Rapidly Correcting Frameshift Mutations in the Mycobacterium tuberculosis orn Gene Produce Reversible Ethambutol Resistance and Small-Colony-Variant Morphology

ABSTRACT We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB) resistance in Mycobacterium tuberculosis that is caused by a reversible frameshift mutation in the M. tuberculosis orn gene. A frameshift mutation in orn produces the small-colony-variant (SCV) phenotype, but this mutation does not change the MICs of any drug for wild-type M. tuberculosis. However, the same orn mutation in a low-level EMB-resistant double embB-aftA mutant (MIC = 8 μg/ml) produces an SCV with an EMB MIC of 32 μg/ml. Reversible resistance is indistinguishable from a drug-persistent phenotype, because further culture of these orn-embB-aftA SCV mutants results in rapid reversion of the orn frameshifts, reestablishing the correct orn open reading frame, returning the culture to normal colony size, and reversing the EMB MIC back to that (8 μg/ml) of the parental strain. Transcriptomic analysis of orn-embB-aftA mutants compared to wild-type M. tuberculosis identified a 27-fold relative increase in the expression of embC, which is a cellular target for EMB. Expression of embC in orn-embB-aftA mutants was also increased 5-fold compared to that in the parental embB-aftA mutant, whereas large-colony orn frameshift revertants of the orn-embB-aftA mutant had levels of embC expression similar to that of the parental embB-aftA strain. Reversible frameshift mutants may contribute to a reversible form of microbiological drug resistance in human tuberculosis.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Influence of the Protein Kinase C Activator Phorbol Myristate Acetate on the Intracellular Activity of Antibiotics against Hemin- and Menadione-Auxotrophic Small-Colony Variant Mutants of Staphylococcus aureus and Their Wild-Type Parental Strain in Human THP-1 Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01031-12 ◽

2012 ◽

Vol 56 (12) ◽

pp. 6166-6174 ◽

Cited By ~ 11

Author(s):

Laetitia G. Garcia ◽

Sandrine Lemaire ◽

Barbara C. Kahl ◽

Karsten Becker ◽

Richard A. Proctor ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Parental Strain ◽

Wild Type ◽

Content Type ◽

Small Colony Variant ◽

Intracellular Activity ◽

Kinase C ◽

Pharmacodynamic Profile ◽

Small Colony ◽

Intracellular Fate

ABSTRACTIn a previous study (L. G. Garcia et al., Antimicrob. Agents Chemother. 56:3700–3711, 2012), we evaluated the intracellular fate ofmenDandhemBmutants (corresponding to menadione- and hemin-dependent small-colony variants, respectively) of the parental COL methicillin-resistantStaphylococcus aureusstrain and the pharmacodynamic profile of the intracellular activity of a series of antibiotics in human THP-1 monocytes. We have now examined the phagocytosis and intracellular persistence of the same strains in THP-1 cells activated by phorbol 12-myristate 13-acetate (PMA) and measured the intracellular activity of gentamicin, moxifloxacin, and oritavancin in these cells. Postphagocytosis intracellular counts and intracellular survival were lower in PMA-activated cells, probably due to their higher killing capacities. Gentamicin and moxifloxacin showed a 5- to 7-fold higher potency (lower static concentrations) against the parental strain, itshemBmutant, and the genetically complemented strain in PMA-activated cells and against themenDstrain in both activated and nonactivated cells. This effect was inhibited when cells were incubated withN-acetylcysteine (a scavenger of oxidant species). In parallel, we observed that the MICs of these drugs were markedly reduced if bacteria had been preexposed to H2O2. In contrast, the intracellular potency of oritavancin was not different in activated and nonactivated cells and was not decreased by the addition ofN-acetylcysteine, regardless of the phenotype of the strains. The oritavancin MIC was also unaffected by preincubation of the bacteria with H2O2. Thus, activation of THP-1 cells by PMA may increase the intracellular potency of certain antibiotics (probably due to synergy with reactive oxygen species), but this effect cannot be generalized to all antibiotics.

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Adaptation Genomics of a Small-Colony Variant in a Pseudomonas chlororaphis 30-84 Biofilm

Applied and Environmental Microbiology ◽

10.1128/aem.02617-14 ◽

2014 ◽

Vol 81 (3) ◽

pp. 890-899 ◽

Cited By ~ 23

Author(s):

Dongping Wang ◽

Robert J. Dorosky ◽

Cliff S. Han ◽

Chien-chi Lo ◽

Armand E. K. Dichosa ◽

...

Keyword(s):

Biofilm Formation ◽

Genetic Alterations ◽

Whole Genome Sequence ◽

Phenotypic Switching ◽

Whole Genome ◽

Pseudomonas Chlororaphis ◽

Wild Type ◽

Content Type ◽

Small Colony Variant ◽

Small Colony

ABSTRACTThe rhizosphere-colonizing bacteriumPseudomonas chlororaphis30-84 is an effective biological control agent against take-all disease of wheat. In this study, we characterize a small-colony variant (SCV) isolated from aP. chlororaphis30-84 biofilm. The SCV exhibited pleiotropic phenotypes, including small cell size, slow growth and motility, low levels of phenazine production, and increased biofilm formation and resistance to antimicrobials. To better understand the genetic alterations underlying these phenotypes, RNA and whole-genome sequencing analyses were conducted comparing an SCV to the wild-type strain. Of the genome's 5,971 genes, transcriptomic profiling indicated that 1,098 (18.4%) have undergone substantial reprograming of gene expression in the SCV. Whole-genome sequence analysis revealed multiple alterations in the SCV, including mutations inyfiR(cyclic-di-GMP production),fusA(elongation factor), andcyoE(heme synthesis) and a 70-kb deletion. Genetic analysis revealed that theyfiRlocus plays a major role in controlling SCV phenotypes, including colony size, growth, motility, and biofilm formation. Moreover, a point mutation in thefusAgene contributed to kanamycin resistance. Interestingly, the SCV can partially switch back to wild-type morphologies under specific conditions. Our data also support the idea that phenotypic switching inP. chlororaphisis not due to simple genetic reversions but may involve multiple secondary mutations. The emergence of these highly adherent and antibiotic-resistant SCVs within the biofilm might play key roles inP. chlororaphisnatural persistence.

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The Endospore-Forming Pathogen Bacillus cereus Exploits a Small Colony Variant-Based Diversification Strategy in Response to Aminoglycoside Exposure

mBio ◽

10.1128/mbio.01172-15 ◽

2015 ◽

Vol 6 (6) ◽

Cited By ~ 9

Author(s):

Elrike Frenzel ◽

Markus Kranzler ◽

Timo D. Stark ◽

Thomas Hofmann ◽

Monika Ehling-Schulz

Keyword(s):

Antibiotic Resistance ◽

Bacillus Cereus ◽

Differential Diagnostics ◽

Wild Type ◽

Content Type ◽

Small Colony Variant ◽

Enhanced Production ◽

Small Colony ◽

First Time ◽

Slow Growing

ABSTRACTBacillus cereusis among the microorganisms most often isolated from cases of food spoilage and causes gastrointestinal diseases as well as nongastrointestinal infections elicited by the emetic toxin cereulide, enterotoxins, and a panel of tissue-destructive virulence factors. This opportunistic pathogen is increasingly associated with rapidly fatal clinical infections especially linked to neonates and immunocompromised individuals. Fatality results from either the misdiagnosis ofB. cereusas a contaminant of the clinical specimen or from failure of antibiotic therapy. Here we report for the first time that exposure to aminoglycoside antibiotics induces a phenotype switching of emeticB. cereussubpopulations to a slow-growing small colony variant (SCV) state. Along with altered antibiotic resistance, SCVs showed distinct phenotypic and metabolic properties, bearing the risk of antibiotic treatment failure and of clinical misdiagnosis by standard identification tests used in routine diagnostic. The SCV subpopulation is characterized by enhanced production of the toxin cereulide, but it does not secrete tissue-destructive and immune system-affecting enzymes such as sphingomyelinase and phospholipase. SCVs showed significantly prolonged persistence and decreased virulence in theGalleria mellonellamodel for bacterial infections, indicating diversification concerning their ecological lifestyle. Importantly, diversification into coexisting wild-type and SCV subpopulations also emerged during amikacin pressure duringin vivoinfection experiments.IMPORTANCEThis study shows for the first time that pathogenic spore-formingB. cereusstrains are able to switch to a so far unreported slow-growing lifestyle, which differs substantially in terms of developmental, phenotypic, metabolic, and virulence traits from the wild-type populations. This underpins the necessity of molecular-based differential diagnostics and a well-chosen therapeutic treatment strategy in clinical environments to combatB. cereusin a tailored manner. The reported induction of SCV in an endospore-forming human pathogen requires further research to broaden our understanding of a yet unexplored antibiotic resistance mechanism in sporulating bacteria. Our work also raises a general question about the ecological meaning of SCV subpopulation emergence and importance of SCV in sporeformer populations as an alternative route, next to sporulation, to cope with stresses encountered in natural niches, such as soil or host interfaces.

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Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01288-09 ◽

2009 ◽

Vol 54 (1) ◽

pp. 103-108 ◽

Cited By ~ 38

Author(s):

Hassan Safi ◽

Robert D. Fleischmann ◽

Scott N. Peterson ◽

Marcus B. Jones ◽

Behnam Jarrahi ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

In Vitro Selection ◽

Gene Mutations ◽

Wild Type ◽

Mutant Selection ◽

Preferential Growth ◽

Allelic Exchange ◽

High Level ◽

Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

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Activation of 2,4-Diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a Putative Dioxygenase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01505-18 ◽

2018 ◽

Vol 63 (1) ◽

Author(s):

Eduard Melief ◽

Shilah A. Bonnett ◽

Edison S. Zuniga ◽

Tanya Parish

Keyword(s):

Mycobacterium Tuberculosis ◽

Mutant Strain ◽

Type Strain ◽

Wild Type Strain ◽

Type A ◽

Wild Type ◽

Metabolite Analysis ◽

Content Type ◽

Data Support ◽

In The Wild

ABSTRACT The diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

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Rv3131, a gene encoding nitroreductase, is essential for metronidazole activation in Mycobacterium tuberculosis under hypoxic condition

10.21203/rs.3.rs-60221/v1 ◽

2020 ◽

Author(s):

Wenzhu Dong ◽

Jin Shi ◽

Ping Chu ◽

Rongmei Liu ◽

Shu’an Wen ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Anaerobic Bacteria ◽

Aerobic Condition ◽

Hypoxic Condition ◽

Significant Inhibition ◽

Wild Type ◽

Minimal Inhibitory Concentrations ◽

High Level ◽

Nitroreductase Activity

Abstract ObjectivesThe impressive potency of metronidazole (MTZ) against anaerobic bacteria indicates the potential for killing anaerobic Mtb. However, how MTZ is activated in Mtb still remains unknown. We aimed to characterize the endogenous nitroreductase responsible for MTZ activation in anaerobic Mtb.MethodsThe minimal inhibitory concentrations (MICs) of Mtb isolates against MTZ were determined by microplate Alamar Blue assay. Intracellular anti-TB activities of MTZ and pyrazinamide (PZA) were tested in THP-1 cells infected by Mycobacterium tuberculosis (Mtb) H37Rv with a multiplicity of infection (MOI) of 10. The nitroreductase activity of purified wild-type Rv3131 and mutants were measured under anaerobic conditions generated by glucose oxidase/catalase system. Two-tailed unpaired Student’s t test was used to compare the difference between various groups.Results180 Mtb isolates (81.8%, 180/220) had MIC values higher than 16 μg/mL, and 40 had MIC values of 16 μg/mL, demonstrating high-level resistance to MTZ under aerobic condition. The number of viable bacteria in macrophages treated with MTZ was dramatically decreased by 71.3% after 5-day MTZ treatment, indicating significant inhibition of MTZ against anaerobic Mtb. In vitro biochemical analysis demonstrated that Rv3131 exhibited the NADPH oxidase activity under anaerobic condition. The substitutions of Cys75Ser and Cys279Ser could maintain 41.7% and 71.1% of enzyme activity compared to wild-type protein, respectively.ConclusionsOur data demonstrate that MTZ has more potent efficacy against intracellular Mtb than PZA. Rv3131 is identified as a nitroreductase enzyme in the activation of MTZ, and Cys75 of Rv3131 is the major active residue for nitroreductase activity.

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Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01184-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5658-5664 ◽

Cited By ~ 48

Author(s):

Soo-Jin Yang ◽

Nagendra N. Mishra ◽

Aileen Rubio ◽

Arnold S. Bayer

Keyword(s):

Staphylococcus Aureus ◽

Single Nucleotide Polymorphisms ◽

Point Mutations ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide ◽

Content Type ◽

Reading Frame ◽

A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

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Extending the Definition of the GyrB Quinolone Resistance-Determining Region in Mycobacterium tuberculosis DNA Gyrase for Assessing Fluoroquinolone Resistance in M. tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.06272-11 ◽

2012 ◽

Vol 56 (4) ◽

pp. 1990-1996 ◽

Cited By ~ 40

Author(s):

Alix Pantel ◽

Stéphanie Petrella ◽

Nicolas Veziris ◽

Florence Brossier ◽

Sylvaine Bastian ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Hot Spot ◽

Dna Gyrase ◽

Quinolone Resistance ◽

Fluoroquinolone Resistance ◽

Wild Type ◽

Content Type ◽

Highly Active ◽

Definition Of

ABSTRACTFluoroquinolone (FQ) resistance is emerging inMycobacterium tuberculosis. The main mechanism of FQ resistance is amino acid substitution within the quinolone resistance-determining region (QRDR) of the GyrA subunit of DNA gyrase, the sole FQ target inM. tuberculosis. However, substitutions in GyrB whose implication in FQ resistance is unknown are increasingly being reported. The present study clarified the role of four GyrB substitutions identified inM. tuberculosisclinical strains, two located in the QRDR (D500A and N538T) and two outside the QRDR (T539P and E540V), in FQ resistance. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to unequivocally clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineeredgyrBalleles by mutagenesis were overexpressed inEscherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. All these substitutions are clearly implicated in FQ resistance, underlining the presence of a hot spot region housing most of the GyrB substitutions implicated in FQ resistance (residues NTE, 538 to 540). These findings help us to refine the definition of GyrB QRDR, which is extended to positions 500 to 540.

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Mutations ingyrAandgyrBamong Fluoroquinolone- and Multidrug-Resistant Mycobacterium tuberculosis Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01049-15 ◽

2016 ◽

Vol 60 (4) ◽

pp. 2090-2096 ◽

Cited By ~ 26

Author(s):

Jung-Yien Chien ◽

Wei-Yih Chiu ◽

Shun-Tien Chien ◽

Chia-Jung Chiang ◽

Chong-Jen Yu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

East Asian ◽

Multidrug Resistant ◽

Molecular Techniques ◽

Content Type ◽

Low Level ◽

High Prevalence ◽

Mdr Tb ◽

High Level

ABSTRACTIn order to correlate the mutations inside the entiregyrAandgyrBgenes with the level of resistance to ofloxacin (OFX) and moxifloxacin (MFX) in isolates of multidrug-resistantMycobacterium tuberculosis(MDR-TB), a total of 111 isolates were categorized into OFX-susceptible (MIC, ≤2 μg/ml) and low-level (MIC, 4 to 8 μg/ml) and high-level (MIC, ≥16 μg/ml) OFX-resistant isolates and MFX-susceptible (MIC, ≤0.5 μg/ml) and low-level (MIC, 1 to 2 μg/ml) and high-level (MIC, ≥4 μg/ml) MFX-resistant isolates. Resistance-associated mutations inside thegyrAgene were found in 30.2% of OFX-susceptible and 72.5% and 72.2% of low-level and high-level OFX-resistant isolates and in 28.6% of MFX-susceptible and 58.1% and 83.9% of low-level and high-level MFX-resistant isolates. Compared with OFX-susceptible isolates, low-level and high-level OFX-resistant isolates had a significantly higher prevalence of mutations atgyrAcodons 88 to 94 (17.0%, 65.0%, and 72.2%, respectively;P< 0.001) and a higher prevalence of thegyrBG512R mutation (0.0%, 2.5%, and 16.7%, respectively;P= 0.006). Similarly, compared with MFX-susceptible isolates, low-level and high-level MFX-resistant isolates had a significantly higher prevalence of mutations atgyrAcodons 88 to 94 (14.3%, 51.6%, and 80.6%, respectively;P< 0.001) as well as a higher prevalence of thegyrBG512R mutation (0.0%, 0.0%, and 12.9%, respectively;P= 0.011). D94G and D94N mutations ingyrAand the G512R mutation ingyrBwere correlated with high-level MFX resistance, while the D94A mutation was associated with low-level MFX resistance. The prevalence of mutations atgyrAcodons 88 to 94 and thegyrBG512R mutation were higher among fluoroquinolone (FQ)-susceptible East Asian (Beijing) and Indo-Oceanic strains than they were among Euro-American strains, implying that molecular techniques to detect FQ resistance may be less specific in areas with a high prevalence of East Asian (Beijing) and Indo-Oceanic strains.

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