Whole genome analysis of extensively drug resistant Mycobacterium tuberculosis strains in Peru

Peru has the highest burden of multidrug-resistant tuberculosis in the Americas region. Since 1999, the annual number of extensively drug-resistant tuberculosis (XDR-TB) Peruvian cases has been increasing, becoming a public health challenge. The objective of this study was to perform genomic characterization of Mycobacterium tuberculosis strains obtained from Peruvian patients with XDR-TB diagnosed from 2011 to 2015 in Peru. Whole genome sequencing (WGS) was performed on 68 XDR-TB strains from different regions of Peru. 58 (85.3%) strains came from the most populated districts of Lima and Callao. Concerning the lineages, 62 (91.2%) strains belonged to the Euro-American Lineage, while the remaining 6 (8.8%) strains belonged to the East-Asian Lineage. Most strains (90%) had high-confidence resistance mutations according to pre-established WHO-confident grading system. Discordant results between microbiological and molecular methodologies were caused by mutations outside the hotspot regions analysed by commercial molecular assays (rpoB I491F and inhA S94A). Cluster analysis using a cut-off ≤ 10 SNPs revealed that only 23 (34%) strains evidenced recent transmission links. This study highlights the relevance and utility of WGS as a high-resolution approach to predict drug resistance, analyse transmission of strains between groups, and determine evolutionary patterns of circulating XDR-TB strains in the country.Revisión por pare

High clustering rates of multidrug-resistant Mycobacterium tuberculosisgenotypes in Panama

Background: Tuberculosis continues to be one of the leading causes of death worldwide and in the American region. Although multidrug-resistant tuberculosis (MDR-TB) remains a threat to TB control in Panama, few studies have focused in typing MDR-TB strains. The aim of our study was to characterize MDR Mycobacterium tuberculosis clinical isolates using PCR-based genetic markers. Methods: From 2002 to 2004, a total of 231 Mycobacterium tuberculosis isolates from TB cases country-wide were screened for antibiotic resistance, and MDR-TB isolates were further genotyped by double repetitive element PCR (DRE-PCR), (GTG)5-PCR and spoligotyping. Results: A total of 37 isolates (0.85%) were resistant to both isoniazid (INH) and rifampicin (RIF). Among these 37 isolates, only two (5.4%) were resistant to all five drugs tested. Dual genotyping using DRE-PCR and (GTG)5-PCR of MDR Mycobacterium tuberculosis isolates revealed eight clusters comprising 82.9% of the MDR-TB strain collection, and six isolates (17.1%) showed unique fingerprints. The spoligotyping of MDR-TB clinical isolates identified 68% as members of the 42 (LAM9) family genotype.Background: Tuberculosis continues to be one of the leading causes of death worldwide and in the American region. Although multidrug-resistant tuberculosis (MDR-TB) remains a threat to TB control in Panama, few studies have focused in typing MDR-TB strains. The aim of our study was to characterize MDR Mycobacterium tuberculosis clinical isolates using PCR-based genetic markers. Methods: From 2002 to 2004, a total of 231 Mycobacterium tuberculosis isolates from TB cases country-wide were screened for antibiotic resistance, and MDR-TB isolates were further genotyped by double repetitive element PCR (DRE-PCR), (GTG)5-PCR and spoligotyping. Results: A total of 37 isolates (0.85%) were resistant to both isoniazid (INH) and rifampicin (RIF). Among these 37 isolates, only two (5.4%) were resistant to all five drugs tested. Dual genotyping using DRE-PCR and (GTG)5-PCR of MDR Mycobacterium tuberculosis isolates revealed eight clusters comprising 82.9% of the MDR-TB strain collection, and six isolates (17.1%) showed unique fingerprints. The spoligotyping of MDR-TB clinical isolates identified 68% as members of the 42 (LAM9) family genotype

High clustering rates of multidrug-resistant Mycobacterium tuberculosisgenotypes in Panama

Background: Tuberculosis continues to be one of the leading causes of death worldwide and in the American region. Although multidrug-resistant tuberculosis (MDR-TB) remains a threat to TB control in Panama, few studies have focused in typing MDR-TB strains. The aim of our study was to characterize MDR Mycobacterium tuberculosis clinical isolates using PCR-based genetic markers. Methods: From 2002 to 2004, a total of 231 Mycobacterium tuberculosis isolates from TB cases country-wide were screened for antibiotic resistance, and MDR-TB isolates were further genotyped by double repetitive element PCR (DRE-PCR), (GTG)5-PCR and spoligotyping. Results: A total of 37 isolates (0.85%) were resistant to both isoniazid (INH) and rifampicin (RIF). Among these 37 isolates, only two (5.4%) were resistant to all five drugs tested. Dual genotyping using DRE-PCR and (GTG)5-PCR of MDR Mycobacterium tuberculosis isolates revealed eight clusters comprising 82.9% of the MDR-TB strain collection, and six isolates (17.1%) showed unique fingerprints. The spoligotyping of MDR-TB clinical isolates identified 68% as members of the 42 (LAM9) family genotype.Background: Tuberculosis continues to be one of the leading causes of death worldwide and in the American region. Although multidrug-resistant tuberculosis (MDR-TB) remains a threat to TB control in Panama, few studies have focused in typing MDR-TB strains. The aim of our study was to characterize MDR Mycobacterium tuberculosis clinical isolates using PCR-based genetic markers. Methods: From 2002 to 2004, a total of 231 Mycobacterium tuberculosis isolates from TB cases country-wide were screened for antibiotic resistance, and MDR-TB isolates were further genotyped by double repetitive element PCR (DRE-PCR), (GTG)5-PCR and spoligotyping. Results: A total of 37 isolates (0.85%) were resistant to both isoniazid (INH) and rifampicin (RIF). Among these 37 isolates, only two (5.4%) were resistant to all five drugs tested. Dual genotyping using DRE-PCR and (GTG)5-PCR of MDR Mycobacterium tuberculosis isolates revealed eight clusters comprising 82.9% of the MDR-TB strain collection, and six isolates (17.1%) showed unique fingerprints. The spoligotyping of MDR-TB clinical isolates identified 68% as members of the 42 (LAM9) family genotype

Transoceanic spreading of pathogenic strains of <i>Vibrio parahaemolyticus</i> with distinctive genetic signatures in the<i> recA</i> gene

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. Consistent multilocus sequence typing for V. parahaemolyticus has shown difficulties in the amplification of the recA gene by PCR associated with a lack of amplification or a larger PCR product than expected. In one strain (090-96, Peru, 1996), the produced PCR product was determined to be composed of two recA fragments derived from different Vibrio species. To better understand this phenomenon, we sequenced the whole genome of this strain. The hybrid recA gene was found to be the result of a fragmentation of the original lineage-specific recA gene resulting from a DNA insertion of approximately 30 kb in length. This insert had a G+C content of 38.8%, lower than that of the average G+C content of V. parahaemolyticus (45.2%), and contained 19 ORFs, including a complete recA gene. This new acquired recA gene deviated 24% in sequence from the original recA and was distantly related to recA genes from bacteria of the Vibrionaceae family. The reconstruction of the original recA gene (recA3) identified the precursor as belonging to ST189, a sequence type reported previously only in Asian countries. The identification of this singular genetic feature in strains from Asia reveals new evidence for genetic connectivity between V. parahaemolyticus populations at both sides of the Pacific Ocean that, in addition to the previously described pandemic clone, supports the existence of a recurrent transoceanic spreading of pathogenic V. parahaemolyticus with the corresponding potential risk of pandemic expansion

Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry techniques are well-suited to high-throughput characterization of natural products, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social molecular networking (GNPS, http://gnps.ucsd.edu), an open-access knowledge base for community wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of ‘living data’ through continuous reanalysis of deposited data

Primers used and <i>V. parahaemolyticus</i> genomic regions investigated.

<p>Primers used and <i>V. parahaemolyticus</i> genomic regions investigated.</p

Transversal clustering of phylogenetic relationships and genomic variation.

<p>Clustering was built using a ClonalFrame tree of MLST allele sequences of 67 <i>V. parahaemolyticus</i> strains, while the VGR data were grouped using a UPGMA algorithm, resulting in a data matrix ordered according to both phylogenetic relationship and the prevalence of VGRs. Solid boxes and open boxes in the appropriate column indicate VGR presence or absence, respectively. Peruvian subset names are shown in red.</p

Phylogenetic tree and recombination events.

<p>A). The phylogenetic relationship of 22 <i>V. parahaemolyticus</i> STs was generated using ClonalFrame by 50% majority-rule consensus visualized using MEGA4. <i>V. alginolyticus</i> was used as an outgroup. Nodes A and B correspond to the recombination events presented in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002210#pntd-0002210-g004" target="_blank">Figure 4B</a>. Red, green and yellow circles denote the place of isolation from Peru, Chile and Asia, respectively. Coalescent units are indicated by the scale bar. B) ClonalFrame representation of the recombination event probability occurring in MLST concatenated is represented on the <i>x</i>-axis with the red line indicating the probability at each locus for an import event on a scale from 0 to 1 (<i>y</i>-axis). Each inferred substitution in the graph is represented by a cross, the intensity of which indicates the posterior probability for that substitution.</p

Minimum spanning tree (MST) of <i>V.</i> parahaemolyticus.

<p>A) MST of 89 MLST allelic profiles of the whole dataset included in this study. The colors are based on the origin of isolation. B) MST of the Peruvian subset that includes 56 strains. The colors are based on the serotyping group. Each circle represents an MLST genotype, and its size is proportional to the number of strains. The numbers along the branch links of MLST genotypes denote the amount of allelic differences.</p