular pathogen is still not clear. However, living agents such as aquatic insects, adult mosquitoes, or other biting arthropods may facilitate transmission.3 Until 2004, surgical debridement with or without subsequent skin grafting was the only therapeutic option, though recurrences were frequently observed.4 The balance between surgery and antibiotics use was shifted when mouse foot-pad model was used for antibiotic susceptibility testing (single dose rifampin and sometimes fluoroquinolones were administered in addition to surgical intervention).5–7 Although monotherapy (strains were isolated from mice after experimental chemotherapy) was effective for certain period but consistent use of antibiotics develop resistance in the microbes.8 To prevent the emergence of resistant strains, combination of antibiotics was used for the treatment. In case of BU, World Health Organization recommended the oral administration of rifampin (10 mg/kg) and intramuscular streptomycin (15 mg/kg) for 8 weeks to reduce the bacterial load.9,10 Recently, the use of rifampin and streptomycin for 2 weeks followed by rifampin and clarithromycin for the next 6 weeks was effective in the treatment of BU.11 The slow growth rate and cultural difficulties of MU12 make this bacterium less favorable for antibiotic susceptibility testing assay. MU ecovar liflandii strain M04–2878, a MU-like Mycobacterium isolated from pipid frog Silurana tropicalis, in a European research laboratory, was reported to be resistant to isoniazid, ethambutol, rifampin, clarithromycin, and ethionamide.13 Recently, a new recombinant bioluminescent assay was successfully applied to evaluate the antibacterial activity in vitro against MU,14 which showed that MU was susceptible to rifampin, streptomycin, and clarithromycin and was resistant to tetracycline, isoniazid, and erythromycin.14 Moreover, it was demonstrated that reserpine, an inhibitor of adenosine triphosphate (ATP)-binding cassette (ABC) transporter, partially restores the susceptibility of MU to tetracycline and erythromycin due to an efflux pump (a putative macrolide ABC transporter) and might be responsible for intrinsic resistance.14
One alternative to in vitro antibiotic susceptibility testing in fastidious bacteria is to predict antibiotic resistance (AR) determinants by whole genome sequence (WGS) in silico analysis as previously reported for bacteria.15 In an effort to guide further therapeutic options, we embarked into MU genome survey to find out genes and mutations potentially associated with AR. The MU Agy99 was isolated in 1999 from an ulcerative lesion on the right elbow of a female patient from the Ga District of Ghana. The genome of MU Agy99 (CP000325) is 5,631,606-base pair long and comprises one main circular chromosome with 64.5% GC content and a 174-kilobase plasmid.16 This virulence plasmid pMUM001 was transferred to MU during its divergence from Mycobacterium marinum.17 ARG-ANNOT database18 and RAST annotation19 were used to find existing and putative new and/or emerging AR genes and point mutations in target genes that could be associated with AR in the genome sequence of MU Agy99 as well as in MU ecovar liflandii and M. marinum strains genomes.
METHODS
The genome sequence of MU Agy99 was retrieved from the NCBI GenBank (CP000325). ARG-ANNOT database18 was used to predict acquired antibiotic resistance genes using BioEdit interface20 and most of additional resistance gene were picked from RAST annotation.19 NCBI BLAST was performed to validate the results obtained from the local blast. Mutational analysis was done by comparing the gene sequences with reported mutational genes.21–27 The AR genes or mutations detected in MU Agy99 were used as a reference to analyze the presence of these AR genes or mutations in closely related genomes which included three genomes of M. marinum (M; NC_010612, Europe; ANPL01000001-4 and MB2; ANPM01000001-3) and MU ecovar liflandii; NC_020133). The Sequence Read Archive data (Illumina pair end reads) for 30 MU genomes (list provided as Supplemental Figure 1 and Supplemental Tables 1 and 2) were retrieved from the NCBI (http://www.ncbi.nlm.nih.gov/sra/website). The reads were assembled using CLC workbench7 (http://www.clcbio.com/website). A local database for all the genes conferring resistance (rpoB, gyrA, gyrB, rpsL, rrs, pncA, embB, inhA, katG, oxyR, ahpC, ndh, and 23S ribosomal RNA [rRNA])21–29 was created in BioEdit.20 The assembled contigs from SRA data of MU genomes were analyzed through blast against the database and blast output was assessed for possible reported mutations.21–29
Phylogenetic trees based on MU rpoB gene sequences were inferred using MEGA.30 The rpoB sequences were extracted from the assembled contigs of SRA data and other genomes under study, multiple sequence alignments were carried out using CLUSTALW and phylogenetic inferences obtained using the neighbor-joining method of MEGA. The bootstrap values of consensus tree were inferred from 500 replicates. The evolutionary distances were computed using the Kimura2 parameter.31 All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option).
RESULTS AND DISCUSSIONS
WGS analysis of MU Agy99 revealed 14 chromosomal AR genes and mutations in pncA and katG genes (Table 1 and Supplemental Table 1). These findings were compared with the genomes of M. marinum (M, Europe and MB2), MU ecovar liflandii and 30 assembled genomes from SRA data (ERR449672) and analysis is presented in Table 1 and Supplemental Tables 1 and 2. Mutations were detected only in katG and pncA genes in 29/30 assembled MU genomes (Supplemental Table 2). Phylogenetic position of all the analyzed genomes is presented in Figure 1.30 Interestingly all AR genes detected in MU AGY99 were also present in the four other genomes under analysis with more than 98% sequence similarity (Table 1). MU encodes an Amber class B metallo-β-lactamase (MBL), an Amber class C β-lactamase and a class A β-lactamase that may likely contribute resistance to β-lactams and resistance to β-lactams in Mycobacterium tuberculosis (BlaC), Mycobacterium smegmatis (BlaS), and Rickettsia felis have been described previously.32,33 Further, we found two aminoglycoside modifying enzymes (AMEs) encoding genes (aac[2′]-Ic and aph) that are known to confer resistance to gentamicin, sisomicin, and fortimicin.34 However, no genes (ant[3′′]-Ia, aadA, and aad[3′′]-9) or mutations in rRNA genes (rpsL, rrs, and rpsD) associated with resistance to streptomycin were found in any of the genome analyzed here (Table 1); and gidB gene (streptomycin resistance) was not detected either. In silico analysis of the 23S rRNA and ribosomal proteins did not show mutations associated with resistance to macrolide compounds in other bacteria.15 However, MU genome encodes a macrolide ABC transporter ATP-binding protein (Table 1 and Supplemental Table 1) exhibiting > 75% nucleotide sequence similarity with the homologous gene in M. tuberculosis H37Rv which may confer resistance to erythromycin/tetracycline.14,35 In M. marinum complex, this protein is highly conserved (100%) and grouped in single clade in most of the assembled genomes except Mu_SRX031535 genome (Supplemental Figure 1). No other gene was found to confer intrinsic resistance to macrolides.33,36 The MU genome contains two copies of dihydropteroate synthase and dihydrofolate reductase that may account for possible resistance to sulfonamide and trimethoprim. This needs to be explored further to clarify whether this bacterium is resistant to such compounds.37 Interestingly, an Arg431Leu mutation in katG, known to confer isoniazid resistance corresponding to position 463 in M. tuberculosis was found in all studied genomes.25,26 Though, Ser315Gly in katG is the most frequent mutation reported in M. tuberculosis,23 we also detected serine in katG gene at position 284 in the alignment for MU, which corresponds to position 315 of M. tuberculosis. The analyzed pncA gene showed amino acid change at position T47A (except all three M. marinum, Mycobacterium liflandii, and Mu_SRX031535) and V125I in all analyzed genomes, which confer resistance to pyrazinamide in M. tuberculosis. Moreover, we analyzed all 18 reported mutations in pncA gene in M. tuberculosis and noticed amino acid change at two positions (T47A and V125I) in MU, though the amino acid change at position 125 was V125I in MU as compared with V125F in M. tuberculosis, thus, further experimental validation would be needed.27 No mutations were detected in inhA, katG, oxyR, ahpC, ndh genes and in the promoter region of inhA (C15T) and ahpC (G32A) associated with isoniazid resistance.29 No mutations were detected in embB and ethA genes to confer resistance to ethambutol and ethanomide respectively.28 Finally we did not find any mutation in the rifampin resistance-determining region of rpoB.29,38 Likewise, the quinolone resistance-determining region of the gyrA and gyrB genes revealed no mutations associated with fluoroquinolone resistance.21,22,39
In silico analysis of chromosomal antibiotic resistance genes and mutations associated with resistance in Mycobacterium ulcerans AGY99 and comparative analysis within the Mycobacterium marinum complex
| Antibiotics class | Size (aa) | Function | Best Hit NCBI | Putative ARG | % Identity | |||
|---|---|---|---|---|---|---|---|---|
| Mycobacterium liflandii | Mycobacterium marinum M | Mycobacterium marinum Europe | M. marinum MB2 | |||||
| Aminoglycoside | 276 | Aminoglycoside 3-phosphotransferase/protein blocks polypeptide synthesis by inhibiting the elongation step | M liflandii 128FXT (5549831_5550562) | aph | 99.73 | 99.05 | 99.18 | 99.32 |
| 181 | Coenzyme A-dependent acetylation of the 2′ hydroxyl or amino group | M. liflandii 128F (1272229_1271684) | aac2-Ic | 99.82 | 99.63 | 99.45 | 99.82 | |
| β-Lactamases | 414 | β-Lactamase class C and other penicillin binding proteins | M. avium 104 (4889836_4891941) | amp | 99.43 | 98.38 | 98.43 | 99.10 |
| 250 | Metallo-β-lactamase superfamily protein | Mycobacterium tuberculosis CDC1551 (3143802_3144554) | aim | 99.87 | 99.60 | 98.41 | 99.34 | |
| 297 | β-Lactamase A | M. marinum M (2568033_2567140) | far | 98.99 | 99.33 | 98.55 | 99.33 | |
| 359 | β-Lactamase/D-alanine carboxypeptidase (transpeptidase superfamily) | M. liflandii 128FXT (2113703- 2114785) | och | 99.32 | 98.64 | 97.27 | 98.77 | |
| 379 | β-Lactamase class C and other penicillin binding proteins | M. marinum M (1958947_1957808) | amp | 99.65 | 99.65 | 99.47 | 99.21 | |
| 225 | Predicted Zn-dependent hydrolase of the β-Lactamase fold | M. liflandii 128FXT (1760783_1760106) | gob | 99.41 | 98.38 | 98.82 | 98.67 | |
| 272 | β-Lactamase class C and other penicillin binding proteins | M. liflandii 128FXT (1391326_1392144) | amp | 99.51 | 99.27 | 99.15 | 99.51 | |
| MLS/TET | 643 | Macrolide ABC transporter ATP-binding protein | Mycobacterium canettii CIPT 140070017 (1825164_1823233) | ery /tet | 99.53 | 99.28 | 98.19 | 99.48 |
| Sulfonamide | 274 | Dihydropteroate synthase | M. liflandii 128FXT (1006149_1006973) | sul | 99.52 | 99.27 | 99.03 | 99.27 |
| 275 | Dihydropteroate synthase | M. marinum M (4661918_4661091) | sul | 99.28 | 99.40 | 97.95 | 99.15 | |
| Trimethoprim | 172 | Disrupts DNA, RNA, and protein synthesis | M. liflandii 128FXT (2431765_2432253) | dfr | 99.45 | 99.64 | 97.27 | 99.61 |
| 182 | Disrupts DNA, RNA, and protein synthesis | Sanguibacter keddieii DSM 10542 (3230564_3231112) | dfr | 99.80 | 100 | 99.39 | 100 | |
| Isoniazid | 708 | Inhibits the synthesis of mycolic acids, an essential component of the bacterial cell wall | M. liflandii 128FXT (2441410-2443536) | katG Arg-431-Leu* | katG Arg-431-Leu (pseudogene) | katG Arg-431-Leu | katG Arg-431-Leu | katG Arg-431-Leu |
| Pyrazinamide | 196 | Disrupted membrane energetics and inhibits membrane transport function at acidic pH | M. liflandii 128FXT (2510652-2511212) | pncA Thr-47-Ala | pncA Thr-47-Ala | pncA Thr-47-Ala | pncA Thr-47-Ala | pncA Thr-47-Ala |
| Val-125-Ile† | Val-125-Ile | Val-125-Ile | Val-125-Ile | Val-125-Ile | ||||
ABC = adenosine triphosphate-binding cassette; ARG = antibiotic resistance gene (not the same gene but closely matched gene).
Codon 431 in M. ulcerans corresponds to codon 463 of M. tuberculosis for katG gene where amino acid change at position Arg463Leu leads to INH resistance.25
The detected codon at position 125 was isoleucine in M. ulcerans, however resistance strains of M. tuberculosis had phenyl alanine in place of valine at 125, no codon changes (Ile-6-The, His-51-Gln, Pro-54-Ser, Phe-58-Leu, Gly-132-Ile etc.)26 were noticed at other regions of pncA gene in MU. Hence, the amino acid change at position 125, predicted as potential mutation for resistance.
Phylogenetic tree based on rpoB gene highlighting the position of Mycobacterium ulcerans (Mu) genomes within the closely related genus Mycobacterium liflandii (Ml), Mycobacterium marinum (Mm), and others. Sequences were aligned using CLUSTALW and phylogenetic inferences obtained using the neighbor-joining method within MEGA program. Numbers at the nodes are bootstrap values obtained by repeating 500 times the analysis to generate a majority consensus tree. The scale bar represents a 1% nucleotide sequence divergence.
Citation: The American Journal of Tropical Medicine and Hygiene 97, 3; 10.4269/ajtmh.16-0478
Phylogenetic tree based on rpoB gene highlighting the position of Mycobacterium ulcerans (Mu) genomes within the closely related genus Mycobacterium liflandii (Ml), Mycobacterium marinum (Mm), and others. Sequences were aligned using CLUSTALW and phylogenetic inferences obtained using the neighbor-joining method within MEGA program. Numbers at the nodes are bootstrap values obtained by repeating 500 times the analysis to generate a majority consensus tree. The scale bar represents a 1% nucleotide sequence divergence.
Citation: The American Journal of Tropical Medicine and Hygiene 97, 3; 10.4269/ajtmh.16-0478
Phylogenetic tree based on rpoB gene highlighting the position of Mycobacterium ulcerans (Mu) genomes within the closely related genus Mycobacterium liflandii (Ml), Mycobacterium marinum (Mm), and others. Sequences were aligned using CLUSTALW and phylogenetic inferences obtained using the neighbor-joining method within MEGA program. Numbers at the nodes are bootstrap values obtained by repeating 500 times the analysis to generate a majority consensus tree. The scale bar represents a 1% nucleotide sequence divergence.
Citation: The American Journal of Tropical Medicine and Hygiene 97, 3; 10.4269/ajtmh.16-0478
CONCLUSIONS
Even if our analysis remains only predictive by in silico analysis such approach was successfully used in our experience for other fastidious and/or intracellular bacteria to shift from empirical to rationale antimicrobial therapy for Bartonella39 and Tropheryma whipplei infections.40 Here, we predict that isoniazid and pyrazinamide are probably ineffective agents against MU complex mycobacteria, whereas rifampin, streptomycin, azithromycin, clarithromycin, and fluoroquinolones currently used for the empirical antibiotic treatment of BU, were predicted to be effective. Very few studies has been conducted on drug resistance MU from humans.7,41 As acquired resistance to these antibiotics might occur by single mutation in target genes during monotherapy, antibiotics combination could be the better alternative to treat BU patients. Finally, we believe that surveillance of a possible emergence of resistance to these antibiotics could be implemented by development of molecular tests easily monitoring any changes in these target genes.
Acknowledgments:
We would like to thank Infectiopole Sud for financial support.
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