Accessory genome contributes to the virulence and resistance of the ocular isolate of Pseudomonas aeruginosa: A complete genome analysis

Bacteria can acquire an accessory genome through the horizontal transfer of genetic elements from non-parental lineages. This leads to rapid genetic evolution allowing traits such as antibiotic resistance and virulence to spread through bacterial communities. The study of complete genomes of bacterial strains helps to understand the genomic traits associated with virulence and antibiotic resistance. We aimed to investigate the complete accessory genome of an ocular isolate of P. aeruginosa. We obtained the complete genome of the ocular isolate strain PA34 of P. aeruginosa utilising genome sequence reads from Illumina and Oxford Nanopore Technology followed by PCR to close any identified gaps. In-depth genomic analysis was performed using various bioinformatics tools. The phenotypic properties of susceptibility to heavy metals and cytotoxicity were determined to confirm expression of certain traits. The complete genome of PA34 includes a chromosome of 6.8 Mbp and two plasmids of 95.4 Kbp (pMKPA34-1) and 26.8 Kbp (pMKPA34-2). PA34 had a large accessory genome of 1,213 genes and had 543 unique genes not present in other strains. These exclusive genes encoded features related to metal and antibiotic resistance, phage integrase and transposons. At least 24 GIs were predicated in the complete chromosome, of which two were integrated into novel sites. Eleven GIs carried virulence factors or replaced pathogenic genes. A bacteriophage carried the aminoglycoside resistance gene (aac(3)-IId). The two plasmids carried other six antibiotic resistance genes. The large accessory genome of this ocular isolate plays a large role in shaping its virulence and antibiotic resistance

Comparative genomics of clinical strains of Pseudomonas aeruginosa strains isolated from different geographic sites

© 2018, The Author(s). The large and complex genome of Pseudomonas aeruginosa, which consists of significant portions (up to 20%) of transferable genetic elements contributes to the rapid development of antibiotic resistance. The whole genome sequences of 22 strains isolated from eye and cystic fibrosis patients in Australia and India between 1992 and 2007 were used to compare genomic divergence and phylogenetic relationships as well as genes for antibiotic resistance and virulence factors. Analysis of the pangenome indicated a large variation in the size of accessory genome amongst 22 stains and the size of the accessory genome correlated with number of genomic islands, insertion sequences and prophages. The strains were diverse in terms of sequence type and dissimilar to that of global epidemic P. aeruginosa clones. Of the eye isolates, 62% clustered together within a single lineage. Indian eye isolates possessed genes associated with resistance to aminoglycoside, beta-lactams, sulphonamide, quaternary ammonium compounds, tetracycline, trimethoprims and chloramphenicols. These genes were, however, absent in Australian isolates regardless of source. Overall, our results provide valuable information for understanding the genomic diversity of P. aeruginosa isolated from two different infection types and countries

Glutathione Activates Type III Secretion System Through Vfr in Pseudomonas aeruginosa

Glutathione (GSH) is the most abundant antioxidant in all living organisms. Previously, we have shown that a deletion mutant in the glutathione synthetase gene (ΔgshB) decreases the expression of type III secretion system (T3SS) genes of Pseudomonas aeruginosa. However, the mechanism remains elusive. In this study, a comprehensive transcriptomic analysis of the GSH-deficient mutant ΔgshAΔgshB was used to elucidate the role of GSH in the pathogenesis of P. aeruginosa. The data show that the expression of genes in T3SS, type VI secretion system (T6SS) and some regulatory genes were impaired. ΔgshAΔgshB was attenuated in a mouse model of acute pneumonia, swimming and swarming motilities, and biofilm formation. Under T3SS inducing conditions, GSH enhanced the expression of T3SS in both wild-type PAO1 and ΔgshAΔgshB, but not in Δvfr. Genetic complementation of Δvfr restored the ability of GSH to induce the expression of T3SS genes. Site-directed mutagenesis based substitution of cysteine residues with alanine in Vfr protein abolished the induction of T3SS genes by GSH, confirming that GSH regulates T3SS genes through Vfr. Exposure to H2O2 decreased free thiol content on Vfr, indicating that the protein was sensitive to redox modification. Importantly, GSH restored the oxidized Vfr to reduced state. Collectively, these results suggest that GSH serves as an intracellular redox signal sensed by Vfr to upregulate T3SS expression in P. aeruginosa. Our work provides new insights into the role of GSH in P. aeruginosa pathogenesis

Diversity and genetics of Australasian dinoflagellates, including Gambierdiscus spp. the causative agent of Ciguatera Fish Poisoning

Marine microbial protists from benthic habitats include species of the genera Gambierdiscus, which can produce polyketide toxins with ecosystem-wide impacts and impacts on human health. Knowledge of the presence, diversity, effects and distribution of benthic dinoflagellates and their toxins is vital both for basic ecological research and for managing potential impacts on human health. However, at present such information is limited. This study designed novel pyrosequencing-based tools that utilised 18S rRNA and mitochondrial cytochrome b barcoding marker genes. Using these tools, the largest set of dinoflagellate cytochrome b gene sequences to date was developed, and dinoflagellate species richness within marine benthic samples from five tropical marine benthic sites were determined. These tools were applied to samples from a temperate marine benthic site from southern NSW and the species Gambierdiscus carpenteri was found to be highly abundant throughout the region. This was unexpected, given that it is primarily considered a tropical species. The significance of finding this species is discussed in the context of long term monitoring data and its potential for toxin production.Species of Gambierdiscus produce two major toxin groups, ciguatoxins (CTXs) and maitotoxins (MTXs), that can accumulate via marine food chains in seafood and ultimately cause ciguatera fish poisoning (CFP) in humans. While the role of CTXs is well established in CFP, little is known about the role of MTXs. This work showed experimentally that MTXs can accumulate in liver and muscle tissues of carnivorous fish (Pagrus auratus). The levels of MTX detected in fish tissues showed for the first time that MTX may accumulate in fish at levels harmful for human consumption. In order to investigate the genetic basis of toxin biosynthesis in species of Gambierdiscus, two comprehensive transcriptomic libraries for the species Gambierdiscus australes and Gambierdiscus belizeanus, which produce MTXs were sequenced and assembled. Data analysis discovered three hundred and seven genes that formed eight clades within a phylogeny of dinoflagellate polyketide synthesis genes, and may be involved in MTX biosynthesis. This is the first step towards identifying prospective genes that may be involved in MTX biosynthesis, and provides a basis for the future development of molecular tools to assess the risk of CFP

Stress resistance development and genome-wide transcriptional response of escherichia coli O157:H7 adapted to sublethal thymol, carvacrol, and trans -cinnamaldehyde

Thymol, carvacrol, and trans-cinnamaldehyde are essential oil (EO) compounds with broad-spectrum antimicrobial activities against foodborne pathogens, including Escherichia coli O157:H7. However, little is known regarding direct resistance and cross-resistance development in E. coli O157:H7 after adaptation to sublethal levels of these compounds, and information is scarce on microbial adaptive responses at a molecular level. The present study demonstrated that E. coli O157:H7 was able to grow in the presence of sublethal thymol (1/2T), carvacrol (1/2C), or trans-cinnamaldehyde (1/2TC), displaying an extended lag phase duration and a lower maximum growth rate. EO-adapted cells developed direct resistance against lethal EO treatments and cross-resistance against heat (58°C) and oxidative (50 mM H2O2) stresses. However, no induction of acid resistance (simulated gastric fluid, pH 1.5) was observed. RNA sequencing revealed a large number (310 to 338) of differentially expressed (adjusted P value [Padj], <0.05; fold change, ≥5) genes in 1/2T and 1/2C cells, while 1/2TC cells only showed 27 genes with altered expression. In accordance with resistance phenotypes, the genes related to membrane, heat, and oxidative stress responses and genes related to iron uptake and metabolism were upregulated. Conversely, virulence genes associated with motility, biofilm formation, and efflux pumps were repressed. This study demonstrated the development of direct resistance and cross-resistance and characterized whole-genome transcriptional responses in E. coli O157:H7 adapted to sublethal thymol, carvacrol, or trans-cinnamaldehyde. The data suggested that caution should be exercised when using EO compounds as food antimicrobials, due to the potential stress resistance development in E. coli O157:H7.Published versio

Polyketide synthase genes and molecular trade-offs in the ichthyotoxic species Prymnesium parvum

Prymnesium parvum is a bloom forming haptophyte that has been responsible for numerous fish kill events across the world. The toxicity of P. parvum has been attributed to the production of large polyketide compounds, collectively called prymnesins, which based on their structure can be divided into A-, B- and C-type. The polyketide chemical nature of prymnesins indicates the potential involvement of polyketide synthases (PKSs) in their biosynthesis. However, little is known about the presence of PKSs in P. parvum as well as the potential molecular trade-offs of toxin biosynthesis. In the current study, we generated and analyzed the transcriptomes of nine P. parvum strains that produce different toxin types and have various cellular toxin contents. Numerous type I PKSs, ranging from 37 to 109, were found among the strains. Larger modular type I PKSs were mainly retrieved from strains with high cellular toxin levels and eight consensus transcripts were present in all nine strains. Gene expression variance analysis revealed potential molecular trade-offs associated with cellular toxin quantity, showing that basic metabolic processes seem to correlate negatively with cellular toxin content. These findings point towards the presence of metabolic costs for maintaining high cellular toxin quantity. The detailed analysis of PKSs in P. parvum is the first step towards better understanding the molecular basis of the biosynthesis of prymnesins and contributes to the development of molecular tools for efficient monitoring of future blooms

Graphene Oxide Mimics Biological Signaling Cue to Rescue Starving Bacteria

There is extensive debate about how 2D nanomaterials such as graphene oxide (GO) affect bacteria. Various effects of GO are proposed, including bacterial growth inhibition or enhancement, killing, and no activity. Herein, we report that GO protects Staphylococcus aureus bacterial cells from death in starvation conditions with up to a 1000-fold improvement in cell viability. Transcriptomic profiling reveals that bacterial cells in starvation conditions generally shut down metabolic activity, while only cells incubated with GO increase production of specific enzymes involved in the glyoxalase detoxification pathway along with repressed autolysis. The oxygen-containing functional groups of GO resemble the molecular structure of methylglyoxal, which bacteria produce to adapt to nutrient imbalances and is detoxified by glyoxalase enzymes. The ability of GO to enable bacterial cell survival in starvation conditions and accompanying cellular responses support that bacterial cells perceive GO as a methylglyoxal-mimicking nanomaterial cue to reshuffle cellular metabolism and defenses.National Research Foundation (NRF)This work was supported by the National Research Foundation of Singapore through a Competitive Research Programme grant (NRF-CRP10-2012-07) and a Proof-of-Concept grant (NRF2015NRF-POC0001-19), the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT) (No. 2020R1C1C1004385), and the Ministry of Education, Youth and Sports grant LL2002 under the ERC CZ program. In addition, this work was supported by the Brain Pool Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2019H1D3A1A01070318)

Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa

Pseudomonas aeruginosa is generally believed to establish biofilm-associated infections under the regulation of the secondary messenger c-di-GMP. To evaluate P. aeruginosa biofilm physiology during ocular infections, comparative transcriptomic analysis was performed on wild-type P. aeruginosa PAO1, a Delta wspF mutant strain (high c-di-GMP levels), and a p(lac)-yhjH-containing strain (low c-di-GMP levels) from mouse corneal infection, as well as in vitro biofilm and planktonic cultures. The c-di-GMP content in P. aeruginosa during corneal infection was monitored using a fluorescent c-di-GMP reporter strain. Biofilm-related genes were induced in in vivo PAO1 compared to in vitro planktonic bacteria. Several diguanylate cyclases and phosphodiesterases were commonly regulated in in vivo PAO1 and in vitro biofilm compared to in vitro planktonic bacteria. Several exopolysaccharide genes and motility genes were induced and downregulated, respectively, in in vivo PAO1 and the in vivo Delta wspF mutant compared to the in vivo p(lac)-yhjH-containing strain. Elevation of c-di-GMP levels in P. aeruginosa began as early as 2 h postinfection. The Delta wspF mutant was less susceptible to host clearance than the p(lac)-yhjH-containing strain and could suppress host immune responses. The type III secretion system (T3SS) was induced in in vivo PAO1 compared to in vitro biofilm bacteria. Delta wspF mutant with a defective T3SS was more susceptible to host clearance than Delta wspF mutant with a functional T3SS. Our study suggests that elevated intracellular c-di-GMP levels and T3SS activity in P. aeruginosa are necessary for establishment of infection and modulation of host immune responses in mouse cornea.ISSN:0019-9567ISSN:1098-552