Microorganisms

13 pages, 2984 KiB

Open AccessArticle

The Influence of Light and Nutrient Starvation on Morphology, Biomass and Lipid Content in Seven Strains of Green Microalgae as a Source of Biodiesel

by Lorenza Rugnini, Catia Rossi, Simonetta Antonaroli, Arnold Rakaj and Laura Bruno

Microorganisms 2020, 8(8), 1254; https://doi.org/10.3390/microorganisms8081254 - 18 Aug 2020

Cited by 17 | Viewed by 3752

Abstract

The development of clean and renewable energy sources is currently one of the most important challenges facing the world. Although research interests in algae-based energy have been increasing in the last decade, only a small percentage of the bewildering diversity exhibited by microalgae [...] Read more.

The development of clean and renewable energy sources is currently one of the most important challenges facing the world. Although research interests in algae-based energy have been increasing in the last decade, only a small percentage of the bewildering diversity exhibited by microalgae has been investigated for biodiesel production. In this work, seven strains of green microalgae belonging to the genera Scenedesmus, Tetradesmus and Desmodesmus were grown in liquid medium with or without a nitrogen (N) source—at two different irradiances (120 ± 20 and 200 ± 20 μmol photons m⁻² s⁻¹)—to evaluate biomass production and FAME (fatty acid methyl esters) content for biodiesel production. The strains of Tetradesmus obliquus and Desmodesmus abundans grown in N-deprived medium showed the highest FAME content (22.0% and 34.6%, respectively); lipid profile characterization highlighted the abundance of saturated FAME (as C16:0 and C18:0) that favors better viscosity (flow properties) and applicability of biodiesel at low temperatures. Light microscopy and confocal laser scanning microscopy observations were employed as a fast method to monitor the vital status of cells and lipid droplet accumulation after Nile red staining in different culture conditions. Full article

(This article belongs to the Section Microbial Biotechnology)

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Figure 1
Biomass productivity (gDW L−1 day−1) obtained during the first run for all the strains employed in this study at the two different irradiances (values are means of 3 measurements ± standard deviation). Full article ">Figure 2
Biomass productivity, as grams of dry weight for liters for day, obtained for the seven strains grown at (a) 120 ± 20 µmol photons m−2 s−1 (L120) and (b) 200 ± 20 µmol photons m−2 s−1 (L200) with (+N) and without (−N) a nitrogen source. Full article ">Figure 3
Light microscopy (LM) observations of S. acuminatus SAG 38.81 grown in BBM+N/L200 (a) at the start and (b) at stationary phase; D. abundans ACUF 283/1.8 in BBM+N/L200 (c) at the start and (d) at stationary phase; D. opoliensis SAG 64.94 in BBM-N/L120 (e) at the start and (f) at stationary phase. bar = 10 µm. Full article ">Figure 4
Confocal laser scanning microscope (CLSM) observations of Desmodesmus abundans. (a–c) cells grown in BBM+N/L120; (d–f) cells grown in BBM-N/L120; (g–i) cells grown in BBM+N/L200; (j–l) cells grown in BBM-N/L200. Colored circles in (b,e,h,k) represent the regions of interest (ROI) of interest (from 1 to 5) studied by spectral analyses in the scan visible region (c,f,i,l). Full article ">Figure 5
Principal component analysis (PCA) performed on a correlation matrix evidencing the relationships among observations (all the strains in all culture conditions) and variables. Biomass means dry weight as grams; production means biomass production as gDW L−1; lipid yield and FAME expressed as %. −/+ indicated the absence/presence of N in BBM medium. Full article ">

23 pages, 1956 KiB

Open AccessArticle

Host–Pathogen Interactions between Xanthomonas fragariae and Its Host Fragaria × ananassa Investigated with a Dual RNA-Seq Analysis

by Michael Gétaz, Joanna Puławska, Theo H.M. Smits and Joël F. Pothier

Microorganisms 2020, 8(8), 1253; https://doi.org/10.3390/microorganisms8081253 - 18 Aug 2020

Cited by 9 | Viewed by 4440

Abstract

Strawberry is economically important and widely grown, but susceptible to a large variety of phytopathogenic organisms. Among them, Xanthomonas fragariae is a quarantine bacterial pathogen threatening strawberry productions by causing angular leaf spots. Using whole transcriptome sequencing, the gene expression of both plant [...] Read more.

Strawberry is economically important and widely grown, but susceptible to a large variety of phytopathogenic organisms. Among them, Xanthomonas fragariae is a quarantine bacterial pathogen threatening strawberry productions by causing angular leaf spots. Using whole transcriptome sequencing, the gene expression of both plant and bacteria in planta was analyzed at two time points, 12 and 29 days post inoculation, in order to compare the pathogen and host response between the stages of early visible and of well-developed symptoms. Among 28,588 known genes in strawberry and 4046 known genes in X. fragariae expressed at both time points, a total of 361 plant and 144 bacterial genes were significantly differentially expressed, respectively. The identified higher expressed genes in the plants were pathogen-associated molecular pattern receptors and pathogenesis-related thaumatin encoding genes, whereas the more expressed early genes were related to chloroplast metabolism as well as photosynthesis related coding genes. Most X. fragariae genes involved in host interaction, recognition, and pathogenesis were lower expressed at late-phase infection. This study gives a first insight into the interaction of X. fragariae with its host. The strawberry plant changed gene expression in order to consistently adapt its metabolism with the progression of infection. Full article

(This article belongs to the Special Issue Integrating Science on Xanthomonas and Xylella for Integrated Plant Disease Management)

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The principle component analysis (PCA) performed with the CummeRbund workflow on differentially expressed genes for (a) Xanthomonas fragariae and (b) Fragaria × ananassa. Three leaf replicates at 12 days post inoculation (dpi) (D12_0, D12_1, D12_2) and three leaf replicate at 29 dpi (D29_0, D29_1, D29_2) were analyzed with principle component for both bacteria and plant and the arrows represent the most-varying direction of the data. Full article ">Figure 2
Volcano plots representing all expressed transcripts. For every transcript, the fold change of 12 days post inoculation (dpi) and 29 dpi was plotted against the p-value for both (a) Xanthomonas fragariae and (b) Fragaria × ananassa. Statistically significant differentially expressed genes, with a Log2 fold change ≥1.5 or ≤−1.5, are depicted as a red dot, and insignificant as black dots. For each organism, the numbers aside the arrows pointing up represent the number of higher expressed genes and the numbers aside arrows pointing down represent the number of lower expressed genes. Full article ">Figure 3
Gene ontology (GO) categories less expressed at 29 days post inoculation (dpi) in Xanthomonas fragariae. Two classes of GO terms, namely biological process and molecular functions in inoculated strawberry plants between 12 and 29 dpi, are shown as a percentage of present genes. Full article ">Figure 4
Gene ontology (GO) categories differentially expressed between 12 and 29 days post inoculation (dpi) in Fragaria × ananassa. The most represented categories from all three classes of GO annotations (i.e., biological process, cellular component, molecular function) are represented as a percentage of genes per categories. Full article ">

18 pages, 2185 KiB

Open AccessArticle

The Absence of C-5 DNA Methylation in Leishmania donovani Allows DNA Enrichment from Complex Samples

by Bart Cuypers, Franck Dumetz, Pieter Meysman, Kris Laukens, Géraldine De Muylder, Jean-Claude Dujardin and Malgorzata Anna Domagalska

Microorganisms 2020, 8(8), 1252; https://doi.org/10.3390/microorganisms8081252 - 18 Aug 2020

Cited by 8 | Viewed by 3345

Abstract

Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In several protozoans, the status of this mechanism remains elusive, such as in Leishmania [...] Read more.

Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In several protozoans, the status of this mechanism remains elusive, such as in Leishmania, the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we showed that the Leishmania donovani genome contains a C-5 DNA methyltransferase (DNMT) from the DNMT6 subfamily, whose function is still unclear, and verified its expression at the RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterized their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite the DNMT6 presence, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites, and 0.0126% at CHH sites at the genomic scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation) allowed enrichment of parasite vs. host DNA using methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of Leishmania promastigote and amastigote DNA with human DNA, resulting in average Leishmania:human enrichments from 62× up to 263×. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing Leishmania clinical samples. Full article

(This article belongs to the Special Issue Leishmania and Leishmaniasis)

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Protein alignment of LdDNMT (LdBPK_251230) and TbDNMT generated with T-coffee, picturing the similarities between the 10 homologous domains of C5 DNA methyltransferases. Black highlights homology, and the red character displays the position of the catalytic cysteine residue. Full article ">Figure 2
RAxML maximum likelihood tree showing the position of trypanosomatid DNMT (DNMT 6) within the DNMT family. Displayed branch bootstrap values are based on 1000 bootstraps. Line thickness is scaled for these bootstrap values. Full article ">Figure 3
CpG, CHG, and CHH genome-wide methylation patterns in (A) Leishmania donovani BPK282 P3 promastigotes (36 chromosomes), (B) Trypanosoma brucei brucei TREU927 (11 chromosomes), and (C) Arabidopsis thaliana Col-0 (5 chromosomes). Data was binned over 10,000 positions to remove local noise and variation. Full article ">Figure 4
DNA/gene copy number based on genomic sequencing depth on chromosome 25 position 465,000–475,000. Both the LdDNMT knock-out (LdDNMT-/-) and LdDNMT overexpressor lines (LdDNMT+) were successful with, respectively, 0 and 64 copies of the gene. The plot shows also that the neighboring genes LdBPK_251220 and LdBPK_251240 were unaffected and had the standard disomic pattern. Full article ">Figure 5
Enrichment (X) of Leishmania DNA in artificial mixtures of Leishmania promastigote DNA and human DNA, with the mixtures ranging from 1:15 to 1:15,000 Leishmania:human DNA. Enrichments were carried out with the NEBNext Microbiome DNA Enrichment Kit (NEB), and the unmethylated Leishmania DNA was enriched on average 263 times. Full article ">

11 pages, 2514 KiB

Open AccessCase Report

First Cases of Natural Infections with Borrelia hispanica in Two Dogs and a Cat from Europe

by Gabriele Margos, Nikola Pantchev, Majda Globokar, Javier Lopez, Jaume Rodon, Leticia Hernandez, Heike Herold, Noelia Salas, Anna Civit and Volker Fingerle

Microorganisms 2020, 8(8), 1251; https://doi.org/10.3390/microorganisms8081251 - 18 Aug 2020

Cited by 8 | Viewed by 3017

Abstract

Canine cases of relapsing fever (RF) borreliosis have been described in Israel and the USA, where two RF species, Borrelia turicatae and Borrelia hermsii, can cause similar clinical signs to the Borrelia persica in dogs and cats reported from Israel, including fever, [...] Read more.

Canine cases of relapsing fever (RF) borreliosis have been described in Israel and the USA, where two RF species, Borrelia turicatae and Borrelia hermsii, can cause similar clinical signs to the Borrelia persica in dogs and cats reported from Israel, including fever, lethargy, anorexia, thrombocytopenia, and spirochetemia. In this report, we describe the first clinical cases of two dogs and a cat from Spain (Cordoba, Valencia, and Seville) caused by the RF species Borrelia hispanica. Spirochetes were present in the blood smears of all three animals, and clinical signs included lethargy, pale mucosa, anorexia, cachexia, or mild abdominal respiration. Laboratory findings, like thrombocytopenia in both dogs, may have been caused by co-infecting pathogens (i.e., Babesia vogeli, confirmed in one dog). Anemia was noticed in one of the dogs and in the cat. Borrelia hispanica was confirmed as an infecting agent by molecular analysis of the 16S rRNA locus. Molecular analysis of housekeeping genes and phylogenetic analyses, as well as successful in vitro culture of the feline isolate confirmed the causative agent as B. hispanica. Full article

(This article belongs to the Special Issue Advance in Tick-Borne Diseases Research)

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Spirochetes in Giemsa-stained blood smears of dog 1 (A, left panel, magnification 150×), dog 2 (A, right panel, magnification 100×, arrow pointing to Borrelia), and 4′,6-diamidino-2-phenylindole (DAPI) stained Borrelia from an in vitro culture of infected cat blood (B). Full article ">Figure 2
Molecular phylogenetic analysis by the maximum likelihood method based on the sequences of four housekeeping loci (clpX, pepX, pyrG, and recG). The tree with the highest log likelihood (−13,651.6897) is shown. The bootstrap value (percentage of trees in which the associated taxa clustered together) is shown next to the branches. The tree is drawn to scale; scale bar = number of substitutions per site. The analysis involved 19 nucleotide sequences. There were a total of 2443 positions in the final dataset. Full article ">Figure 3
Molecular phylogenetic analysis by maximum likelihood method 16S rRNA. The tree with the highest log likelihood (−1091.6698) is shown. The bootstrap value (percentage of trees in which the associated taxa are clustered together) is shown next to the branches. The tree is drawn to scale; scale bar = number of substitutions per site. The analysis involved 43 nucleotide sequences, and the GenBank accession number, species names, and isolate names are given. There were a total of 418 positions in the final dataset. The subtree containing 17 B. burgdorferi sensu lato genospecies is collapsed for clarity. Full article ">

18 pages, 1736 KiB

Open AccessReview

Progress in Developing Inhibitors of SARS-CoV-2 3C-Like Protease

by Qingxin Li and CongBao Kang

Microorganisms 2020, 8(8), 1250; https://doi.org/10.3390/microorganisms8081250 - 18 Aug 2020

Cited by 96 | Viewed by 10278

Abstract

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral outbreak started in late 2019 and rapidly became a serious health threat to the global population. COVID-19 was declared a pandemic by the World Health Organization in [...] Read more.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral outbreak started in late 2019 and rapidly became a serious health threat to the global population. COVID-19 was declared a pandemic by the World Health Organization in March 2020. Several therapeutic options have been adopted to prevent the spread of the virus. Although vaccines have been developed, antivirals are still needed to combat the infection of this virus. SARS-CoV-2 is an enveloped virus, and its genome encodes polyproteins that can be processed into structural and nonstructural proteins. Maturation of viral proteins requires cleavages by proteases. Therefore, the main protease (3 chymotrypsin-like protease (3CL^pro) or M^pro) encoded by the viral genome is an attractive drug target because it plays an important role in cleaving viral polyproteins into functional proteins. Inhibiting this enzyme is an efficient strategy to block viral replication. Structural studies provide valuable insight into the function of this protease and structural basis for rational inhibitor design. In this review, we describe structural studies on the main protease of SARS-CoV-2. The strategies applied in developing inhibitors of the main protease of SARS-CoV-2 and currently available protein inhibitors are summarized. Due to the availability of high-resolution structures, structure-guided drug design will play an important role in developing antivirals. The availability of high-resolution structures, potent peptidic inhibitors, and diverse compound scaffolds indicate the feasibility of developing potent protease inhibitors as antivirals for COVID-19. Full article

(This article belongs to the Special Issue Antiviral Drug Discovery and Development in the Twenty-First Century)

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Genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (a) Viral proteins encoded by the viral genome. The nonstructural proteins (nsps), structural proteins, and accessory proteins (Orf3a to Orf9b) are shown. (b) Membrane topology of several nonstructural proteins. The transmembrane domains of proteins are shown as cylinders. Arrows indicate cleavage sites of: papain-like cysteine protease (PLpro; red) and 3 chymotrypsin-like protease (3CLpro; blue). Other nonstructural proteins are shown as spheres. The sphere has not been drawn to actual scale of individual proteins. More information can be obtained from <a href="https://viralzone.expasy.org/764" target="_blank">https://viralzone.expasy.org/764</a>. Full article ">Figure 2
Structure of SARS-CoV-2 3CLpro. The N-terminal seven residues (N-finger), domains I, II, III, and the linker of domains II and III of both protomers are shown in red, light blue, wheat, green, and purple, respectively. The linker in the two protomers is shown in ribbon mode. Other domains in one protomer are shown in surface mode except the linker region, and corresponding domains in the other protomer are shown in ribbon mode. The structure (PDB ID 6Y2G) is used in this figure. Full article ">Figure 3
De novo drug discovery versus drug repurposing. The time required from hit identification to lead optimization (upper panel) is saved in drug repurposing (lower panel). In the case of SARS-CoV-2 3CLpro, virtual screening, biochemical, and cell-based assays were applied to identify protease inhibitors from FDA-approved drugs. The duration required in individual processes is based on [<a href="#B59-microorganisms-08-01250" class="html-bibr">59</a>], which gives detailed information for drug repurposing. It is worth mentioning that the timeline for COVID-19 might be different from other diseases due to its pandemic status. HTS, high throughput screening. FDA, the Food and Drug Administration. Full article ">Figure 4
The substrate binding site of SARS-CoV-2 3CLpro. (a) A cocrystal structure of SARS-CoV-2 3CLpro with an inhibitor (N3) is shown. (b) Surface charge analysis of the active site of the protease. The structure (PDB ID 6LU7) is shown using PyMOL (<a href="https://pymol.org/2/" target="_blank">https://pymol.org/2/</a>). The protease in the absence (a) and presence (b) is shown in the same orientation. The inhibitor is shown as green sticks. Only domains I and II of one protomer of the protease is shown for clarity. Full article ">Figure 5
Some peptidic inhibitors of SARS-CoV-2. Structures and their half maximal inhibitory concentration values/ half-maximal effective concentration (IC50s/EC50s) against SARS-CoV-2 3CLpro are shown. The binding site 11a with SARS-CoV-2 3CLpro is shown. The inhibitor 11a is shown as sticks, and the protease is shown as a surface. S1 and S2 indicate the binding sites for P1 and P2 residues of the inhibitor. Full article ">Figure 6
Compounds identified through high-throughput screening. Structures, half maximal inhibitory concentration values/ half-maximal effective concentration (IC50s/EC50s) (when applicable) of compounds are shown. Please refer to [<a href="#B54-microorganisms-08-01250" class="html-bibr">54</a>] for more details. Full article ">

20 pages, 2253 KiB

Open AccessFeature PaperReview

Mycobacteriosis in Aquatic Invertebrates: A Review of Its Emergence

by Nadav Davidovich, Danny Morick and Francesca Carella

Microorganisms 2020, 8(8), 1249; https://doi.org/10.3390/microorganisms8081249 - 17 Aug 2020

Cited by 16 | Viewed by 15176

Abstract

Mycobacteriosis is a chronic bacterial disease reported in aquatic and terrestrial animals, including humans. The disease affects a wide range of cultured and wild organisms worldwide. Mycobacteriosis is well-known in aquatic vertebrates (e.g., finfish, marine mammals), while in the last few years, reports [...] Read more.

Mycobacteriosis is a chronic bacterial disease reported in aquatic and terrestrial animals, including humans. The disease affects a wide range of cultured and wild organisms worldwide. Mycobacteriosis is well-known in aquatic vertebrates (e.g., finfish, marine mammals), while in the last few years, reports of its presence in aquatic invertebrates have been on the rise, for both freshwater and marine species. The number of cases is likely to increase as a result of increased awareness, surveillance and availability of diagnostic methods. Domestication of wild aquatic species and the intensification of modern aquaculture are also leading to an increase in the number of reported cases. Moreover, climate changes are affecting fresh and marine aquatic ecosystems. The increasing reports of mycobacteriosis in aquatic invertebrates may also be influenced by global climate warming, which could contribute to the microbes’ development and survival rates, pathogen transmission and host susceptibility. Several species of the genus Mycobacterium have been diagnosed in aquatic invertebrates; a few of them are significant due to their wide host spectrum, economic impact in aquaculture, and zoonotic potential. The impact of mycobacteriosis in aquatic invertebrates is probably underestimated, and there is currently no effective treatment other than facility disinfection. In this review, we provide an overview of the diversity of mycobacterial infections reported in molluscs, crustaceans, cnidarians, echinoderms and sponges. We highlight important issues relating to its pathological manifestation, diagnosis and zoonotic considerations. Full article

(This article belongs to the Section Public Health Microbiology)

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15 pages, 1755 KiB

Open AccessArticle

Niche Differentiation of Active Methane-Oxidizing Bacteria in Estuarine Mangrove Forest Soils in Taiwan

by Yo-Jin Shiau, Chiao-Wen Lin, Yuanfeng Cai, Zhongjun Jia, Yu-Te Lin and Chih-Yu Chiu

Microorganisms 2020, 8(8), 1248; https://doi.org/10.3390/microorganisms8081248 - 17 Aug 2020

Cited by 14 | Viewed by 3940

Abstract

Mangrove forests are one of the important ecosystems in tropical coasts because of their high primary production, which they sustain by sequestering a substantial amount of CO₂ into plant biomass. These forests often experience various levels of inundation and play an important [...] Read more.

Mangrove forests are one of the important ecosystems in tropical coasts because of their high primary production, which they sustain by sequestering a substantial amount of CO₂ into plant biomass. These forests often experience various levels of inundation and play an important role in CH₄ emissions, but the taxonomy of methanotrophs in these systems remains poorly understood. In this study, DNA-based stable isotope probing showed significant niche differentiation in active aerobic methanotrophs in response to niche differentiation in upstream and downstream mangrove soils of the Tamsui estuary in northwestern Taiwan, in which salinity levels differ between winter and summer. Methylobacter and Methylomicrobium-like Type I methanotrophs dominated methane-oxidizing communities in the field conditions and were significantly ¹³C-labeled in both upstream and downstream sites, while Methylobacter were well adapted to high salinity and low temperature. The Type II methanotroph Methylocystis comprised only 10–15% of all the methane oxidizers in the upstream site but less than 5% at the downstream site under field conditions. ¹³C-DNA levels in Methylocystis were significantly lower than those in Type I methanotrophs, while phylogenetic analysis further revealed the presence of novel methane oxidizers that are phylogenetically distantly related to Type Ia in fresh and incubated soils at a downstream site. These results suggest that Type I methanotrophs display niche differentiation associated with environmental differences between upstream and downstream mangrove soils. Full article

(This article belongs to the Special Issue Microbial Cycling of Atmospheric Trace Gases)

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Map of studied mangrove sites (black stars) in the Tamsui estuary, Taiwan. The grey star indicates the site from a previous study [<a href="#B19-microorganisms-08-01248" class="html-bibr">19</a>]. Full article ">Figure 2
CH4 oxidation potential in the studied upstream (Guandu) and downstream (Bali) mangrove soils. Bars with the same letters are not significantly different at p = 0.05 based on Tukey’s honestly significant difference (HSD) comparison. Full article ">Figure 3
Number of pmoA copies in the studied upstream (Guandu) and downstream (Bali) mangrove soils. Bars with the same letters are not significantly different at p = 0.05 based on Tukey’s HSD comparison. The capital letters indicate the statistical results from pmoA genes before and after incubation at one site, and the lower case letters indicate the statistical results from pmoA genes between the sites and seasons in the fresh or incubated soils. Full article ">Figure 4
Relative abundance of the pmoA gene from 12CH4- and 13CH4-incubated soils in upstream (Guandu) and downstream (Bali) mangrove forests in winter (a,c) and in summer (b,d) at different density fractions (fractions 2–14). Full article ">Figure 5
The relative abundance of methanotrophic communities identified with the pmoA genes in the average of triplicate fresh soils under field conditions and in 13C-DNA from 13CH4-enriched microcosms of (a) upstream (Guandu) and (b) downstream (Bali) mangrove forest soils, Taipei, Taiwan. Two regional peak fractions of pmoA genes were found in the 13CH4-amended Bali mangrove soils in one season (<a href="#microorganisms-08-01248-f004" class="html-fig">Figure 4</a>), so the pmoA genes in both fractions were sequenced to identify the potential active methanotrophs. Full article ">Figure 6
The relative abundance of methanotrophic communities identified with the 16S rRNA genes in fresh and 13CH4-enriched (a) upstream (Guandu) and (b) downstream (Bali) mangrove forest soils, Taipei, Taiwan. Two regional peak fractions of pmoA genes were found in the 13CH4-amended Bali mangrove soils in one season (<a href="#microorganisms-08-01248-f004" class="html-fig">Figure 4</a>), so the 16S rRNA in both fractions were sequenced to identify the potential active methanotrophs. Full article ">Figure 7
Canonical correspondence analysis (CCA) between methanotrophs and physiochemical properties in mangrove forests in Tamsui, Taipei, Taiwan. (SbOC: soluble organic C; SbON: soluble organic N; NH4+: ammonium; NO3-: nitrate; TDN: total dissolved N; PMN: potential mineralizable N; TOC: total organic C; TN: total N.). Full article ">

13 pages, 316 KiB

Open AccessReview

Antimicrobial Prosthetic Surfaces in the Oral Cavity—A Perspective on Creative Approaches

by Jorge L. Garaicoa, Amber M. Bates, Gustavo Avila-Ortiz and Kim A. Brogden

Microorganisms 2020, 8(8), 1247; https://doi.org/10.3390/microorganisms8081247 - 17 Aug 2020

Cited by 17 | Viewed by 3294

Abstract

Replacement of missing teeth is an essential component of comprehensive dental care for patients suffering of edentulism. A popular option is implant-supported restorations. However, implant surfaces can become colonized with polymicrobial biofilms containing Candida species that may compromise peri-implant health. To prevent this, [...] Read more.

Replacement of missing teeth is an essential component of comprehensive dental care for patients suffering of edentulism. A popular option is implant-supported restorations. However, implant surfaces can become colonized with polymicrobial biofilms containing Candida species that may compromise peri-implant health. To prevent this, implant components may be treated with a variety of coatings to create surfaces that either repel the attachment of viable microorganisms or kill microorganisms on contact. These coatings may consist of nanoparticles of pure elements (more commonly silver, copper, and zinc), sanitizing agents and disinfectants (quaternary ammonium ions and chlorhexidine), antibiotics (cefalotin, vancomycin, and gentamicin), or antimicrobial peptides (AMPs). AMPs in bioactive coatings have a number of advantages. They elicit a protective action against pathogens, inhibit the formation of biofilms, are less toxic to host tissues, and do not prompt inflammatory responses. Furthermore, many of these coatings may involve unique delivery systems to direct their antimicrobial capacity against pathogens, but not commensals. Coatings may also contain multiple antimicrobial substances to widen antimicrobial activity across multiple microbial species. Here, we compiled relevant information about a variety of creative approaches used to generate antimicrobial prosthetic surfaces in the oral cavity with the purpose of facilitating implant integration and peri-implant tissue health. Full article

(This article belongs to the Special Issue The Role of the Oral Mucosa and Salivary Gland in Anti-Fungal Immunity)

20 pages, 935 KiB

Open AccessArticle

Association of the Gut Microbiota with Weight-Loss Response within a Retail Weight-Management Program

by Samitinjaya Dhakal, Lacey McCormack and Moul Dey

Microorganisms 2020, 8(8), 1246; https://doi.org/10.3390/microorganisms8081246 - 16 Aug 2020

Cited by 22 | Viewed by 4978

Abstract

Retail programs offer popular weight-loss options amid the ongoing obesity crisis. However, research on weight-loss outcomes within such programs is limited. This prospective-cohort observational study enrolled 58 men and women between ages 20 and 72 years from a retail program to assess the [...] Read more.

Retail programs offer popular weight-loss options amid the ongoing obesity crisis. However, research on weight-loss outcomes within such programs is limited. This prospective-cohort observational study enrolled 58 men and women between ages 20 and 72 years from a retail program to assess the influence of client features on energy-restriction induced weight-loss response. DESeq2 in R-studio, a linear regression model adjusting for significantly correlating covariates, and Wilcoxon signed-rank and Kruskal–Wallis for within- and between-group differences, respectively, were used for data analyses. An average 10% (~10 kg) reduction in baseline-weight along with lower total-, android-, gynoid-, and android:gynoid-fat were observed at Week 12 (all, p < 0.05). Fifty percent of participants experienced a higher response, losing an average of 14.5 kg compared to 5.9 kg in the remaining low-response group (p < 0.0001). Hemoglobin-A1C (p = 0.005) and heart rate (p = 0.079) reduced in the high-response group only. Fat mass and A1C correlated when individuals had high android:gynoid fat (r = 0.55, p = 0.008). Gut-microbial β-diversity was associated with BMI, body fat%, and android-fat (all, p < 0.05). Microbiota of the high-response group had a higher baseline OTU-richness (p = 0.02) as well as differential abundance and/or associations with B. eggerthi, A. muciniphila, Turicibacter, Prevotella, and Christensenella (all, p/p_adj < 0.005). These results show that intestinal microbiota as well as sex and body composition differences may contribute to variable weight-loss response. This highlights the importance of various client features in the context of real-world weight control efforts. Full article

(This article belongs to the Special Issue The Human Gut Microbiome, Diets and Health)

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25 pages, 2469 KiB

Open AccessArticle

Ibuprofen Degradation and Associated Bacterial Communities in Hyporheic Zone Sediments

by Cyrus Rutere, Kirsten Knoop, Malte Posselt, Adrian Ho and Marcus A. Horn

Microorganisms 2020, 8(8), 1245; https://doi.org/10.3390/microorganisms8081245 - 16 Aug 2020

Cited by 29 | Viewed by 5047

Abstract

Ibuprofen, a non-steroidal anti-inflammatory pain reliever, is among pharmaceutical residues of environmental concern ubiquitously detected in wastewater effluents and receiving rivers. Thus, ibuprofen removal potentials and associated bacteria in the hyporheic zone sediments of an impacted river were investigated. Microbially mediated ibuprofen degradation [...] Read more.

Ibuprofen, a non-steroidal anti-inflammatory pain reliever, is among pharmaceutical residues of environmental concern ubiquitously detected in wastewater effluents and receiving rivers. Thus, ibuprofen removal potentials and associated bacteria in the hyporheic zone sediments of an impacted river were investigated. Microbially mediated ibuprofen degradation was determined in oxic sediment microcosms amended with ibuprofen (5, 40, 200, and 400 µM), or ibuprofen and acetate, relative to an un-amended control. Ibuprofen was removed by the original sediment microbial community as well as in ibuprofen-enrichments obtained by re-feeding of ibuprofen. Here, 1-, 2-, 3-hydroxy- and carboxy-ibuprofen were the primary transformation products. Quantitative real-time PCR analysis revealed a significantly higher 16S rRNA abundance in ibuprofen-amended relative to un-amended incubations. Time-resolved microbial community dynamics evaluated by 16S rRNA gene and 16S rRNA analyses revealed many new ibuprofen responsive taxa of the Acidobacteria, Actinobacteria, Bacteroidetes, Gemmatimonadetes, Latescibacteria, and Proteobacteria. Two ibuprofen-degrading strains belonging to the genera Novosphingobium and Pseudomonas were isolated from the ibuprofen-enriched sediments, consuming 400 and 300 µM ibuprofen within three and eight days, respectively. The collective results indicated that the hyporheic zone sediments sustain an efficient biotic (micro-)pollutant degradation potential, and hitherto unknown microbial diversity associated with such (micro)pollutant removal. Full article

(This article belongs to the Special Issue Terrestrial Ecotoxicology- How Biocides of Building Materials Impact Soil Microbial Communities)

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Degradation of ibuprofen in oxic hyporheic zone sediment microcosms. Plots (A–D) correspond to sediment amended with ibuprofen concentrations of 5, 40, 200, and 400 μM, respectively. Plots (E–H) correspond to sediment amended with both 1 mM acetate and ibuprofen concentrations of 5, 40, 200, and 400 μM, respectively. Values are the arithmetic means of triplicate oxic incubations. Error bars indicate standard deviations. Some standard deviations are smaller than the symbol size and therefore not apparent. Arrows indicate the time of refeeding of microcosms with ibuprofen (A–D) and acetate and ibuprofen (E–H), respectively. Red and blue arrows indicate sampling of the sediment for nucleic acid extraction after the third and fifth refeeding, respectively. Full article ">Figure 2
Principal coordinate analysis of the Bray–Curtis dissimilarity metric showing the effect of ibuprofen and ibuprofen/acetate treatments on the bacterial community composition based on OTUs from the 16S rRNA gene (panel A) and 16S rRNA (panel B). Sample code: A, amended with 1 mM acetate and ibuprofen per feeding; 0, 5, 40, 200, and 400 indicate supplemental ibuprofen concentrations of 0, 5, 40, 200, and 400 μM, respectively, given per feeding; 0′, 3′, and 5′, correspond to samples obtained at the start of the incubation, and after the third and fifth refeeding, respectively. Sampling times for unamended controls were according to those of the 400 μM ibuprofen treatment. Full article ">Figure 3
Linear discriminant analysis (LDA) scores on the DNA and RNA levels for families that were more (A) or less (B) abundant in treatments with ibuprofen relative to non-supplemented controls and displayed a consistent response on the DNA and RNA level. Full article ">Figure 4
Log2fold change of ibuprofen-responsive OTUs summed up for all OTUs affiliating with the same (sub-) phylum (based on data from <a href="#app1-microorganisms-08-01245" class="html-app">Table S2</a>). A, DNA; B, RNA (cDNA). OTUs significantly enriched by ibuprofen relative to unamended controls sampled at the same time point had a Log2-fold change >0 at p-adj < 0.05. IBU40 and IBU400, ibuprofen amendment with 40 and 400 μM ibuprofen, respectively. IBA40 and IBA400, ibuprofen amendment with 40 and 400 μM ibuprofen, respectively, together with 1 mM acetate. Full article ">Figure 5
16S rRNA (cDNA) to 16S rRNA gene ratios determined by qPCR for selected taxa stimulated by ibuprofen in the 400 μM treatment (<a href="#microorganisms-08-01245-f001" class="html-fig">Figure 1</a> and <a href="#microorganisms-08-01245-f004" class="html-fig">Figure 4</a>) as an indicator of taxon-specific activity. Values are the arithmetic means of triplicate incubations. Error bars indicate the standard deviation but are smaller than the symbol size and therefore not apparent. Sample code: 0 and 400 indicate supplemental ibuprofen concentrations in μM given per feeding; 3′ and 5′ correspond to samples obtained after the third and fifth refeeding, respectively. Sampling times for unamended controls were according to those of the 400 μM ibuprofen treatment. Full article ">Figure 6
Effect of ibuprofen on the relative abundance of OTUs in 16S rRNA gene (DNA) and 16S rRNA (RNA or cDNA)-derived amplicon libraries from oxic hyporheic zone sediment microcosms (<a href="#microorganisms-08-01245-f001" class="html-fig">Figure 1</a>) affiliating with ibuprofen-degrading strains Novosphingobium CN1 (A) and Pseudomonas MAH1 (B), and the capacity of both strains to degrade ibuprofen under oxic conditions. The grey box indicates unsupplemented oxic control microcosms. Values represent the arithmetic means of triplicates, and error bars indicate standard deviations. Filled and open circles, DNA and RNA (cDNA) level, respectively; filled squares, ibuprofen concentration. Sample code: A, amended with 1 mM acetate and ibuprofen per feeding; 0, 5, 40, 200, and 400 indicate supplemental ibuprofen concentrations in μM given per feeding; 0′, 3′, and 5′ correspond to samples obtained at the start of the incubation, and after the third and fifth refeeding, respectively. Sampling times for unamended controls were according to those of the 400 μM ibuprofen treatment. Full article ">

19 pages, 7243 KiB

Open AccessArticle

Assessing the Pyloric Caeca and Distal Gut Microbiota Correlation with Flesh Color in Atlantic Salmon (Salmo salar L., 1758)

by Chan D. H. Nguyen, Gianluca Amoroso, Tomer Ventura and Abigail Elizur

Microorganisms 2020, 8(8), 1244; https://doi.org/10.3390/microorganisms8081244 - 16 Aug 2020

Cited by 19 | Viewed by 9456

Abstract

The Atlantic salmon (Salmo salar L., 1758) is a temperate fish species native to the northern Atlantic Ocean. The distinctive pink–red flesh color (i.e., pigmentation) significantly affects the market price. Flesh paleness leads to customer dissatisfaction, a loss of competitiveness, a drop [...] Read more.

The Atlantic salmon (Salmo salar L., 1758) is a temperate fish species native to the northern Atlantic Ocean. The distinctive pink–red flesh color (i.e., pigmentation) significantly affects the market price. Flesh paleness leads to customer dissatisfaction, a loss of competitiveness, a drop in product value and, consequently, severe economic losses. This work extends our knowledge on salmonid carotenoid dynamics to include the interaction between the gut microbiota and flesh color. A significant association between the flesh color and abundance of specific bacterial communities in the gut microbiota suggests that color may be affected either by seeding resilient beneficial bacteria or by inhibiting the negative effect of pathogenic bacteria. We sampled 96 fish, which covered all phenotypes of flesh color, including the average color and the evenness of color of different areas of the fillet, at both the distal intestine and the pyloric caeca of each individual, followed by 16S rRNA sequencing at the V3-V4 region. The microbiota profiles of these two gut regions were significantly different; however, there was a consistency in the microbiota, which correlated with the flesh color. Moreover, the pyloric caeca microbiota also showed high correlation with the evenness of the flesh color (beta diversity index, PERMANOVA, p = 0.002). The results from the pyloric caeca indicate that Carnobacterium, a group belonging to the lactic acid bacteria, is strongly related to the flesh color and the evenness of the color between the flesh areas. Full article

(This article belongs to the Section Gut Microbiota)

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16 pages, 2695 KiB

Open AccessArticle

Improving Biodegradation of Clofibric Acid by Trametes pubescens through the Design of Experimental Tools

by Claudia Veronica Ungureanu, Lidia Favier and Gabriela Elena Bahrim

Microorganisms 2020, 8(8), 1243; https://doi.org/10.3390/microorganisms8081243 - 15 Aug 2020

Cited by 12 | Viewed by 2411

Abstract

Clofibric acid (CLF) is the main pharmacologically active metabolite in composition of the pharmaceutical products used for controlling blood lipid content. This xenobiotic compound is highly persistent in the aquatic environment and passes unchanged or poorly transformed in wastewater treatment plants. A white-rot [...] Read more.

Clofibric acid (CLF) is the main pharmacologically active metabolite in composition of the pharmaceutical products used for controlling blood lipid content. This xenobiotic compound is highly persistent in the aquatic environment and passes unchanged or poorly transformed in wastewater treatment plants. A white-rot fungal strain of Trametes pubescens was previously selected, for its ability for clofibric acid biodegradation (up to 30%) during cultivation in submerged system under aerobic conditions at an initial CLF concentration of 15 mg L⁻¹. Plackett-Burman design (PBD) and response surface methodology (RSM) were used for experimental planning, mathematical modelling and statistical analysis of data of the biotechnological process of CLF biotransformation by Trametes pubescens fungal strain. After optimization, the capacity of the selected Trametes pubescens strain to degrade CLF was increased by cultivation in a liquid medium containing 3 g·L⁻¹ yeast extract, 15 g·L⁻¹ peptone, 5 g·L⁻¹ glucose and mineral salts, inoculated at 2% (v/v) vegetative inoculum and cultivated at pH 5.5, during 14 days at 25 °C and 135 rpm. In these optimized biotechnological conditions, the CLF biotransformation yield was 60%. Full article

(This article belongs to the Section Environmental Microbiology)

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Pareto chart for the effect of independent variables on CLF biotransformation by Trametes pubescens based on PBD analysis. Full article ">Figure 2
Parity plot of experimental vs. predicted values of CLF biodegradation by Trametes pubescens. Full article ">Figure 3
Contour plot (a) and three-dimensional surface plot (b) showing the effect between inoculum concentration and concentration of yeast extract on CLF biodegradation by Trametes pubescens. Full article ">Figure 4
Contour plot (a) and three-dimensional surface plot (b) showing the effect between inoculum concentration and concentration of peptone on CLF biodegradation by Trametes pubescens. Full article ">Figure 5
Contour plot (a) and three-dimensional surface plot (b) showing the effect between incubation time and concentration of yeast extract on CLF biodegradation by Trametes pubescens. Full article ">Figure 6
Contour plot (a) and three-dimensional surface plot (b) showing the effect between incubation time and inoculum concentration on CLF biodegradation by Trametes pubescens Full article ">

18 pages, 2033 KiB

Open AccessArticle

Do Metabolomics and Taxonomic Barcode Markers Tell the Same Story about the Evolution of Saccharomyces sensu stricto Complex in Fermentative Environments?

by Luca Roscini, Angela Conti, Debora Casagrande Pierantoni, Vincent Robert, Laura Corte and Gianluigi Cardinali

Microorganisms 2020, 8(8), 1242; https://doi.org/10.3390/microorganisms8081242 - 15 Aug 2020

Cited by 3 | Viewed by 2735

Abstract

Yeast taxonomy was introduced based on the idea that physiological properties would help discriminate species, thus assuming a strong link between physiology and taxonomy. However, the instability of physiological characteristics within species configured them as not ideal markers for species delimitation, shading the [...] Read more.

Yeast taxonomy was introduced based on the idea that physiological properties would help discriminate species, thus assuming a strong link between physiology and taxonomy. However, the instability of physiological characteristics within species configured them as not ideal markers for species delimitation, shading the importance of physiology and paving the way to the DNA-based taxonomy. The hypothesis of reconnecting taxonomy with specific traits from phylogenies has been successfully explored for Bacteria and Archaea, suggesting that a similar route can be traveled for yeasts. In this framework, thirteen single copy loci were used to investigate the predictability of complex Fourier Transform InfaRed spectroscopy (FTIR) and High-performance Liquid Chromatography–Mass Spectrometry (LC-MS) profiles of the four historical species of the Saccharomyces sensu stricto group, both on resting cells and under short-term ethanol stress. Our data show a significant connection between the taxonomy and physiology of these strains. Eight markers out of the thirteen tested displayed high correlation values with LC-MS profiles of cells in resting condition, confirming the low efficacy of FTIR in the identification of strains of closely related species. Conversely, most genetic markers displayed increasing trends of correlation with FTIR profiles as the ethanol concentration increased, according to their role in the cellular response to different type of stress. Full article

(This article belongs to the Special Issue Yeast in Winemaking)

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Genotypic vs. phenotypic FTIR and LC-MS description of S. bayanus, S. pastorianus, S. paradoxus and S. cerevisiae reference strains. Hierarchical clustering of S. bayanus CBS 380, S. pastorianus CBS 1538, S. paradoxus CBS 432 and S. cerevisiae CBS 1171 control samples (0% ethanol). (A) Clustering obtained analyzing concatenated ITS_LSU sequences; distances were inferred with the Maximum Composite Likelihood method and expressed as number of base substitutions per site. The Neighbor-Joining method was used to reconstruct the tree. (B) Clustering obtained analyzing FTIR spectra considering the regions from 3200 to 2800 cm−1 (fatty acids) and from 1800 to 1200 cm−1 (amides and mixed region). (C) Clustering obtained analyzing LC-MS spectra using Spearman’s distance measure and Ward’s algorithm. Hierarchical clustering of phenotypes was performed. Full article ">Figure 2
Correlation between taxonomic markers and phenotypic responses of control samples of S. bayanus, S. pastorianus, S. paradoxus and S. cerevisiae reference strains. Correlation coefficients obtained from the comparison between the distance matrices calculated on the basis of each of the thirteen loci employed in the study and those obtained analyzing FTIR (A) and LC-MS (B) spectra of strains in resting condition are reported. Distances were calculated using dist and dist.dna functions included in R-Ape package (<a href="https://cran.r-project.org/web/packages/ape/index.html" target="_blank">https://cran.r-project.org/web/packages/ape/index.html</a>). Correlation analysis was carried out using cor.test function included in the R-Vegan package (<a href="https://CRAN.R-project.org/package=vegan" target="_blank">https://CRAN.R-project.org/package=vegan</a>). Full article ">Figure 3
Trends of significative correlations between taxonomic markers and whole FTIR spectra from cells under short-term ethanol stress. Correlations between the distances of the IR spectra from cells challenged with four different ethanol concentrations (0%, 8%, 12% and 16%) and those obtained considering each taxonomic marker under study were reported as trends of Percentages of Significative Wavelengths (PSW). Markers were grouped according to their correlation trend in: low intensity responses (A); high intensity responses (B); and non-monotonous responses (C). Full article ">Figure 4
Correlations between taxonomic markers and characteristic spectral regions of the FTIR spectrum of both control and stressed cells. Maximum correlation values between each taxonomic marker and fatty acids (W1), amides (W2), mixed (W3) and carbohydrates (W4) spectral areas are reported for both control (A) and stressed (B) cells. The latter analysis was carried out grouping together data from the three stressing conditions tested. Colored labels refer to markers displaying correlation values higher than 0.75 threshold, represented by the red dashed lines. Full article ">Figure 5
Heatmaps of the top significantly altered metabolites and pathways of S. bayanus CBS 380, S. pastorianus CBS 1538, S. paradoxus CBS 432 and S. cerevisiae 1171 strains after 1hr exposure to increasing ethanol concentrations. (A–D) Heatmaps of the top significantly altered metabolites at ethanol concentrations (v/v) of: 0% (A); 8% (B); 12% (C); and 16% (D). The colored boxes indicate the relative concentrations of the corresponding metabolite in each group under study. The color scale is log2 transformed value and indicates relatively high (red) and low (green) metabolite levels. (E–H) The most important altered pathways in the response of the four strains to ethanol (v/v) at: 0% (E); 8% (F); 12% (G); and 16% (H). y-axis, -log(p) represents metabolic pathways containing metabolites that are significantly different between the four strains; x-axis, pathway impact represents the impact of these significantly changed metabolites on the overall pathway, based on their position/number within the pathway. Full article ">

18 pages, 3944 KiB

Open AccessArticle

Microbial Diversity of Fermented Greek Table Olives of Halkidiki and Konservolia Varieties from Different Regions as Revealed by Metagenomic Analysis

by Konstantina Argyri, Agapi I. Doulgeraki, Evanthia Manthou, Athena Grounta, Anthoula A. Argyri, George-John E. Nychas and Chrysoula C. Tassou

Microorganisms 2020, 8(8), 1241; https://doi.org/10.3390/microorganisms8081241 - 14 Aug 2020

Cited by 34 | Viewed by 5799

Abstract

Current information from conventional microbiological methods on the microbial diversity of table olives is insufficient. Next-generation sequencing (NGS) technologies allow comprehensive analysis of their microbial community, providing microbial identity of table olive varieties and their designation of origin. The purpose of this study [...] Read more.

Current information from conventional microbiological methods on the microbial diversity of table olives is insufficient. Next-generation sequencing (NGS) technologies allow comprehensive analysis of their microbial community, providing microbial identity of table olive varieties and their designation of origin. The purpose of this study was to evaluate the bacterial and yeast diversity of fermented olives of two main Greek varieties collected from different regions—green olives, cv. Halkidiki, from Kavala and Halkidiki and black olives, cv. Konservolia, from Magnesia and Fthiotida—via conventional microbiological methods and NGS. Total viable counts (TVC), lactic acid bacteria (LAB), yeast and molds, and Enterobacteriaceae were enumerated. Microbial genomic DNA was directly extracted from the olives’ surface and subjected to NGS for the identification of bacteria and yeast communities. Lactobacillaceae was the most abundant family in all samples. In relation to yeast diversity, Phaffomycetaceae was the most abundant yeast family in Konservolia olives from the Magnesia region, while Pichiaceae dominated the yeast microbiota in Konservolia olives from Fthiotida and in Halkidiki olives from both regions. Further analysis of the data employing multivariate analysis allowed for the first time the discrimination of cv. Konservolia and cv. Halkidiki table olives according to their geographical origin. Full article

(This article belongs to the Special Issue Food Microbial Diversity)

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16 pages, 1018 KiB

Open AccessArticle

Drug Resistance Determinants in Clinical Isolates of Enterococcus faecalis in Bangladesh: Identification of Oxazolidinone Resistance Gene optrA in ST59 and ST902 Lineages

by Sangjukta Roy, Meiji Soe Aung, Shyamal Kumar Paul, Salma Ahmed, Nazia Haque, Emily Rahman Khan, Tridip Kanti Barman, Arup Islam, Sahida Abedin, Chand Sultana, Anindita Paul, Muhammad Akram Hossain, Noriko Urushibara, Mitsuyo Kawaguchiya, Ayako Sumi and Nobumichi Kobayashi

Microorganisms 2020, 8(8), 1240; https://doi.org/10.3390/microorganisms8081240 - 14 Aug 2020

Cited by 14 | Viewed by 3277

Abstract

Enterococcus faecalis is one of the major causes of urinary tract infection, showing acquired resistance to various classes of antimicrobials. The objective of this study was to determine the prevalence of drug resistance and its genetic determinants for E. faecalis clinical isolates in [...] Read more.

Enterococcus faecalis is one of the major causes of urinary tract infection, showing acquired resistance to various classes of antimicrobials. The objective of this study was to determine the prevalence of drug resistance and its genetic determinants for E. faecalis clinical isolates in north-central Bangladesh. Among a total of 210 E. faecalis isolates, isolated from urine, the resistance rates to erythromycin, levofloxacin, and gentamicin (high level) were 85.2, 45.7, and 11.4%, respectively, while no isolates were resistant to ampicillin, vancomycin and teicoplanin. The most prevalent resistance gene was erm(B) (97%), and any of the four genes encoding aminoglycoside modifying enzyme (AME) were detected in 99 isolates (47%). The AME gene aac(6′)-Ie-aph(2”)-Ia was detected in 46 isolates (21.9%) and was diverse in terms of IS256-flanking patterns, which were associated with resistance level to gentamicin. Tetracycline resistance was ascribable to tet(M) (61%) and tet(L) (38%), and mutations in the quinolone resistance-determining region of both GyrA and ParC were identified in 44% of isolates. Five isolates (2.4%) exhibited non-susceptibility to linezolide (MIC, 4 μg/mL), and harbored the oxazolidinone resistance gene optrA, which was located in a novel genetic cluster containing the phenicol exporter gene fexA. The optrA-positive isolates belonged to ST59, ST902, and ST917 (CC59), while common lineages of other multiple drug-resistant isolates were ST6, ST28, CC16, and CC116. The present study first revealed the prevalence of drug resistance determinants of E. faecalis and their genetic profiles in Bangladesh. Full article

(This article belongs to the Section Antimicrobial Agents and Resistance)

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Schematic representation of the IS256 flanking patterns of aac(6′)-Ie-aph(2”)-Ia (A-D) detected in E. faecalis isolates in the present study. A, Tn4001-like structure containing IS256 (IS256-L and -R) at both ends; B-D, Tn4001-truncated structure lacking IS256 at the 3′-end, 5′-end, and both ends, respectively. Intact open reading frame of aac(6′)-Ie-aph(2”)-Ia is shown as a blue box with an arrow indicating the transcription direction. The pseudogene in pattern C3 indicates the incomplete gene that lacks the 5′-end region including the start codon. E. faecalis isolate ID and MIC to GEN are shown on the right. Full article ">Figure 2
Schematic representation of the genetic background of optrA in the E. faecalis strain SJ82 (uppermost) and the genetic organization or components similar to that of SJ82 in other strains reported previously [<a href="#B30-microorganisms-08-01240" class="html-bibr">30</a>] or available in GenBank database. Prototype of the fexA–optrA cluster in the pE349 of E. faecalis strain E349 [<a href="#B22-microorganisms-08-01240" class="html-bibr">22</a>] is shown at the bottom. Arrows indicate the transcription direction of genes. Arrows of RNase J are shown in black and blue, representing different sequences. Gene names are shown above arrows, and the strain names are indicated on the right. Full article ">

13 pages, 1417 KiB

Open AccessArticle

EMS-Induced Mutagenesis of Clostridium carboxidivorans for Increased Atmospheric CO₂ Reduction Efficiency and Solvent Production

by Naoufal Lakhssassi, Azam Baharlouei, Jonas Meksem, Scott D. Hamilton-Brehm, David A. Lightfoot, Khalid Meksem and Yanna Liang

Microorganisms 2020, 8(8), 1239; https://doi.org/10.3390/microorganisms8081239 - 14 Aug 2020

Cited by 9 | Viewed by 3283

Abstract

Clostridium carboxidivorans (P7) is one of the most important solvent-producing bacteria capable of fermenting syngas (CO, CO₂, and H₂) to produce chemical commodities when grown as an autotroph. This study aimed to develop ethyl methanesulfonate (EMS)-induced P7 mutants that [...] Read more.

Clostridium carboxidivorans (P7) is one of the most important solvent-producing bacteria capable of fermenting syngas (CO, CO₂, and H₂) to produce chemical commodities when grown as an autotroph. This study aimed to develop ethyl methanesulfonate (EMS)-induced P7 mutants that were capable of growing in the presence of CO₂ as a unique source of carbon with increased solvent formation and atmospheric CO₂ reduction to limit global warming. Phenotypic analysis including growth and end product characterization of the P7 wild type (WT) demonstrated that this strain grew better at 25 °C than 37 °C when CO₂ served as the only source of carbon. In the current study, 55 mutagenized P7-EMS mutants were developed by using 100 mM and 120 mM EMS. Interestingly, using a forward genetic approach, three out of the 55 P7-EMS mutants showed a significant increase in ethanol, butyrate, and butanol production. The three P7-EMS mutants presented on average a 4.68-fold increase in concentrations of ethanol when compared to the P7-WT. Butyric acid production from 3 P7-EMS mutants contained an average of a 3.85 fold increase over the levels observed in the P7-WT cultures under the same conditions (CO₂ only). In addition, one P7-EMS mutant presented butanol production (0.23 ± 0.02 g/L), which was absent from the P7-WT under CO₂ conditions. Most of the P7-EMS mutants showed stability of the obtained end product traits after three transfers. Most importantly, the amount of reduced atmospheric CO₂ increased up to 8.72 times (0.21 g/Abs) for ethanol production and up to 8.73 times higher (0.16 g/Abs) for butyrate than the levels contained in the P7-WT. Additionally, to produce butanol, the P7-EMS_III-J mutant presented 0.082 g/Abs of CO₂ reduction. This study demonstrated the feasibility and effectiveness of employing EMS mutagenesis in generating solvent-producing anaerobic bacteria mutants with improved and novel product formation and increased atmospheric CO₂ reduction efficiency. Full article

(This article belongs to the Special Issue Anaerobes in Biogeochemical Cycles)

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Growth comparison of Clostridium carboxidivorans P7 wild type employing two different temperatures 25 °C and 37 °C in the optimized 1754-B medium. (A) Optical density, (B) Cell count. Full article ">Figure 2
End product identified for Clostridium carboxidivorans wild type P7 in the 1754-B modified medium. Butyrate production was detected, but not until after day 26. Full article ">Figure 3
Correlation between the number of colonies and ethyl methanesulfonate (EMS) concentrations. Eight ethyl methanesulfonate (EMS) concentrations have been used to mutagenize the Clostridium carboxidivorans P7 wild type culture. Concentrations above 140mM were lethal for the P7 strain. Mutational rates show an LD50 of 74.89mM of EMS treatment. 40 (purple box) and 15 (brown box) EMS mutants were randomly isolated from 100 mM and 120mM EMS treatment, respectively. Full article ">Figure 4
End products identified for the mutagenized Clostridium carboxidivorans P7-EMS mutants. Three out of 55 P7-EMS mutants presented different products and product profiles when compared to the wild type P7. The three mutants (P7III-J, P7III-R, and P7III-P) showed increased ethanol production, in addition, butyrate acid production was observed in all three mutants, but not in the P7-WT when CO2 is provided as the only source of carbon. The purple line represents ethanol production of the P7 wild type. Full article ">Figure 5
Acid and Alcohol end product (g/L) comparison between the three P7-EMS and the P7-WT. The three P7-EMS mutants (P7III-J, P7III-R, and P7III-P) showed decreased formic acid production during four weeks of growth, and increased ethanol production, in addition, butyrate acid production was observed in all three mutants, but not in the P7-WT when CO2 is provided as the only source of carbon. Full article ">Figure 6
End products identified for the three P7-EMS mutants on CO2 after three transfers. All three P7-EMS mutants presented stable and increased ethanol, butyrate acid, and butanol production when compared to the P7 wild type. Full article ">

15 pages, 4761 KiB

Open AccessArticle

Impact of Cyanidin-3-Glucoside on Gut Microbiota and Relationship with Metabolism and Inflammation in High Fat-High Sucrose Diet-Induced Insulin Resistant Mice

by Fei Huang, Ruozhi Zhao, Min Xia and Garry X. Shen

Microorganisms 2020, 8(8), 1238; https://doi.org/10.3390/microorganisms8081238 - 14 Aug 2020

Cited by 41 | Viewed by 3561

Abstract

The present study assessed the effects of freeze-dried cyanidin-3-glucoside (C3G), an anthocyanin enriched in dark-red berries, compared to Saskatoon berry powder (SBp) on metabolism, inflammatory markers and gut microbiota in high fat-high sucrose (HFHS) diet-induced insulin-resistant mice. Male C57 BL/6J mice received control, [...] Read more.

The present study assessed the effects of freeze-dried cyanidin-3-glucoside (C3G), an anthocyanin enriched in dark-red berries, compared to Saskatoon berry powder (SBp) on metabolism, inflammatory markers and gut microbiota in high fat-high sucrose (HFHS) diet-induced insulin-resistant mice. Male C57 BL/6J mice received control, HFHS, HFHS + SBp (8.0 g/kg/day) or HFHS + C3G (7.2 mg/kg/day, equivalent C3G in SBp) diet for 11 weeks. The HFHS diet significantly increased plasma levels of glucose, cholesterol, triglycerides, insulin resistance and inflammatory markers. The HFHS + SBp diet increased the Bacteroidetes/Firmicutes (B/F) ratio and relative abundance of Muriculaceae family bacteria in mouse feces detected using 16S rRNA gene sequencing. The HFHS + SBp or HFHS + C3G diet attenuated glucose, lipids, insulin resistance and inflammatory markers, and increased the B/F ratio and Muriculaceae relative abundance compared to the HFHS diet alone. The relative abundances of Muriculaceae negatively correlated with body weight, glucose, lipids, insulin resistance and inflammatory mediators. Functional predication analysis suggested that the HFHS diet upregulated gut bacteria genes involved in inflammation, and downregulated bacteria involved in metabolism. C3G and SBp partially neutralized HFHS diet-induced alterations of gut bacteria. The results suggest that C3G is a potential prebiotic, mitigating HFHS diet-induced disorders in metabolism, inflammation and gut dysbiosis, and that C3G contributes to the metabolic beneficial effects of SBp. Full article

(This article belongs to the Section Gut Microbiota)

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Figure 1
Effects of Cyanidin-3-glucoside (C3G) supplemented in a high fat-high sucrose (HFHS) diet on the body weight and food intake of mice. Male C57 BL/J6 mice (6 weeks of age) were randomized into 4 groups and received the following diets for 11 weeks: control (CTL) group: control diet, HFHS group: HFHS diet, Saskatoon berry powder (SBp) group: SBp (8.0 g/kg/day) supplemented in the HFHS diet, C3G group: C3G (7.2 mg/kg/day) supplemented in the HFHS diet. Body weights and food intake were measured every two weeks up to 10 weeks. (A) Body weights, (B) daily food intake. The values are expressed as mean ± standard deviation (SD) g (n = 8/group). *, **: p < 0.05 or 0.01 HFHS versus CTL group, +, ++: p < 0.05 or 0.01 SBp versus CTL group, ^^: p < 0.01 C3G versus CTL group. Full article ">Figure 2
Levels of glucose, cholesterol and triglycerides in plasma of mice fed with HFHS diets supplemented with or without C3G. The dietary regimen was the same as described in the legend of <a href="#microorganisms-08-01238-f001" class="html-fig">Figure 1</a>. Fasting plasma glucose, cholesterol and triglycerides were measured biochemically using assay kits. Values are expressed as mean ± SD mg/dL (n = 8/group). **: p < 0.01 versus the control (CTL) group; +, ++: p < 0.05 or 0.01 versus the HFHS group. Full article ">Figure 3
Effects of HFHS diets supplemented with C3G on insulin and insulin resistance in mice. The experimental regimen was described in the legend of <a href="#microorganisms-08-01238-f001" class="html-fig">Figure 1</a>. The levels of fasting plasma insulin (ng/mL) were measured at indicated time points (A). Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated according to the levels of glucose and insulin in the same plasma samples (B). Values are expressed as mean ± SD (n = 8/group). **: p < 0.01 versus the control (CTL) group; ++: p < 0.05 or 0.01 versus the HFHS group. Full article ">Figure 4
Levels of inflammatory regulators in plasma of mice receiving the HFHS diet supplemented with C3G. The experimental regimen was described in the legend of <a href="#microorganisms-08-01238-f001" class="html-fig">Figure 1</a>. The levels of monocyte chemotactic protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) were analyzed in plasma collected before tissue harvesting using enzyme-linked immunosorbent assay (ELISA) kits for mouse MCP-1 (A) or PAI-1 (B). Values are expressed as mean ± SD ng/mL (n = 8/group). **: p < 0.01 versus the control (CTL) group; ++: p < 0.05 or 0.01 versus the HFHS group. Full article ">Figure 5
Effect of the HFHS diet supplemented with C3G on β-diversity of gut microbiota in mice. The experimental regimen was described in the legend of <a href="#microorganisms-08-01238-f001" class="html-fig">Figure 1</a>. Principle component analysis (PCA) was based on Bray–Curtis dissimilarities between all sample sets (weighted by taxon abundance). Full article ">Figure 6
Effects of HFHS diet supplemented with or without the relative abundance of Bacteroidetes and Firmicutes and their ratios. The experimental regimen was described in the legend of <a href="#microorganisms-08-01238-f001" class="html-fig">Figure 1</a>. (A) Relative abundance (%) of Bacteroidetes in gut microbial composition. (B) Relative abundance (%) of Firmicutes in gut microbial composition. (C) Ratio of Bacteroidetes over Firmicutes (B/F) in gut microbiota. (D) Ratio of Firmicutes over Bacteroidetes (F/B) in gut microbiota. Values are expressed as mean ± SD (%) (n = 8/group). *, **: p < 0.05 or 0.01 versus the control (CTL) group; +, ++: p < 0.05 or 0.01 versus the HFHS group. Full article ">Figure 7
Effect of four HFHS (H) diets supplemented with C3G on the relative abundance of gut family bacteria. The experimental regimen was described in the legend of <a href="#microorganisms-08-01238-f001" class="html-fig">Figure 1</a>. (A) Statistical differences among mice with different diets (analysis of variance (ANOVA) and post-hoc Tukey test), (B) correlation heatmap of relative abundance of gut microbiota on family level with physiological and biochemical parameters, (C) extended error bar plot (STAMP tool) showing difference in mean relative abundance between the SBp (S) group and HFHS group, (D) mean proportion and difference in mean proportion of family bacteria (STAMP tool) between the C3G group and the HFHS group (mean ± SD). ∗: p < 0.05 in overall ANOVA result, ●: p < 0.05 in the HFHS (H) group versus the control (CTL) group (H/CTL), ■: p < 0.05 in the SBp (S) group versus the CTL group (S/CTL), ▲: p < 0.05 in the C3G group versus the CTL group (C3G/CTL), ○: p < 0.05 in the S group versus the H group (S/H), □: p < 0.05 in the C3G group versus the H group (C3G/H), △: p < 0.05 in the C3G group versus the S group (C3G/S). *, **, ***: p < 0.05 or 0.01 or 0.001 in positive (blue) or negative (red) correlations between the abundance of each gut family bacteria and physiological or biochemical variables. Full article ">Figure 8
Effects of HFHS diets supplemented with and without C3G on metagenome functional activity in gut microbiota based on the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). The experimental regimen was the same as described in legend of <a href="#microorganisms-08-01238-f001" class="html-fig">Figure 1</a>. Differences in relative abundance (%) in each selected pathway among various dietary groups are in the form of a bar plot. Values are expressed as mean value (n = 8/group). **: p < 0.01, showing ANOVA results among the four groups. Full article ">

26 pages, 1299 KiB

Open AccessReview

Patents on Endophytic Fungi for Agriculture and Bio- and Phytoremediation Applications

by Humberto E. Ortega, Daniel Torres-Mendoza and Luis Cubilla-Rios

Microorganisms 2020, 8(8), 1237; https://doi.org/10.3390/microorganisms8081237 - 14 Aug 2020

Cited by 44 | Viewed by 5672

Abstract

Plant endophytic fungi spend all or part of their lives inside host tissues without causing disease symptoms. They can colonize the plant to protect against predators, pathogens and abiotic stresses generated by drought, salinity, high concentrations of heavy metals, UV radiation and temperature [...] Read more.

Plant endophytic fungi spend all or part of their lives inside host tissues without causing disease symptoms. They can colonize the plant to protect against predators, pathogens and abiotic stresses generated by drought, salinity, high concentrations of heavy metals, UV radiation and temperature fluctuations. They can also promote plant growth through the biosynthesis of phytohormones and nutrient acquisition. In recent years, the study of endophytic fungi for biological control of plant diseases and pests has been intensified to try to reduce the ecological and public health impacts due the use of chemicals and the emergence of fungicide resistance. In this review, we examine 185 patents related to endophytic fungi (from January 1988 to December 2019) and discuss their applicability for abiotic stress tolerance and growth promotion of plants, as agents for biocontrol of herbivores and plant pathogens and bio- and phytoremediation applications. Full article

(This article belongs to the Section Microbial Biotechnology)

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Graphical abstract

5 pages, 240 KiB

Open AccessCase Report

Compassionate Use of Cefiderocol to Treat a Case of Prosthetic Joint Infection Due to Extensively Drug-Resistant Enterobacter hormaechei

by Soline Siméon, Laurent Dortet, Frédérique Bouchand, Anne-Laure Roux, Rémy A. Bonnin, Clara Duran, Jean-Winoc Decousser, Simon Bessis, Benjamin Davido, Grégory Sorriaux and Aurélien Dinh

Microorganisms 2020, 8(8), 1236; https://doi.org/10.3390/microorganisms8081236 - 13 Aug 2020

Cited by 23 | Viewed by 3071

Abstract

We report the case of a 67-year old man with a right knee prosthetic joint infection due to extensively drug-resistant Enterobacter hormaechei. The resistance phenotype was due to the overproduction of the intrinsic cephalosporinase (ACT-5) associated with the production of three acquired [...] Read more.

We report the case of a 67-year old man with a right knee prosthetic joint infection due to extensively drug-resistant Enterobacter hormaechei. The resistance phenotype was due to the overproduction of the intrinsic cephalosporinase (ACT-5) associated with the production of three acquired β-lactamases (CTX-M-15, TEM-1B and OXA-1), and a putative membrane decreased permeability. He was first treated with colistin-tigecyclin due to adverse drug reactions; treatment was switched to cefiderocol for a 12-week antibiotic duration, with a favorable outcome. Full article

(This article belongs to the Special Issue Molecular Epidemiology of Antimicrobial Resistance)

19 pages, 969 KiB

Open AccessArticle

Evaluation of the Diagnostic Potential of Recombinant Coxiella burnetii Com1 in an ELISA for the Diagnosis of Q Fever in Sheep, Goats and Cattle

by Mareike Stellfeld, Claudia Gerlach, Ina-Gabriele Richter, Peter Miethe, Dominika Fahlbusch, Birgitta Polley, Reinhard Sting, Martin Pfeffer, Heinrich Neubauer and Katja Mertens-Scholz

Microorganisms 2020, 8(8), 1235; https://doi.org/10.3390/microorganisms8081235 - 13 Aug 2020

Cited by 13 | Viewed by 3906

Abstract

Coxiella burnetii is the causative agent of Q fever, a zoonosis infecting domestic ruminants and humans. Currently used routine diagnostic tools offer limited sensitivity and specificity and symptomless infected animals may be missed. Therefore, diagnostic tools of higher sensitivity and specificity must be [...] Read more.

Coxiella burnetii is the causative agent of Q fever, a zoonosis infecting domestic ruminants and humans. Currently used routine diagnostic tools offer limited sensitivity and specificity and symptomless infected animals may be missed. Therefore, diagnostic tools of higher sensitivity and specificity must be developed. For this purpose, the C. burnetii outer membrane protein Com1 was cloned and expressed in Escherichia coli. The His-tagged recombinant protein was purified and used in an indirect enzyme-linked immunosorbent assay (ELISA). Assay performance was tested with more than 400 positive and negative sera from sheep, goats and cattle from 36 locations. Calculation of sensitivity and specificity was undertaken using receiver operating characteristic (ROC) curves. The sensitivities and specificities for sheep were 85% and 68% (optical density at 450nm, OD₄₅₀ cut-off value 0.32), for goats 94% and 77% (OD₄₅₀ cut-off value 0.23) and for cattle 71% and 70% (OD₄₅₀ cut-off value 0.18), respectively. These results correspond to excellent, outstanding and acceptable discrimination of positive and negative sera. In summary, recombinant Com1 can provide a basis for more sensitive and specific diagnostic tools in veterinary medicine. Full article

(This article belongs to the Section Public Health Microbiology)

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Figure 1

Figure 1
Analysis of purified recombinant Com1 protein with His-tag. Ni-NTA purified recombinant Com1 protein (predicted molecular weight 29.9 kDa, 30 µg per lane) was separated using a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (a) For Western blot analysis mouse anti-6xHis monoclonal antibody (1:5000, Clontech Laboratories, Inc., Mountain View, CA, USA) was used and traced with alkaline phosphatase labeled goat anti-mouse secondary antibody (1:5000, Sigma-Aldrich, St. Louis, MO, USA). (c) Recombinant Com1 protein was visualized using colloidal Coomassie Blue. Lane (a,c), purified recombinant Com1 protein; Lane (b), protein marker with size in kDa (Thermo Fisher Scientific, Waltham, MA, USA). Full article ">Figure 2
Plot of sensitivity versus 1–specificity for all possible cut-off values. Dots show OD450 values of ovine field sera (blue dots), caprine field sera (green triangles) and bovine field sera (yellow squares). Red line shows random distribution in contrast. Full article ">Figure A1
Vector map of recombinant pCR™8/GW/TOPO™ (Gateway, Invitrogen) with cloned com1 (pCR8-com1). Full article ">Figure A2
Cloned com1. Amplification of com1 (762 bp) without the 5′-terminal signal sequence (1–60 bp) but including the 3′-terminal stop codon (759–762) from genomic DNA of C. burnetii Nine Mile Phase II RSA 439. Arrows show com1 CBU_1910 F and com1 CBU_1910 R primers used for amplification. Full article ">Figure A3
Vector map of recombinant expression vector pET300/NT-DEST (Invitrogen) with cloned com1 cloned into (pET300-com1). Full article ">Figure A4
Cloned and expressed Com1 protein. (a) Insertion of com1 into pET300/NT-DEST vector for expression of recombinant Com1 of 29.9 kDa with His-tag. (b) Expression of His-tagged com1 as recombinant Com1 protein. ATG, vector encoded start codon; RBS, vector encoded ribosome binding site; 6xHis, vector encoded His-tag. Axis counting base pairs in relation to com1 <a href="#microorganisms-08-01235-f0A2" class="html-fig">Figure A2</a>. Full article ">

18 pages, 3826 KiB

Open AccessArticle

Four New Genes of Cyanobacterium Synechococcus elongatus PCC 7942 Are Responsible for Sensitivity to 2-Nonanone

by Olga A. Koksharova, Alexandra A. Popova, Vladimir A. Plyuta and Inessa A. Khmel

Microorganisms 2020, 8(8), 1234; https://doi.org/10.3390/microorganisms8081234 - 13 Aug 2020

Cited by 3 | Viewed by 3851

Abstract

Microbial volatile organic compounds (VOCs) are cell metabolites that affect many physiological functions of prokaryotic and eukaryotic organisms. Earlier we have demonstrated the inhibitory effects of soil bacteria volatiles, including ketones, on cyanobacteria. Cyanobacteria are very sensitive to ketone action. To investigate the [...] Read more.

Microbial volatile organic compounds (VOCs) are cell metabolites that affect many physiological functions of prokaryotic and eukaryotic organisms. Earlier we have demonstrated the inhibitory effects of soil bacteria volatiles, including ketones, on cyanobacteria. Cyanobacteria are very sensitive to ketone action. To investigate the possible molecular mechanisms of the ketone 2-nonanone influence on cyanobacterium Synechococcus elongatus PCC 7942, we applied a genetic approach. After Tn5-692 transposon mutagenesis, several 2-nonanone resistant mutants have been selected. Four different mutant strains were used for identification of the impaired genes (Synpcc7942_1362, Synpcc7942_0351, Synpcc7942_0732, Synpcc7942_0726) that encode correspondingly: 1) a murein-peptide ligase Mpl that is involved in the biogenesis of cyanobacteria cell wall; 2) a putative ABC transport system substrate-binding proteins MlaD, which participates in ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane by aberrantly localized phospholipids transport from outer to inner membranes of bacterial cells; 3) a conserved hypothetical protein that is encoding by gene belonging to phage gene cluster in Synechococcus elongatus PCC 7942 genome; 4) a protein containing the VRR-NUC (virus-type replication-repair nuclease) domain present in restriction-modification enzymes involved in replication and DNA repair. The obtained results demonstrated that 2-nonanone may have different targets in Synechococcus elongatus PCC 7942 cells. Among them are proteins involved in the biogenesis and functioning of the cyanobacteria cell wall (Synpcc7942_1362, Synpcc7942_0351, Synpcc7942_0732) and protein participating in stress response at DNA restriction-modification level (Synpcc7942_0726). This paper is the first report about the genes that encode protein products, which can be affected by 2-nonanone. Full article

(This article belongs to the Section Environmental Microbiology)

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Figure 1
This scheme represents the genetic constructions that have been made by using the deleted copies of the target genes for site-directed mutagenesis of Synechococcus wild type cells. (A) Construction of a plasmid pΔNR401. Deleted copy of the Synpcc7942_1362 gene was amplified and cloned into the pRL498 vector in EcoRI sites. Blue arrows correspond to primers. (B) Construction of a plasmid pΔNR385. Deleted copy of Synpcc7942_0726 gene was amplified and cloned into pJet1.2/blunt vector. The fragment was digested in BglII/BamHI sites and cloned into pRL498 for further conjunction. Blue arrows correspond to primers. (C) Gel electrophoresis of the PCR products of the target genes. Lanes: 1 Synpcc7942_1362 (for ΔNR401), 2 Synpcc7942_0351 (for ΔNR365), 3 Synpcc7942_0726 (for ΔNR385), and 4 Synpcc7942_0732 (for ΔNR359), L-GeneRuler 1 kb Plus DNA ladder. Full article ">Figure 2
This scheme shows the transposon Tn5-692 localization in genomic regions of Synechococcus NR-mutants that were resistant to 2-nonanone. (A) Transposon insertion in Synpcc7942_1362 gene in NR401(Tn) mutant. (B) Transposon insertion in Synpcc7942_0726 gene in NR385(Tn) mutant. (C) Transposon insertion in Synpcc7942_0351 gene in NR365(Tn) mutant. (D) Transposon insertion in Synpcc7942_0732 gene in ΔNR359(Tn) mutant. Full article ">Figure 3
Network of ABB57392.1 protein (Synpcc7942_1362) and its protein partners according to STRING (<a href="https://string-db.org" target="_blank">https://string-db.org</a>). In this figure: (1) protein ABB57391.1 (Synpcc7942_1361) is a conserved hypothetical protein that contains a signal peptide and 8 pentapeptide repeats; (2) ABB57390.1 (Synpcc7942_1360) is cell envelope-related transcriptional attenuator, polyisoprenyl-teichoic acid--peptidoglycan teichoic acid transferase [EC:2.7.8.-]; (3) protein ABB57393.1 (Synpcc7942_1363) is uncharacterized protein. Full article ">Figure 4
Network of ABB56383.1 protein (Synpcc7942_0351) and its protein partners according to STRING (<a href="https://string-db.org" target="_blank">https://string-db.org</a>) is shown. In this figure: (1) protein ABB56410.1 (Synpcc7942_0378) is MlaE, ABC transporter phospholipid/cholesterol/gamma-HCH transport system permease protein; (2) protein ABB56382.1 (Synpcc7942_0350) is MlaF, phospholipid/cholesterol/gamma-HCH transport system ATP-binding protein; (3) protein ABB57818.1 (Synpcc7942_1788) is translocation and assembly module TamB protein; (4) protein ABB56590.1 (Synpcc7942_0558) is conserved hypothetical protein, chaperone-like protein; (5) protein ABB57973.1 (Synpcc7942_1943) is cell division protein Ftn2-like. Full article ">Figure 5
Network of ABB56764.1 protein (Synpcc7942_0732) and its protein partners according to STRING (<a href="https://string-db.org" target="_blank">https://string-db.org</a>) is shown. In this figure: protein ABB56765.1 (Synpcc7942_0733) is the Phage portal protein, which belongs to the lambda family; protein ABB56766.1 (Synpcc7942_0734) is putative DNA primase/helicase. Full article ">Figure 6
Network of ABB56758.1 protein (Synpcc7942_0726) and its protein partners according to STRING (<a href="https://string-db.org" target="_blank">https://string-db.org</a>) is presented. In this figure: the protein ABB56756.1 (Synpcc7942_0724) and the protein ABB56760.1 (Synpcc7942_0728) are conserved hypothetical proteins; the protein ABB56757.1 (Synpcc7942_0725) is DEAD/DEAH box helicase-like enzyme; the protein ABB56759.1 (Synpcc7942_0727) is putative DNA primase/helicase. Full article ">Figure 7
This figure shows the effect of 2-nonanone on the wild type of Synechococcus and the insertion of mutants ΔNR401 and ΔNR385. (A) Control plate. The growth of the wild type and mutant cells is similar. (B) 100 µmol of 2-nononone was added. The growth of the wild type cells is inhibited. The mutants ΔNR401 and ΔNR385 demonstrated growth at the ketone presence. Full article ">Figure 8
The wild-type and mutant phenotypes of Synechococcus. (A) Mutant ΔNR385. (B) The wild type of Synechococcus. (C) Mutant ΔNR401. Full article ">Figure 9
Modes of 2-nonanone action on cyanobacteria cell, (some figure elements are adapted from [<a href="#B47-microorganisms-08-01234" class="html-bibr">47</a>,<a href="#B48-microorganisms-08-01234" class="html-bibr">48</a>]). Full article ">

13 pages, 1336 KiB

Open AccessArticle

Rapid Detection and Antibiotic Susceptibility of Uropathogenic Escherichia coli by Flow Cytometry

by Alexandra Mihaela Velican, Luminiţa Măruţescu, Crina Kamerzan, Violeta Corina Cristea, Otilia Banu, Elvira Borcan and Mariana-Carmen Chifiriuc

Microorganisms 2020, 8(8), 1233; https://doi.org/10.3390/microorganisms8081233 - 13 Aug 2020

Cited by 10 | Viewed by 3415

Abstract

Background: Early preliminary data on antibiotic resistance patterns available before starting the empiric therapy of urinary tract infections (UTIs) in patients with risk factors for acquiring antibiotic resistance could improve both clinical and epidemiological outcomes. The aim of the present study was two-fold: [...] Read more.

Background: Early preliminary data on antibiotic resistance patterns available before starting the empiric therapy of urinary tract infections (UTIs) in patients with risk factors for acquiring antibiotic resistance could improve both clinical and epidemiological outcomes. The aim of the present study was two-fold: (i) to assess the antibiotic susceptibility of uropathogenic Escherichia coli isolates, exhibiting different antibiotic resistance phenotypes, directly in artificially contaminated urine samples using a flow cytometry (FC) based protocol; (ii) to optimize the protocol on urine samples deliberately contaminated with bacterial suspensions prepared from uropathogenic E. coli strains. Results: The results of the FC based antimicrobial susceptibility testing (AST) protocol were compared with the reference AST methods results (disk diffusion and broth microdilution) for establishing the sensitivity and specificity. The proposed FC protocol allowed the detection and quantification of uropathogenic E. coli strains susceptibility to nitrofurantoin, trimethoprim–sulfamethoxazole, ciprofloxacin, and ceftriaxone within 4 h after the inoculation of urine specimens. The early availability of preliminary antibiotic susceptibility results provided by direct analysis of clinical specimens could essentially contribute to a more targeted emergency therapy of UTIs in the anticipation of AST results obtained by reference methodology. Conclusions: This method will increase the therapeutic success rate and help to prevent the emergence and dissemination of drug resistant pathogens. Full article

(This article belongs to the Special Issue Rapid Diagnosis of Microbial Pathogens)

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Figure 1
Fluorescence distributions for two Escherichia coli isolates grown in artificially contaminated urine treated with antibiotics (nitrofurantoin 150 μg/mL, ciprofloxacin 4 μg/mL, ceftriaxone 4 μg/mL, and trimethoprim–sulfamethoxazole 100 μg/mL). The grey area of the histograms indicates the green fluorescence of untreated bacteria, and the white area with blue contour shows the green fluorescence of bacterial population treated with antibiotics. The fraction of bacterial population with increased green fluorescence in urine sample is expressed in percentages (M1). The SI was defined as the ratio of the median fluorescence of cells treated with antibiotics versus the median fluorescence of untreated cells. The upper histograms correspond to an E. coli isolate susceptible to nitrofurantoin (minimum inhibitory concentration (MIC) ≤ 18 μg/mL) and resistant to ceftriaxone (MIC ≥ 4 μg/mL), trimethoprim–sulfamethoxazole (cotrimoxazole) (MIC ≥ 100 μg/mL), and ciprofloxacin (MIC ≥ 4 μg/mL), while the below ones to an E. coli isolate susceptible to nitrofurantoin (MIC ≤ 18 μg/mL) and trimethoprim–sulfamethoxazole (MIC ≤ 25 μg/mL) and resistant to ceftriaxone (MIC ≥ 4 μg/mL) and ciprofloxacin (MIC ≥ 4 μg/mL). All these results were in agreement with the standard cultivation method. Full article ">Figure 2
Fluorescence distribution for E. coli 127, E. coli 428, and E. coli 547 isolates. These strains were classified as susceptible to ceftriaxone, ciprofloxacin, and trimethoprim–sulfamethoxazole, respectively, and resistant by standard cultivation method. These results were considered as false-negative. Full article ">Figure 3
Fluorescence distribution for E. coli 2432 and E. coli 491 isolates. These strains were classified as resistant to trimethoprim–sulfamethoxazole and, respectively, to ceftriaxone by flow cytometry (FC) and susceptible by cultivation method. These results were considered as false-positive. Full article ">

9 pages, 1090 KiB

Open AccessCommunication

Genomic Insight of VIM-harboring IncA Plasmid from a Clinical ST69 Escherichia coli Strain in Italy

by Vittoria Mattioni Marchetti, Ibrahim Bitar, Aurora Piazza, Alessandra Mercato, Elena Fogato, Jaroslav Hrabak and Roberta Migliavacca

Microorganisms 2020, 8(8), 1232; https://doi.org/10.3390/microorganisms8081232 - 12 Aug 2020

Cited by 5 | Viewed by 3293

Abstract

Background: VIM (Verona Integron-encoded Metallo-beta-lactamase) is a member of the Metallo-Beta-Lactamases (MBLs), and is able to hydrolyze all beta-lactams antibiotics, except for monobactams, and including carbapenems. Here we characterize a VIM-producing IncA plasmid isolated from a clinical ST69 Escherichia coli strain from [...] Read more.

Background: VIM (Verona Integron-encoded Metallo-beta-lactamase) is a member of the Metallo-Beta-Lactamases (MBLs), and is able to hydrolyze all beta-lactams antibiotics, except for monobactams, and including carbapenems. Here we characterize a VIM-producing IncA plasmid isolated from a clinical ST69 Escherichia coli strain from an Italian Long-Term Care Facility (LTCF) inpatient. Methods: An antimicrobial susceptibility test and conjugation assay were carried out, and the transferability of the bla_VIM-type gene was confirmed in the transconjugant. Whole-genome sequencing (WGS) of the strain 550 was performed using the Sequel I platform. Genome assembly was performed using “Microbial Assembly”. Genomic analysis was conducted by uploading the contigs to ResFinder and PlasmidFinder databases. Results: Assembly resulted in three complete circular contigs: the chromosome (4,962,700 bp), an IncA plasmid (p550_IncA_VIM_1; 162,608 bp), harboring genes coding for aminoglycoside resistance (aac(6′)-Ib4, ant(3″)-Ia, aph(3″)-Ib, aph(3′)-XV, aph(6)-Id), beta-lactam resistance (bla_SHV-12, bla_VIM-1), macrolides resistance (mph(A)), phenicol resistance (catB2), quinolones resistance (qnrS1), sulphonamide resistance (sul1, sul2), and trimethoprim resistance (dfrA14), and an IncK/Z plasmid (p550_IncB_O_K_Z; 100,306 bp), free of antibiotic resistance genes. Conclusions: The increase in reports of IncA plasmids bearing different antimicrobial resistance genes highlights the overall important role of IncA plasmids in disseminating carbapenemase genes, with a preference for the bla_VIM-1 gene in Italy. Full article

(This article belongs to the Special Issue Bacterial Responses to Environmental Stress and Their Specific Contribution to Escherichia coli and Vibrio spp. Survival and Virulence)

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14 pages, 1363 KiB

Open AccessArticle

Enhancement of the Molecular and Serological Assessment of Hepatitis E Virus in Milk Samples

by Ibrahim M. Sayed, Ahmed R. A. Hammam, Mohamed Salem Elfaruk, Khalid A. Alsaleem, Marwa A. Gaber, Amgad A. Ezzat, Eman H. Salama, Amal A. Elkhawaga and Mohamed A. El-Mokhtar

Microorganisms 2020, 8(8), 1231; https://doi.org/10.3390/microorganisms8081231 - 12 Aug 2020

Cited by 14 | Viewed by 3699

Abstract

Hepatitis E virus (HEV) infection is endemic in developing and developed countries. HEV was reported to be excreted in the milk of ruminants, raising the possibility of transmission of HEV infection through the ingestion of contaminated milk. Therefore, the detection of HEV markers [...] Read more.

Hepatitis E virus (HEV) infection is endemic in developing and developed countries. HEV was reported to be excreted in the milk of ruminants, raising the possibility of transmission of HEV infection through the ingestion of contaminated milk. Therefore, the detection of HEV markers in milk samples becomes pivotal. However, milk includes inhibitory components that affect HEV detection assays. Previously it was reported that dilution of milk matrix improves the performance of HEV molecular assay, however, the dilution of milk samples is not the best strategy especially when the contaminated milk sample has a low HEV load. Therefore, the objective of this study is to compare the effect of extraction procedures on the efficiency of HEV RNA detection in undiluted milk samples. In addition, we assessed the effect of the removal of milk components such as fats and casein on the performance of the molecular and serological assays of HEV. Phosphate buffered saline (PBS) and different milk matrices (such as whole milk, skim milk, and milk serum) were inoculated with different HEV inoculums and subjected to two different extraction procedures. Method A includes manual extraction using spin column-based extraction, while method B includes silica-based automated extraction. Method A was more sensitive than method B in the whole milk and skim milk matrices with a LoD_95% of 300 IU/mL, and virus recovery yield of 47%. While the sensitivity and performance of method B were significantly improved using the milk serum matrix, with LoD_95% of 96 IU/mL. Interestingly, retesting HEV positive milk samples using the high sensitivity assay based on method B extraction and milk serum matrix increased the HEV RNA detection rate to 2-fold. Additionally, the performance of HEV serological assays such as anti-HEV IgG and HEV Ag in the milk samples was improved after the removal of the fat globules from the milk matrix. In conclusion, HEV RNA assay is affected by the components of milk and the extraction procedure. Removal of inhibitory substances, such as fat and casein from the milk sample increased the performance of HEV molecular and serological assays which will be suitable for the low load HEV milk with no further dilutions. Full article

(This article belongs to the Special Issue Enteric Virus Detection: Recent Developments and Application in Food and Waters)

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Figure 1
A schematic diagram showing the study design of the effect of extraction procedures and milk components on hepatitis E virus (HEV) molecular assays. Full article ">Figure 2
Schematic design showing the experiment design “Effect of fat removal on the performance of HEV serology assay.” Full article ">Figure 3
(A) Comparing the effect of two extraction procedures on the recovery of HEV from the whole milk matrix. Method A includes spin column-based manual extraction, and method B includes automated extraction using magnetic beads. Comparison of HEV recovery in method A vs. method B, ** indicates p < 0.01 as determined by unpaired two-tailed Student’s t-test. (B) The recovery yield of Internal Positive Control (IPC) added to each sample was calculated for the two extraction procedures. Method A represented here includes data of high pure viral nucleic acid. * indicates p < 0.05 as determined by unpaired Student’s t-test. Full article ">Figure 4
(A) Comparing the effect of extraction procedures and milk matrices on the recovery of HEV from the whole milk matrix. Comparison of HEV recovery in skim milk matrix and milk serum matrix using method B, * indicates p < 0.05 as determined by Tukey’s multiple comparison tests. (B) The recovery yield of IPC. Comparison of IPC recovery in the skim milk matrix and milk serum matrix using method B * indicates p < 0.05 as determined by Tukey’s multiple comparison tests. Full article ">Figure 5
Effect of fat removal on the performance of HEV serology assay. (Anti HEV IgG (A) and HEV Ag (B) were retested in the cow and goat milk samples before (whole milk) and after (skim milk) the removal of fat globules. Negative whole milk samples and negative skim samples were run in the same assay, and the cut off was calculated. S/C.O. means absorbance/ cut off. ** indicates p < 0.01 as determined by unpaired Student’s t-test. Full article ">

11 pages, 3136 KiB

Open AccessArticle

Involvement of the cbb₃-Type Terminal Oxidase in Growth Competition of Bacteria, Biofilm Formation, and in Switching between Denitrification and Aerobic Respiration

by Igor Kučera and Vojtěch Sedláček

Microorganisms 2020, 8(8), 1230; https://doi.org/10.3390/microorganisms8081230 - 12 Aug 2020

Cited by 8 | Viewed by 2709

Abstract

Paracoccus denitrificans has a branched electron transport chain with three terminal oxidases transferring electrons to molecular oxygen, namely aa₃-type and cbb₃-type cytochrome c oxidases and ba₃-type ubiquinol oxidase. In the present study, we focused on strains expressing [...] Read more.

Paracoccus denitrificans has a branched electron transport chain with three terminal oxidases transferring electrons to molecular oxygen, namely aa₃-type and cbb₃-type cytochrome c oxidases and ba₃-type ubiquinol oxidase. In the present study, we focused on strains expressing only one of these enzymes. The competition experiments showed that possession of cbb₃-type oxidase confers significant fitness advantage during oxygen-limited growth and supports the biofilm lifestyle. The aa₃-type oxidase was shown to allow rapid aerobic growth at a high oxygen supply. Activity of the denitrification pathway that had been expressed in cells grown anaerobically with nitrate was fully inhibitable by oxygen only in wild-type and cbb₃ strains, while in strains aa₃ and ba₃ dinitrogen production from nitrate and oxygen consumption occurred simultaneously. Together, the results highlight the importance of the cbb₃-type oxidase for the denitrification phenotype and suggest a way of obtaining novel bacterial strains capable of aerobic denitrification. Full article

(This article belongs to the Section Microbial Biotechnology)

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Figure 1
Relative fitness (W) of P. denitrificans strains versus the Pd9312 (ba3) strain as a reference. The results are from five separate experiments and represent means ± standard deviation (S.D.). Left bars, separate cultures; right bars, 1:1 mixed culture. The asterisk denotes statistically significant difference between the respective mixed culture and separate cultures (paired t-test, p < 0.01). There is no significant difference (analysis of variance (ANOVA), p = 0.48) in the W values for separate cultures (left bars) and none of these values differs significantly from 1 (one sample t-test, p > 0.05). Full article ">Figure 2
Planktonic bacterial growth (left bars) and biofilm formation (right bars) in polystyrene Petri dishes after 72 h. Data are normalized to wild-type values. Bar heights show mean values of five replicates, error bars show standard deviations. Means represented by left bars do not statistically differ (ANOVA, p = 0.12), while right bar values differ significantly from each other (t-test, p < 0.01). Full article ">Figure 3
Dynamics of O2 consumption and production of N2 from nitrate in wild-type and single-oxidase strains of P. denitrificans. O2 and 15N2 concentrations were monitored by membrane inlet mass spectrometry (MIMS) at m/z 32 and 30 respectively. The measuring chamber was filled up with 5 mL of 0.1 M sodium phosphate pH 7.3, containing 5 mM sodium succinate, 1 mM 15N-NaNO3 and 0.24 mM O2. The arrow indicates addition of 100 μL of suspension of anaerobically grown cells (5 mg dry weight). Full article ">Figure 4
Kinetics of O2 and N2O respiration in wild-type and ba3 single-route strain of P. denitrificans. O2 and N2O concentrations were monitored by MIMS at m/z 32 and 30 respectively. The measuring chamber was filled up with 5 mL of 0.1 M sodium phosphate pH 7.3, containing 5 mM sodium succinate, 0.24 mM O2 and 0.24 mM N2O. The experiment was started by the addition of anaerobically grown cells (5 mg dry weight) at zero time. Full article ">Figure 5
Electron acceptor-induced transient oxidation of cytochromes c. A closed 1-cm cuvette contained 3 mL of 5 mM succinate in 0.1 M sodium phosphate buffer (pH 7.3, 30 °C). At zero time, 9.3 mg dry weight of anaerobically grown cells were added and the time course of cytochrome c reduction was measured by dual wavelength spectroscopy at the wavelength pair 550 minus 535 nm. 0.24 mM O2 and/or 0.17 mM nitrate were present initially or at the times indicated by arrows. FeCy stands for potassium ferricyanide, added to attain the fully oxidized level. Full article ">

17 pages, 5678 KiB

Open AccessArticle

Import of Entamoeba histolytica Mitosomal ATP Sulfurylase Relies on Internal Targeting Sequences

by Herbert J. Santos, Yoko Chiba, Takashi Makiuchi, Saki Arakawa, Yoshitaka Murakami, Kentaro Tomii, Kenichiro Imai and Tomoyoshi Nozaki

Microorganisms 2020, 8(8), 1229; https://doi.org/10.3390/microorganisms8081229 - 12 Aug 2020

Cited by 2 | Viewed by 3551

Abstract

Mitochondrial matrix proteins synthesized in the cytosol often contain amino (N)-terminal targeting sequences (NTSs), or alternately internal targeting sequences (ITSs), which enable them to be properly translocated to the organelle. Such sequences are also required for proteins targeted to mitochondrion-related organelles (MROs) that [...] Read more.

Mitochondrial matrix proteins synthesized in the cytosol often contain amino (N)-terminal targeting sequences (NTSs), or alternately internal targeting sequences (ITSs), which enable them to be properly translocated to the organelle. Such sequences are also required for proteins targeted to mitochondrion-related organelles (MROs) that are present in a few species of anaerobic eukaryotes. Similar to other MROs, the mitosomes of the human intestinal parasite Entamoeba histolytica are highly degenerate, because a majority of the components involved in various processes occurring in the canonical mitochondria are either missing or modified. As of yet, sulfate activation continues to be the only identified role of the relic mitochondria of Entamoeba. Mitosomes influence the parasitic nature of E. histolytica, as the downstream cytosolic products of sulfate activation have been reported to be essential in proliferation and encystation. Here, we investigated the position of the targeting sequence of one of the mitosomal matrix enzymes involved in the sulfate activation pathway, ATP sulfurylase (AS). We confirmed by immunofluorescence assay and subcellular fractionation that hemagluttinin (HA)-tagged EhAS was targeted to mitosomes. However, its ortholog in the δ-proteobacterium Desulfovibrio vulgaris, expressed as DvAS-HA in amoebic trophozoites, indicated cytosolic localization, suggesting a lack of recognizable mitosome targeting sequence in this protein. By expressing chimeric proteins containing swapped sequences between EhAS and DvAS in amoebic cells, we identified the ITSs responsible for mitosome targeting of EhAS. This observation is similar to other parasitic protozoans that harbor MROs, suggesting a convergent feature among various MROs in favoring ITS for the recognition and translocation of targeted proteins. Full article

(This article belongs to the Special Issue Virulence and Parasitism of Parasitic Protozoa)

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21 pages, 1485 KiB

Open AccessReview

COVID-19 Is a Multifaceted Challenging Pandemic Which Needs Urgent Public Health Interventions

by Carlo Contini, Elisabetta Caselli, Fernanda Martini, Martina Maritati, Elena Torreggiani, Silva Seraceni, Fortunato Vesce, Paolo Perri, Leonzio Rizzo and Mauro Tognon

Microorganisms 2020, 8(8), 1228; https://doi.org/10.3390/microorganisms8081228 - 12 Aug 2020

Cited by 33 | Viewed by 18463

Abstract

Until less than two decades ago, all known human coronaviruses (CoV) caused diseases so mild that they did not stimulate further advanced CoV research. In 2002 and following years, the scenario changed dramatically with the advent of the new more pathogenic CoVs, including [...] Read more.

Until less than two decades ago, all known human coronaviruses (CoV) caused diseases so mild that they did not stimulate further advanced CoV research. In 2002 and following years, the scenario changed dramatically with the advent of the new more pathogenic CoVs, including Severe Acute Respiratory Syndome (SARS-CoV-1), Middle Eastern respiratory syndrome (MERS)-CoV, and the new zoonotic SARS-CoV-2, likely originated from bat species and responsible for the present coronavirus disease (COVID-19), which to date has caused 15,581,007 confirmed cases and 635,173 deaths in 208 countries, including Italy. SARS-CoV-2 transmission is mainly airborne via droplets generated by symptomatic patients, and possibly asymptomatic individuals during incubation of the disease, although for the latter, there are no certain data yet. However, research on asymptomatic viral infection is currently ongoing worldwide to elucidate the real prevalence and mortality of the disease. From a clinical point of view, COVID-19 would be defined as “COVID Planet “ because it presents as a multifaceted disease, due to the large number of organs and tissues infected by the virus. Overall, based on the available published data, 80.9% of patients infected by SARS-CoV-2 develop a mild disease/infection, 13.8% severe pneumonia, 4.7% respiratory failure, septic shock, or multi-organ failure, and 3% of these cases are fatal, but mortality parameter is highly variable in different countries. Clinically, SARS-CoV-2 causes severe primary interstitial viral pneumonia and a “cytokine storm syndrome”, characterized by a severe and fatal uncontrolled systemic inflammatory response triggered by the activation of interleukin 6 (IL-6) with development of endothelitis and generalized thrombosis that can lead to organ failure and death. Risk factors include advanced age and comorbidities including hypertension, diabetes, and cardiovascular disease. Virus entry occurs via binding the angiotensin-converting enzyme 2 (ACE2) receptor present in almost all tissues and organs through the Spike (S) protein. Currently, SARS-CoV-2 infection is prevented by the use of masks, social distancing, and improved hand hygiene measures. This review summarizes the current knowledge on the main biological and clinical features of the SARS-CoV-2 pandemic, also focusing on the principal measures taken in some Italian regions to face the emergency and on the most important treatments used to manage the COVID-19 pandemic. Full article

(This article belongs to the Special Issue COVID-19: Focusing on Epidemiologic, Virologic, and Clinical Studies)

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17 pages, 2844 KiB

Open AccessArticle

Gene Expression and Photophysiological Changes in Pocillopora acuta Coral Holobiont Following Heat Stress and Recovery

by Rosa Celia Poquita-Du, Yi Le Goh, Danwei Huang, Loke Ming Chou and Peter A. Todd

Microorganisms 2020, 8(8), 1227; https://doi.org/10.3390/microorganisms8081227 - 12 Aug 2020

Cited by 17 | Viewed by 4679

Abstract

The ability of corals to withstand changes in their surroundings is a critical survival mechanism for coping with environmental stress. While many studies have examined responses of the coral holobiont to stressful conditions, its capacity to reverse responses and recover when the stressor [...] Read more.

The ability of corals to withstand changes in their surroundings is a critical survival mechanism for coping with environmental stress. While many studies have examined responses of the coral holobiont to stressful conditions, its capacity to reverse responses and recover when the stressor is removed is not well-understood. In this study, we investigated among-colony responses of Pocillopora acuta from two sites with differing distance to the mainland (Kusu (closer to the mainland) and Raffles Lighthouse (further from the mainland)) to heat stress through differential expression analysis of target genes and quantification of photophysiological metrics. We then examined how these attributes were regulated after the stressor was removed to assess the recovery potential of P. acuta. The fragments that were subjected to heat stress (2 °C above ambient levels) generally exhibited significant reduction in their endosymbiont densities, but the extent of recovery following stress removal varied depending on natal site and colony. There were minimal changes in chl a concentration and maximum quantum yield (Fv/Fm, the proportion of variable fluorescence (Fv) to maximum fluorescence (Fm)) in heat-stressed corals, suggesting that the algal endosymbionts’ Photosystem II was not severely compromised. Significant changes in gene expression levels of selected genes of interest (GOI) were observed following heat exposure and stress removal among sites and colonies, including Actin, calcium/calmodulin-dependent protein kinase type IV (Camk4), kinesin-like protein (KIF9), and small heat shock protein 16.1 (Hsp16.1). The most responsive GOIs were Actin, a major component of the cytoskeleton, and the adaptive immune-related Camk4 which both showed significant reduction following heat exposure and subsequent upregulation during the recovery phase. Our findings clearly demonstrate specific responses of P. acuta in both photophysiological attributes and gene expression levels, suggesting differential capacity of P. acuta corals to tolerate heat stress depending on the colony, so that certain colonies may be more resilient than others. Full article

(This article belongs to the Special Issue The Human Epoch: Cnidarians Holobiont Responses from Physiology to Epigenetic)

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11 pages, 565 KiB

Open AccessArticle

Diversity, Antibiotic Resistance, and Biofilm-Forming Ability of Enterobacteria Isolated from Red Meat and Poultry Preparations

by Rosa Capita, Ana Castaño-Arriba, Cristina Rodríguez-Melcón, Gilberto Igrejas, Patricia Poeta and Carlos Alonso-Calleja

Microorganisms 2020, 8(8), 1226; https://doi.org/10.3390/microorganisms8081226 - 12 Aug 2020

Cited by 10 | Viewed by 3764

Abstract

A total of 44 samples of beef, pork, and poultry preparations were tested. Average counts (log cfu/g) of enterobacteria were 1.99 ± 0.99 (beef preparations), 1.96 ± 1.44 (pork), 2.09 ± 0.92 (chicken), and 2.17 ± 1.06 (turkey) (p > 0.05). Two [...] Read more.

A total of 44 samples of beef, pork, and poultry preparations were tested. Average counts (log cfu/g) of enterobacteria were 1.99 ± 0.99 (beef preparations), 1.96 ± 1.44 (pork), 2.09 ± 0.92 (chicken), and 2.17 ± 1.06 (turkey) (p > 0.05). Two hundred enterobacterial strains were identified and 13 genera (21 species) were distinguished, including species that are a significant cause of infection. The most common genera were Escherichia (32.5% of strains), Serratia (17.0%), Hafnia (12.5%), and Salmonella (12.0%). Isolates were screened by disc diffusion for susceptibility to 15 antibiotics. A total of 126 strains (63% of the isolates) were multirresistant (having resistance to two or more antibiotics), 46 (23%) were resistant to one antibiotic, and 28 (14%) were sensitive to all antibiotics. The average number of resistances per strain was 2.53 ± 2.05. A higher (p < 0.05) average number of resistances was observed in strains from turkey (3.14 ± 2.55) than in strains from beef (2.15 ± 1.22), pork (2.16 ± 1.39), or chicken (2.44 ± 2.22). At least 50% of strains showed resistance or reduced susceptibility to ampicillin, cefotaxime, ceftazidime, or streptomycin, considered to be “critically important” antimicrobial agents in human medicine. Seventy-nine strains (39.5%), 60 strains (30.0%), and 46 strains (23.0%) were weak, moderate, and strong biofilm producers (crystal violet assay), respectively. This investigation provides evidence that bacteria from red meat and poultry preparations pose major potential risk to consumers. Full article

(This article belongs to the Section Food Microbiology)

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17 pages, 4939 KiB

Open AccessArticle

Lactobacillus Mucosae Strain Promoted by a High-Fiber Diet in Genetic Obese Child Alleviates Lipid Metabolism and Modifies Gut Microbiota in ApoE^-/- Mice on a Western Diet

by Tianyi Jiang, Huan Wu, Xin Yang, Yue Li, Ziyi Zhang, Feng Chen, Liping Zhao and Chenhong Zhang

Microorganisms 2020, 8(8), 1225; https://doi.org/10.3390/microorganisms8081225 - 12 Aug 2020

Cited by 25 | Viewed by 4351

Abstract

Supplementation of probiotics is a promising gut microbiota-targeted therapeutic method for hyperlipidemia and atherosclerosis. However, the selection of probiotic candidate strains is still empirical. Here, we obtained a human-derived strain, Lactobacillus mucosae A1, which was shown by metagenomic analysis to be promoted by [...] Read more.

Supplementation of probiotics is a promising gut microbiota-targeted therapeutic method for hyperlipidemia and atherosclerosis. However, the selection of probiotic candidate strains is still empirical. Here, we obtained a human-derived strain, Lactobacillus mucosae A1, which was shown by metagenomic analysis to be promoted by a high-fiber diet and associated with the amelioration of host hyperlipidemia, and validated its effect on treating hyperlipidemia and atherosclerosis as well as changing structure of gut microbiota in ApoE^-/- mice on a Western diet. L. mucosae A1 attenuated the severe lipid accumulation in serum, liver and aortic sinus of ApoE^-/- mice on a Western diet, while it also reduced the serum lipopolysaccharide-binding protein content of mice, reflecting the improved metabolic endotoxemia. In addition, L. mucosae A1 shifted the gut microbiota structure of ApoE^-/- mice on a Western diet, including recovering a few members of gut microbiota enhanced by the Western diet. This study not only suggests the potential of L. mucosae A1 to be a probiotic in the treatment of hyperlipidemia and atherosclerosis, but also highlights the advantage of such function-based rather than taxonomy-based strategies for the selection of candidate strains for the next generation probiotics. Full article

(This article belongs to the Special Issue The Human Gut Microbiome, Diets and Health)

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Figure 1
Identification and genomic characterization of L. mucosae A1. (A) Electron micrograph of A1 strain. The scale bar is 5 μm. (B) Phylogenetic relationships of the A1 strain with its relatives based on 16S rRNA gene sequences. The nearest neighbor of A1 strain was L. mucosae LM1. Escherichia coli U5/41 was used as an outgroup. The tree was constructed using the Neighbor-Joining method in MEGA6. The bar indicates sequence divergence. (C) Functional categories in the clusters of orthologous groups (COG) analysis of L. mucosae A1. The numbers out the brackets on the circle represents the numbers of genes in each COG category. The numbers in the brackets are the percentages of genes in the corresponding categories to the total genes identified by COG database. Those COGs have more than 5% of the total genes are marked with an asterisk. Full article ">Figure 2
L. mucosae A1 alleviated lipid accumulation of ApoE-/- mice fed with a Western diet. (A) Body weight gain measured at the 13th week as the percentage of baseline weight for each mouse. (B) Epididymal, subcutaneous, retroperitoneal, mesenteric and total adipose tissue weight (ratio to body weight). (C) Representative photomicrographs of hematoxylin and eosin (H&E)-stained sections of epididymal adipose tissue (eAT) under 100× magnification and mean cell area of adipocyte in eAT. The scale bar is 200 μm. (D) The level of total cholesterol and triglyceride in serum collected at the 4th, 8th and 13th week. (E) Liver weight (ratio to body weight). (F) The level of total cholesterol and triglyceride in liver. (G) Representative photomicrographs of H&E-stained sections of liver under 100× magnification and volume density of liver steatosis. The scale bar is 200 μm. All the data are shown as means ± s.e.m. NC: n = 7, WD: n = 8, WD + LM: n = 9. Values of each group were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 3
L. mucosae A1 protected Western diet-fed ApoE-/- mice from atherosclerosis. (A) Representative photomicrographs of Oil Red O-stained sections of aortic sinus under 40× magnification and area ratio of atherosclerotic plaque. The scale bar is 500 μm. (B) The level of lipopolysaccharide-binding protein (LBP) in serum. (C) The level of trimethylamine (TMA) and trimethylamine-N-oxide (TMAO) in serum collected at the 4th, 8th and 13th week. All the data are shown as means ± s.e.m. NC: n = 7, WD: n =8, WD + LM: n =9. Values of each group were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 4
Modulation of gut microbiota in ApoE-/- mice after administration of a Western diet with or without L. mucosae A1 for 13 weeks. (A) Amplicon sequence variants (ASVs) richness of gut microbiota. (B) Shannon index of gut microbiota. (C) Principal coordinate analysis (PCoA) plot of gut microbiota based on weighted Unifrac distance and weighted Unifrac distance from WD group and WD + LM group to NC group. Data for ASVs richness, Shannon index and distance are shown as means ± s.e.m. Number of mice per group: NC: 7, WD: 8, WD + LM: 9. Values for ASVs richness and Shannon index of each group were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Values for distance were analyzed by unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. (D) 47 ASVs identified as key variables for differentiation between the gut microbiota of WD + LM group and that of WD group by sparse partial least squares discriminant analysis (sPLS-DA) model (shown in <a href="#app1-microorganisms-08-01225" class="html-app">Figure S8</a>). Left, the heatmap represents the normalized and log2-transformed relative abundance of the ASVs in each sample. The ASVs were clustered by the ward.D method. Middle, the changing direction of 47 ASVs in NC group and WD+LM group compared to WD group according to sPLS-DA models (shown in <a href="#app1-microorganisms-08-01225" class="html-app">Figures S7 and S8</a>). Redness and blueness indicate the relative abundance of ASVs were more and less, respectively, compared to WD group. The relative abundance of key ASVs were compared between groups by Mann–Whitney U test. * p < 0.05, ** p < 0.01, *** p < 0.001. Right, Spearman correlations between the host parameters related to lipid metabolism as well as atherosclerosis and the relative abundance of 47 ASVs. Colors red and blue denote positive and negative association, respectively. The intensity of the colors represents the degree of association between the abundances of ASVs and host parameters. p values of spearman correlations were adjusted by false discovery rate (FDR). * adjusted p < 0.05, ** adjusted p < 0.01, *** adjusted p < 0.001. The genus-level taxonomic classifications of the ASVs are shown. For all the figures, NC: n = 7, WD: n = 8, WD+LM: n = 9. Full article ">

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