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Biology subjects

Sheen, P.

Publications and source records attributed to Sheen, P..

6 recordsLinked to original sources

Tiled Amplicon Sequencing Enables Culture-free Whole-Genome Sequencing of Pathogenic Bacteria From Clinical Specimens

Pathogen sequencing is an important tool for disease surveillance and demonstrated its high value during the COVID-19 pandemic. Viral sequencing during the pandemic allowed us to track disease spread, quickly identify new variants, and guide the development of vaccines. Tiled amplicon sequencing, in which a panel of primers is used for multiplex amplification of fragments across an entire genome, was the cornerstone of SARS-CoV-2 sequencing. The speed, reliability, and cost-effectiveness of this method led to its implementation in academic and public health laboratories across the world and adaptation to a broad range of viral pathogens. However, similar methods are not available for larger bacterial genomes, for which whole-genome sequencing typically requires in vitro culture. This increases costs, error rates and turnaround times. The need to culture poses particular problems for medically important bacteria such as Mycobacterium tuberculosis, which are slow to grow and challenging to culture. As a proof of concept, we developed two novel whole-genome amplicon panels for M. tuberculosis and Streptococcus pneumoniae. Applying our amplicon panels to clinical samples, we show the ability to classify pathogen subgroups and to reliably identify markers of drug resistance without culturing. Development of this work in clinical settings has the potential to dramatically reduce the time of diagnosis of drug resistance for multiple drugs in parallel, enabling earlier intervention for high priority pathogens.

genomics↗

Assembly and phylogeographic analysis of novel Taenia solium mitochondrial genomes reveal further differentiation between and within Asian and African-American genotypes

BackgroundTaenia solium is a parasite that hampers human health, causing taeniasis and cysticercosis. The genetic variability in its mitochondrial genome is related to the geographical origin of the specimen. Two main genotypes have been identified: The Asian and the African-American. The geographic genetic variability is expected to cause different clinical manifestations. Thus, characterizing differences between and within genotypes is crucial for completing the epidemiology of T. solium diseases. Methods/Principal FindingsHere, two Peruvian (one complete and one partial; 7,811X and 42X of coverage, respectively) and one Mexican (complete, 3,395X) T. solium mitochondrial genomes were assembled using the Chinese reference. Variant calling with respect to the reference was performed. Thirteen SNPs that involved a change in the amino acid physicochemical nature were identified. Those were present in all the assembled genomes and might be linked to differences in aerobic respiration efficiency between Latin American (African-American) and Asian genotypes. Then, phylogeographic studies were conducted using Cytochrome C oxidase subunit I and cytochrome B from these genomes and other isolates. The analysis showed that Indonesian samples are the most ancient and related to the modern T. solium ancestor of the Asian genotype. Finally, a consistent subdivision of the African-American genotype into two subgroups was found. One subgroup relates to East African countries, while the other is West Africa. The East African linage suggests a previously unnoticed influence of the Indian Ocean trade in the genetic structure of Latin America T. solium. Conclusions/SignificanceOverall, this study reports novel mitochondrial genomes valuable for further studies. New Latin American SNPs were identified and suggest metabolic differences between parasites of the Asian and African-American genotypes. Moreover, the phylogeographic analysis revealed differences within each genotype that shed light on T. soliums historical spread. Overall, the results represent an important step in completing T. solium genetic epidemiology. Author SummaryTaenia solium is a human parasite that causes taeniasis and cysticercosis. Eradicated from developed countries, they are still a public health problem in developing nations. T. solium differences in the mitochondrial genetic material depend on its geographical origin. This is expected to cause different clinical manifestations. Despite the importance of genetics to the epidemiology of T. solium diseases, few efforts have been made to assemble and compare their genomes. We aimed to help fill this knowledge gap by assembling three mitochondrial genomes from Latin America and comparing them to the Chinese reference. Additionally, two genes from the Latin American genomes and from other isolates were employed to assess T. solium genetic distribution. We found thirteen mutations with respect to the Chinese genome present in all Latin American samples, which involved a change in the amino acid physicochemical nature. Those might be causing metabolic differences between Asian and Latin American parasites that could change their affinity to specific human tissues. Moreover, we determined that Indonesian samples are the most ancient and related to the modern T. solium ancestor. Finally, we identified a previously unnoticed influence of East African countries in T. solium phylogeny, with which our assembled genomes are closely related.

genetics↗

Silencing of an efflux pump coding gene decreases the efflux rate of pyrazinoic acid in Mycobacterium smegmatis

BackgroundTuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB). The recommended treatment for TB is based on the use of first-line drugs, including pyrazinamide (PZA). PZA is also a drug used in the treatment of multidrug-resistant TB (MDR-TB) because of its main effect against the latent stage. The main cause of resistance to PZA is mutations in the pncA gene, which compromise the activity of the encoded enzyme pyrazinamidase (PZAse), which hydrolyzes PZA into POA, the active antituberculosis molecule. The mechanism of action of PZA requires that POA is expelled from the bacterium by an efflux mechanism. After that, if the extracellular medium is sufficiently acidic, POA is protonated and returns to the cytosol, releasing the proton and repeating the cycle, resulting lethal to the bacteria. The efflux pump responsible for extruding the POA to the extracellular environment is not yet known. Mycobacterium smegmatis is naturally resistant to PZA and has a 900-fold faster POA efflux rate than MTB, and has the advantage to be a faster growing mycobacterium. MethodsIn the present study we have silenced the transcription of several genes encoding efflux pumps in M. smegmatis by CRISPRi (CRISPR interference). These genes (MSMEG_0250, MSMEG_3815, MSMEG_0241, MSMEG_5046 and MSMEG_0410) were homologous to efflux pump genes in MTB. POA efflux rate was measured, and a quantitative Waynes test was performed after silencing each gene. ResultsSilencing of MSMEG_0250, resulted in approximately 5-fold decrease in the POA efflux rate in M. smegmatis (P<0.0001). None of the other silenced genes showed a notable decrease in POA efflux rate.

molecular biology↗

Low-cost 3D-printed inverted microscope to detect Mycobacterium tuberculosis in a MODS culture

BackgroundThe MODS is an important assay for early diagnosis of tuberculosis and drug susceptibility. MODS is based in the microscopic observation, underneath, of the characteristic cords of Mycobacterium tuberculosis colonies grown in liquid media. An inverted optical microscope is required to observe and interpret MODS cultures. Unfortunately, the cost of commercial inverted microscopes is not affordable in low resource settings in developing countries. MethodologyTo perform a diagnosis of tuberculosis using the MODS assay, images with modest quality are enough for proper interpretation. Therefore, the use of a high cost commercial inverted optical microscope is not indispensable. In this study, we designed a prototype of an optical inverted microscope created with a 3D printer and based on a smartphone. The system was evaluated by comparison of manual interpretations of 226 TB positive MODS culture images and 207 negative MODS culture images. SignificanceThe prototype resulted in a low-cost inverted optical microscope, with simple functioning, and whose parts have been manufactured using 3D printing techniques. The quality of the images was good enough and achieved a 100% concordance between the manual inspection with the developed microscope, and the standard diagnostics of MODS.

microbiology↗

EXPRESSION OF Mycobacterium tuberculosis RpsA IN Mycobacterium smegmatis INCREASES SUSCEPTIBILITY TO PYRAZINAMIDE

Pyrazinamide (PZA) is one of the most important drugs used in combined antituberculous therapy. After the drug enters Mycobacterium tuberculosis it is hydrolyzed by pyrazinamidase (PZAse) to the bactericidal molecule pyrazinoic acid (POA). Ribosomal protein S1 (RpsA) was recently identified as a possible target of PZA based on its binding activity to POA and capacity to inhibit trans-translation. However, its role is not completely understood. It has been proposed that Mycobacterium smegmatis RpsA is not capable of binding POA, unlike M. tuberculosis RpsA. This may be due to the different amino acid sequence in the carboxy-terminal region of the two molecules: in M. smegmatis RpsA it is much closer to the sites that may interact with POA than in M. tuberculosis RpsA. These differences could be contributing, along with the presence of highly active POA efflux, to the natural resistance to PZA in M. smegmatis. To further understand the mechanisms of action of PZA and the role of RpsA in PZA susceptibility, we evaluated the effect of complementing M. tuberculosis RpsA expression in M. smegmatis using pNIT mycobacterial non-integrative expression vector and then performed a PZA susceptibility test determining the minimum inhibitory concentration (MIC) of PZA. It was expected that chimeric ribosomes comprising M. tuberculosis RpsA may be present and may affect PZA susceptibility. Our results showed a reduction in PZA MIC in M. smegmatis complemented with overexpressed M. tuberculosis RpsA compared to non-overexpressed M. smegmatis (468 {micro}g/mL and >7500 {micro}g/mL respectively).

microbiology↗

Bioactive compounds from Chrysosporium multifidum, a fungus isolated from Hermetia illucens gut microbiota

The gut microbiota of insects contains a wide range of organisms that protect them against the attack of pathogens by releasing various types of bioactive compounds. In the present study, we report the isolation and identification of the fungus Chrysosporium multifidum as a component of the microbiota from the larval gut of Hermetia illucens. Extract from the broth culture of C. multifidum showed moderate activity on a strain of methicillin-resistant Staphylococcus aureus (MRSA). The bioguided isolation of the extract resulted in the characterization of six -pyrone derivatives (1-6) and one diketopiperazines (7), among them 5,6-dihydro-4-methoxy-6-(1-oxopentyl)-2H-pyran-2-one (4) showed the best activity (IC50 = 11.4 {+/-} 0.7 {micro}g/ml and MIC = 62.5 g/ml).

microbiology↗