Endotracheal tube microbiome in hospitalized patients defined largely by hospital environment.

BACKGROUND: Studies of the respiratory tract microbiome primarily focus on airway and lung microbial diversity, but it is still unclear how these microbial communities may be affected by intubation and long periods in intensive care units (ICU), an aspect that today could aid in the understanding of COVID19 progression and disease severity. This study aimed to explore and characterize the endotracheal tube (ETT) microbiome by analyzing ETT-associated microbial communities. METHODS: This descriptive study was carried out on adult patients subjected to invasive mechanical ventilation from 2 to 21 days. ETT samples were obtained from 115 patients from ICU units in two hospitals. Bacteria isolated from endotracheal tubes belonging to the ESKAPE group were analyzed for biofilm formation using crystal violet quantification. Microbial profiles were obtained using Illumina sequencing of 16S rRNA gene. RESULTS: The ETT microbiome was mainly composed by the phyla Proteobacteria, Firmicutes and Bacteroidetes. Microbiome composition correlated with the ICU in which patients were hospitalized, while intubation time and diagnosis of ventilator-associated pneumonia (VAP) did not show any significant association. CONCLUSION: These results suggest that the ICU environment, or medical practices, could be a key to microbial colonization and have a direct influence on the ETT microbiomes of patients that require mechanical ventilation.

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.

Heterogeneous fitness landscape cues, pknG low expression, and phthiocerol dimycocerosate low production of Mycobacterium tuberculosis ATCC25618 rpoB S450L in enriched broth.

Multidrug-resistant tuberculosis (isoniazid/rifampin[RIF]-resistant TB) ravages developing countries. Fitness is critical in clinical outcomes. Previous studies on RIF-resistant TB (RR-TB) showed competitive fitness gains and losses, with rpoB-S450L as the most isolated/fit mutation. This study measured virulence/resistance genes, phthiocerol dimycocerosate (PDIM) levels and their relationship with rpoB S450L ATCC25618 RR-TB strain fitness. After obtaining 10 different RR-TB GenoType MTBDRplus 2.0-genotyped isolates (with nontyped, S441, H445 and S450 positions), only one S450L isolate (R9, rpoB-S450L ATCC 25618, RR 1 µg/mL) was observed, with H445Y being the most common. A competitive fitness in vitro assay with wild-type (wt) ATCC 25618: R9 1:1 in 50 mL Middlebrook 7H9/OADC was performed, and generation time (G) in vitro and relative fitness were obtained. mRNA and PDIM were extracted on log and stationary phases. Fitness decreased in rpoB S450L and H445Y strains, with heterogeneous fitness cues in three biological replicas of rpoB-S450L: one high and two low fitness replicas. S450L strain had significant pknG increase. Compared with S450L, wt-rpoB showed increased polyketide synthase ppsA expression and high PDIM peak measured by HPLC-MS in log phase compared to S450L. This contrasts with previously increased PDIM in other RR-TB isolates.

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.

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.

Mycofier: a new machine learning-based classifier for fungal ITS sequences.

BACKGROUND: The taxonomic and phylogenetic classification based on sequence analysis of the ITS1 genomic region has become a crucial component of fungal ecology and diversity studies. Nowadays, there is no accurate alignment-free classification tool for fungal ITS1 sequences for large environmental surveys. This study describes the development of a machine learning-based classifier for the taxonomical assignment of fungal ITS1 sequences at the genus level. RESULTS: A fungal ITS1 sequence database was built using curated data. Training and test sets were generated from it. A Naïve Bayesian classifier was built using features from the primary sequence with an accuracy of 87 % in the classification at the genus level. CONCLUSIONS: The final model was based on a Naïve Bayes algorithm using ITS1 sequences from 510 fungal genera. This classifier, denoted as Mycofier, provides similar classification accuracy compared to BLASTN, but the database used for the classification contains curated data and the tool, independent of alignment, is more efficient and contributes to the field, given the lack of an accurate classification tool for large data from fungal ITS1 sequences. The software and source code for Mycofier are freely available at https://github.com/ldelgado-serrano/mycofier.git .

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.

Identificación de genes de Mycobacterium tuberculosis que se expresan bajo condiciones de estrés anaeróbico / Identification of Mycobacterium tuberculosis gen differentially expresed during anaerobic stress conditions

Objetivo: identificar genes de M. tuberculosis que se inducen bajo condiciones de limitación de oxigeno para estudiar su posterior efecto sobre la viabilidad del patógeno. Materiales y métodos: se crecieron cultivos de la cepa no patógena M. smegmatis mc²155 en cajas de microcultivo bajo condiciones de estrés anaeróbico. Se construyó una biblioteca genómica de M. tuberculosis H37Rv en el plasmado pGFP, la cual fue introducida dentro de M. smegmatis. A partir de observación de las células de M. smegmatis recombinantes bajo luz UV se identificaron clones que indujeran la expresión del gen reportero bajo condiciones de limitación de oxígeno. Los plásmidos recombinantes de estos clones fueron aislados y los insertos secuenciados. Las secuencias obtenidas fueron analizadas comparándolas con el genoma completo de M. tuberculosis. Resultados: Se estandarizaron las condiciones in vitro para realizar estudios por estrés anaeróbico usando la micobacteria M. smegmatis. El tamizaje de la genoteca de M. tuberculosis dentro de M. smegmatis llevó a la identificación de 3 posibles fragmentos genómicos que inducen la expresión de gfp en condiciones de estrés anaeróbico. La secuencia de estos insertos reveló que contenían posibles secuencias reguladoras, dos de ellas corriente arriba de los genes para las proteínas hipotéticas Rv2603 y Rv3267 de M. tuberculosis y la otra para un marco abierto de lectura no identificado. Conclusiones: Utilizando librerías genómicas y una micobacteria no patógena como M. smegmatis es posible identificar genes de M. tuberculosis posiblemente involucrados en resistencia a condiciones de estrés

Utilidad de la biopsia pulmonar abierta en pediatría 11 años de experiencia en el Hospital Santa Clara

Mediante una revisión retrospectiva se evaluó la utilidad de la biopsia pulmonar abierta (BPA) sobre el diagnóstico y tratamiento de las neumopátias crónicas en los niños. Se recolectaron 33 registros de Historia Clínica. La edad promedio de los pacientes fué de 4.16 años con predominio del sexo masculino (4.1). La mayoría de los pacientes tenía síntomas respiratorios crónicos que no pudieron encasillarse en un diagnóstico adecuado utilizando pruebas menos invasivas. Cerca del 40 por ciento de los casos tenía infiltrados readiológicos de tipo intersticial. Una proporción significativa de pacientes no tenía una valoracion paraclínica satisfactoria, en especial sobre su estado inmunológico y sobre métodos para descartar patología infecciosa. La BPA permitió un cambio en el diagnóstico en el 48.5 por ciento de los casos y generó una modificación terapéutica en un porcentaje similar de pacientes, aunque estas variaciones no estuvieron asociadas a una franca mejoría, dado que solamente el 36.4 por ciento de los pacientes evolucionó satisfactoriamente. La frecuencia de complicaciones alcanzó un 12.1 por ciento las cuales fueron superadas rápidamente y no se registraron casos de mortalidad. La BPA en nuestro medio es un procedimiento quirúrgico seguro, sin embargo, no siempre aporta los resultados esperados para definir un diagnóstico certero