Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Murray, S. A., Ruvindy, R., Kohli, G. S., Anderson, D. M., & Brosnahan, M. L. Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1. Scientific Reports, 9(1), (2019): 14532, doi:10.1038/s41598-019-51074-3.Alexandrium catenella (formerly A. tamarense Group 1, or A. fundyense) is the leading cause of Paralytic Shellfish Poisoning in North and South America, Europe, Africa, Australia and Asia. The quantification of A.catenella via sxtA, a gene involved in Paralytic Shellfish Toxin synthesis, may be a promising approach, but has not been evaluated in situ on blooms of A. catenella, in which cell abundances may vary from not detectable to in the order of 106 cells L−1. In this study, we compared sxtA assay performance to a qPCR assay targeted to a species-specific region of ribosomal DNA (rDNA) and an established fluorescent in situ hybridization (FISH) microscopy method. Passing-Bablok regression analyses revealed the sxtA assay to overestimate abundances when <5 cell equivalents A. catenella DNA were analysed, but otherwise was closer to microscopy estimates than the rDNA assay, which overestimated abundance across the full range of concentrations analysed, indicative of a copy number difference between the bloom population and a culture used for assay calibration a priori. In contrast, the sxtA assay performed more consistently, indicating less copy number variation. The sxtA assay was generally reliable, fast and effective in quantifying A. catenella and was predictive of PST contamination of shellfish.We thank the Australian Research Council for Funding (FT120100704). We thank Chowdhury Sarowar for the toxicity measurements, at the Sydney Institute of Marine Science. Support to MB and DA was provided by MIT Sea Grant (NA14OAR4170077) and the Woods Hole Center for Oceans and Human Health (National Science Foundation award OCE-1840381 and National Institute of Environmental Health Sciences award 1-P01-ES028938–01). We are grateful for assistance from David Kulis, Claire Mullen, and Isaac Rosenthal for assistance in the collection and processing of Salt Pond samples

Evolutionary distinctiveness of fatty acid and polyketide synthesis in eukaryotes

© 2016 International Society for Microbial Ecology All rights reserved. Fatty acids, which are essential cell membrane constituents and fuel storage molecules, are thought to share a common evolutionary origin with polyketide toxins in eukaryotes. While fatty acids are primary metabolic products, polyketide toxins are secondary metabolites that are involved in ecologically relevant processes, such as chemical defence, and produce the adverse effects of harmful algal blooms. Selection pressures on such compounds may be different, resulting in differing evolutionary histories. Surprisingly, some studies of dinoflagellates have suggested that the same enzymes may catalyse these processes. Here we show the presence and evolutionary distinctiveness of genes encoding six key enzymes essential for fatty acid production in 13 eukaryotic lineages for which no previous sequence data were available (alveolates: dinoflagellates, Vitrella, Chromera; stramenopiles: bolidophytes, chrysophytes, pelagophytes, raphidophytes, dictyochophytes, pinguiophytes, xanthophytes; Rhizaria: chlorarachniophytes, haplosporida; euglenids) and 8 other lineages (apicomplexans, bacillariophytes, synurophytes, cryptophytes, haptophytes, chlorophyceans, prasinophytes, trebouxiophytes). The phylogeny of fatty acid synthase genes reflects the evolutionary history of the organism, indicating selection to maintain conserved functionality. In contrast, polyketide synthase gene families are highly expanded in dinoflagellates and haptophytes, suggesting relaxed constraints in their evolutionary history, while completely absent from some protist lineages. This demonstrates a vast potential for the production of bioactive polyketide compounds in some lineages of microbial eukaryotes, indicating that the evolution of these compounds may have played an important role in their ecological success

Role of Modular Polyketide Synthases in the Production of Polyether Ladder Compounds in Ciguatoxin-Producing Gambierdiscus polynesiensis and G. excentricus (Dinophyceae).

Gambierdiscus, a benthic dinoflagellate, produces ciguatoxins that cause the human illness Ciguatera. Ciguatoxins are polyether ladder compounds that have a polyketide origin, indicating that polyketide synthases (PKS) are involved in their production. We sequenced transcriptomes of Gambierdiscus excentricus and Gambierdiscus polynesiensis and found 264 contigs encoding single domain ketoacyl synthases (KS; G. excentricus: 106, G. polynesiensis: 143) and ketoreductases (KR; G. excentricus: 7, G. polynesiensis: 8) with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 24 contigs (G. excentricus: 3, G. polynesiensis: 21) encoding multiple PKS domains (forming typical type I PKSs modules) were found. The proposed structure produced by one of these megasynthases resembles a partial carbon backbone of a polyether ladder compound. Seventeen contigs encoding single domain KS, KR, s-malonyltransacylase, dehydratase and enoyl reductase with sequence similarity to type II fatty acid synthases (FAS) in plants were found. Type I PKS and type II FAS genes were distinguished based on the arrangement of domains on the contigs and their sequence similarity and phylogenetic clustering with known PKS/FAS genes in other organisms. This differentiation of PKS and FAS pathways in Gambierdiscus is important, as it will facilitate approaches to investigating toxin biosynthesis pathways in dinoflagellates.Versión del edito

Metrological performance of optical coordinate measuring machines under industrial conditions

Species of the genus Gambierdiscus are epiphytic dinoflagellates well known from tropical coral reef areas at water temperatures from 24 to 29 °C. Gambierdiscus spp. are able to produce ciguatoxins (CTXs) known to bioaccumulate in fish, and the ingestion of tropical fish that accumulated CTXs and possibly also maitotoxins (MTXs) can cause ciguatera fish poisoning (CFP) in humans. In Australia, ciguatera poisonings have been reported in tropical parts of Queensland and the Northern Territory. Here, we report for the first time the seasonal abundance (April–May 2012/13) of Gambierdiscus spp. (up to 6565–8255 cells g^-1 wet weight algae) from Merimbula and Wagonga Inlets in temperate southern New South Wales, Australia (37° S) at water temperatures of 16.5–17 °C. These are popular shellfish aquaculture and recreational fisheries areas with no reports of ciguatera poisoning. Sequencing of a region of the 28S rRNA gene led to the conclusive identification of Gambierdiscus carpenteri. The cells differed however from the Belize type description, including the absence of a thecal groove, dorsal rostrum and variable hatchet- to rectangular-shaped 2' plate, and were morphologically more similar to Gambierdiscus toxicus. To study the dinoflagellate community structure in detail, a pyrosequencing approach based on the 18S rRNA gene was applied, which confirmed the presence of a single Gambierdiscus species only. Neither CTXs nor MTXs were detected in natural bloom material by LC–MS/MS; however, the extracts were found to be toxic via mouse-bioassay, with symptoms suggestive of poisoning by MTX-like compounds. Understanding the abundance of Gambierdiscus populations in areas with no apparent human health impacts is important towards defining the alternate conditions where sparse populations can create ciguatera problems

Characterization of a novel multidrug resistance plasmid pSGB23 isolated from Salmonella enterica subspecies enterica serovar Saintpaul

Abstract Background Salmonella enterica subspecies enterica serovar Saintpaul (S. Saintpaul) is an important gut pathogen which causes salmonellosis worldwide. Although intestinal salmonellosis is usually self-limiting, it can be life-threatening in children, the elderlies and immunocompromised patients. Appropriate antibiotic treatment is therefore required for these patients. However, the efficacy of many antibiotics on S. enterica infections has been greatly compromised due to spreading of multidrug resistance (MDR) plasmids, which poses serious threats on public health and needs to be closely monitored. In this study, we sequenced and fully characterized an S. enterica MDR plasmid pSGB23 isolated from chicken. Results Complete genome sequence analysis revealed that S. Saintpaul strain SGB23 harbored a 254 kb megaplasmid pSGB23, which carries 11 antibiotic resistance genes responsible for resistance to 9 classes of antibiotics and quaternary ammonium compounds that are commonly used to disinfect food processing facilities. Furthermore, we found that pSGB23 carries multiple conjugative systems, which allow it to spread into other Enterobacteriaceae spp. by self-conjugation. It also harbors multiple types of replicons and plasmid maintenance and addictive systems, which explains its broad host range and stable inheritance. Conclusions We report here a novel MDR plasmid pSGB23 harboured by S. enterica. To our knowledge, it carried the greatest number of antibiotic resistance genes with the broadest range of resistance spectrum among S. enterica MDR plasmids identified so far. The isolation of pSGB23 from food sources is worrisome, while surveillance on its further spreading will be carried out based on the findings reported in this study

Reduced Intracellular c-di-GMP Content Increases Expression of Quorum Sensing-Regulated Genes in Pseudomonas aeruginosa

Cyclic-di-GMP (c-di-GMP) is an intracellular secondary messenger which controls the biofilm life cycle in many bacterial species. High intracellular c-di-GMP content enhances biofilm formation via the reduction of motility and production of biofilm matrix, while low c-di-GMP content in biofilm cells leads to increased motility and biofilm dispersal. While the effect of high c-di-GMP levels on bacterial lifestyles is well studied, the physiology of cells at low c-di-GMP levels remains unclear. Here, we showed that Pseudomonas aeruginosa cells with high and low intracellular c-di-GMP contents possessed distinct transcriptome profiles. There were 535 genes being upregulated and 432 genes downregulated in cells with low c-di-GMP, as compared to cells with high c-di-GMP. Interestingly, both rhl and pqs quorum-sensing (QS) operons were expressed at higher levels in cells with low intracellular c-di-GMP content compared with cells with higher c-di-GMP content. The induced expression of pqs and rhl QS required a functional PqsR, the transcriptional regulator of pqs QS. Next, we observed increased production of pqs and rhl-regulated virulence factors, such as pyocyanin and rhamnolipids, in P. aeruginosa cells with low c-di-GMP levels, conferring them with increased intracellular survival rates and cytotoxicity against murine macrophages. Hence, our data suggested that low intracellular c-di-GMP levels in bacteria could induce QS-regulated virulence, in particular rhamnolipids that cripple the cellular components of the innate immune system

High abundance of the potentially maitotoxic dinoflagellate Gambierdiscus carpenteri in temperate waters of New South Wales, Australia

Species of the genus Gambierdiscus are epiphytic dinoflagellates well known from tropical coral reef areas at water temperatures from 24 to 29°C. Gambierdiscus spp. are able to produce ciguatoxins (CTXs) known to bioaccumulate in fish, and the ingestion of tropical fish that accumulated CTXs and possibly also maitotoxins (MTXs) can cause ciguatera fish poisoning (CFP) in humans. In Australia, ciguatera poisonings have been reported in tropical parts of Queensland and the Northern Territory. Here, we report for the first time the seasonal abundance (April-May 2012/13) of Gambierdiscus spp. (up to 6565-8255cellsg-1 wet weight algae) from Merimbula and Wagonga Inlets in temperate southern New South Wales, Australia (37°S) at water temperatures of 16.5-17°C. These are popular shellfish aquaculture and recreational fisheries areas with no reports of ciguatera poisoning. Sequencing of a region of the 28S rRNA gene led to the conclusive identification of Gambierdiscus carpenteri. The cells differed however from the Belize type description, including the absence of a thecal groove, dorsal rostrum and variable hatchet- to rectangular-shaped 2' plate, and were morphologically more similar to Gambierdiscus toxicus. To study the dinoflagellate community structure in detail, a pyrosequencing approach based on the 18S rRNA gene was applied, which confirmed the presence of a single Gambierdiscus species only. Neither CTXs nor MTXs were detected in natural bloom material by LC-MS/MS; however, the extracts were found to be toxic via mouse-bioassay, with symptoms suggestive of poisoning by MTX-like compounds. Understanding the abundance of Gambierdiscus populations in areas with no apparent human health impacts is important towards defining the alternate conditions where sparse populations can create ciguatera problems. © 2014 Elsevier B.V

Supplementary Figures and Tables from The rapid <i>in vivo</i> evolution of <i>Pseudomonas aeruginosa</i> in ventilator-associated pneumonia patients leads to attenuated virulence

<i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that causes severe airway infections in humans. These infections are usually difficult to treat and associated with high mortality rate. While colonizing the human airways, <i>P. aeruginosa</i> could accumulate genetic mutations that often lead to its better adaptability to the host environment. Understanding these evolutionary traits may provide important clues for the development of effective therapies to treat <i>P. aeruginosa</i> infections. In this study, 25 <i>P. aeruginosa</i> isolates were longitudinally sampled from the airways of four ventilator-associated pneumonia (VAP) patients. Pacbio and Illumina sequencing were used to analyse the <i>in vivo</i> evolutionary trajectories of these isolates. Our analysis showed that positive selection dominantly shaped <i>P. aeruginosa</i> genomes during VAP infections and led to three convergent evolution events, including loss-of-function mutations of <i>lasR</i> and <i>mpl</i>, and a pyoverdine-deficient phenotype. Specifically, <i>lasR</i> encodes one of the major transcriptional regulators in quorum sensing, whereas <i>mpl</i> encodes an enzyme responsible for recycling cell wall peptidoglycan. We also found that <i>P. aeruginosa</i> isolated at late stages of VAP infections produce less elastase and are less virulent <i>in vivo</i> than their earlier isolated counterparts, suggesting the short-term <i>in vivo</i> evolution of <i>P. aeruginosa</i> leads to attenuated virulence