Corrigendum: Iron deprivation enhances transcriptional responses to in vitro growth arrest of Mycobacterium tuberculosis.

[This corrects the article DOI: 10.3389/fmicb.2022.956602.].

Iron deprivation enhances transcriptional responses to in vitro growth arrest of Mycobacterium tuberculosis.

The establishment of Mycobacterium tuberculosis (Mtb) long-term infection in vivo depends on several factors, one of which is the availability of key nutrients such as iron (Fe). The relation between Fe deprivation inside and outside the granuloma, and the capacity of Mtb to accumulate lipids and persist in the absence of growth is not well understood. In this context, current knowledge of how Mtb modifies its lipid composition in response to growth arrest, depending on iron availability, is scarce. To shed light on these matters, in this work we compare genome-wide transcriptomic and lipidomic profiles of Mtb at exponential and stationary growth phases using cultures with glycerol as a carbon source, in the presence or absence of iron. As a result, we found that transcriptomic responses to growth arrest, considered as the transition from exponential to stationary phase, are iron dependent for as many as 714 genes (iron-growth interaction contrast, FDR <0.05), and that, in a majority of these genes, iron deprivation enhances the magnitude of the transcriptional responses to growth arrest in either direction. On the one hand, genes whose upregulation upon growth arrest is enhanced by iron deprivation were enriched in functional terms related to homeostasis of ion metals, and responses to several stressful cues considered cardinal features of the intracellular environment. On the other hand, genes showing negative responses to growth arrest that are stronger in iron-poor medium were enriched in energy production processes (TCA cycle, NADH dehydrogenation and cellular respiration), and key controllers of ribosomal activity shut-down, such as the T/A system mazE6/F6. Despite of these findings, a main component of the cell envelope, lipid phthiocerol dimycocerosate (PDIM), was not detected in the stationary phase regardless of iron availability, suggesting that lipid changes during Mtb adaptation to non-dividing phenotypes appear to be iron-independent. Taken together, our results indicate that environmental iron levels act as a key modulator of the intensity of the transcriptional adaptations that take place in the bacterium upon its transition between dividing and dormant-like phenotypes in vitro.

The Lack of the TetR-Like Repressor Gene BCG_2177c (Rv2160A) May Help Mycobacteria Overcome Intracellular Redox Stress and Survive Longer Inside Macrophages When Surrounded by a Lipid Environment.

Mycobacteria, like other microorganisms, survive under different environmental variations by expressing an efficient adaptive response, oriented by regulatory elements, such as transcriptional repressors of the TetR family. These repressors in mycobacteria also appear to be related to cholesterol metabolism. In this study, we have evaluated the effect of a fatty acid (oleic-palmitic-stearic)/cholesterol mixture on some phenotypic and genotypic characteristics of a tetR-mutant strain (BCG_2177c mutated gene) of M. bovis BCG, a homologous of Rv2160A of M. tuberculosis. In order to accomplish this, we have analyzed the global gene expression of this strain by RNA-seq and evaluated its neutral-lipid storage capacity and potential to infect macrophages. We have also determined the macrophage response by measuring some pro- and anti-inflammatory cytokine expressions. In comparison with wild-type microorganisms, we showed that the mutation in the BCG_2177c gene did not affect the growth of M. bovis BCG in the presence of lipids but it probably modified the structure/composition of its cell envelope. Compared to with dextrose, an overexpression of the transcriptome of the wild-type and mutant strains was observed when these mycobacteria were cultured in lipids, mainly at the exponential phase. Twelve putative intracellular redox balance maintenance genes and four others coding for putative transcriptional factors (including WhiB6 and three TetR-like) were the main elements repeatedly overexpressed when cultured in the presence of lipids. These genes belonged to the central part of what we called the "genetic lipid signature" for M. bovis BCG. We have also found that all these mycobacteria genotypic changes affected the outcome of BCG-infected macrophages, being the mutant strain most adapted to persist longer inside the host. This high persistence result was also confirmed when mutant-infected macrophages showed overexpression of the anti-inflammatory cytokine TGF-ß versus pro-inflammatory cytokines. In summary, the lack of this TetR-like repressor expression, within a lipid environment, may help mycobacteria overcome intracellular redox stress and survive longer inside their host.

Close Related Drug-Resistance Beijing Isolates of Mycobacterium tuberculosis Reveal a Different Transcriptomic Signature in a Murine Disease Progression Model.

Mycobacterium tuberculosis (MTB) lineage 2/Beijing is associated with high virulence and drug resistance worldwide. In Colombia, the Beijing genotype has circulated since 1997, predominantly on the pacific coast, with the Beijing-Like SIT-190 being more prevalent. This genotype conforms to a drug-resistant cluster and shows a fatal outcome in patients. To better understand virulence determinants, we performed a transcriptomic analysis with a Beijing-Like SIT-190 isolate (BL-323), and Beijing-Classic SIT-1 isolate (BC-391) in progressive tuberculosis (TB) murine model. Bacterial RNA was extracted from mice lungs on days 3, 14, 28, and 60. On average, 0.6% of the total reads mapped against MTB genomes and of those, 90% against coding genes. The strains were independently associated as determined by hierarchical cluster and multidimensional scaling analysis. Gene ontology showed that in strain BL-323 enriched functions were related to host immune response and hypoxia, while proteolysis and protein folding were enriched in the BC-391 strain. Altogether, our results suggested a differential bacterial transcriptional program when evaluating these two closely related strains. The data presented here could potentially impact the control of this emerging, highly virulent, and drug-resistant genotype.

Dissecting industrial fermentations of fine flavour cocoa through metagenomic analysis.

The global demand for fine-flavour cocoa has increased worldwide during the last years. Fine-flavour cocoa offers exceptional quality and unique fruity and floral flavour attributes of high demand by the world's elite chocolatiers. Several studies have highlighted the relevance of cocoa fermentation to produce such attributes. Nevertheless, little is known regarding the microbial interactions and biochemistry that lead to the production of these attributes on farms of industrial relevance, where traditional fermentation methods have been pre-standardized and scaled up. In this study, we have used metagenomic approaches to dissect on-farm industrial fermentations of fine-flavour cocoa. Our results revealed the presence of a shared core of nine dominant microorganisms (i.e. Limosilactobacillus fermentum, Saccharomyces cerevisiae, Pestalotiopsis rhododendri, Acetobacter aceti group, Bacillus subtilis group, Weissella ghanensis group, Lactobacillus_uc, Malassezia restricta and Malassezia globosa) between two farms located at completely different agro-ecological zones. Moreover, a community metabolic model was reconstructed and proposed as a tool to further elucidate the interactions among microorganisms and flavour biochemistry. Our work is the first to reveal a core of microorganisms shared among industrial farms, which is an essential step to process engineering aimed to design starter cultures, reducing fermentation times, and controlling the expression of undesirable phenotypes.

Recovery and functional validation of hidden soil enzymes in metagenomic libraries.

The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.

Hypoxia Is Not a Main Stress When Mycobacterium tuberculosis Is in a Dormancy-Like Long-Chain Fatty Acid Environment.

The capacity of Mycobacterium tuberculosis (Mtb) to sense, respond and adapt to a variable and hostile environment within the host makes it one of the most successful human pathogens. During different stages of infection, Mtb is surrounded by a plethora of lipid molecules and current evidence points out the relevance of fatty acids during the infectious process. In this study, we have compared the transcriptional response of Mtb to hypoxia in cultures supplemented with a mix of even long-chain fatty acids or dextrose as main carbon sources. Using RNA sequencing, we have identified differential expressed genes in early and late hypoxia, defined according to the in vitro Wayne and Hayes model, and compared the results with the exponential phase of growth in both carbon sources. We show that the number of genes over-expressed in the lipid medium was quite low in both, early and late hypoxia, relative to conditions including dextrose, with the exception of transcripts of stable and non-coding RNAs, which were more expressed in the fatty acid medium. We found that sigB and sigE were over-expressed in the early phase of hypoxia, confirming their pivotal role in early adaptation to low oxygen concentration independently of the carbon source. A drastic contrast was found with the transcriptional regulatory factors at early hypoxia. Only 2 transcriptional factors were over-expressed in early hypoxia in the lipid medium compared to 37 that were over-expressed in the dextrose medium. Instead of Rv0081, known to be the central regulator of hypoxia in dextrose, Rv2745c (ClgR), seems to play a main role in hypoxia in the fatty acid medium. The low level of genes associated to the stress-response during their adaptation to hypoxia in fatty acids, suggests that this lipid environment makes hypoxia a less stressful condition for the tubercle bacilli. Taken all together, these results indicate that the presence of lipid molecules shapes the metabolic response of Mtb to an adaptive state for different stresses within the host, including hypoxia. This fact could explain the success of Mtb to establish long-term survival during latent infection.

Bioelectrochemical Detection of Mycobacterium tuberculosis ESAT-6 in an Antibody-Based Biomicrosystem.

Bioelectrochemical sensing of Mycobacterium tuberculosis through electro-immunosensors is a promising technique to detect relevant analytes. In general, immunosensors require the formation of organic assemblies by the adsorption of molecular constituents. Moreover, they depend on the correct immobilization of the bio-recognition element in the biosensor. These procedures cannot be easily monitored without the use of invasive methods. In this work, an impedance analysis technique was used, as a non-invasive method, to measure and differentiate the manufacturing stages of the sensors. Biomicrosystems were fabricated through physical vapor deposition (PVD) of 80 nm Au nanolayers on 35 µm copper surfaces. Later, the surface was modified through thiolation methods generating a self-assembled-monolayer (SAM) with 20 mM 4-aminothiophenol (4-ATP) on which a polyclonal antibody (pAb) was covalently attached. Using impedance analysis, every step of the electro-immunosensor fabrication protocol was characterized using 40 independent replicas. Results showed that, compared to the negative controls, distilled water, and 0.5 µg/mL HSA, a maximum variation of 171% between each replica was achieved when compared to samples containing 0.5 µg/mL of ESAT-6 M. tuberculosis immunodominant protein. Therefore, this development validates a non-invasive method to electrically monitor the assembly process of electro-immunosensors and a tool for its further measure for detection of relevant antigens.

Clonación, expresión y caracterización de una nueva esterasa derivada de metagenomas de suelos agrícolas colombianos / Cloning, expression and characterization of a novel esterase derived from colombian agricultural soils metagenomes

El presente trabajo tuvo como objetivo la bioprospección de ADN metagenómico derivado de comunidades microbianas asociadas a un agroecosistema de importancia nacional. Este análisis permitió realizar la producción, expresión, purificación y caracterización de una enzima novedosa con actividad esterasa. Esta enzima, denominada LipM, había sido previamente identificada en clones metagenómicos derivados de suelos dedicados al cultivo de papa criolla (Solanum pureja), mediante secuencia de nueva generación y análisis bioinformáticos. La secuencia codificante de la enzima fue clonada en el vector pBADgiii y expresada en E. coli como sistema de expresión, lo que permitió optimizar el proceso de producción recombinante y su posterior purificación. Funcionalmente la enzima presentó una mayor afinidad por sustratos de p-nitrofenil con ácidos grasos de cadena corta (

The present work had as a main objective to bioprospect metagenomic DNA from microbial communities associated with an agro-ecosystem of national importance. This analysis allowed the production, expression, purification and characterization of a novel enzyme with esterase activity. This enzyme, named here as LipM, was previously identified in metagenomic clones derived from soils dedicated to creole potato (Solanum pureja) crops by means of next-generation sequencing and bioinformatics analyses. The coding sequence of the enzyme was cloned into pBADgiii vector and expressed in E. coli as an expression system, allowing to optimize its recombinant production process and its further purification. The enzyme functionally showed a greater affinity for p-nitrophenyl substrates with short-chain fatty acids (

Development of a genetic tool for functional screening of anti-malarial bioactive extracts in metagenomic libraries.

BACKGROUND: The chemical treatment of Plasmodium falciparum for human infections is losing efficacy each year due to the rise of resistance. One possible strategy to find novel anti-malarial drugs is to access the largest reservoir of genomic biodiversity source on earth present in metagenomes of environmental microbial communities. METHODS: A bioluminescent P. falciparum parasite was used to quickly detect shifts in viability of microcultures grown in 96-well plates. A synthetic gene encoding the Dermaseptin 4 peptide was designed and cloned under tight transcriptional control in a large metagenomic insert context (30 kb) to serve as proof-of-principle for the screening platform. RESULTS: Decrease in parasite viability consistently correlated with bioluminescence emitted from parasite microcultures, after their exposure to bacterial extracts containing a plasmid or fosmid engineered to encode the Dermaseptin 4 anti-malarial peptide. CONCLUSIONS: Here, a new technical platform to access the anti-malarial potential in microbial environmental metagenomes has been developed.

Respiratory tract clinical sample selection for microbiota analysis in patients with pulmonary tuberculosis.

BACKGROUND: Changes in respiratory tract microbiota have been associated with diseases such as tuberculosis, a global public health problem that affects millions of people each year. This pilot study was carried out using sputum, oropharynx, and nasal respiratory tract samples collected from patients with pulmonary tuberculosis and healthy control individuals, in order to compare sample types and their usefulness in assessing changes in bacterial and fungal communities. FINDINGS: Most V1-V2 16S rRNA gene sequences belonged to the phyla Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Fusobacteria, with differences in relative abundances and in specific taxa associated with each sample type. Most fungal ITS1 sequences were classified as Ascomycota and Basidiomycota, but abundances differed for the different samples. Bacterial and fungal community structures in oropharynx and sputum samples were similar to one another, as indicated by several beta diversity analyses, and both differed from nasal samples. The only difference between patient and control microbiota was found in oropharynx samples for both bacteria and fungi. Bacterial diversity was greater in sputum samples, while fungal diversity was greater in nasal samples. CONCLUSIONS: Respiratory tract microbial communities were similar in terms of the major phyla identified, yet they varied in terms of relative abundances and diversity indexes. Oropharynx communities varied with respect to health status and resembled those in sputum samples, which are collected from tuberculosis patients only due to the difficulty in obtaining sputum from healthy individuals, suggesting that oropharynx samples can be used to analyze community structure alterations associated with tuberculosis.

Global adaptation to a lipid environment triggers the dormancy-related phenotype of Mycobacterium tuberculosis.

UNLABELLED: Strong evidence supports the idea that fatty acids rather than carbohydrates are the main energy source of Mycobacterium tuberculosis during infection and latency. Despite that important role, a complete scenario of the bacterium's metabolism when lipids are the main energy source is still lacking. Here we report the development of an in vitro model to analyze adaptation of M. tuberculosis during assimilation of long-chain fatty acids as sole carbon sources. The global lipid transcriptome revealed a shift toward the glyoxylate cycle, the overexpression of main regulators whiB3, dosR, and Rv0081, and the increased expression of several genes related to reductive stress. Our evidence showed that lipid storage seems to be the selected mechanism used by M. tuberculosis to ameliorate the assumed damage of reductive stress and that concomitantly the bacilli acquired a slowed-growth and drug-tolerant phenotype, all characteristics previously associated with the dormant stage. Additionally, intergenic regions were also detected, including the unexpected upregulation of tRNAs that suggest a new role for these molecules in the acquisition of a drug-tolerant phenotype by dormant bacilli. Finally, a set of lipid signature genes for the adaptation process was also identified. This in vitro model represents a suitable condition to illustrate the participation of reductive stress in drugs' activity against dormant bacilli, an aspect scarcely investigated to date. This approach provides a new perspective to the understanding of latent infection and suggests the participation of previously undetected molecules. IMPORTANCE: Mycobacterium tuberculosis establishes long-lasting highly prevalent infection inside the human body, called latent tuberculosis. The known involvement of fatty acids is changing our understanding of that silent infection; however, question of how tubercle bacilli globally adapt to a lipid-enriched environment is still an unanswered. With the single change of providing fatty acids as carbon sources, the bacilli switch on their program related to dormant stage: slowed growth, accumulation of lipid bodies, and development of drug tolerance. In this stage, unexpected and previously unknown participants were found to play putatively important roles during the process. For the first time, this work compares the global transcriptomics of bacteria by using strand-specific RNA sequencing under two different growth conditions. This study suggests novel targets for the control of tuberculosis and provides a new straightforward in vitro model that could help to test the activity of drugs against dormant bacilli from a novel perspective.

Computational modeling and preliminary iroN, fepA, and cirA gene expression in Salmonella Enteritidis under iron-deficiency-induced conditions.

Salmonellosis outbreaks in Europe, the United States, and Latin America have been associated with contaminated food derivatives including meat from the poultry industry. Salmonella grown under iron-limiting conditions has the capability to increase concentration of several iron-regulated outer-membrane proteins to augment the acquisition of the metal. These proteins have been proved to have immunogenic properties. Our aim was to increase the relative expression of iroN, fepA, and cirA in Salmonella Enteritidis domestic strain. Furthermore, we proposed a 3-dimensional structure model for each protein to predict and locate antigenic peptides. Our eventual objective is to produce an effective vaccine against regional avian salmonellosis. Two simple factorial designs were carried out to discriminate between 2 nitrogen sources and determine chelating-agent addition timing to augment relative gene expression. Two antigenic peptides located at the external face of each protein and 2 typical domains of iron-regulated outer-membrane proteins, plug and TonB-dep-Rec, were identified from the 3-dimensional models. Tryptone was selected as the best nitrogen source based on growth rate (µx = 0.36 h(-1)) and biomass productivity (Px = 0.9 g•h(-1)•L(-1)) as determined by a general factorial design. Optimum timing for chelating agent addition was in the middle of the log phase, which allowed relative expressions at 4 h of culture. Increase in iroN, fepA, and cirA relative expression was favored by the length of log phase and the addition of chelating agent, which decreased chelating toxicity and enhanced cell growth rate.

Composition andffunction of the microbial commuity related with the nitrogen ccycling on the potato rhizosphere / Composición y función de la comunidad microbiana relacionada con el ciclaje de nitrógeno en la rizosfera de Solanum tuberosum (BAHUIN) grupo phureja

In the S. tuberosum group phureja crops, mineral fertilizer and organic amendments are applied to meet the plants´ nutritional demands, however the effect of such practices on the associated rizospheric microbial communities are still unknown. Nitrogen plays an important role in agricultural production, and a great diversity of microorganisms regulates its transformation in the soil, affecting its availability for the plant. The aim of this study was to assess the structure of microbial communities related with the N cycle of S. tuberosum group phureja rizospheric soil samples, with contrasting physical-chemical properties and fertilization strategy. Few significant differences between the community composition at the phylum level were found, only Planctomycetes phylum was different between samples of different soil type and fertilization strategy. However, the analysis of nitrogen-associated functional groups made by ribotyping characterization, grouped soils in terms of such variables in a similar way to the physical-chemical properties. Major differences between soil samples were typified by higher percentages of the ribotypes from nitrite oxidation, nitrogen fixation and denitrification on organic amendment soils. Our results suggest that, the dominant rhizosphere microbial composition is very similar between soils, possibly as a result of population´s selection mediated by the rhizosphere effect. However, agricultural management practices in addition to edaphic properties of sampled areas, appear to affect some functional groups associated with the nitrogen cycling, due to differences found on soil´s physicalchemical properties, like the concentration of ammonium that seems to have an effect regulating the distribution and activity of nitrogen related functional groups in the S. tuberosum rhizosphere.

Fertilización mineral y enmiendas orgánicas son aplicadas para satisfacer las demandas nutricionales de los cultivos de S. tuberosum grupo phureja . Sin embargo, el efecto de esas prácticas sobre la comunidad microbiana asociada a la rizósfera aún no se conocen. El nitrógeno juega un papel importante en la producción agrícola y una gran diversidad de microorganismos regulan su transformación en el suelo, afectando su disponibilidad para la planta. El objeto de este estudio fue determinar la composición de la comunidad microbiana de la rizósfera de S. tuberosum grupo phureja , asociada con el ciclo del nitrógeno, en muestras de suelo contrastantes en sus propiedades fisicoquímicas y estrategia de fertilización. Pocas diferencias significativas entre la composición de la comunidad microbiana a nivel de phylum fueron encontradas, Í°nicamente el phylum Planctomycetes fue diferente entre las muestras de suelos con estrategias de fertilización diferentes. Sin embargo, el análisis de grupos funcionales asociados al nitrógeno llevado a cabo por la caracterización de ribotipificación, agrupó los suelos en términos de esas variables en una forma similar a las propiedades fisicoquímicas del suelo. Diferencias mayores entre las muestras de suelo fueron tipificadas por los altos porcentajes de ribotipos asociados a la oxidación de nitrito, fijación de nitrógeno y denitrificación sobre los suelos con enmiendas orgánicas. Nuestros resultados sugieren que la composición microbiana dominante es muy similar entre suelos, posiblemente como resultado de la selección de poblaciones mediada por el efecto rizosférico. Sin embargo, las prácticas del manejo agrícola en conjunto con las propiedades del suelo en las áreas muestreadas, parecen afectar algunos grupos funcionales asociados con el ciclo de nitrógeno, debido a las diferencias encontradas en las propiedades fisicoquímicas del suelo, como la concentración de amonio que parece tener un efecto regulando la distribución y actividad de los grupos funcionales relacionados con el ciclo del nitrógeno en la rizosfera de S. tuberosum.

Global study of IS6110 in a successful Mycobacterium tuberculosis strain: clues for deciphering its behavior and for its rapid detection.

The Mycobacterium tuberculosis insertion sequence IS6110, besides being a very useful tool in molecular epidemiology, seems to have an impact on the biology of bacilli. In the present work, we mapped the 12 points of insertion of IS6110 in the genome of a successful strain named M. tuberculosis Zaragoza (which has been referred to as the MTZ strain). This strain, belonging to principal genetic group 3, caused a large unsuspected tuberculosis outbreak involving 85 patients in Zaragoza, Spain, in 2001 to 2004. The mapping of the points of insertion of IS6110 in the genome of the Zaragoza strain offers clues for a better understanding of the adaptability and virulence of M. tuberculosis. Surprisingly, the presence of one copy of IS6110 was found in Rv2286c, as was recently described for a successful Beijing sublineage. As a result of this analysis, a rapid method for detecting this particular M. tuberculosis strain has been designed.

Tuberculosis-spoligo-rifampin-isoniazid typing: an all-in-one assay technique for surveillance and control of multidrug-resistant tuberculosis on Luminex devices.

As a follow-up of the "spoligoriftyping" development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.

IS-seq: a novel high throughput survey of in vivo IS6110 transposition in multiple Mycobacterium tuberculosis genomes.

BACKGROUND: The insertion element IS6110 is one of the main sources of genomic variability in Mycobacterium tuberculosis, the etiological agent of human tuberculosis. Although IS 6110 has been used extensively as an epidemiological marker, the identification of the precise chromosomal insertion sites has been limited by technical challenges. Here, we present IS-seq, a novel method that combines high-throughput sequencing using Illumina technology with efficient combinatorial sample multiplexing to simultaneously probe 519 clinical isolates, identifying almost all the flanking regions of the element in a single experiment. RESULTS: We identified a total of 6,976 IS6110 flanking regions on the different isolates. When validated using reference strains, the method had 100% specificity and 98% positive predictive value. The insertions mapped to both coding and non-coding regions, and in some cases interrupted genes thought to be essential for virulence or in vitro growth. Strains were classified into families using insertion sites, and high agreement with previous studies was observed. CONCLUSIONS: This high-throughput IS-seq method, which can also be used to map insertions in other organisms, extends previous surveys of in vivo interrupted loci and provides a baseline for probing the consequences of disruptions in M. tuberculosis strains.

Multicentre laboratory validation of the colorimetric redox indicator (CRI) assay for the rapid detection of extensively drug-resistant (XDR) Mycobacterium tuberculosis.

OBJECTIVES: To perform a multicentre study to evaluate the performance of the colorimetric redox indicator (CRI) assay and to establish the MICs and critical concentrations of rifampicin, isoniazid, ofloxacin, kanamycin and capreomycin. METHODS: The study was carried out in two phases. Phase I determined the MIC of each drug. Phase II established critical concentrations for the five drugs tested by the CRI assay compared with the conventional proportion method. RESULTS: Phase I: a strain was considered resistant by the CRI assay if the MIC was ≥0.5 mg/L for rifampicin, ≥0.25 mg/L for isoniazid, ≥4.0 mg/L for ofloxacin and ≥5.0 mg/L for kanamycin and capreomycin. Sensitivity was 99.1% for isoniazid and 100% for the other drugs and specificity was 97.9% for capreomycin and 100% for the other drugs. Phase II: the critical concentration was 0.5 mg/L for rifampicin, 0.25 mg/L for isoniazid, 2.0 mg/L for ofloxacin and 2.5 mg/L for kanamycin and capreomycin giving an overall accuracy of 98.4%, 96.6%, 96.7%, 98.3% and 90%, respectively. CONCLUSIONS: Results demonstrate that the CRI assay is an accurate method for the rapid detection of XDR Mycobacterium tuberculosis. The CRI assay is faster than the conventional drug susceptibility testing method using solid medium, has the same turnaround time as the BACTEC MGIT 960 system, but is less expensive, and could be an adequate method for low-income countries.

Evaluation of the BD MGIT TBc Identification Test (TBc ID), a rapid chromatographic immunoassay for the detection of Mycobacterium tuberculosis complex from liquid culture.

The BACTEC MGIT 960 system is increasingly used to culture Mycobacterium tuberculosis. We evaluated the performance of the new immunochromatographic assay BD MGIT TBc Identification Test (TBc ID) for the rapid identification of M. tuberculosis complex in clinical samples when performed directly from BACTEC MGIT 960 culture positive for acid-fast bacilli (AFB). Of 92 cultures evaluated, the sensitivity and specificity of the TBc ID test was 98.5% and 100%, respectively compared to sequencing of the 16S rRNA gene. One culture that was TBc ID test negative but that was identified as M. tuberculosis by 16S rRNA sequencing was confirmed to have a mutation in the mpt64 gene. The TBc ID test is an easy and sensitive method for the identification of M. tuberculosis complex in liquid culture medium, does not require a high level of skills, neither any additional specific equipment and gives results in 15 min, which provide a good alternative for the rapid identification of M. tuberculosis complex in liquid medium.

Rifampin-isoniazid oligonucleotide typing: an alternative format for rapid detection of multidrug-resistant Mycobacterium tuberculosis.

A reverse line blot DNA hybridization format for rapid detection of multidrug-resistant tuberculosis was developed. Simultaneous detection of rifampin and isoniazid resistance in clinical isolates of Mycobacterium tuberculosis was based on the same amplification/reverse hybridization principle of the widely used spoligotyping. The test involved probing nine DNA regions that are targets of common drug resistance-associated mutations in the genes rpoB, katG, and inhA. Addition of quaternary amine tetramethyl ammonium chloride to the hybridization buffer promoted multiple hybrid formations at a single annealing temperature irrespective of the different GC contents of probes. The assay was standardized using 20 well-documented strains from the Institute of Tropical Medicine (Belgium) and evaluated blindly in a central laboratory with 100 DNA samples that were obtained from cultured clinical isolates and shipped dried from three other countries. Compared with drug susceptibility testing, both sensitivity and specificity for rifampin resistance detection were 93.0% while for isoniazid the values were 87.7% and 97.7%, respectively. Compared with sequencing and GenoType MTBDRplus methods, sensitivity and specificity reached 96.4% and 95.5% for rifampin and 92.7% and 100% for isoniazid. Altogether, 40/45 (89%) multidrug-resistant isolates were correctly identified. Advantages of this in-house development include versatility, capacity to run up to 41 samples by triplicate in a single run, and reuse of the membrane at least 10 times. These features substantially reduce cost per reaction and make the assay an attractive tool for use in reference laboratories of countries that have a high burden of multidrug-resistant tuberculosis but that cannot afford expensive commercial tests because of limited resources.