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Journal of Bacteriology Jan 2022The transcriptomes of Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phases of planktonic growth were evaluated by...
The transcriptomes of Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phases of planktonic growth were evaluated by direct RNA sequencing on the nanopore platform and compared with established short-read cDNA sequencing on the Illumina platform. Fifty to ninety percent of the sense RNAs turned out to be rRNA molecules, followed by similar proportions of mRNA transcripts and noncoding RNAs. The two platforms detected similar proportions of uncharged tRNAs and 29 yet-undescribed antisense tRNAs. For example, the rarest arginine codon was paired with the most abundant tRNA, and the tRNA gene is missing for the most frequent arginine codon. More than 90% of the antisense RNA molecules were complementary to a coding sequence. The antisense RNAs were evenly distributed in the genomes. Direct RNA sequencing identified more than 4,000 distinct nonoverlapping antisense RNAs during exponential and stationary growth. Besides highly expressed small antisense RNAs less than 200 bases in size, a population of longer antisense RNAs was sequenced that covered a broad range (a few hundred to thousands of bases) and could be complementary to a contig of several genes. In summary, direct RNA sequencing identified yet-undescribed RNA molecules and an unexpected composition of the pools of tRNAs and sense and antisense RNAs. Genome-wide gene expression of bacteria is commonly studied by high-throughput sequencing of size-selected cDNA fragment libraries of reverse-transcribed RNA preparations. However, the depletion of rRNAs, enzymatic reverse transcription, and the fragmentation, size selection, and amplification during library preparation lead to inevitable losses of information about the initial composition of the RNA pool. We demonstrate that direct RNA sequencing on the Nanopore platform can overcome these limitations. Nanopore sequencing of total RNA yielded novel insights into the Pseudomonas aeruginosa transcriptome that-if replicated in other species-will change our view of the bacterial RNA world. The discovery of sense-antisense pairs of transfer-messenger RNA (tmRNA), tRNAs, and mRNAs indicates a further and unknown level of gene regulation in bacteria.
Topics: Gene Expression Regulation, Bacterial; Genome, Bacterial; Genome-Wide Association Study; Nanopore Sequencing; Pseudomonas aeruginosa; RNA, Bacterial; Transcriptome
PubMed: 34780302
DOI: 10.1128/JB.00418-21 -
The European Respiratory Journal Oct 2020
Topics: Allografts; Bronchiolitis Obliterans; Humans; Lung; Lung Transplantation; Pseudomonas; Pseudomonas aeruginosa; Retrospective Studies; Risk Factors
PubMed: 33004442
DOI: 10.1183/13993003.01968-2020 -
Frontiers in Cellular and Infection... 2023Biofilms are viscoelastic materials that are a prominent public health problem and a cause of most chronic bacterial infections, in large part due to their resistance to... (Review)
Review
Biofilms are viscoelastic materials that are a prominent public health problem and a cause of most chronic bacterial infections, in large part due to their resistance to clearance by the immune system. Viscoelastic materials combine both solid-like and fluid-like mechanics, and the viscoelastic properties of biofilms are an emergent property of the intercellular cohesion characterizing the biofilm state (planktonic bacteria do not have an equivalent property). However, how the mechanical properties of biofilms are related to the recalcitrant disease that they cause, specifically to their resistance to phagocytic clearance by the immune system, remains almost entirely unstudied. We believe this is an important gap that is ripe for a large range of investigations. Here we present an overview of what is known about biofilm infections and their interactions with the immune system, biofilm mechanics and their potential relationship with phagocytosis, and we give an illustrative example of one important biofilm-pathogen () which is the most-studied in this context. We hope to inspire investment and growth in this relatively-untapped field of research, which has the potential to reveal mechanical properties of biofilms as targets for therapeutics meant to enhance the efficacy of the immune system.
Topics: Phagocytes; Phagocytosis; Biofilms; Kinetics; Pseudomonas aeruginosa
PubMed: 36875516
DOI: 10.3389/fcimb.2023.1102199 -
Microbial Biotechnology Jan 2022To meet the needs of synthetic biologists, DNA assembly methods have transformed from simple 'cut-and-paste' procedures to highly advanced, standardised assembly...
To meet the needs of synthetic biologists, DNA assembly methods have transformed from simple 'cut-and-paste' procedures to highly advanced, standardised assembly techniques. Implementing these standardised DNA assembly methods in biotechnological research conducted in non-model hosts, including Pseudomonas putida and Pseudomonas aeruginosa, could greatly benefit reproducibility and predictability of experimental results. SEVAtile is a Type IIs-based assembly approach, which enables the rapid and standardised assembly of genetic parts - or tiles - to create genetic circuits in the established SEVA-vector backbone. Contrary to existing DNA assembly methods, SEVAtile is an easy and straightforward method, which is compatible with any vector, both SEVA- and non-SEVA. To prove the efficiency of the SEVAtile method, a three-vector system was successfully generated to independently co-express three different proteins in P. putida and P. aeruginosa. More specifically, one of the vectors, pBGDes, enables genomic integration of assembled circuits in the Tn7 landing site, while self-replicatory vectors pSTDesX and pSTDesR enable inducible expression from the XylS/Pm and RhaRS/PrhaB expression systems, respectively. Together, we hope these vector systems will support research in both the microbial SynBio and Pseudomonas field.
Topics: DNA; Genetic Vectors; Plasmids; Pseudomonas; Pseudomonas putida; Reproducibility of Results
PubMed: 34651450
DOI: 10.1111/1751-7915.13922 -
ELife Sep 2021Rapidly switching between similar antibiotics may help to slow down the evolution of resistance.
Rapidly switching between similar antibiotics may help to slow down the evolution of resistance.
Topics: Anti-Bacterial Agents; Pharmaceutical Preparations; Pseudomonas aeruginosa
PubMed: 34499033
DOI: 10.7554/eLife.72562 -
Scientific Reports Mar 2022Exudate production is a natural part of the wound healing process, however levels of exudate need to be appropriately managed to maintain a moist wound environment which...
Exudate production is a natural part of the wound healing process, however levels of exudate need to be appropriately managed to maintain a moist wound environment which supports healing. An overly-exuding wound creates an environment favourable to bacterial growth. In recent years, a significant increase in commercially available superabsorbent dressings have become available which claim to absorb and retain excess exudate and its components. However, the effectiveness of these dressings in sequestering and retaining bacteria and host-derived proteins has not been compared. We have therefore investigated several superabsorbent dressings for their ability to absorb and retain bacteria (Staphylococcus aureus and Pseudomonas aeruginosa), their impact on bacterial viability, and their ability to sequester matrix metalloproteinases (MMP)-2 and 9 over 7 days. Whilst all dressings could sequester bacteria, some dressings internalised bacteria more effectively. There was considerable variation in bacterial viability within the dressings' core, as well as differences in bacterial retention. Some dressings effectively internalised and retained bacteria over time, whereas other dressings retained significantly less. These differences were reflected visually using scanning electron microscopy. Most dressings fully sequestered MMP-2 and 9. These data illustrate differences in the ability of superabsorbent dressings to absorb and retain exudate and its components.
Topics: Bandages; Exudates and Transudates; Metalloproteases; Pseudomonas aeruginosa; Wound Healing
PubMed: 35306513
DOI: 10.1038/s41598-022-08361-3 -
Microbiology Spectrum Apr 2022In human medicine, antibiotics have been widely used to treat microbial infections. The extensive use of antibiotics is a leading cause of antibiotic resistance....
In human medicine, antibiotics have been widely used to treat microbial infections. The extensive use of antibiotics is a leading cause of antibiotic resistance. Currently, the influence of the use of antibiotics on the ocular surface microbiome in the course of keratitis treatment remains to be explored in depth. We performed metagenomic analyses in a cohort of 26 healthy controls (HCs), 28 keratitis patients (KPs) who received antibiotics [KP (abx+) group], and 12 KPs who were antibiotic naive [KP (abx-) group]. We identified that the dissimilarities in microbial community structure (Bray-Curtis and Jaccard analyses) between the KP (abx+) group and the HC group were greater than those between the KP (abx-) group and the HC group. Pseudomonas lactis, P. aeruginosa, Pseudomonas sp. , Pseudomonas sp. , Corynebacterium simulans, Streptococcus pyogenes, Finegoldia magna, and Aspergillus oryzae had no statistically significant differences between the KP (abx+) and KP (abx-) groups but did have statistically significant differences between the KP (abx+) and HC groups and between the KP (abx-) and HC groups. Among them, Pseudomonas lactis, P. aeruginosa, Pseudomonas sp. , and Pseudomonas sp. were identified as possible hosts carrying multidrug-resistant genes. The total abundance and number of antibiotic resistance genes (ARGs) were greater in the KP (abx+) group than in the HC and KP (abx-) groups. The functional profile analysis of ocular surface microbiota revealed that pathogenesis-related functional pathways and virulence functions were enriched in KPs. In conclusion, our results show that empirical antibiotic treatment in KPs leads to increases in the antibiotic resistance of ocular surface microbiota. Treatment for keratitis is based on appropriate antimicrobial therapy. A direct correlation between antibiotic use and the extent of antibiotic resistance has been reported. Therefore, knowledge of the antibiotic resistance patterns of ocular surface microbial flora in KPs is important for clinical treatment. To the best of our knowledge, this is the first study to use metagenomic approaches to investigate the associations between ophthalmic antibiotic use and the ocular surface microbiome of KPs. Monitoring the microbiota and antibiotic resistome profiles for the ocular surface has huge potential to help ophthalmologists choose the appropriate antibiotics and will thereby improve the efficacy of treatment regimens, which has important implications for reducing the development of antibiotic resistance of the ocular surface to a certain extent.
Topics: Anti-Bacterial Agents; Drug Resistance, Microbial; Humans; Keratitis; Microbiota; Pseudomonas; Pseudomonas aeruginosa
PubMed: 35293804
DOI: 10.1128/spectrum.02162-21 -
Nature Microbiology Dec 2023Counting viable cells is a universal practice in microbiology. The colony-forming unit (CFU) assay has remained the gold standard to measure viability across...
Counting viable cells is a universal practice in microbiology. The colony-forming unit (CFU) assay has remained the gold standard to measure viability across disciplines, but it is time-intensive and resource-consuming. Here we describe the geometric viability assay (GVA) that replicates CFU measurements over 6 orders of magnitude while reducing over 10-fold the time and consumables required. GVA computes a sample's viable cell count on the basis of the distribution of embedded colonies growing inside a pipette tip. GVA is compatible with Gram-positive and Gram-negative planktonic bacteria (Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis), biofilms and fungi (Saccharomyces cerevisiae). Laborious CFU experiments such as checkerboard assays, treatment time-courses and drug screens against slow-growing cells are simplified by GVA. The ease and low cost of GVA evinces that it can replace existing viability assays and enable viability measurements at previously impractical scales.
Topics: Colony Count, Microbial; Escherichia coli; Biofilms; Gram-Negative Bacteria; Pseudomonas aeruginosa
PubMed: 37919425
DOI: 10.1038/s41564-023-01513-9 -
Cell Metabolism Jun 2020Itaconate is an immunometabolite with anti-inflammatory and anti-microbial properties. Riquelme et al. (2020) demonstrate that pathogenic Pseudomonas aeruginosa drives...
Itaconate is an immunometabolite with anti-inflammatory and anti-microbial properties. Riquelme et al. (2020) demonstrate that pathogenic Pseudomonas aeruginosa drives itaconate production by macrophages, which it then uses as a carbon source for biofilm formation, allowing it to persist during infection and suppress inflammation.
Topics: Biofilms; Pseudomonas; Pseudomonas aeruginosa; Succinates
PubMed: 32492389
DOI: 10.1016/j.cmet.2020.05.005 -
Biotechnology and Bioengineering Jun 2022Biofilms are communities of bacterial cells encased in a self-produced polymeric matrix and exhibit high tolerance towards environmental stress. Despite the plethora of...
Biofilms are communities of bacterial cells encased in a self-produced polymeric matrix and exhibit high tolerance towards environmental stress. Despite the plethora of research on biofilms, most biofilm models are produced using mono-interface culture in static flow conditions, and knowledge of the effects of interfaces and mechanical forces on biofilm development remains fragmentary. This study elucidated the effects of air-liquid (ALI) or liquid-liquid (LLI) interfaces and mechanical shear forces induced by airflow and hydrodynamic flow on biofilm growing using a custom-designed dual-channel microfluidic platform. Results from this study showed that comparing biofilms developed under continuous nutrient supply and shear stresses free condition to those developed with limited nutrient supply, ALI biofilms were four times thicker, 60% less permeable, and 100 times more resistant to antibiotics, while LLI biofilms were two times thicker, 20% less permeable, and 100 times more resistant to antibiotics. Subjecting the biofilms to mechanical shear stresses affected the biofilm structure across the biofilm thickness significantly, resulting in generally thinner and denser biofilm compared to their controlled biofilm cultured in the absence of shear stresses, and the ALI and LLI biofilm's morphology was vastly different. Biofilms developed under hydrodynamic shear stress also showed increased antibiotic resistance. These findings highlight the importance of investigating biofilm growth and its mechanisms in realistic environmental conditions and demonstrate a feasible approach to undertake this study using a novel platform.
Topics: Anti-Bacterial Agents; Biofilms; Hydrodynamics; Pseudomonas aeruginosa; Stress, Mechanical
PubMed: 35274289
DOI: 10.1002/bit.28077