David Drissner

Bio: David Drissner is an academic researcher. The author has contributed to research in topics: Phyllosphere & Plasmid. The author has an hindex of 6, co-authored 11 publications receiving 87 citations.

Complete genome sequence of Pseudomonas citronellolis P3B5, a candidate for microbial phyllo-remediation of hydrocarbon-contaminated sites

Abstract: Pseudomonas citronellolis is a Gram negative, motile gammaproteobacterium belonging to the order Pseudomonadales and the family Pseudomonadaceae. We isolated strain P3B5 from the phyllosphere of basil plants (Ocimum basilicum L.). Here we describe the physiology of this microorganism, its full genome sequence, and detailed annotation. The 6.95 Mbp genome contains 6071 predicted protein coding sequences and 96 RNA coding sequences. P. citronellolis has been the subject of many studies including the investigation of long-chain aliphatic compounds and terpene degradation. Plant leaves are covered by long-chain aliphates making up a waxy layer that is associated with the leaf cuticle. In addition, basil leaves are known to contain high amounts of terpenoid substances, hinting to a potential nutrient niche that might be exploited by P. citronellolis. Furthermore, the isolated strain exhibited resistance to several antibiotics. To evaluate the potential of this strain as source of transferable antibiotic resistance genes on raw consumed herbs we therefore investigated if those resistances are encoded on mobile genetic elements. The availability of the genome will be helpful for comparative genomics of the phylogenetically broad pseudomonads, in particular with the sequence of the P. citronellolis type strain PRJDB205 not yet publicly available. The genome is discussed with respect to a phyllosphere related lifestyle, aliphate and terpenoid degradation, and antibiotic resistance.

MiniTn7-transposon delivery vectors for inducible or constitutive fluorescent protein expression in Enterobacteriaceae.

Abstract: Here we present the generation and function of two sets of bacterial plasmids that harbor fluorescent genes encoding either blue, cyan, yellow or red fluorescent proteins. In the first set, protein expression is controlled by the strong and constitutive nptII promoter whereas in the second set, the strong tac promoter was chosen that underlies LacIq regulation. Furthermore, the plasmids are mobilizable, contain Tn 7 transposons and a temperature-sensitive origin of replication. Using Escherichia coli S17-1 as donor strain, the plasmids allow fast and convenient Tn 7 -transposon delivery into many enterobacterial hosts, such as the here-used E. coli O157:H7. This procedure omits the need of preparing competent recipient cells and antibiotic resistances are only transiently conferred to the recipients. As the fluorescence proteins show little to no overlap in fluorescence emission, the constructs are well suited for the study of multicolored synthetic bacterial communities during biofilm production or in host colonization studies, e.g. of plant surfaces. Furthermore, tac promoter-reporter constructs allow the generation of so-called reproductive success reporters, which allow to estimate past doublings of bacterial individuals after introduction into environments, emphasizing the role of individual cells during colonization.

MALDI-TOF mass spectroscopy of yeasts and filamentous fungi for research and diagnostics in the agricultural value chain

Abstract: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; MALDI biotyping) has become a standard tool for the accurate, rapid, and economical identification of pathogens in the clinical diagnostics laboratory. The method is continuously being improved, and new applications for distinguishing strains, identifying metabolites or functional characteristics (e.g., antibiotic resistance), and detecting microbes directly in patient samples have been developed. Adopting these methods in other disciplines than clinical diagnostics, for example, in agriculture, food safety and quality testing, or ecology, will open up new opportunities for diagnostics and research. This review focuses on MALDI-TOF MS approaches for the identification of yeasts and filamentous fungi. In contrast to bacterial diagnostics, MALDI biotyping of fungi is more challenging and less established. We thus start by discussing the role of MALDI-TOF MS as a tool for species identification; in particular with respect to DNA-based identification methods. The review then highlights the value of custom-made reference spectra for MALDI biotyping and points out recent advancements of MALDI-TOF MS, mainly from the field of clinical diagnostics that may be adopted and used for fungal diagnostic challenges. The overview ends with a summary of MALDI-TOF MS studies of yeasts and filamentous fungi of agricultural relevance.

Dynamics of culturable mesophilic bacterial communities of three fresh herbs and their production environment

Abstract: Aim Investigate dynamics of culturable mesophilic bacteria and selected food contaminating bacteria from three herbs and their production environment. Methods and Results Marjoram, basil, and thyme were investigated during one growing season by sampling plants, organic fertilizers, soil, irrigation water, and marketed products. Mesophilic bacteria and selected food contaminating bacteria (Escherichia coli, Enterococcus spp., Bacillus cereus group) were cultured and identified by MALDI biotyping. Culturable mesophilic bacteria on marjoram and basil plants decreased over time by two orders of magnitude starting at above 106 colony forming units per gram (CFU per g), while they remained constant on thyme (~ 104 CFU per g). Compared to the last field sample, mesophilic bacteria were increased on all market-ready products by one order of magnitude. Marjoram and basil were dominated by B. cereus group, Enterobacter spp., and Pseudomonas spp., thyme by Bacillus spp. and Pseudomonas spp. All selected food contaminating bacteria were detected in soil and reservoir-sourced irrigation water, whereas in municipal water, only B. cereus group and rarely Enterococcus spp. were found. E. coli was detected only on young marjoram and basil plants (5 × 102 and 5 × 101 CFU per g, respectively), whereas Enterococcus spp. and B. cereus group were consistently detected on these two herbs. Thyme plants only contained B. cereus group consistently (above 103 CFU per g). Marketed marjoram and thyme contained Enterococcus spp. (5 × 102 and 104 CFU per g) and B. cereus group (~ 5 × 102 CFU per g), while no selected food contaminating bacteria were found on marketed basil. Conclusions Overall, culturable mesophilic bacteria were dominated by Pseudomonas spp. and Bacillus spp., with increased numbers on market-ready products. Selected food contaminating bacteria were readily detectable, however, only the B. cereus group was found throughout in all systems. Significance and Impact of the Study Insight into composition and development of mesophilic bacterial communities and selected food contaminating bacteria of fresh herbs contributes to estimating consumer exposure. This article is protected by copyright. All rights reserved.

Transferable Extended-Spectrum β-Lactamase (ESBL) Plasmids in Enterobacteriaceae from Irrigation Water.

Abstract: Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae are classified as serious threats to human health by the US Centers for Disease Control and Prevention Water used for irrigation of fresh produce can transmit such resistant bacteria directly to edible plant parts We screened ESBL-producing Escherichia coli, Enterobacter cloacae, and Citrobacter freundii isolated from irrigation water for their potential to transmit resistance to antibiotic-susceptible E coli All strains were genome-sequenced and tested in vitro for transmission of resistance to third-generation cephalosporins on solid agar as well as in liquid culture Of the 19 screened isolates, five ESBL-producing E coli were able to transfer resistance with different efficiency to susceptible recipient E coli Transconjugant strains were sequenced for detection of transferred antibiotic resistance genes (ARGs) and compared to the known ARG pattern of their respective donors Additionally, phenotypic resistance patterns were obtained for both transconjugant and corresponding donor strains, confirming ESBL-producing phenotypes of all obtained transconjugants

FEMS microbiology letters

Abstract: All aspects of microbiology, including virology, are covered. Areas of special interest include: physiology, biochemistry and genetics (including molecular biology and 'omic' studies); biotechnology and synthetic biology; pathogenicity (including medical, veterinary and plant pathogens particularly those relating to food security); environmental microbiology (including ecophysiology, ecogenomics and meta-omic studies); virology; food microbiology (from food production and spoilage to food-borne pathogens); taxonomy and systematics (including publication of novel species and taxonomic reclassifications), and professional development (including education, training, CPD, research assessment frameworks, research metrics, best-practice and history of microbiology).

Selection for antimicrobial resistance is reduced when embedded in a natural microbial community

Abstract: Antibiotic resistance has emerged as one of the most pressing, global threats to public health. In single-species experiments selection for antibiotic resistance occurs at very low antibiotic concentrations. However, it is unclear how far these findings can be extrapolated to natural environments, where species are embedded within complex communities. We competed isogenic strains of Escherichia coli, differing exclusively in a single chromosomal resistance determinant, in the presence and absence of a pig faecal microbial community across a gradient of antibiotic concentration for two relevant antibiotics: gentamicin and kanamycin. We show that the minimal selective concentration was increased by more than one order of magnitude for both antibiotics when embedded in the community. We identified two general mechanisms were responsible for the increase in minimal selective concentration: an increase in the cost of resistance and a protective effect of the community for the susceptible phenotype. These findings have implications for our understanding of the evolution and selection of antibiotic resistance, and can inform future risk assessment efforts on antibiotic concentrations.

Pushing the limits of de novo genome assembly for complex prokaryotic genomes harboring very long, near identical repeats.

Abstract: Generating a complete, de novo genome assembly for prokaryotes is often considered a solved problem. However, we here show that Pseudomonas koreensis P19E3 harbors multiple, near identical repeat pairs up to 70 kilobase pairs in length, which contained several genes that may confer fitness advantages to the strain. Its complex genome, which also included a variable shufflon region, could not be de novo assembled with long reads produced by Pacific Biosciences' technology, but required very long reads from Oxford Nanopore Technologies. Importantly, a repeat analysis, whose results we release for over 9600 prokaryotes, indicated that very complex bacterial genomes represent a general phenomenon beyond Pseudomonas. Roughly 10% of 9331 complete bacterial and a handful of 293 complete archaeal genomes represented this 'dark matter' for de novo genome assembly of prokaryotes. Several of these 'dark matter' genome assemblies contained repeats far beyond the resolution of the sequencing technology employed and likely contain errors, other genomes were closed employing labor-intense steps like cosmid libraries, primer walking or optical mapping. Using very long sequencing reads in combination with assembly algorithms capable of resolving long, near identical repeats will bring most prokaryotic genomes within reach of fast and complete de novo genome assembly.

Competition assays and physiological experiments of soil and phyllosphere yeasts identify Candida subhashii as a novel antagonist of filamentous fungi.

Abstract: While recent advances in next generation sequencing technologies have enabled researchers to readily identify countless microbial species in soil, rhizosphere, and phyllosphere microbiomes, the biological functions of the majority of these species are unknown. Functional studies are therefore urgently needed in order to characterize the plethora of microorganisms that are being identified and to point out species that may be used for biotechnology or plant protection. Here, we used a dual culture assay and growth analyses to characterise yeasts (40 different isolates) and their antagonistic effect on 16 filamentous fungi; comprising plant pathogens, antagonists, and saprophytes. Overall, this competition screen of 640 pairwise combinations revealed a broad range of outcomes, ranging from small stimulatory effects of some yeasts up to a growth inhibition of more than 80% by individual species. On average, yeasts isolated from soil suppressed filamentous fungi more strongly than phyllosphere yeasts and the antagonistic activity was a species-/isolate-specific property and not dependent on the filamentous fungus a yeast was interacting with. The isolates with the strongest antagonistic activity were Metschnikowia pulcherrima, Hanseniaspora sp., Cyberlindnera sargentensis, Aureobasidium pullulans, Candida subhashii, and Pichia kluyveri. Among these, the soil yeasts (C. sargentensis, A. pullulans, C. subhashii) assimilated and/or oxidized more di-, tri- and tetrasaccharides and organic acids than yeasts from the phyllosphere. Only the two yeasts C. subhashii and M. pulcherrima were able to grow with N-acetyl-glucosamine as carbon source. The competition assays and physiological experiments described here identified known antagonists that have been implicated in the biological control of plant pathogenic fungi in the past, but also little characterised species such as C. subhashii. Overall, soil yeasts were more antagonistic and metabolically versatile than yeasts from the phyllosphere. Noteworthy was the strong antagonistic activity of the soil yeast C. subhashii, which had so far only been described from a clinical sample and not been studied with respect to biocontrol. Based on binary competition assays and growth analyses (e.g., on different carbon sources, growth in root exudates), C. subhashii was identified as a competitive and antagonistic soil yeast with potential as a novel biocontrol agent against plant pathogenic fungi.

Chromatic Bacteria - A Broad Host-Range Plasmid and Chromosomal Insertion Toolbox for Fluorescent Protein Expression in Bacteria.

Abstract: Differential fluorescent labelling of bacteria has become instrumental for many aspects of microbiological research, such as the study of biofilm formation, bacterial individuality, evolution, and bacterial behaviour in complex environments. We designed a variety of plasmids, each bearing one of eight unique, constitutively expressed fluorescent protein genes in conjunction with one of four different antibiotic resistance combinations. The fluorophores mTagBFP2, mTurquoise2, sGFP2, mClover3, sYFP2, mOrange2, mScarlet-I, and mCardinal, encoding for blue, cyan, green, green-yellow, yellow, orange, red, and far-red fluorescent proteins, respectively, were combined with selectable markers conferring tetracycline, gentamicin, kanamycin, and/or chloramphenicol resistance. These constructs were cloned into three different plasmid backbones: a broad host-range plasmid, a Tn5 transposon delivery plasmid, and a Tn7 transposon delivery plasmid. The utility of the plasmids and transposons was tested in bacteria from the phyla Actinobacteria, Proteobacteria, and Bacteroidetes. We were able to tag representatives from the phylum Proteobacteria at least via our Tn5 transposon delivery system. The present study enables labelling bacteria with a set of plasmids available to the community. One potential application of fluorescently-tagged bacterial species is the study of bacteria-bacteria, bacteria-host, and bacteria-environment interactions.