Map of the region deleted in the <i>M. tuberculosis</i> H37Rv reference strain as a result of the exposure to isoniazid.

<p>The region analyzed spans from positions 5′-2150314 to 5′-2159943 of the <i>M. tuberculosis</i> H37Rv genome sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034538#pone.0034538-Cole1" target="_blank">[14]</a>, adapted from Tuberculist, 2010, <a href="http://tuberculist.epfl.ch/" target="_blank">http://tuberculist.epfl.ch/</a>. The area delimited corresponds to the fragment deleted in strain H37Rv_INH(a)3.</p

Average quantification of the relative expression level, by RT-qPCR, of the genes that code for efflux pumps in <i>M. tuberculosis</i> in the two independent isoniazid exposure processes.

<p><b>Legend:</b> (a)1: exposition process A, first passage; (b)1: exposition process B, first passage; (a)26: exposition process A, passage twenty six; (b)26: exposition process B, passage twenty six. The relative expression of the efflux pump genes was assessed by comparison of the relative quantity of the respective mRNA in the presence of isoniazid to the non-exposed strain. Each culture was assayed in triplicate using total RNA obtained from three independent cultures. A level of relative expression equal to 1 indicates that the expression level was identical to the strain that was being compared. Genes showing expression levels equal or above four, when compared to the non-exposed strain, were considered to be overexpressed and are shown in bold.</p

The effect of serial passages in a constant concentration of isoniazid (0.1 µg/ml) on the INH MIC and the number of days required for detection of growth.

<p><b>Legend:</b> INH: isoniazid; RIF: rifampicin; INH (a)/(b): adaptation processes to isoniazid A and B, respectively.</p

MIC determination and susceptibility testing for the strains exposed to isoniazid in the presence and absence of efflux inhibitors.

<p><b>Legend:</b> INH: isoniazid; INH (a): exposure process A; INH (b): exposure process B. S: susceptible; R: resistant. Values in bold correspond to full reversion of the INH resistance phenotype. EIs were used at ½ of their MIC. MICs for the EIs (passage 1): thioridazine (TZ): H37Rv and 359/03: 15 µg/ml, 401/06 and 267/05: 30 µg/ml; chlorpromazine (CPZ): 30 µg/ml for all strains; verapamil (VP): 256 µg/ml for all strains. MICs for the EIs (passage 26): TZ: H37Rv and 359/03: 15 µg/ml, 401/06(a)26: 15 µg/ml; 401/06(b)26: 30 µg/ml; 267/05: 30 µg/ml; CPZ: MIC of 30 µg/ml for all strains except H37Rv INH(a)26: 15 µg/ml; VP: 256 µg/ml for all strains except H37Rv INH(a)26 and 401/06: 128 µg/ml.</p

Additional file 1: of One nanoprobe, two pathogens: gold nanoprobes multiplexing for point-of-care

Table S1. Multiplex PCR primers and Au-nanoprobes sequences. Figure S1. Electrophoretic analysis on 1% agarose gel: In lane 1 was used Thermo Scientific GeneRuler DNA Ladder Mix; Lane 2 and 3 Multiplex PCR products of M. tuberculosis and P. falciparum, respectively; Lane 4 Multiplex PCR product of M. tuberculosis and P. falciparum, Lane 5 negative control of the PCR reaction. Figure S2. Physical characterization of the synthesized gold nanoparticles. A) Transmission electron microscopy (TEM) imaging and inset size histogram frequency from ≈ 400 AuNPs counting; B) UV-Vis spectrum of spherical AuNPs, with a characteristic maximum absorption peak, C) Dynamic light scattering (DLS) measurements to define the hydrodynamic diameter of the AuNPs and zeta-potential (ζ-potential) as AuNP surface charge indicator. For DLS it was performed 3 runs per sample with 2000 measurements each and for ζ-potential a total of 5 runs per sample with 250 measurements. Figure S3. Aggregation profiles of the synthesized Au-nanoprobes. Aggregation measured as the ratio of localized surface plasmon resonance intensity at 525 nm and 600 nm for increasing salt concentrations (MgCl2). The minimum amount of salt required to cause aggregation was determined based on each Au-nanoprobe aggregation profiles. Ratio <1 was considered for full aggregation, 60 mM. Figure S4. Single pathogen Au-nanoprobe specificity analysis using synthetic targets. A 1 pmol concentration of synthetic targets (see Table S1) were individually tested with a MgCl2 concentration of 60 mM

Accumulation of EtBr by the <i>M. tuberculosis</i> strains tested.

<p>The figure shows the accumulation of EtBr by the strains from adaptation process A as an example. The values at bold type correspond to the higher concentration of EtBr that cells can handle without detectable accumulation. The dotted line corresponds to the assay run using the EtBr concentrations for which influx-efflux are at equilibrium, in the presence of the EI verapamil, at sub-inhibitory concentrations. Panel (A): Parental strains (passage #0); Panel (B) strains after first passage with INH and Panel (C); strains after 26 passages with INH. INH: isoniazid.</p

Genotypic characterization of the strains and derived cultures exposed to isoniazid (adaptation process A).

<p><b>Legend:</b> INH: isoniazid; RIF: rifampicin; wt: wild type; Δ: deletion of <i>katG</i> gene; S: serine; L: leucine. MIRU-VNTR profile A: 2,4,2,2,3,4,2,3,2,3,2,4,2,2,6,1,6,3,1,3,1,7,2,2; profile B: 2,4,4,2,3,4,3,3,2,4,2,4,2,2,6,1,5,3,1,3,1,5,2,2; profile C: 2,1,4,2,1,3,2,3,2,2,2,5,1,2,6,1,6,3,3,3,2,4,2,2.</p

Sequences of the primers used in the RT-qPCR assays.

<p>FW: forward; RV: reverse.</p

Schematic representation of exposure of strain H37Rv to 0.1 µg/ml INH using the BACTEC™ MGIT™ 960 and characterization assays performed at selected points.

<p>For each strain, exposure to INH was done in duplicate, in two independent assays - processes A and B. INH(a): exposure process A; INH(b): exposure process B; INH: isoniazid; EI: efflux inhibitor. Vertical arrows represent transfer to new MGIT tubes containing 0.1 µg/ml INH. Seq: nucleotide sequence determination for specific fragments of the genes involved in the resistance to INH; AST: susceptibility testing to all first line antibiotics. MIC_INH: minimum inhibitory concentration determination of isoniazid. Note: This same procedure, here depicted as an example, was carried out for isoniazid exposure of each strain involved in this study.</p