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Microorganisms
Volume 8
Issue 3
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Microorganisms, Volume 8, Issue 3 (March 2020) – 149 articles

Cover Story (view full-size image): Siboglinidae worms are known to host chemosynthetic endosymbionts in a dedicated trophosome organ. However, little is known about their tube as a potential niche for other microorganisms. In this study, siboglinids sampled from four mud volcanoes in the Gulf of Cádiz (El Cid MV, Bonjardim MV, Al Gacel MV, and Anastasya MV) revealed that the tube was colonized by a thick microbial biofilm. This external biofilm of the tubes was mostly composed of cell-aggregations of methanotrophic bacteria, but other morphotypes such as filamentous, prosthecate, spirillum-like and rod-shaped bacteria were also observed. Yet, these microorganisms seem to influence in the structure and composition of the tube. Thus, siboglinids’ tubes remarkably increase the microbial biomass related to the worms and provide an additional microbial niche in deep-sea ecosystems.View this paper.
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18 pages, 1863 KiB  
Review
The Essential Co-Option of Uracil-DNA Glycosylases by Herpesviruses Invites Novel Antiviral Design
by Renos Savva
Microorganisms 2020, 8(3), 461; https://doi.org/10.3390/microorganisms8030461 - 24 Mar 2020
Cited by 5 | Viewed by 4480
Abstract
Vast evolutionary distances separate the known herpesviruses, adapted to colonise specialised cells in predominantly vertebrate hosts. Nevertheless, the distinct herpesvirus families share recognisably related genomic attributes. The taxonomic Family Herpesviridae includes many important human and animal pathogens. Successful antiviral drugs targeting Herpesviridae are [...] Read more.
Vast evolutionary distances separate the known herpesviruses, adapted to colonise specialised cells in predominantly vertebrate hosts. Nevertheless, the distinct herpesvirus families share recognisably related genomic attributes. The taxonomic Family Herpesviridae includes many important human and animal pathogens. Successful antiviral drugs targeting Herpesviridae are available, but the need for reduced toxicity and improved efficacy in critical healthcare interventions invites novel solutions: immunocompromised patients presenting particular challenges. A conserved enzyme required for viral fitness is Ung, a uracil-DNA glycosylase, which is encoded ubiquitously in Herpesviridae genomes and also host cells. Research investigating Ung in Herpesviridae dynamics has uncovered an unexpected combination of viral co-option of host Ung, along with remarkable Subfamily-specific exaptation of the virus-encoded Ung. These enzymes apparently play essential roles, both in the maintenance of viral latency and during initiation of lytic replication. The ubiquitously conserved Ung active site has previously been explored as a therapeutic target. However, exquisite selectivity and better drug-like characteristics might instead be obtained via targeting structural variations within another motif of catalytic importance in Ung. The motif structure is unique within each Subfamily and essential for viral survival. This unique signature in highly conserved Ung constitutes an attractive exploratory target for the development of novel beneficial therapeutics. Full article
(This article belongs to the Special Issue Herpesviruses: Virus-Host Interaction)
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Figure 1
<p>DNA pyrimidines and their interconversion. The pyrimidine uracil as a deoxynucleotide is a precursor in thymidine biosynthesis. Deoxyuridine can accrue in DNA by misincorporation under conditions of nucleotide pool perturbation and imbalance such as during viral replication; its unique structure can create dysfunction in gene regulation. The DNA base cytosine is also converted to 5-methylcytosine in epigenetic imprinting. Both cytosine and 5-methylcytosine can convert in situ to uracil and to thymine, respectively, via spontaneous loss of the 4-amino group under ambient cellular conditions. This conversion in the context of the original base pair is considered promutagenic and must be repaired to retain sequence fidelity upon replication. Enzymes listed by KEGG Orthology are: 2.1.1.37 (cytosine-5)-methyltransferase; 2.1.1.45 thymidylate synthase; 2.7.1.21 thymidine kinase; 2.7.4.6 nucleoside diphosphate kinase; 2.7.4.9 thymidylate kinase; 3.1.3.89 HD-domain 5′-nucleotidase; 3.5.4.5 cytidine deaminase; 3.5.4.12 dCMP deaminase.</p> Full article ">Figure 2
<p>The base excision repair pathway. (<b>A</b>) Uracil spontaneously occurs in DNA due to the deamination of cytosine bases. If left uncorrected, the uracil-containing strand will give rise to a permanently mutated daughter strand upon replication. (<b>B</b>) A uracil-DNA glycosylase (UDG) cleaves the uracil base from the DNA backbone, leaving an abasic site in the DNA. (<b>C</b>) An AP-endonuclease (APE) cleaves the phosphodiester backbone, 5′ of an abasic site [AP refers to apyrimidinic, or lacking a pyrimidine; it can also refer to apurinic in other contexts]. (<b>D</b>) A type-B DNA polymerase (POLB), typically involved in short-patch DNA repair, resynthesises several bases complementary to the undamaged strand while displacing the cleaved strand beginning at the nick generated by AP-endonuclease. (<b>E</b>) A Flap-Endonuclease (FEN) cleaves off the displaced DNA strand after the B-type DNA polymerase has dissociated, leaving behind a nick in the DNA duplex. (<b>F</b>) A DNA ligase (LIG) seals the nick to return DNA to its canonical pre-damaged state.</p> Full article ">Figure 3
<p>UDG can contextually disrupt DNA integrity. The destructive effect on DNA chain integrity after the action of uracil-DNA glycosylase (UDG) and AP-endonuclease (APE) during initiation of BER, on uracil-containing DNA in situations where: (<b>a</b>) Uracil occurs in a single-stranded DNA molecule, in which case there could be stalling or disruption of DNA replication, or of ssDNA-transfer processes such as in bacterial conjugation or in virus replication or packaging. (<b>b</b>) There are unusually high levels of uracil in duplex DNA, in which case backbone breaks will be enzymatically created on both strands of dsDNA at close proximity. At short oligonucleotide lengths, the hydrogen bonding forces between complementary base pairs are insufficient to resist the thermal disintegration of the duplex, and the consequence is chain fragmentation that is incompatible with high-fidelity DNA repair.</p> Full article ">Figure 4
<p>The roles of Ung in cellular programs. On the left side of the schematic: Innate cellular immunity. Ung acts upon spontaneously arising or enzymatically generated uracil residues. For random chemical events, Ung initiates DNA repair by base excision repair (BER); in the pathogen response, UDG follows APOBEC3 enzymes that deaminate cytosine in pathogen DNA to cause hyperuracilation. On the right side of the schematic: Development of humoral immunity. Ung acts downstream of the cytidine deaminase AID, which generates uracil residues in (i) Immunoglobulin variable chain regions to initiate somatic hyper mutation (SHM), generating variable chain diversity; and (ii) immunoglobulin class switch regions to initiate class switch recombination (CSR), permitting successful antibody variable chains to be deployed via alternative antibody scaffolds, e.g., IgM to IgG. The role of Ung and APE are the same as in BER, but the different pathways (error-prone repair, or non-homologous end joining) deploy alternative suites of proteins downstream to create the intended outcome.</p> Full article ">Figure 5
<p>Sequence and structure alignment of the herpesvirus adapted motif central to Ung catalysis. (<b>a</b>) Multiple sequence alignment of the Ung minor groove DNA intercalation loop motif, generated via structural superposition of protein chains: UNG2 (Human), UL2 (HHV-1), and ORF46 / HHV8GK18_gp50 (HHV-8), using the program Chimera: PDB accession codes are used (UNG2 = 1SSP; UL2 = 1LAU; ORF46 = 5NNU, chain A). Ung sequences for which there is currently no deposited molecular structure (i.e., β-herpesviruses) are indicated in blue font (beta 1 = HHV-5; beta 2 = HHV-6A; beta 3 = HHV-6B; beta 4 = HHV-7). Residue positions lacking structural equivalence are shown in lower case with gross differences in grey font. The Ung catalytic leucine residue is in dark red font as are (in lower case) positionally equivalent betaherpesvirus residues. (<b>b</b>) Cartoon structure excerpts of the aligned region only, overlaid (from 1LAU, 1SSP and 5NNU). (<b>c</b>) The sequence aligned is displayed as a darker shaded region of the entire Ung molecule in its biological contexts: (<b>i</b>) 5NNU: ORF46 (HHV-8) Ung [chain A] in complex with dsDNA [chains S and T] (<b>ii</b>) 1SSP: UNG2 (Human) [chain E] in complex with dsDNA [chains A and B], (<b>iii</b>) 1UDH: UNG2 [chain E] in complex with Ugi [chain I], a protein mimic of DNA that specifically targets Ung. Highlighted in dark red is the Ung catalytic leucine (i),(ii),(iii) and catalysed DNA residue (i),(ii); highlighted in dark blue (i) and unique to gammaherpesvirus Ung is a key residue of an exaptation that precisely deforms DNA (also highlighted dark blue) as a possible signal essential to viral DNA replication assembly in gammaherpesviruses: This novel exaptation, shown for 5NNU (HHV-8 Ung), is conserved in HHV-4 (deposited as pdb structure 2J8X – not shown).</p> Full article ">Figure 6
<p>The latent and lytic phase of herpesviruses and relevance to uracil-DNA glycosylase. The simplified schematic depicts a cell cytosolic compartment (outer ellipse) with a nucleus (interior ellipse). Subfamily and virus are indicated in black text with uracil-DNA glycosylase encoded by that virus in blue text. Placement of text indicates the location UDG is currently thought to act (latent phase in the nucleus, and lytic phase in the cytosol) and the type of evidence supporting the premise (cyan text). Host cell encoded UNG2 is indicated with the named virus subfamily in dark red text, and placement indicates the phase of action (latency reactivation).</p> Full article ">
19 pages, 1962 KiB  
Article
First Report on the Prevalence and Subtype Distribution of Blastocystis sp. in Edible Marine Fish and Marine Mammals: A Large Scale-Study Conducted in Atlantic Northeast and on the Coasts of Northern France
by Nausicaa Gantois, Angélique Lamot, Yuwalee Seesao, Colette Creusy, Luen-Luen Li, Sébastien Monchy, Sadia Benamrouz-Vanneste, Jacky Karpouzopoulos, Jean-Luc Bourgain, Célia Rault, Fabien Demaret, Martha Baydoun, Magali Chabé, Emilie Fréalle, Cécile-Marie Aliouat-Denis, Mélanie Gay, Gabriela Certad and Eric Viscogliosi
Microorganisms 2020, 8(3), 460; https://doi.org/10.3390/microorganisms8030460 - 24 Mar 2020
Cited by 27 | Viewed by 3460
Abstract
Blastocystis is frequently identified in humans and animal hosts and exhibits a large genetic diversity with the identification of 17 subtypes (STs). Despite its zoonotic potential, its prevalence and ST distribution in edible marine fish and marine mammals remain unknown. A large-scale survey [...] Read more.
Blastocystis is frequently identified in humans and animal hosts and exhibits a large genetic diversity with the identification of 17 subtypes (STs). Despite its zoonotic potential, its prevalence and ST distribution in edible marine fish and marine mammals remain unknown. A large-scale survey was thus conducted by screening 345 fish caught in Atlantic Northeast and 29 marine mammals stranded on the coasts of northern France for the presence of the parasite using real-time Polymerase Chain Reaction PCR. The prevalence of the parasite was about 3.5% in marine fish. These animals were mostly colonized by poikilotherm-derived isolates not identified in humans and corresponding to potential new STs, indicating that fish are natural hosts of Blastocystis. Marine fishes are also carriers of human STs and represent a likely limited source of zoonotic transmission. 13.8% of the marine mammals tested were colonized and 6 different STs were identified including 3 potential new STs. The risk of zoonotic transmission through marine mammals is insignificant due to the lack of repeated contact with humans. The present survey represents the first data regarding the prevalence and ST distribution of Blastocystis in marine fish and marine mammals and provides new insights into its genetic diversity, host range and transmission. Full article
(This article belongs to the Section Parasitology)
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<p>Detailed boundaries of the fishing sub-areas within the Atlantic Northeast zone 27 (<b>A</b>) and the geographical location of marine mammal stranding sites (<b>B</b>). The number of fishes collected for the selected species in each sub-area is indicated as well as the number of marine mammals and the corresponding species for each stranded site on the coasts of northern France.</p> Full article ">Figure 2
<p>Unrooted maximum likelihood cladogram based on SSU rDNA sequences depicting relationships between <span class="html-italic">Blastocystis</span> sp. isolates. Only the tree with the highest log likelihood (−2762.8734) is shown. For each sequence extracted from the databases, its accession number, the potential name of the corresponding isolate and its host are indicated. Numbers near the individual nodes correspond to BP (left of the slash) and BPP (right of the slash) given by the two different tree reconstruction methods (Maximum Likelihood/MrBAYES). The asterisks designate nodes with BPs or BPPs below 50% or 0.5, respectively. The sequences obtained in the present study are shown in bold and in boxes.</p> Full article ">Figure 3
<p>Hematoxylin and eosin-stained section of the bowel of herring CH-II-7. The presence in large numbers of round bodies (arrows) is suggestive of <span class="html-italic">Blastocystis</span> sp. vacuolar forms located in the tissue. Scale bar = 100 µm.</p> Full article ">
12 pages, 1646 KiB  
Article
Unraveling the Fungal Community Associated with Leaf Spot on Crataegus sp.
by Sonia Salazar-Cerezo, María de la Cruz Meneses-Sánchez, Rebeca D. Martínez-Contreras and Nancy Martínez-Montiel
Microorganisms 2020, 8(3), 459; https://doi.org/10.3390/microorganisms8030459 - 24 Mar 2020
Cited by 3 | Viewed by 3447
Abstract
Crataegus sp. is a tree that grows in temperate zones with worldwide distribution and is commonly known in Mexico as tejocote. The use of products derived from Crataegus in traditional medicine, food, and cosmetics has increased over the last few years and the [...] Read more.
Crataegus sp. is a tree that grows in temperate zones with worldwide distribution and is commonly known in Mexico as tejocote. The use of products derived from Crataegus in traditional medicine, food, and cosmetics has increased over the last few years and the relevance of this plant has also grown. Here, we report a disease that was observed in tejocote plants that grew both in the wild and in greenhouses in Puebla (Mexico). The disease was characterized by necrotic spots on the leaf ranging from brown to reddish tones that were accompanied by structures on the back of the leaf. Furthermore, we investigated the fungal genera associated with infected leaves in wild tejocote plants, from which we recovered Alternaria sp., Aureobasidium sp., Dreschlera sp., Fusarium sp., Paecilomyces sp. and Ulocladium sp. genera. Inoculation on healthy Crataegus sp. plants with isolate UAP140 showed similar symptoms as observed in nature, while inoculation with UAP127 resulted in the development of necrotic lesions in the leaf. The identity of these isolates was further studied through the phylogenetic analysis of the ribosomal DNA internal transcribed spacer (ITS) region, where isolate UAP140 showed the highest identity with Fusarium equiseti and isolate UAP127 was similar to Alternaria arborescens. To our knowledge, this is the first report of a characteristic disease developed in Crataegus sp. plants in Mexico where the fungal community associated to the lesion was analyzed. Further studies would be necessary to determine the ecological and environmental implications of the microbiome on the appearance and development of the disease. Full article
(This article belongs to the Section Environmental Microbiology)
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<p>Lesions observed on leaves of <span class="html-italic">Crataegus</span> sp. plants. Samples were observed using the microscope with the 10X objective. The lesion evolved as a yellow and brown spot on the surface covering different parts of the leaf (compare (<b>A</b>), (<b>B</b>) and (<b>C</b>); scale bar = 0.5 cm). An enlarged view of the structures can be observed in (<b>D</b>) with the 40X objective; scale bar = 0.5 mm.</p> Full article ">Figure 2
<p>Abundance of fungal genera isolated from leaf disease in <span class="html-italic">Crataegus</span><b>.</b> The number of fungal isolates corresponding to the different genera identified phenotypically is shown. Isolate distribution is presented for the surface (blue), for injured tissue (orange) and for the structures (gray).</p> Full article ">Figure 3
<p>Abundance of fungal genera isolated from diseased and healthy leaves in <span class="html-italic">Crataegus</span>. Fungal distribution identified in the endophytic and epiphytic population in healthy (green) and sick (yellow) leaves is shown.</p> Full article ">Figure 4
<p>Pathogenicity test in healthy <span class="html-italic">Crataegus</span> sp. trees. The image shows injuries produced by isolates UAP140 (<b>A</b>), UAP168 (<b>B</b>) and UAP127 (<b>C</b>) in three-month-old trees. Control trees remained uninfected 35 days after infection (<b>D</b>). Scale bar = 0.5 mm.</p> Full article ">Figure 5
<p>Phylogenetic relationship for isolate UAP140. The tree with the highest log likelihood is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved six <span class="html-italic">Fusarium</span> reference nucleotide sequences with a total of 100 replicates in the bootstrap analysis, while three non-related fungal species were used as the outgroup.</p> Full article ">
21 pages, 5058 KiB  
Article
Epidemiological Surveillance of Norovirus and Rotavirus in Sewage (2016–2017) in Valencia (Spain)
by Cristina Santiso-Bellón, Walter Randazzo, Alba Pérez-Cataluña, Susana Vila-Vicent, Roberto Gozalbo-Rovira, Carlos Muñoz, Javier Buesa, Gloria Sanchez and Jesús Rodríguez Díaz
Microorganisms 2020, 8(3), 458; https://doi.org/10.3390/microorganisms8030458 - 24 Mar 2020
Cited by 49 | Viewed by 5508
Abstract
The aim of the present study was to perform the molecular epidemiology of rotaviruses and noroviruses detected in sewage samples from a large wastewater facility from the city of Valencia, Spain. A total of 46 sewage samples were collected over a one-year period [...] Read more.
The aim of the present study was to perform the molecular epidemiology of rotaviruses and noroviruses detected in sewage samples from a large wastewater facility from the city of Valencia, Spain. A total of 46 sewage samples were collected over a one-year period (September 2016 to September 2017). Norovirus and rotavirus were detected and quantified by RT-qPCR, genotyped by semi-nested RT-PCR and further characterized by sequencing and phylogenetic analyses. Noroviruses and rotaviruses were widely distributed in sewage samples (69.6% for norovirus GI, 76.0% norovirus GII, and 71.7% rotaviruses) and viral loads varied from 4.33 to 5.75 log PCRU/L for norovirus GI, 4.69 to 6.95 log PCRU/L for norovirus GII, and 4.08 to 6.92 log PCRU/L for rotavirus. Overall, 87.5% (28/32) of GI noroviruses could not be genotyped, 6.25% (2/32) of the samples contained GI.2 genotype, and another 6.25% (2/32) were positive for GI.4 genotype. The most common genotype of GII noroviruses was GII.2 (40%, 14/35), followed by GII.6 (8.6%, 3/35) and GII.17 (5.7%, 2/35) while the remaining GII strains could not be typed (45.7%, 16/35). Rotavirus VP4 genotype P[8] was the only one found in 19 out of 33 rotavirus-positive samples (57.7%). G2 was the most prevalent rotavirus VP7 genotype (15.2%, 5/33) followed by G3, G9, and G12, with two positive samples for each genotype (6.1%, 2/33). In one sample both G1 and G2 genotypes were detected simultaneously (3%). The results presented here show that the surveillance of noroviruses and rotaviruses in sewage is useful for the study of their transmission in the population and their molecular epidemiology. Full article
(This article belongs to the Special Issue Enteric Virus Detection: Recent Developments and Application in Food and Waters)
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<p>Overview of the concentrations and genotypes of enteric viruses detected in sewage. Boxplots show median concentrations (log PCRU/L) with the 25th and 75th percentile values of norovirus GI (<b>A</b> panel in dark blue), norovirus GII (<b>B</b> panel in light blue), and rotavirus (<b>C</b> panel in green). Viral genotypes are indicated according to the month in which the sample was sampled.</p> Full article ">Figure 1 Cont.
<p>Overview of the concentrations and genotypes of enteric viruses detected in sewage. Boxplots show median concentrations (log PCRU/L) with the 25th and 75th percentile values of norovirus GI (<b>A</b> panel in dark blue), norovirus GII (<b>B</b> panel in light blue), and rotavirus (<b>C</b> panel in green). Viral genotypes are indicated according to the month in which the sample was sampled.</p> Full article ">Figure 2
<p>Molecular phylogenetic analysis of norovirus GI capsid. The evolutionary history was inferred using the Maximum Likelihood method based on the Kimura 2-parameter model [<a href="#B42-microorganisms-08-00458" class="html-bibr">42</a>] with a bootstrap of 1000 replicates. The tree is drawn to scale, the branch lengths measure the number of substitutions per site. The analysis included 18 nucleotide sequences. There were 276 positions in the final dataset including nucleotides from 5386 to 5662.</p> Full article ">Figure 3
<p>Molecular phylogenetic analysis of norovirus GII capsid. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model [<a href="#B42-microorganisms-08-00458" class="html-bibr">42</a>] with a bootstrap of 1000 replicates. The tree is drawn to scale, the branch lengths measure the number of substitutions per site. The analysis included 43 nucleotide sequences. There were 273 positions in the final dataset including nucleotides from 5113 to 5386. a, b, and c: Boxes that contain identical sequences.</p> Full article ">Figure 4
<p>Molecular phylogenetic analysis of P[8] rotavirus (VP4 gene). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model [<a href="#B43-microorganisms-08-00458" class="html-bibr">43</a>] with a bootstrap of 1000 replicates. The tree is drawn to scale, the branch lengths measure the number of substitutions per site. The analysis included 32 nucleotide sequences. There were 127 positions in the final dataset. including nucleotides from 180 to 307. a, b, and c: Boxes containing identical sequences.</p> Full article ">Figure 5
<p>Molecular phylogenetic analysis of G1 rotavirus (VP7 gene). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model [<a href="#B43-microorganisms-08-00458" class="html-bibr">43</a>] with a bootstrap of 1000 replicates. The tree is drawn to scale, the branch lengths measure the number of substitutions per site. The analysis included 16 nucleotide sequences. There were 416 positions in the final dataset including nucleotides from 358 to 774.</p> Full article ">Figure 6
<p>Molecular phylogenetic analysis of G2 rotavirus (VP7 gene). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model [<a href="#B43-microorganisms-08-00458" class="html-bibr">43</a>] with a bootstrap of 1000 replicates. The tree is drawn to scale, the branch lengths measure the number of substitutions per site. The analysis included 18 nucleotide sequences. There was a total of 460 positions in the final dataset including nucleotides from 424 to 884.</p> Full article ">Figure 7
<p>Molecular phylogenetic analysis of G3 rotavirus (VP7 gene). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model [<a href="#B43-microorganisms-08-00458" class="html-bibr">43</a>] with a bootstrap of 1000 replicates. The tree is drawn to scale, the branch lengths measure the number of substitutions per site. The analysis included 10 nucleotide sequences. There was a total of 159 positions in the final dataset including nucleotides from 774 to 933.</p> Full article ">Figure 8
<p>Molecular phylogenetic analysis of G9 rotavirus (VP7 gene). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model [<a href="#B43-microorganisms-08-00458" class="html-bibr">43</a>] with a bootstrap of 1000 replicates. The tree is drawn to scale, the branch lengths measure the number of substitutions per site. The analysis included 11 nucleotide sequences. There was a total of 159 positions in the final dataset including nucleotides from 774 to 933.</p> Full article ">Figure 9
<p>Molecular phylogenetic analysis of G12 rotavirus (VP7 gene). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model [<a href="#B43-microorganisms-08-00458" class="html-bibr">43</a>] with a bootstrap of 1000 replicates. The tree is drawn to scale, the branch lengths measure the number of substitutions per site. The analysis involved 16 nucleotide sequences. There was a total of 378 positions in the final dataset including nucleotides from 584 to 926.</p> Full article ">
7 pages, 216 KiB  
Article
Multicenter Evaluation of the C6 Lyme ELISA Kit for the Diagnosis of Lyme Disease
by Silvia Zannoli, Michela Fantini, Simona Semprini, Barbara Marchini, Barbara Ceccarelli, Monica Sparacino, Pasqua Schiavone, Anna Belgrano, Maurizio Ruscio, Martina Gobbetti, Maira Nicoletti, Eva Robatscher, Elisabetta Pagani and Vittorio Sambri
Microorganisms 2020, 8(3), 457; https://doi.org/10.3390/microorganisms8030457 - 24 Mar 2020
Cited by 1 | Viewed by 3122
Abstract
Lyme disease (LD), caused by infection with Borrelia burgdorferi, is the most common tick-borne infection in many regions of Eurasia. Antibody detection is the most frequently used laboratory test, favoring a two-step serodiagnostic algorithm; immunoenzymatic detection of antibodies to C6 has been [...] Read more.
Lyme disease (LD), caused by infection with Borrelia burgdorferi, is the most common tick-borne infection in many regions of Eurasia. Antibody detection is the most frequently used laboratory test, favoring a two-step serodiagnostic algorithm; immunoenzymatic detection of antibodies to C6 has been shown to perform similarly to a standard two-step workflow. The aim of this study was the performance evaluation of the C6 Lyme ELISA kit compared to a standard two-step algorithm in three laboratories located in the northeastern region of Italy which cater to areas with different LD epidemiology. A total of 804 samples were tested, of which 695 gave concordant results between C6 testing and routine workflow (564 negative, 131 positive). Wherever available, clinical presentation and additional laboratory tests were analyzed to solve discrepancies. The C6 based method showed a good concordance with the standard two-step algorithm (Cohen’s κ = 0.619), however, the distribution of discrepancies seems to point towards a slightly lower specificity of C6 testing, which is supported by literature and could impact on patient management. The C6 ELISA, therefore, is not an ideal stand-alone test; however, if integrated into a two-step algorithm, it might play a part in achieving a sensitive, specific laboratory diagnosis of LD. Full article
(This article belongs to the Special Issue Advance in Tick-Borne Diseases Research)
18 pages, 3343 KiB  
Article
Fungal Microbiota of Sea Buckthorn Berries at Two Ripening Stages and Volatile Profiling of Potential Biocontrol Yeasts
by Juliana Lukša, Iglė Vepštaitė-Monstavičė, Violeta Apšegaitė, Laima Blažytė-Čereškienė, Ramunė Stanevičienė, Živilė Strazdaitė-Žielienė, Bazilė Ravoitytė, Dominykas Aleknavičius, Vincas Būda, Raimondas Mozūraitis and Elena Servienė
Microorganisms 2020, 8(3), 456; https://doi.org/10.3390/microorganisms8030456 - 23 Mar 2020
Cited by 16 | Viewed by 4543 | Correction
Abstract
Sea buckthorn, Hippophae rhamnoides L., has considerable potential for landscape reclamation, food, medicinal, and cosmetics industries. In this study, we analyzed fungal microorganism populations associated with carposphere of sea buckthorn harvested in Lithuania. An amplicon metagenomic approach based on the ITS2 region [...] Read more.
Sea buckthorn, Hippophae rhamnoides L., has considerable potential for landscape reclamation, food, medicinal, and cosmetics industries. In this study, we analyzed fungal microorganism populations associated with carposphere of sea buckthorn harvested in Lithuania. An amplicon metagenomic approach based on the ITS2 region of fungal rDNA was used to reveal the ripening-affected fungal community alterations on sea buckthorn berries. According to alpha and beta diversity analyses, depending on the ripening stage, sea buckthorn displayed significantly different fungal communities. Unripe berries were shown to be prevalent by Aureobasidium, Taphrina, and Cladosporium, while ripe berries were dominated by Aureobasidium and Metschnikowia. The selected yeast strains from unripe and mature berries were applied for volatile organic compounds identification by gas chromatography and mass spectrometry techniques. It was demonstrated that the patterns of volatiles of four yeast species tested were distinct from each other. The current study for the first time revealed the alterations of fungal microorganism communities colonizing the surface of sea buckthorn berries at different ripening stages. The novel information on specific volatile profiles of cultivable sea buckthorn-associated yeasts with a potential role in biocontrol is important for the development of the strategies for plant cultivation and disease management, as well as for the improvement of the quality and preservation of the postharvest berries. Management of the fungal microorganisms present on the surface of berries might be a powerful instrument for control of phytopathogenic and potentially antagonistic microorganisms affecting development and quality of the berries. Full article
(This article belongs to the Special Issue Applying Metaorganism Studies to the Fruit Microbiome: A New Frontier)
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Figure 1

Figure 1
<p>Principal coordinate analysis (PCoA) of the relative abundance of fungal microorganism amplicon sequence variants (ASVs) associated with unripe (UB) and ripe (RB) sea buckthorn berries. PCoA plot is based on unweighted UniFrac distance metrics.</p> Full article ">Figure 2
<p>Fungal microorganism community distribution on sea buckthorn unripe (UB) and ripe (RB) berries. Relative abundance of sequences classified at the phylum (<b>A</b>), class (<b>B</b>), family (<b>C</b>), and genus (<b>D</b>) level. The taxonomic groups comprising less than 1% of the total composition were assigned to “Other”.</p> Full article ">Figure 3
<p>Venn diagram illustrating the number of unique and shared amplicon sequence variants (ASVs) among sea buckthorn samples at different ripening stages. UB – unripe sea buckthorn berries, RB – ripe berries.</p> Full article ">Figure 4
<p>Heatmap of fungal microorganism’s unique amplicon sequence variants (ASVs) abundance on sea buckthorn. The color intensity is proportional to the relative abundance of fungal microorganism ASVs.</p> Full article ">Figure 5
<p>Identification of yeasts isolated from sea buckthorn surface.</p> Full article ">Figure 6
<p>Associations between blends and their components of four species of yeasts populating sea buckthorn berries. Associations are visualized by principal component analysis (PCA). (<b>A</b>) Distribution of odor blends released by <span class="html-italic">C. wieringae</span> (Cw, diamond shape), <span class="html-italic">A. pullulans</span> (Ap, triangle shape), <span class="html-italic">M. pulcherrima</span> (Mp, square shape), and <span class="html-italic">H. uvarum</span> (Hu, circle shape). n=5 for each species. (<b>B</b>) Distribution of volatile compounds composing the blends. Stars represent the volatile compounds indicated by numbers. Names of the compounds are listed in <a href="#microorganisms-08-00456-t002" class="html-table">Table 2</a>.</p> Full article ">
13 pages, 1680 KiB  
Article
Activity of Cinnamaldehyde on Quorum Sensing and Biofilm Susceptibility to Antibiotics in Pseudomonas aeruginosa
by Sanjida Halim Topa, Enzo A. Palombo, Peter Kingshott and Linda L. Blackall
Microorganisms 2020, 8(3), 455; https://doi.org/10.3390/microorganisms8030455 - 23 Mar 2020
Cited by 50 | Viewed by 5641
Abstract
Quorum sensing (QS) plays an important role during infection for the opportunistic human pathogen Pseudomonas aeruginosa. Quorum sensing inhibition (QSI) can disrupt this initial event of infection without killing bacterial cells, and thus QS inhibitors have been suggested as novel approaches for [...] Read more.
Quorum sensing (QS) plays an important role during infection for the opportunistic human pathogen Pseudomonas aeruginosa. Quorum sensing inhibition (QSI) can disrupt this initial event of infection without killing bacterial cells, and thus QS inhibitors have been suggested as novel approaches for anti-infective therapy. Cinnamaldehyde (CAD) is a P. aeruginosa biofilm inhibitor and disperser of preformed biofilms. In this study, the combined use of CAD and colistin (COL) revealed a synergistic activity, but this was not the case for CAD combined with carbenicillin, tobramycin (TOB), or erythromycin in checkerboard assays for P. aeruginosa. CAD demonstrated QSI activity by repression of the expression of lasB, rhlA and pqsA in GFP reporter assays. Approximately 70% reduction in GFP production was observed with the highest CAD concentration tested in all the QS reporter strains. TOB also showed strong QSI when combined with CAD in reporter assays. Combination treatments revealed an additive activity of CAD with COL and TOB in biofilm inhibition (75.2% and 83.9%, respectively) and preformed biofilm dispersion (~90% for both) when compared to the individual treatments. Therefore, a proposed method to mitigate P. aeruginosa infection is a combination therapy of CAD with COL or CAD with TOB as alternatives to current individual drug therapies. Full article
(This article belongs to the Special Issue Natural Antimicrobial Compounds)
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<p>Growth curve of <span class="html-italic">P. aeruginosa</span> isolates in the presence of 5.9 mM CAD that were passaged for 1 day (●) or 23 days (▲). The results are the average of three independent experiments in parallel, and error bars indicate ± standard deviations.</p> Full article ">Figure 2
<p>Dose–response curves of CAD incubated with (<b>a</b>) <span class="html-italic">P. aeruginosa</span> PAO1-<span class="html-italic">lasB-gfp</span>; (<b>b</b>) <span class="html-italic">P. aeruginosa</span> PAO1-<span class="html-italic">rhlA-gfp</span> and (<b>c</b>) <span class="html-italic">P. aeruginosa</span> PAO1-<span class="html-italic">pqsA-gfp</span>. ×, 3 mM CAD; ▲, 1.5 mM CAD; ◆, 0.8 mM CAD; ●, A7; ■, untreated. (<b>d</b>) Green fluorescent protein (GFP) inhibition percent with varying levels of CAD at 7 h: 0.8 mM CAD (white bars); 1.5 mM CAD (black bars); 3 mM CAD (downward diagonal bars). A7 (gray bars) is a QS inhibitor. Data represent the average of three independent experiments, and error bars indicate ± standard deviations. <span class="html-italic">p</span> values of ≤ 0.05. Relative fluorescence units (RFU) were normalized to OD<sub>600</sub> for all reporter assays.</p> Full article ">Figure 3
<p>The effect of combined CAD and tobramycin (TOB) treatment on quorum sensing (QS). (<b>a</b>) <span class="html-italic">P. aeruginosa</span> PAO1-<span class="html-italic">lasB-gfp</span>; (<b>b</b>) <span class="html-italic">P. aeruginosa</span> PAO1-<span class="html-italic">rhlA-gfp</span> and (<b>c</b>) <span class="html-italic">P. aeruginosa</span> PAO1-<span class="html-italic">pqsA-gfp</span>. ◆, CAD; ▲, TOB ●; CAD-TOB; ■, untreated. (<b>d</b>) GFP inhibition % with CAD and TOB at 7 h. CAD (white bars); TOB (black bars); CAD + TOB (downward diagonal bars). Data represent the average of three independent experiments, and error bars indicate ± standard deviation. <span class="html-italic">p</span> values of ≤ 0.05. RFU were normalized to OD<sub>600</sub> for all reporter assays.</p> Full article ">Figure 4
<p>CAD-, colistin (COL)-, TOB-, CAD-COL- and CAD-TOB-mediated inhibition of biofilm formation according to the crystal violet (CV) assay. Data represents the average of six technical replicates from three independent experiments, and error bars indicate ± standard deviation. <span class="html-italic">p</span> values of &lt; 0.05.</p> Full article ">Figure 5
<p>Effect of CAD, TOB, COL, CAD-TOB and CAD-COL on preformed biofilms. Data represent the average of six technical replicates from three independent experiments, and error bars indicate ± standard deviations. <span class="html-italic">p</span> values of &lt; 0.05.</p> Full article ">
20 pages, 1796 KiB  
Article
Formate Utilization by the Crenarchaeon Desulfurococcus amylolyticus
by Ipek Ergal, Barbara Reischl, Benedikt Hasibar, Lokeshwaran Manoharan, Aaron Zipperle, Günther Bochmann, Werner Fuchs and Simon K.-M. R. Rittmann
Microorganisms 2020, 8(3), 454; https://doi.org/10.3390/microorganisms8030454 - 23 Mar 2020
Cited by 6 | Viewed by 5624
Abstract
Formate is one of the key compounds of the microbial carbon and/or energy metabolism. It owes a significant contribution to various anaerobic syntrophic associations, and may become one of the energy storage compounds of modern energy biotechnology. Microbial growth on formate was demonstrated [...] Read more.
Formate is one of the key compounds of the microbial carbon and/or energy metabolism. It owes a significant contribution to various anaerobic syntrophic associations, and may become one of the energy storage compounds of modern energy biotechnology. Microbial growth on formate was demonstrated for different bacteria and archaea, but not yet for species of the archaeal phylum Crenarchaeota. Here, we show that Desulfurococcus amylolyticus DSM 16532, an anaerobic and hyperthermophilic Crenarchaeon, metabolises formate without the production of molecular hydrogen. Growth, substrate uptake, and production kinetics on formate, glucose, and glucose/formate mixtures exhibited similar specific growth rates and similar final cell densities. A whole cell conversion experiment on formate revealed that D. amylolyticus converts formate into carbon dioxide, acetate, citrate, and ethanol. Using bioinformatic analysis, we examined whether one of the currently known and postulated formate utilisation pathways could be operative in D. amylolyticus. This analysis indicated the possibility that D. amylolyticus uses formaldehyde producing enzymes for the assimilation of formate. Therefore, we propose that formate might be assimilated into biomass through formaldehyde dehydrogenase and the oxidative pentose phosphate pathway. These findings shed new light on the metabolic versatility of the archaeal phylum Crenarchaeota. Full article
(This article belongs to the Section Environmental Microbiology)
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<p>Growth curves of <span class="html-italic">D. amylolyticus</span> on formate, glucose, and glucose/formate at different concentrations. A slightly higher µ could be obtained when glucose/formate was used as substrate. All the substrate concentrations are given as C-mmol L<sup>−1</sup>. A negative (un-inoculated) control and positive–negative (inoculated into medium where formic acid was omitted) control were performed in each set and no growth was observed.</p> Full article ">Figure 2
<p>End point gas composition of <span class="html-italic">D. amylolyticus</span> grown at different concentrations of formate, glucose, and glucose/formate at the end of the cultivation. The results indicate that the CO<sub>2</sub> production is very low in the cultures grown on formate compared to cultures grown on glucose or glucose/formate. All the substrate concentrations are given as C-mmol L<sup>−1</sup>.</p> Full article ">Figure 3
<p>Growth, substrate uptake, and production kinetics of <span class="html-italic">D. amylolyticus</span> on 100 C-mmol L<sup>−1</sup> formate. The results indicate that CO<sub>2</sub> and citric acid were produced and consumed completely during the cultivation and only after the consumption of CO<sub>2</sub> and citric acid, acetic acid was produced.</p> Full article ">Figure 4
<p>Headspace gas composition of <span class="html-italic">D. amylolyticus</span>, from experiments performed in triplicates, at the end points of the whole cell conversion experiment. The experiment was designed and performed to be able to measure the cumulative gas accumulation in the serum bottle headspace. Despite the application of high cell density in the whole cell conversion experiment no H<sub>2</sub> accumulation was detected.</p> Full article ">Figure 5
<p>Comparison of genetic organization of (<b>A</b>) the fdh complex subunits in <span class="html-italic">T. onnurineus</span>, and (<b>B</b>) the pyruvate formate lyase (PFL) complex subunits in <span class="html-italic">E. coli</span> and <span class="html-italic">A. woodii</span> to the genetic organisation in <span class="html-italic">D. amylolyticus</span>.</p> Full article ">Figure 6
<p>Schematic illustration of the proposed route for formate assimilation in <span class="html-italic">D. amylolyticus</span>. The first part of the cycle is formaldehyde production from formate, which might be catalysed by formaldehyde dehydrogenase (FoDH), or formyl/acetyl transferase (F/AT) and aldehyde dehydrogenase (ADH). The second part of the cycle represents formaldehyde assimilation and ribulose 5-phosphate (Ru5P) regeneration via ribulose monophosphate pathway (RuMP) and oxidative pentose phosphate pathway (OPPP). Formaldehyde could be fixed by Ru5P to form D-arabino-3-hexulose-6-phosphate (A3H6P) by 3-hexulose-6-phosphate synthase (HPS) (1) and then isomerized to fructose 6-phosphate (F6P) by 6-phospho-3-hexuloisomerase (PHI) (2). In the genome of <span class="html-italic">D. amylolyticus,</span> only gene was found for an HPS-PHI-fused bifunctional enzyme (1-2). F6P is further isomerized to glucose-6-phosphate (G6P) by glucose-6-phosphate isomerase (3). Later, G6P is oxidized to Ru5P by glucose-6-phosphate dehydrogenase (4) and 6-phosphogluconate dehydrogenase (5).</p> Full article ">
18 pages, 1688 KiB  
Article
Lipid Production from Sugarcane Top Hydrolysate and Crude Glycerol with Rhodosporidiobolus fluvialis Using a Two-Stage Batch-Cultivation Strategy with Separate Optimization of Each Stage
by Jeerapan Boonyarit, Pirapan Polburee, Bongkot Khaenda, Zongbao K. Zhao and Savitree Limtong
Microorganisms 2020, 8(3), 453; https://doi.org/10.3390/microorganisms8030453 - 23 Mar 2020
Cited by 9 | Viewed by 3242
Abstract
Lipids from oleaginous microorganisms, including oleaginous yeasts, are recognized as feedstock for biodiesel production. A production process development of these organisms is necessary to bring lipid feedstock production up to the industrial scale. This study aimed to enhance lipid production of low-cost substrates, [...] Read more.
Lipids from oleaginous microorganisms, including oleaginous yeasts, are recognized as feedstock for biodiesel production. A production process development of these organisms is necessary to bring lipid feedstock production up to the industrial scale. This study aimed to enhance lipid production of low-cost substrates, namely sugarcane top and biodiesel-derived crude glycerol, by using a two-stage cultivation process with Rhodosporidiobolus fluvialis DMKU-SP314. In the first stage, sugarcane top hydrolysate was used for cell propagation, and in the second stage, cells were suspended in a crude glycerol solution for lipid production. Optimization for high cell mass production in the first stage, and for high lipid production in the second stage, were performed separately using a one-factor-at-a-time methodology together with response surface methodology. Under optimum conditions in the first stage (sugarcane top hydrolysate broth containing; 43.18 g/L total reducing sugars, 2.58 g/L soy bean powder, 0.94 g/L (NH4)2SO4, 0.39 g/L KH2PO4 and 2.5 g/L MgSO4 7H2O, pH 6, 200 rpm, 28 °C and 48 h) and second stage (81.54 g/L crude glycerol, pH 5, 180 rpm, 27 °C and 196 h), a high lipid concentration of 15.85 g/L, a high cell mass of 21.07 g/L and a high lipid content of 73.04% dry cell mass were obtained. Full article
(This article belongs to the Section Microbial Biotechnology)
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<p>Cell mass (<span style="color:#008080">■</span>), lipid content (<span style="color:#FF6600">■</span>) and lipid concentration (<span style="color:#009900">■</span>) by <span class="html-italic">R. fluvialis</span> DMKU-SP314 cultivated in a STH medium in various additional nitrogen compounds (<b>A</b>) at the end of the first stage (120 h) and (<b>B</b>) at the end of the second stage (240 h) in 70 g/L of CG solution with a pH of 5.5 while incubated at 150 rpm and 28 °C throughout the cultivation. Data are presented as mean value ± standard deviation. The different, same and overlapping lower-case letters mean significantly different, no-significantly different, and on-significantly different of their overlapping according to Duncan’s multiple range test at <span class="html-italic">p</span> ≤ 0.05.</p> Full article ">Figure 2
<p>Time-course of cell mass (<span style="color:#0070C0">●</span>), lipid content (<span style="color:#00B050">▲</span>) and lipid concentration (<span style="color:#833C0B">■</span>) of <span class="html-italic">R. fluvialis</span> DMKU-SP314 cultivated in a STH medium consisting of 70 g/L TRS, 2.58 g/L SBP, 0.94 g/L (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 0.39 g/L KH<sub>2</sub>PO<sub>4</sub>, 2.5 g/L MgSO<sub>4</sub> 7H<sub>2</sub>O, with an initial pH of 6, a temperature of 28 °C and a shaking speed of 200 rpm in the first stage. In the second stage, the cells were re-suspended in 70 g/L of a CG solution with a pH of 5.5 and incubated at 150 rpm and 28 °C at different shift times (<b>A</b>) 48 h (<b>B</b>) 72 h (<b>C</b>) 96 h and (<b>D</b>) 120 h. Data are presented as mean value ± standard deviation.</p> Full article ">Figure 3
<p>The effect of temperature on <b>(A)</b> cell mass production (g/L) when cultivated in a STH medium supplemented with SBP at 120 h in a temperature gradient incubator (15.0–35.5 °C). The effect of temperature on <b>(B)</b> lipid concentration (g/L) when cultivated in a CG solution for 196 h (after cultivation in the first stage for 48 h) in a temperature gradient incubator (17.0–37.8 °C). Data were presented as mean value ± standard deviation.</p> Full article ">
11 pages, 1467 KiB  
Review
Bridging the Gap: A Role for Campylobacter jejuni Biofilms
by Greg Tram, Christopher J. Day and Victoria Korolik
Microorganisms 2020, 8(3), 452; https://doi.org/10.3390/microorganisms8030452 - 23 Mar 2020
Cited by 31 | Viewed by 5153
Abstract
Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the developed world. Cases of Campylobacteriosis are common, as the organism is an avian commensal and is passed on to humans through contaminated poultry meat, water, and food preparation areas. Although typically a [...] Read more.
Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the developed world. Cases of Campylobacteriosis are common, as the organism is an avian commensal and is passed on to humans through contaminated poultry meat, water, and food preparation areas. Although typically a fastidious organism, C. jejuni can survive outside the avian intestinal tract until it is able to reach a human host. It has long been considered that biofilms play a key role in transmission of this pathogen. The aim of this review is to examine factors that trigger biofilm formation in C. jejuni. A range of environmental elements have been shown to initiate biofilm formation, which are then affected by a suite of intrinsic factors. We also aim to further investigate the role that biofilms may play in the life cycle of this organism. Full article
(This article belongs to the Special Issue Foodborne Pathogen Campylobacter)
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<p>A scanning electron micrograph of biofilm formed by <span class="html-italic">Campylobacter jejuni</span> strain 11168-O under 800× magnification. These biofilms exhibit the archetypal biofilm architecture with cells encased in an exuded extracellular matrix. <span class="html-italic">C. jejuni</span> has been shown to form biofilms under a variety of conditions and plays a large role in survival under harsh conditions.</p> Full article ">Figure 2
<p>Processes upregulated and downregulated by CsrA. The global post-translational regulator CsrA has been shown to impact a wide range of survival factors in <span class="html-italic">C. jejuni</span> and may be an important regulator of biofilm formation.</p> Full article ">Figure 3
<p>A scanning electron micrograph of <span class="html-italic">C. jejuni</span> biofilm formed by strain 11168-O in chicken caecum at 200× magnification. These biofilms were formed throughout the caecum and suggest that biofilms formed by <span class="html-italic">C. jejuni</span> affect survival in the avian intestinal tract.</p> Full article ">
15 pages, 2942 KiB  
Article
Comparative Proteomics Analysis Reveals New Features of the Oxidative Stress Response in the Polyextremophilic Bacterium Deinococcus radiodurans
by Lihua Gao, Zhengfu Zhou, Xiaonan Chen, Wei Zhang, Min Lin and Ming Chen
Microorganisms 2020, 8(3), 451; https://doi.org/10.3390/microorganisms8030451 - 23 Mar 2020
Cited by 21 | Viewed by 5772
Abstract
Deinococcus radiodurans is known for its extreme resistance to ionizing radiation, oxidative stress, and other DNA-damaging agents. The robustness of this bacterium primarily originates from its strong oxidative resistance mechanisms. Hundreds of genes have been demonstrated to contribute to oxidative resistance in D. [...] Read more.
Deinococcus radiodurans is known for its extreme resistance to ionizing radiation, oxidative stress, and other DNA-damaging agents. The robustness of this bacterium primarily originates from its strong oxidative resistance mechanisms. Hundreds of genes have been demonstrated to contribute to oxidative resistance in D. radiodurans; however, the antioxidant mechanisms have not been fully characterized. In this study, comparative proteomics analysis of D. radiodurans grown under normal and oxidative stress conditions was conducted using label-free quantitative proteomics. The abundances of 852 of 1700 proteins were found to significantly differ between the two groups. These differential proteins are mainly associated with translation, DNA repair and recombination, response to stresses, transcription, and cell wall organization. Highly upregulated expression was observed for ribosomal proteins such as RplB, Rpsl, RpsR, DNA damage response proteins (DdrA, DdrB), DNA repair proteins (RecN, RecA), and transcriptional regulators (members of TetR, AsnC, and GntR families, DdrI). The functional analysis of proteins in response to oxidative stress is discussed in detail. This study reveals the global protein expression profile of D. radiodurans in response to oxidative stress and provides new insights into the regulatory mechanism of oxidative resistance in D. radiodurans. Full article
(This article belongs to the Special Issue Extremophiles and Extremozymes in Academia and Industries)
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<p>Survival phenotype plate assay of <span class="html-italic">Deinococcus radiodurans</span> exposed to 80 mM of H<sub>2</sub>O<sub>2</sub> for different shock times (0, 10, 20, 30, 50 min). CK: untreated sample control. The survival rate is expressed as the percentage of the number of colonies in the H<sub>2</sub>O<sub>2</sub>-treated samples compared with that in the untreated sample control. The survival rates of different samples were obtained from 10<sup>−3</sup> dilution with three independent repeats.</p> Full article ">Figure 2
<p>Transmission electron microscopy (TEM) images of <span class="html-italic">D. radiodurans</span> cells (<b>A</b>) under normal growth conditions and (<b>B</b>) following exposure to 80 mM H<sub>2</sub>O<sub>2</sub> for 30 min. Bar: 500 nm. The red arrow points to the cell wall and cell membrane.</p> Full article ">Figure 3
<p>Comparison of the fold change in expression levels of differential proteins detected by Proteomic and Quantitative Real-Time PCR (qRT-PCR). The qRT-PCR data are presented as the mean ± SD values of triplicate reactions for each gene transcript. The selected proteins belong to different functional categories; the annotation of each gene is detailed in <a href="#app1-microorganisms-08-00451" class="html-app">Supplementary Table S1</a>.</p> Full article ">Figure 4
<p>Gene ontology (GO) enrichment analysis of differentially expressed proteins between DC (control samples) and DH (treated samples). The vertical histogram represents the number of differential proteins enriched in each GO term; the red line represents the enrichment degree (−log<sub>10</sub> <span class="html-italic">p</span> value) of each GO term. The 45 most enriched GO terms are shown.</p> Full article ">Figure 5
<p>Top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway classification histograms for differential proteins in DH versus DC. The x-axis represents the enriched KEGG pathway, and the y-axis represents the number of proteins that were significantly upregulated or downregulated expressed in DH versus DC.</p> Full article ">Figure 6
<p>The protein–protein interaction (PPI) network of oxidative stress response proteins in <span class="html-italic">D. radiodurans</span> revealed by functional protein association network (STRING) analysis. A total of 336 differential proteins are shown in the PPI network, with 11 modules indicated in different colors. Module 1: translation; Module 2: DNA repair; Module 3: cell wall organization; Module 4: cellular response to gamma radiation; Module 5: catalytic activity; Module 6: regulation of transcription; Module 7: ATP/GTP/DNA/RNA/protein binding; Module 8: membrane and transport; Module 9: oxidation–reduction process; Module 10: unclassified; Module 11: protein folding and degradation.</p> Full article ">
10 pages, 468 KiB  
Article
Liver Transudate, a Potential Alternative to Detect Anti-Hepatitis E Virus Antibodies in Pigs and Wild Boars (Sus scrofa)
by Alejandro Navarro, Carmen Bárcena, Pilar Pozo, Alberto Díez-Guerrier, Irene Martínez, Coral Polo, Clara Duque, David Rodríguez-Lázaro, Joaquín Goyache and Nerea García
Microorganisms 2020, 8(3), 450; https://doi.org/10.3390/microorganisms8030450 - 23 Mar 2020
Cited by 7 | Viewed by 3247
Abstract
In recent years, cases of hepatitis E virus (HEV) infection have increased in Europe in association with the consumption of contaminated food, mainly from pork products but also from wild boars. The animal’s serum is usually tested for the presence of anti-HEV antibodies [...] Read more.
In recent years, cases of hepatitis E virus (HEV) infection have increased in Europe in association with the consumption of contaminated food, mainly from pork products but also from wild boars. The animal’s serum is usually tested for the presence of anti-HEV antibodies and viral RNA but, in many cases such as during hunting, an adequate serum sample cannot be obtained. In the present study, liver transudate was evaluated as an alternative matrix to serum for HEV detection. A total of 125 sera and liver transudates were tested by enzyme-linked immunosorbent assay at different dilutions (1:2, 1:10, 1:20), while 58 samples of serum and liver transudate were checked for the presence of HEV RNA by RT-qPCR. Anti- HEV antibodies were detected by ELISA in 68.0% of the serum samples, and in 61.6% of the undiluted transudate, and in 70.4%, 56.8%, and 44.8% of 1:2, 1:10, or 1:20 diluted transudate, respectively. The best results were obtained for the liver transudate at 1:10 dilution, based on the Kappa statistic (0.630) and intraclass correlation coefficient (0.841). HEV RNA was detected by RT-qPCR in 22.4% of the serum samples and 6.9% of the transudate samples, all samples used for RT-qPCR were positive by ELISA. Our results indicate that liver transudate may be an alternative matrix to serum for the detection of anti-HEV antibodies. Full article
(This article belongs to the Special Issue Hepatitis E Virus, an Emergent Foodborne Pathogen? Public Health Implications)
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<p>Box plot diagram showing the optical densities (OD) for serum and undiluted or diluted liver transudate (1:2, 1:10, or 1:20).</p> Full article ">
18 pages, 972 KiB  
Review
Citrus Postharvest Green Mold: Recent Advances in Fungal Pathogenicity and Fruit Resistance
by Yulin Cheng, Yunlong Lin, Haohao Cao and Zhengguo Li
Microorganisms 2020, 8(3), 449; https://doi.org/10.3390/microorganisms8030449 - 23 Mar 2020
Cited by 68 | Viewed by 7678
Abstract
As the major postharvest disease of citrus fruit, postharvest green mold is caused by the necrotrophic fungus Penicillium digitatum (Pd), which leads to huge economic losses worldwide. Fungicides are still the main method currently used to control postharvest green mold in [...] Read more.
As the major postharvest disease of citrus fruit, postharvest green mold is caused by the necrotrophic fungus Penicillium digitatum (Pd), which leads to huge economic losses worldwide. Fungicides are still the main method currently used to control postharvest green mold in citrus fruit storage. Investigating molecular mechanisms of plant–pathogen interactions, including pathogenicity and plant resistance, is crucial for developing novel and safer strategies for effectively controlling plant diseases. Despite fruit–pathogen interactions remaining relatively unexplored compared with well-studied leaf–pathogen interactions, progress has occurred in the citrus fruit–Pd interaction in recent years, mainly due to their genome sequencing and establishment or optimization of their genetic transformation systems. Recent advances in Pd pathogenicity on citrus fruit and fruit resistance against Pd infection are summarized in this review. Full article
(This article belongs to the Special Issue Plant Microbial Interactions)
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<p>Overview of identified genes (red-colored) involved in the pathogenicity of <span class="html-italic">Penicillium digitatum</span>.</p> Full article ">Figure 2
<p>Overview of resistance responses in citrus fruit against <span class="html-italic">Penicillium digitatum</span> infection. Solid and dotted arrows indicate direct and tentative stimulatory modification, respectively.</p> Full article ">
28 pages, 3630 KiB  
Article
Whole Genome Sequencing Differentiates Presumptive Extended Spectrum Beta-Lactamase Producing Escherichia coli along Segments of the One Health Continuum
by Emelia H. Adator, Matthew Walker, Claudia Narvaez-Bravo, Rahat Zaheer, Noriko Goji, Shaun R. Cook, Lisa Tymensen, Sherry J. Hannon, Deirdre Church, Calvin W. Booker, Kingsley Amoako, Celine A. Nadon, Ron Read and Tim A. McAllister
Microorganisms 2020, 8(3), 448; https://doi.org/10.3390/microorganisms8030448 - 22 Mar 2020
Cited by 23 | Viewed by 5693
Abstract
Antimicrobial resistance (AMR) has important implications for the continued use of antibiotics to control infectious diseases in both beef cattle and humans. AMR along the One Health continuum of the beef production system is largely unknown. Here, whole genomes of presumptive extended-spectrum β-lactamase [...] Read more.
Antimicrobial resistance (AMR) has important implications for the continued use of antibiotics to control infectious diseases in both beef cattle and humans. AMR along the One Health continuum of the beef production system is largely unknown. Here, whole genomes of presumptive extended-spectrum β-lactamase E. coli (ESBL-EC) from cattle feces (n = 40), feedlot catch basins (n = 42), surrounding streams (n = 21), a beef processing plant (n = 4), municipal sewage (n = 30), and clinical patients (n = 25) are described. ESBL-EC were isolated from ceftriaxone selective plates and subcultured on ampicillin selective plates. Agreement of genotype-phenotype prediction of AMR ranged from 93.2% for ampicillin to 100% for neomycin, trimethoprim/sulfamethoxazole, and enrofloxacin resistance. Overall, β-lactam (100%; blaEC, blaTEM-1, blaSHV, blaOXA, blaCTX-M-), tetracycline (90.1%; tet(A), tet(B)) and folate synthesis (sul2) antimicrobial resistance genes (ARGs) were most prevalent. The ARGs tet(C), tet(M), tet(32), blaCTX-M-1, blaCTX-M-14, blaOXA-1, dfrA18, dfrA19, catB3, and catB4 were exclusive to human sources, while blaTEM-150, blaSHV-11–12, dfrA12, cmlA1, and cmlA5 were exclusive to beef cattle sources. Frequently encountered virulence factors across all sources included adhesion and type II and III secretion systems, while IncFIB(AP001918) and IncFII plasmids were also common. Specificity and prevalence of ARGs between cattle-sourced and human-sourced presumptive ESBL-EC likely reflect differences in antimicrobial use in cattle and humans. Comparative genomics revealed phylogenetically distinct clusters for isolates from human vs. cattle sources, implying that human infections caused by ESBL-EC in this region might not originate from beef production sources. Full article
(This article belongs to the Special Issue Antimicrobial Resistance in Livestock)
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<p>Phenotypic antimicrobial resistance profiles of presumptive extended-spectrum β-lactamase (ESBL) <span class="html-italic">E. coli</span> obtained from different sources as described in materials and methods. OXYT—tetracycline; AMPI—ampicillin; CTIO—ceftiofur; STEP—streptomycin; SULF—sulfisoxazole; TMSZ—trimethoprim/sulfamethoxazole; FLOR—florfenicol; ENRO—enrofloxacin; CTZD—ceftazidime; AMCL—amoxicillin/clavulanic acid; NMYN—neomycin; MDR—multidrug resistance. MDR referred to resistance to two or more classes of antibiotics.</p> Full article ">Figure 2
<p>Heat map of resistance genes associated with various classes of antimicrobials as detected by the whole genome sequences of 162 presumptive ESBL-producing <span class="html-italic">E. coli</span> across a One Health continuum: Isolates originated from multiple segments of the One Health continuum including human clinical, municipal sewage, beef processing plant, cattle feces, catch basin, and surrounding streams. <span class="html-italic">aac-(6′)-Ib-cr</span> classified under aminoglycoside also encodes quinolone resistance. The overall prevalence of each determinant is displayed as a numeric percentage at the bottom of each determinant.</p> Full article ">Figure 3
<p>Phylogenetic tree generated on the basis of single-nucleotide polymorphisms (SNPs) of the core genes of 162 <span class="html-italic">E. coli</span> isolates obtained from multiple segments of the beef production system and human-associated isolates with reference genome <span class="html-italic">E. coli</span> str. K-12 substr. MG1655 (GenBank accession # GI: 545778205/U00096.3). Inner, middle ring, and outer rings are representative of isolate sources, phenotypic antimicrobial, and multidrug resistance, respectively.</p> Full article ">Figure 4
<p>Minimum spanning tree (MST) based on whole-genome multi-locus sequence typing (wgMLST) profiles of 162 presumptive ESBL-producing <span class="html-italic">E. coli</span> genomes along the One Health continuum from cattle feces, catch basins, surface streams, beef processing plants, municipal sewage, and humans: The MST includes a total of 9580 wgMLST loci and was constructed with BioNumerics (version 7.6.2). Each circle corresponds to a unique wgMLST profile and is colored based on sample origin. The size of the circle is proportional to the number of isolates sharing the same wgMLST profile, while the branch lengths correspond to the number of allele differences between isolates. Boxes highlight three major clusters of isolates of cattle and human origin.</p> Full article ">Figure 5
<p>Blast atlas (A—circular) showing sequences similarity and diversity among true ESBL and presumptive non-ESBL <span class="html-italic">E. coli</span> using <span class="html-italic">E. coli</span> MG1655 as a reference: Isolates shown in this analysis are all cattle-derived (cattle feces, catch basins, surface water, and processing plant) and do not include any human-derived presumptive ESBL-EC because WGS did not identify any non-ESBL-EC from humans or municipal sewage sources based on the presence of <span class="html-italic">bla</span><sub>SHV</sub>, <span class="html-italic">bla</span><sub>TEM</sub>, <span class="html-italic">bla</span><sub>OXA</sub>, or <span class="html-italic">bla</span><sub>CTX-M</sub>. Inner ring comprises GC skew, GC content, and sequence of reference <span class="html-italic">E. coli</span> MG1655, while middle and outer rings comprise ESBL and non-ESBL <span class="html-italic">E. coli</span>, respectively.</p> Full article ">Figure 6
<p>Percentage prevalence of specific sequence types (ST10 to ST7618 and unknown STs) identified in multiple segments of the One Health continuum using the <span class="html-italic">E. coli</span> Achtman scheme.</p> Full article ">Figure 7
<p>Prevalence of plasmid replicons per source of presumptive ESBL-producing <span class="html-italic">E. coli</span> from cattle feces, catch basins, surface streams, municipal sewage, humans, and beef processing along a One Health continuum.</p> Full article ">
15 pages, 2708 KiB  
Article
Identification and in vitro Characterization of a Novel Phage Endolysin that Targets Gram-Negative Bacteria
by Jaewoo Bai, Sangmi Lee and Sangryeol Ryu
Microorganisms 2020, 8(3), 447; https://doi.org/10.3390/microorganisms8030447 - 21 Mar 2020
Cited by 21 | Viewed by 5094
Abstract
Most double-stranded (ds) DNA phages utilize holin proteins to secrete endolysin for host peptidoglycan lysis. In contrast, several holin-independent endolysins with secretion sequences or signal-arrest-release (SAR) sequences are secreted via the Sec pathway. In this study, we characterized a novel lysis protein (M4Lys) [...] Read more.
Most double-stranded (ds) DNA phages utilize holin proteins to secrete endolysin for host peptidoglycan lysis. In contrast, several holin-independent endolysins with secretion sequences or signal-arrest-release (SAR) sequences are secreted via the Sec pathway. In this study, we characterized a novel lysis protein (M4Lys) encoded by the dsDNA phage BSPM4, whose lysis function is not dependent on either holin or the Sec pathway in vitro. In silico analysis of M4Lys revealed that it contains a putative virion protein domain and an unusual C-terminal transmembrane domain (TMD). Turbidity reduction assays and liquid chromatography-mass spectrometry using purified peptidoglycan showed that the virion protein domain of M4Lys has peptidoglycan lysis activity. In vitro overproduction of M4Lys in Escherichia coli revealed that M4Lys alone caused rapid cell lysis. Treatment of E. coli with a Sec inhibitor did not inhibit the lysis activity of M4Lys, indicating that the Sec pathway is not involved in M4Lys-mediated cell lysis. Truncation of the TMD eliminated the cell lysis phenomenon, while production of the TMD alone did not induce the cell lysis. All these findings demonstrate that M4Lys is a novel endolysin that has a unique mosaic structure distinct from other canonical endolysins and the TMD plays a critical role in M4Lys-mediated in vitro cell lysis. Full article
(This article belongs to the Section Food Microbiology)
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Figure 1

Figure 1
<p>Schematic representation of the phage BSPM4 lysis cassette and analysis of the lysis kinetics by its putative lysis proteins. (<b>A</b>) Schematic representation of the phage BSPM4 lysis cassette; (<b>B</b>) Growth kinetics of the <span class="html-italic">E. coli</span> strains producing the putative lysis protein A (M4Lys) and the putative lysis protein B (ORF_37). Closed circles, empty vector; closed triangles, production of ORF_37; closed squares, production of M4Lys; reverse closed triangles, co-production of ORF_37 and M4Lys. Lysis activity of each gene was investigated by 0.1 mM IPTG induction and the turbidity of the cells was observed. Experiments for the growth kinetics were performed in triplicate. The results of each treatment are represented by the mean ± standard deviation.</p> Full article ">Figure 2
<p>Sec machinery is not involved in M4Lys-mediated cell lysis in vitro. Cell lysis kinetics of M4Lys in the presence of the Sec translocase inhibitor, NaN<sub>3</sub>. In all experiments, expression of M4Lys from pETDuet-1 plasmid in <span class="html-italic">E. coli</span> cells was induced by adding 0.1 mM of IPTG, except for the controls. Open circles, no NaN<sub>3</sub> without IPTG (control 1); closed circles, 10 mM NaN<sub>3</sub> without IPTG (control 2); closed squares, no NaN<sub>3</sub>; closed triangles, 1 mM NaN<sub>3</sub>; reverse triangles, 5 mM NaN<sub>3</sub>; and diamonds, 10 mM NaN<sub>3.</sub> All experiments for the cell lysis kinetics were performed in triplicate and the results of each treatment are represented by the mean ± standard deviation.</p> Full article ">Figure 3
<p>Domain analysis of M4Lys and its peptidoglycan degradation activity. (<b>A</b>) Domain analysis of M4Lys. The numbers represent amino acid positions; (<b>B</b>) Charge distribution of the TMD region of M4Lys. Positive and negative charges are represented as + and -, respectively, above the amino acid sequences. Numbers under the amino acid sequences indicate amino acid position; (<b>C</b>) Turbidity reduction assay of purified M4LysΔTMD using <span class="html-italic">E. coli</span> peptidoglycan. Closed circles, buffer treatment (control); closed squares, 0.4 mM M4LysΔTMD treatment; and closed triangles, 1.6 mM M4LysΔTMD treatment. Turbidity reduction assays were performed in triplicate and the results of each treatment are represented by the mean ± standard deviation.</p> Full article ">Figure 4
<p>Determination of M4Lys cleavage sites. Analysis of <span class="html-italic">E. coli</span> MG1655 peptidoglycan digested by (<b>A</b>) mutanolysin and (<b>B</b>) mutanolysin with purified M4LysΔTMD. Soluble muropeptides were reduced and analyzed by RP-HPLC coupled to MS. Peaks corresponding to m/z values matching previously identified muropeptides are numbered. The fragmentation pattern (peaks 1, 2, and 3) is typical of Tri (L-Ala-D-Glu-m-DAP), Tetra (L-Ala-D-Glu-m-DAP-D-Ala), and Tetra-Tetra muropeptides, respectively. The fragmentation event leading to the loss of a nonreduced GlcNAc residue (203.078, theoretical mass; peaks 4 and 5) indicates the N-acetylmuramidase activity of M4LysΔTMD. Cleavage of D-Glu-mDAP crosslink (peak 6) shows the endopeptidase activity of M4LysΔTMD; (<b>C</b>) Inferred structures, theoretical monoisotopic masses, and theoretical and observed m/z values of individual peaks are tabulated. M<sup>R</sup>, reduced MurNAc; G, GlcNAc; Di, m-DAP (meso-diaminopimelic acid)-D-Ala; Tri, L-Ala-D-Glu-m-DAP; and Tetra, L-Ala-D-Glu-m-DAP-D-Ala.</p> Full article ">Figure 5
<p>Functional analysis of the M4Lys transmembrane domain. (<b>A</b>) Catalytic domain (M4LysΔTMD), C-terminal TMD of M4Lys (M4TMD), and a putative lysis protein B (ORF_37) were produced separately or together in <span class="html-italic">E. coli</span> BL21 (DE3) cells. Closed circles, empty vector; closed square, expression of M4LysΔTMD; open square, coexpression of M4LysΔTMD and ORF_37; and reverse closed triangles, expression of TMD; (<b>B</b>) Lysis activity of various deletion mutants compared with the full-length M4Lys. Closed circles, empty vector; closed squares, full-length M4Lys; closed triangles, 11 amino acid deletion in TMD of M4Lys (M4Lys<sub>1–226</sub>); reverse closed triangles, 12 amino acid deletion in M4Lys TMD (M4Lys<sub>1–225</sub>); and closed diamonds, 13 amino acid deletion of M4Lys TMD (M4Lys<sub>1–224</sub>). Truncation of 13 amino acids from the M4Lys sequence eliminated the lytic activity. All protein expression was induced by adding 0.1 mM IPTG. Experiments for cell lysis kinetics were performed in triplicate and the results of each treatment are represented by the mean ± standard deviation.</p> Full article ">Figure 6
<p>Amino acid sequence alignments of M4Lys with other endolysins. (<b>A</b>) Phylogenetic analysis of endolysins from Gram-negative bacteria phages. Each group corresponds to a different functional domain: group I, V superfamily; group II, D-alanyl-D-alanine-carboxypeptidase; group III, <span class="html-italic">N</span>-acetyl muramoyl L-alanine amidase; group IV, glycoside hydrolase family 19; group V, <span class="html-italic">N</span>-acetylmuramidase; and group VI, glycoside hydrolase family 24, respectively; (<b>B</b>) Amino acid sequence alignments of the M4Lys protein of phage BSPM4 with other putative lysis proteins of relevant phages including SPN19, iEPS5, FSLSP088, and Chi. Conserved and identical residues are shaded in gray and black, respectively.</p> Full article ">Figure 6 Cont.
<p>Amino acid sequence alignments of M4Lys with other endolysins. (<b>A</b>) Phylogenetic analysis of endolysins from Gram-negative bacteria phages. Each group corresponds to a different functional domain: group I, V superfamily; group II, D-alanyl-D-alanine-carboxypeptidase; group III, <span class="html-italic">N</span>-acetyl muramoyl L-alanine amidase; group IV, glycoside hydrolase family 19; group V, <span class="html-italic">N</span>-acetylmuramidase; and group VI, glycoside hydrolase family 24, respectively; (<b>B</b>) Amino acid sequence alignments of the M4Lys protein of phage BSPM4 with other putative lysis proteins of relevant phages including SPN19, iEPS5, FSLSP088, and Chi. Conserved and identical residues are shaded in gray and black, respectively.</p> Full article ">
12 pages, 1350 KiB  
Review
Do Foliar Endophytes Matter in Litter Decomposition?
by Emily R. Wolfe and Daniel J. Ballhorn
Microorganisms 2020, 8(3), 446; https://doi.org/10.3390/microorganisms8030446 - 21 Mar 2020
Cited by 33 | Viewed by 6090
Abstract
Litter decomposition rates are affected by a variety of abiotic and biotic factors, including the presence of fungal endophytes in host plant tissues. This review broadly analyzes the findings of 67 studies on the roles of foliar endophytes in litter decomposition, and their [...] Read more.
Litter decomposition rates are affected by a variety of abiotic and biotic factors, including the presence of fungal endophytes in host plant tissues. This review broadly analyzes the findings of 67 studies on the roles of foliar endophytes in litter decomposition, and their effects on decomposition rates. From 29 studies and 1 review, we compiled a comprehensive table of 710 leaf-associated fungal taxa, including the type of tissue these taxa were associated with and isolated from, whether they were reported as endo- or epiphytic, and whether they had reported saprophytic abilities. Aquatic (i.e., in-stream) decomposition studies of endophyte-affected litter were significantly under-represented in the search results (p < 0.0001). Indicator species analyses revealed that different groups of fungal endophytes were significantly associated with cool or tropical climates, as well as specific plant host genera (p < 0.05). Finally, we argue that host plant and endophyte interactions can significantly influence litter decomposition rates and should be considered when interpreting results from both terrestrial and in-stream litter decomposition experiments. Full article
(This article belongs to the Special Issue Fungal Endophytes and Their Interactions with Plants)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>The 15 most frequently reported taxa that have measurable decomposition or saprophytic ability (e.g., cellulase secretion, etc.). Bars represent the number of times members of a taxon were reported. Taxa identified as “undetermined” or “Fungal sp.” are excluded.</p> Full article ">Figure 2
<p>The 15 most frequently reported taxa from leaf litter (e.g., senescent tissue, dead leaves, dried leaves, fresh litter, and decaying litter). Bars represent the number of times members of a taxon were reported. Taxa identified as “undetermined” or “Fungal sp.” are excluded.</p> Full article ">Figure 3
<p>These are the 15 taxa most frequently reported as endophytic (leaves were surface-sterilized). Bars represent the number of times members of a taxon were reported. Taxa identified as “undetermined” or “Fungal sp.” are excluded.</p> Full article ">Figure 4
<p>Infographic of factors that influence endophyte-mediated litter decomposition.</p> Full article ">
11 pages, 1839 KiB  
Article
Evaluation of Cyprinid Herpesvirus 2 Latency and Reactivation in Carassius gibel
by Wenjun Chai, Lin Qi, Yujun Zhang, Mingming Hong, Ling Jin, Lijuan Li and Junfa Yuan
Microorganisms 2020, 8(3), 445; https://doi.org/10.3390/microorganisms8030445 - 21 Mar 2020
Cited by 17 | Viewed by 3190
Abstract
Cyprinid herpesvirus 2 (CyHV-2, species Cyprinid herpesvirus 2) causes severe mortality in ornamental goldfish, crucian carp (Carassius auratus), and gibel carp (Carassius gibelio). It has been shown that the genomic DNA of CyHV-2 could be detected in subclinical [...] Read more.
Cyprinid herpesvirus 2 (CyHV-2, species Cyprinid herpesvirus 2) causes severe mortality in ornamental goldfish, crucian carp (Carassius auratus), and gibel carp (Carassius gibelio). It has been shown that the genomic DNA of CyHV-2 could be detected in subclinical fish, which implied that CyHV-2 could establish persistent infection. In this study, the latency of CyHV-2 was investigated in the survival fish after primary infection. CyHV-2 genomic DNA was detected in multiple tissues of acute infection samples; however, detection of CyHV-2 DNA was significantly reduced in fish recovered from the primary infection on day 300 postinfection. No active viral gene transcription, such as DNA polymerase and ORF99, was detected in recovered fish. Following temperature stress, an increase of CyHV-2 DNA copy numbers and gene transcription were observed in tissues examined, which suggests that CyHV-2 was reactivated under stress. In addition, a cell line (GCBLat1) derived from the brain tissue from CyHV-2-exposed fish harbored CyHV-2 genome but did not produce infectious virions under normal culture conditions. However, CyHV-2 replication and viral gene transcription occurred when GCBLat1 cells were treated with trichostatin A (TSA) or phorbol 12-myristate 13-acetate (TPA). It suggests CyHV-2 can remain latent in vitro and can reactivate under stress condition. Full article
(This article belongs to the Section Molecular Microbiology and Immunology)
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Figure 1

Figure 1
<p>Detection of CyHV-2 DNA and gene transcription from moribund fish or surviving fish after primary infection with CyHV-2: Quantification of the CyHV-2 DNA in tissues from moribund fish (<b>A</b>) or surviving fish (<b>C</b>) by qPCR. White bars and black bars represent two moribund fish A1 and A2 in panel A and two surviving fish in panel C, respectively. CyHV-2 DNA copies were calculated according to the standard curve based on the series of diluted plasmids containing the DNA polymerase gene and were expressed as copies per µg of total DNA (copies/µg DNA). Data in A and C are presented as the mean of triplicates ± SD of each tested tissue sample from two representative fish. RT-PCR analysis of total RNA harvested from moribund fish (<b>B</b>) or surviving fish (<b>D</b>) tissues after primary infection with CyHV-2 in the presence (RT+) or absence (RT-) of reverse transcriptase: The amplicons of <span class="html-italic">DNA pol</span> (362 bp) and <span class="html-italic">ORF99</span> (513 bp) are indicated. Lanes 1 to 6 represent the tissue of head-kidney, gills, midbrain, medulla oblongata, heart, and spleen, respectively. The amplicons of β-actin gene are used as an internal control to ensure that comparable levels of input RNA were used in RT-PCR. One representative experiment of three is shown.</p> Full article ">Figure 2
<p>Detection of CyHV-2 DNA and gene transcription in the moribund fish from temperature stress: (<b>A</b>) Quantification of the CyHV-2 DNA from three moribund fish suffered temperature stress by qPCR. White (1), gray (2), and black (3) bars respectively represent the different moribund fish. CyHV-2 DNA copies were calculated similarly, as described in <a href="#microorganisms-08-00445-f001" class="html-fig">Figure 1</a>. The DNA copies shown are the mean of triplicates ± SD. (<b>B</b>) RT-PCR analysis of total RNA harvested from moribund fish in the presence (RT+) or absence (RT-) of reverse transcriptase: The amplicons of <span class="html-italic">DNA pol</span> (362 bp) and <span class="html-italic">ORF99</span> (513 bp) are indicated. Lanes 1 to 6 respectively represent the tissue of the head, kidney, gills, midbrain, medulla oblongata, heart, and spleen, which were sampled from the same fish. The amplicons of β-actin gene are used as an internal control to ensure that comparable levels of input RNA were used in RT-PCR. One representative fish of three is shown.</p> Full article ">Figure 3
<p>Characteristics of the GCBlat1 cell line: (<b>A</b>) Morphology of GCBlat1 cells at passage 80. Scale bars = 100 μm. (<b>B</b>) Growth curves of the GCBlat1 cell line at passage 30 at 28 °C within 4 days. Y-axis represents cell numbers of the mean ± standard deviation of three independent experiments. (<b>C</b>) Morphological characteristics of chromosomes arrested in metaphase at passage 36. (<b>D</b>) Frequency distribution of chromosomes: The <span class="html-italic">x</span>-axis represents the number of chromosomes; the <span class="html-italic">y</span>-axis represents the number of cells analyzed.</p> Full article ">Figure 4
<p>Examination of CyHV-2 DNA and gene transcription in the GCBlat1 cell line: (<b>A</b>) PCR analysis of the CyHV-2 DNA from GCBlat1 cells at indicated passages. Amplicons of <span class="html-italic">DNA pol</span> (362 bp) and <span class="html-italic">ORF 99</span> (513 bp) are indicated. (<b>B</b>) qPCR analysis of the CyHV-2 DNA in GCBlat1 cells at indicated passages: CyHV-2 DNA copies were calculated similarly as described above. The data shown are the mean of triplicates ± SD. (<b>C</b>) RT-PCR analysis of total RNA harvested from GCBlat1 cells of passage 32: Lane 1, β-actin; lane 2, <span class="html-italic">DNA pol</span> (362 bp); lane 3, <span class="html-italic">ORF99</span> (513 bp); M, DNA ladder. One representative experiment of three is shown.</p> Full article ">Figure 5
<p>Examination of CyHV-2 DNA and gene transcription in GCBlat1 cells upon chemicals treatment: Cell viabilities of GCBlat1 cells were measured by an MTT assay following treatments with trichostatin A (TSA) (<b>A</b>) or phorbol 12-myristate 13-acetate (TPA) (<b>B</b>) (80, 40, 20, 10, and 5 μM) or dimethyl sulfoxide (DMSO) for 48 h. (<b>C</b>) qPCR analysis of the CyHV-2 DNA from GCBlat1 cells with TSA, TPA, DMSO, or without treatment (nc). CyHV-2 DNA copies were calculated similarly as described above. Data presented as the mean ± SD (<span class="html-italic">n</span> = 3). (<b>D</b>) RT-PCR analysis of total RNA harvested from treated and untreated GCBlat1 cells: The amplicons of <span class="html-italic">DNA pol</span> (362 bp) and <span class="html-italic">ORF99</span> (513 bp) are indicated. The amplicons of β-actin gene are used as an internal control to ensure that comparable levels of input RNA were used in RT-PCR. M: DNA ladder; NC: negative control for PCR; lane 1: mock-treated GCBlat1 cells; lane 2: DMSO-treated GCBlat1 cells; lane 3: TPA-treated GCBlat1 cells; lane 4: TSA-treated GCBlat1 cells. One representative experiment of three is shown here. * <span class="html-italic">p</span> &lt; 0.05</p> Full article ">
12 pages, 1518 KiB  
Article
Prevalence, Antimicrobial Resistance, Virulence Genes and Genetic Diversity of Salmonella Isolated from Retail Duck Meat in Southern China
by Zhengquan Chen, Jie Bai, Shaojun Wang, Xibin Zhang, Zeqiang Zhan, Haiyan Shen, Hongxia Zhang, Junping Wen, Yuan Gao, Ming Liao and Jianmin Zhang
Microorganisms 2020, 8(3), 444; https://doi.org/10.3390/microorganisms8030444 - 21 Mar 2020
Cited by 42 | Viewed by 4210
Abstract
Salmonella is an important cause of foodborne diseases. This study was undertaken to investigate the prevalence, serotype distribution, antimicrobial resistance, virulence genes, and genetic diversity of Salmonella isolates recovered from fresh duck meat obtained from retail markets in Southern China. In total, 365 [...] Read more.
Salmonella is an important cause of foodborne diseases. This study was undertaken to investigate the prevalence, serotype distribution, antimicrobial resistance, virulence genes, and genetic diversity of Salmonella isolates recovered from fresh duck meat obtained from retail markets in Southern China. In total, 365 samples of fresh duck meat were collected from retail markets in six different cities of Guangdong Province between May 2017 and April 2019. High levels of Salmonella contamination were detected in duck meat (151/365, 41.4%). Twenty-six different Salmonella serotypes were identified: S. Corvallis (n = 25, 16.6%), S. Kentucky (n = 22, 14.6%) and S. Agona (n = 20, 13.3%) were the most prevalent serotypes. All isolates were resistant to at least one antibiotic and 133 (88.1%) isolates exhibited multidrug resistance (MDR). Most (86.1%) Salmonella isolates carried seven classes of virulence-associated genes. This study showed the diversity of Salmonella serotypes and genotypes and the high prevalence of MDR isolates carrying multiple virulence-associated genes among isolates from duck meat obtained from retail markets in Southern China. Isolates from different districts had similar pulsed-field gel electrophoresis (PFGE) patterns indicating that circulating foodborne Salmonella constitutes a potential public health issue across different districts. Full article
(This article belongs to the Section Food Microbiology)
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<p>Serotype distribution of <span class="html-italic">Salmonella</span> isolates (<span class="html-italic">n</span> = 151).</p> Full article ">Figure 2
<p>Dendrogram of PFGE patterns of 151 <span class="html-italic">Salmonella</span> isolates from retail duck meats in Southern China showing the antibiotic resistance phenotype, resistance genes and virulence gene patterns. A black box indicates carrier-related genes.</p> Full article ">
19 pages, 11196 KiB  
Article
Unlocking the Microbiome Communities of Banana (Musa spp.) under Disease Stressed (Fusarium wilt) and Non-Stressed Conditions
by Manoj Kaushal, Rony Swennen and George Mahuku
Microorganisms 2020, 8(3), 443; https://doi.org/10.3390/microorganisms8030443 - 20 Mar 2020
Cited by 40 | Viewed by 10839
Abstract
We assessed the diversity, structure, and assemblage of bacterial and fungal communities associated with banana plants with and without Fusarium oxysporum f. sp. cubense (Foc) symptoms. A total of 117,814 bacterial and 17,317 fungal operational taxonomy units (OTUs) were identified in the rhizosphere, [...] Read more.
We assessed the diversity, structure, and assemblage of bacterial and fungal communities associated with banana plants with and without Fusarium oxysporum f. sp. cubense (Foc) symptoms. A total of 117,814 bacterial and 17,317 fungal operational taxonomy units (OTUs) were identified in the rhizosphere, roots, and corm of the host plant. Results revealed that bacterial and fungal microbiota present in roots and corm primarily emanated from the rhizosphere. The composition of bacterial communities in the rhizosphere, roots, and corm were different, with more diversity observed in the rhizosphere and less in the corm. However, distinct sample types i.e., without (asymptomatic) and with (symptomatic) Fusarium symptoms were the major drivers of the fungal community composition. Considering the high relative abundance among samples, we identified core microbiomes with bacterial and fungal OTUs classified into 20 families and colonizing distinct plant components of banana. Our core microbiome assigned 129 bacterial and 37 fungal genera to known taxa. Full article
(This article belongs to the Special Issue Plant Microbial Interactions)
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<p>Banana plants under non-stressed (asymptomatic) and disease stressed conditions (symptomatic) caused by <span class="html-italic">Fusarium oxysporum</span>.</p> Full article ">Figure 2
<p>Factors driving the microbiota composition of bacterial and fungal communities in organs, locations, and sample types. The principal coordinate analyses (PCoA) of pairwise Euclidean distance matrixes of filtered operational taxonomy unit (OTU) tables. PCoA analyses were performed considering all 12 samples of 16S and ITS based on three properties, i.e., sample organ (rhizosphere, roots, and corm of banana), location (Arusha and Kilimanjaro), and sample type (asymptomatic and symptomatic). For each group, the same graph was differentially colored to emphasize the influence of sample type and location of sample in the community assemblage.</p> Full article ">Figure 2 Cont.
<p>Factors driving the microbiota composition of bacterial and fungal communities in organs, locations, and sample types. The principal coordinate analyses (PCoA) of pairwise Euclidean distance matrixes of filtered operational taxonomy unit (OTU) tables. PCoA analyses were performed considering all 12 samples of 16S and ITS based on three properties, i.e., sample organ (rhizosphere, roots, and corm of banana), location (Arusha and Kilimanjaro), and sample type (asymptomatic and symptomatic). For each group, the same graph was differentially colored to emphasize the influence of sample type and location of sample in the community assemblage.</p> Full article ">Figure 3
<p>Rhizosphere, root, and corm samples of banana showing differences in their compositions and structure in bacterial and fungal communities of both locations. Principal coordinate analysis (PCoA) of pairwise, showing jackknife-supported confidence ellipsoids. The first three principal axes are shown. Principal coordinate analysis based on Euclidean distances. A: Arusha; C: Corm; H: Asymptomatic; I: Symptomatic; K: Kilimanjaro; R: Roots; S: Rhizosphere.</p> Full article ">Figure 4
<p>Venn diagram of the banana rhizosphere, roots, and corm. The observed OTUs for each treatment were produced in the UCLUST algorithm to show the shared and unique OTUs. Only the most abundant OTUs among all the samples were represented. A: Arusha; C: Corm; H: Asymptomatic; I: Symptomatic; K: Kilimanjaro; R: Roots; S: Rhizosphere.</p> Full article ">Figure 4 Cont.
<p>Venn diagram of the banana rhizosphere, roots, and corm. The observed OTUs for each treatment were produced in the UCLUST algorithm to show the shared and unique OTUs. Only the most abundant OTUs among all the samples were represented. A: Arusha; C: Corm; H: Asymptomatic; I: Symptomatic; K: Kilimanjaro; R: Roots; S: Rhizosphere.</p> Full article ">Figure 5
<p>An OTU table heat map showing taxonomy assignment for each OTU from sample. The OTU heatmap displays OTU counts per sample, where the counts are colored based on the contribution of each OTU to the total OTU count present in the sample (blue: contributes low percentage of OTUs to sample; red: contributes high percentage of OTUs). The table based on taxonomy assignment is filtered the OTU table by number (10,000 for bacteria and 5,000 for fungi) of counts per OTU. A: Arusha; C: Corm; H: Asymptomatic; I: Symptomatic; K: Kilimanjaro; R: Roots; S: Rhizosphere.</p> Full article ">Figure 6
<p>Relative abundance of OTU belonging to core and non-core OTUs. Blue bars represent relative abundance of core OTUs whereas red bars represent relative abundance of non-core OTUs. A: Arusha; C: Corm; H: Asymptomatic; I: Symptomatic; K: Kilimanjaro; R: Roots; S: Rhizosphere.</p> Full article ">Figure 7
<p>Distinct core colonizers pattern of banana samples. The heatmap display a distribution pattern of core OTUs across sample types based on relative abundance (column z-score). For plotting heatmap displaying core-colonization pattern the z-scores of relative abundance of top 20 core OTUs (family level) were considered. Samples were hierarchically grouped based on the pairwise distances. A: Arusha; C: Corm; H: Asymptomatic; I: Symptomatic; K: Kilimanjaro; R: Roots; S: Rhizosphere.</p> Full article ">Figure 8
<p>Untapped diversity in bacterial and fungal communities. The relative abundance of core OTUs is demonstrated by dots in a sample type. Dot representing OTUs where, black dots represent OTUs assigned as plant growth promoter, red dots represent OTUs unassigned as plant growth promoter, and green dots represent OTUs not assigned to a defined genus. Majority of core OTUs exhibit no functional indications in association with hosts. A: Arusha; C: Corm; H: Asymptomatic; I: Symptomatic; K: Kilimanjaro; R: Roots; S: Rhizosphere.</p> Full article ">
22 pages, 334 KiB  
Article
Potential PGPR Properties of Cellulolytic, Nitrogen-Fixing, Phosphate-Solubilizing Bacteria in Rehabilitated Tropical Forest Soil
by Amelia Tang, Ahmed Osumanu Haruna, Nik Muhamad Ab. Majid and Mohamadu Boyie Jalloh
Microorganisms 2020, 8(3), 442; https://doi.org/10.3390/microorganisms8030442 - 20 Mar 2020
Cited by 72 | Viewed by 9431
Abstract
In the midst of the major soil degradation and erosion faced by tropical ecosystems, rehabilitated forests are being established to avoid the further deterioration of forest lands. In this context, cellulolytic, nitrogen-fixing (N-fixing), phosphate-solubilizing bacteria are very important functional groups in regulating the [...] Read more.
In the midst of the major soil degradation and erosion faced by tropical ecosystems, rehabilitated forests are being established to avoid the further deterioration of forest lands. In this context, cellulolytic, nitrogen-fixing (N-fixing), phosphate-solubilizing bacteria are very important functional groups in regulating the elemental cycle and plant nutrition, hence replenishing the nutrient content in forest soils. As is the case for other potential plant growth-promoting (PGP) rhizobacteria, these functional bacteria could have cross-functional abilities or beneficial traits that are essential for plants and can improve their growth. This study was conducted to isolate, identify, and characterize selected PGP properties of these three functional groups of bacteria from tropical rehabilitated forest soils at Universiti Putra Malaysia Bintulu Sarawak Campus, Malaysia. The bacteria were isolated based on their colonial growth on respective functional media, identified using both molecular and selected biochemical properties, and were assessed for their functional quantitative activities as well as PGP properties based on seed germination tests and indole-3-acetic acid (IAA) production. Out of the 15 identified bacterial isolates that exhibited beneficial phenotypic traits, a third belong to the genus Burkholderia and a fifth to Stenotrophomonas sp., with both genera consisting of members from two different functional groups. The results of the experiments confirm the multiple PGP traits of some selected bacterial isolates based on their respective high functional activities, root and shoot lengths, and seedling vigor improvements when bacterized on mung bean seeds, as well as significant IAA production. The results of this study suggest that these functional bacterial strains could potentially be included in bio-fertilizer formulations for crop growth on acid soils. Full article
(This article belongs to the Section Environmental Microbiology)
18 pages, 2554 KiB  
Article
Taxonomic Characterization and Secondary Metabolite Analysis of NEAU-wh3-1: An Embleya Strain with Antitumor and Antibacterial Activity
by Han Wang, Tianyu Sun, Wenshuai Song, Xiaowei Guo, Peng Cao, Xi Xu, Yue Shen and Junwei Zhao
Microorganisms 2020, 8(3), 441; https://doi.org/10.3390/microorganisms8030441 - 20 Mar 2020
Cited by 6 | Viewed by 3370
Abstract
Cancer is a serious threat to human health. With the increasing resistance to known drugs, it is still urgent to find new drugs or pro-drugs with anti-tumor effects. Natural products produced by microorganisms have played an important role in the history of drug [...] Read more.
Cancer is a serious threat to human health. With the increasing resistance to known drugs, it is still urgent to find new drugs or pro-drugs with anti-tumor effects. Natural products produced by microorganisms have played an important role in the history of drug discovery, particularly in the anticancer and anti-infective areas. The plant rhizosphere ecosystem is a rich resource for the discovery of actinomycetes with potential applications in pharmaceutical science, especially Streptomyces. We screened Streptomyces-like strains from the rhizosphere soil of wheat (Triticum aestivum L.) in Hebei province, China, and thirty-nine strains were obtained. Among them, the extracts of 14 isolates inhibited the growth of colon tumor cell line HCT-116. Strain NEAU-wh-3-1 exhibited better inhibitory activity, and its active ingredients were further studied. Then, 16S rRNA gene sequence similarity studies showed that strain NEAU-wh3-1 with high sequence similarities to Embleya scabrispora DSM 41855T (99.65%), Embleya hyalina MB891-A1T (99.45%), and Streptomyces lasii 5H-CA11T (98.62%). Moreover, multilocus sequence analysis based on the five other house-keeping genes (atpD, gyrB, rpoB, recA, and trpB) and polyphasic taxonomic approach comprising chemotaxonomic, phylogenetic, morphological, and physiological characterization indicated that the isolate should be assigned to the genus Embleya and was different from its closely related strains, therefore, it is proposed that strain NEAU-wh3-1 may be classified as representatives of a novel species of the genus Embleya. Furthermore, active substances in the fermentation broth of strain NEAU-wh-3-1 were isolated by bioassay-guided analysis and identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses. Consequently, one new Zincophorin analogue together with seven known compounds was detected. The new compound showed highest antitumor activity against three human cell lines with the 50% inhibition (IC50) values of 8.8–11.6 μg/mL and good antibacterial activity against four pathogenic bacteria, the other known compounds also exhibit certain biological activity. Full article
(This article belongs to the Special Issue Actinobacteria and Myxobacteria—Important Resources for Novel Antibiotics 2.0)
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<p>Antitumor activities of extracts obtained from fourteen isolates against human colon tumor cell line HCT-116.</p> Full article ">Figure 2
<p>Scanning electron micrograph of spore chains of strain NEAU-wh3-1 grown on ISP 3 agar for 2 weeks at 28 °C.</p> Full article ">Figure 3
<p>Neighbor-joining tree showing the phylogenetic position of strain NEAU-wh3-1 (1487 bp) and related taxa based on 16S rRNA gene sequences. Bootstrap values &gt; 50% (based on 1000 replications) are shown at branch points. <span class="html-italic">Arthrobacter globiformis</span> DSM 20124<sup>T</sup> (M23411) was used as an outgroup. <span class="html-italic">Asterisks</span> indicate branches also recovered in the maximum-likelihood tree; Bar, 0.01 substitutions per nucleotide position.</p> Full article ">Figure 4
<p>Neighbor-joining tree based on multilocus sequence analysis (MLSA) analysis of the concatenated partial sequences (1979 bp) from five housekeeping genes (<span class="html-italic">atp</span>D, <span class="html-italic">gyr</span>B, <span class="html-italic">rec</span>A, <span class="html-italic">rpo</span>B, and <span class="html-italic">trp</span>B) of strain NEAU-wh3-1 (in bold) with related taxa. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. Asterisks indicate branches also recovered in the maximum-likelihood tree; Bar, 0.05 substitutions per nucleotide position.</p> Full article ">Figure 5
<p>The structures of compounds <b>1</b>–<b>8.</b> Compound <b>1</b> was isolated as white solid with <math display="inline"><semantics> <mrow> <msubsup> <mrow> <mo>[</mo> <mi>α</mi> <mo>]</mo> </mrow> <mi>D</mi> <mrow> <mn>25</mn> </mrow> </msubsup> </mrow> </semantics></math> + 15 (c 0.043, EtOH) and UV (EtOH) λmax nm (log ε): 202 (4.53). Its molecular formula was established as C<sub>31</sub>H<sub>56</sub>O<sub>7</sub> by HR-ESI-MS at <span class="html-italic">m/z</span> 539.3942 [M-H]<sup>-</sup> (calcd 539.3953 as C<sub>31</sub>H<sub>55</sub>O<sub>7</sub>). The IR spectrum revealed hydroxyl absorption at 3320 cm<sup>−1</sup> and carbonyl absorption at 1735 cm<sup>−1</sup>, as well as methyl and methylene absorptions at 2953 cm<sup>−1</sup> and 2924 cm<sup>−1</sup>.</p> Full article ">Figure 6
<p>2D nuclear magnetic resonance (NMR) correlations of compound <b>1.</b></p> Full article ">
8 pages, 901 KiB  
Brief Report
Seroprevalence in Bats and Detection of Borrelia burgdorferi in Bat Ectoparasites
by Arinjay Banerjee, Kaushal Baid, Taylor Byron, Alyssa Yip, Caleb Ryan, Prasobh Raveendran Thampy, Hugh Broders, Paul Faure and Karen Mossman
Microorganisms 2020, 8(3), 440; https://doi.org/10.3390/microorganisms8030440 - 20 Mar 2020
Cited by 7 | Viewed by 4660
Abstract
The role of bats in the enzootic cycle of Lyme disease and relapsing fever-causing bacteria is a matter of speculation. In Canada, Borrelia burgdorferi sensu stricto (ss) is the genospecies that is responsible for most cases of Lyme disease in humans. In this [...] Read more.
The role of bats in the enzootic cycle of Lyme disease and relapsing fever-causing bacteria is a matter of speculation. In Canada, Borrelia burgdorferi sensu stricto (ss) is the genospecies that is responsible for most cases of Lyme disease in humans. In this study, we determined if big brown bats, Eptesicus fuscus, have been exposed to spirochetes from the genus Borrelia. We collected serum from 31 bats and tested them for the presence of anti-Borrelia burgdorferi antibodies using a commercial enzyme-linked immunosorbent assay (ELISA). We detected cross-reactive antibodies to Borrelia spp. in 14 of 31 bats. We confirmed the ELISA data using a commercial immunoblot assay. Pooled sera from ELISA-positive bats also cross-reacted with Borrelia antigens coated on the immunoblot strips, whereas pooled sera from ELISA-negative bats did not bind to Borrelia spp. antigens. Furthermore, to identify if bat ectoparasites, such as mites, can carry Borrelia spp., we analyzed DNA from 142 bat ectoparasites that were collected between 2003 and 2019. We detected DNA for the Borrelia burgdorferi flaB gene in one bat mite, Spinturnix americanus. The low detection rate of Borrelia burgdorferi DNA in bat ectoparasites suggests that bats are not reservoirs of this bacterium. Data from this study also raises intriguing questions about Borrelia infections in bats, including the role of humoral immunity and the ability of bats to be infected with Borrelia burgdorferi. This study can lead to more sampling efforts and controlled laboratory studies to identify if bats can be infected with Borrelia burgdorferi and the role of bat ectoparasites, such as S. americanus, in the transmission of this spirochete. Furthermore, we outlined reagents that can be used to adapt ELISA kits and immunoblot strips for use with bat sera. Full article
(This article belongs to the Special Issue Bats in Infectiology Research—Novel Tools and New Findings)
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<p>Detection of antibodies to <span class="html-italic">Borrelia</span> spp. in <span class="html-italic">Eptesicus fuscus</span> and the <span class="html-italic">flaB</span> gene segment in bat ectoparasites. We extracted sera from wild-caught and laboratory-bred <span class="html-italic">E. fuscus</span> to detect antibodies to <span class="html-italic">Borrelia</span>. <b>A</b>. Sites of bat collection. Bats were later kept in a captive research colony. <b>B</b>. Bar graph: antibody levels detected in <span class="html-italic">E. fuscus</span> serum samples using a commercial ELISA kit specific for anti-<span class="html-italic">Borrelia burgdorferi</span> antibodies. Human control sera were assayed in parallel. The cut-off for bat samples was set at 0.475 and is represented by the dotted line. <b>C</b>. Reactivity of pooled bat (<span class="html-italic">E. fuscus</span>) and control human sera with antigens from <span class="html-italic">B. burgdorferi, Borrelia afzelii</span>, and <span class="html-italic">Borrelia garinii</span>. See manufacturer’s manual for antigen details (EUROIMMUN, Germany). <b>D</b>. Ectoparasites collected from bats in Canada. <b>E</b>. Detection of <span class="html-italic">Borrelia burgdorferi flaB</span> gene fragment in a bat ectoparasite. The maximum likelihood tree (1000 bootstraps) showing the phylogenetic relationship of the <span class="html-italic">Borrelia burgdorferi flaB</span> gene segment detected in 1 of 142 bat ectoparasites that were collected between 2003 and 2019 is shown. The tree is drawn to scale, with branch lengths measured by the number of substitutions per site. The percentage of trees in which the associated taxa are clustered together is shown next to the branches. Values over 60 are shown. Evolutionary analysis was conducted in MEGA7. The following terms are used: SD (standard deviation); Ba (<span class="html-italic">Borrelia afzelii</span>); Bb (<span class="html-italic">Borrelia burgdorferi</span>); Bg (<span class="html-italic">Borrelia garinii</span>).</p> Full article ">
15 pages, 571 KiB  
Article
Lactobacillus paracasei A13 and High-Pressure Homogenization Stress Response
by Lorenzo Siroli, Giacomo Braschi, Samantha Rossi, Davide Gottardi, Francesca Patrignani and Rosalba Lanciotti
Microorganisms 2020, 8(3), 439; https://doi.org/10.3390/microorganisms8030439 - 20 Mar 2020
Cited by 22 | Viewed by 3569
Abstract
Sub-lethal high-pressure homogenization treatments applied to Lactobacillus paracasei A13 demonstrated to be a useful strategy to enhance technological and functional properties without detrimental effects on the viability of this strain. Modification of membrane fatty acid composition is reported to be the main regulatory [...] Read more.
Sub-lethal high-pressure homogenization treatments applied to Lactobacillus paracasei A13 demonstrated to be a useful strategy to enhance technological and functional properties without detrimental effects on the viability of this strain. Modification of membrane fatty acid composition is reported to be the main regulatory mechanisms adopted by probiotic lactobacilli to counteract high-pressure stress. This work is aimed to clarify and understand the relationship between the modification of membrane fatty acid composition and the expression of genes involved in fatty acid biosynthesis in Lactobacillus paracasei A13, before and after the application of different sub-lethal hyperbaric treatments. Our results showed that Lactobacillus paracasei A13 activated a series of reactions aimed to control and stabilize membrane fluidity in response to high-pressure homogenization treatments. In fact, the production of cyclic fatty acids was counterbalanced by the unsaturation and elongation of fatty acids. The gene expression data indicate an up-regulation of the genes accA, accC, fabD, fabH and fabZ after high-pressure homogenization treatment at 150 and 200 MPa, and of fabK and fabZ after a treatment at 200 MPa suggesting this regulation of the genes involved in fatty acids biosynthesis as an immediate response mechanism adopted by Lactobacillus paracasei A13 to high-pressure homogenization treatments to balance the membrane fluidity. Although further studies should be performed to clarify the modulation of phospholipids and glycoproteins biosynthesis since they play a crucial role in the functional properties of the probiotic strains, this study represents an important step towards understanding the response mechanisms of Lactobacillus paracasei A13 to sub-lethal high-pressure homogenization treatments. Full article
(This article belongs to the Section Food Microbiology)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Schematic representation of the conserved fatty acids biosynthesis pathway based on the <span class="html-italic">Lb. paracasei</span> ATCC334 fatty acids synthesis model. In red are highlighted the genes considered in this study. Acc genes (<span class="html-italic">accA</span>, <span class="html-italic">accB</span>, <span class="html-italic">accC</span>, <span class="html-italic">accD</span>) are involved in the malonyl-CoA synthesis. Malonyl-ACP Malonyl-CoA:ACP transacylase (<span class="html-italic">fabD</span>) catalyze the coenzyme A substitution with an acyl carrier protein (ACP). Malonyl-ACP is condensed by 3-oxoacyl-ACP synthase (<span class="html-italic">fabH</span>) to acetyl-CoA, producing a β-ketoacyl-ACP. Ketoacyl-ACP reductase (<span class="html-italic">fabG</span>) starts with a reduction and is followed by a dehydration executed by 3-hydroxyacyl-ACP- dehydratase (<span class="html-italic">fabZ</span>). A second reduction by enoyl-ACP-reductase (<span class="html-italic">fabK</span>) produces acyl-ACP. In order to continue the elongation reaction, acyl-ACPs are dehydrated by 3-oxoacyl-ACP synthase (<span class="html-italic">fabF</span>) and the elongation chain cycle starts again. Once sufficiently elongated, acyl-ACPs are processed by different hydrolytic enzymes depending on the chain length. Medium-chain fatty acids (i.e., C:12) are released by the esterase activity of the Medium-chain acyl-ACP hydrolase (<span class="html-italic">mch</span>).</p> Full article ">Figure 2
<p>Relative gene expression (RGE) index of <span class="html-italic">Lb. paracasei</span> fatty acids biosynthesis genes accA, accB, <span class="html-italic">accD</span>, <span class="html-italic">fabD</span>, <span class="html-italic">fabH</span>, <span class="html-italic">fabG</span>, <span class="html-italic">fabZ</span>, <span class="html-italic">fabK</span> and <span class="html-italic">fabF</span> in relation to homogenization pressure. RGE index was calculated according to the model proposed by Pfaffl [<a href="#B24-microorganisms-08-00439" class="html-bibr">24</a>] and reviewed by Muller et al. [<a href="#B25-microorganisms-08-00439" class="html-bibr">25</a>] using <span class="html-italic">ileS</span> (Isoleucine tRNA ligase) as reference gene and homogenization at 0.1 MPa as the control condition. The results are the average of three replicates and for the same gene, column followed by different superscript letters are significantly different <span class="html-italic">p</span> &lt; 0.05. Error bars indicate ± SD.</p> Full article ">
19 pages, 2685 KiB  
Article
The Interplay between Mucosal Microbiota Composition and Host Gene-Expression is Linked with Infliximab Response in Inflammatory Bowel Diseases
by Nikolas Dovrolis, George Michalopoulos, George E. Theodoropoulos, Kostantinos Arvanitidis, George Kolios, Leonardo A. Sechi, Aristidis G. Eliopoulos and Maria Gazouli
Microorganisms 2020, 8(3), 438; https://doi.org/10.3390/microorganisms8030438 - 20 Mar 2020
Cited by 52 | Viewed by 4780
Abstract
Even though anti-TNF therapy significantly improves the rates of remission in inflammatory bowel disease (IBD) patients, there is a noticeable subgroup of patients who do not respond to treatment. Dysbiosis emerges as a key factor in IBD pathogenesis. The aim of the present [...] Read more.
Even though anti-TNF therapy significantly improves the rates of remission in inflammatory bowel disease (IBD) patients, there is a noticeable subgroup of patients who do not respond to treatment. Dysbiosis emerges as a key factor in IBD pathogenesis. The aim of the present study is to profile changes in the gut microbiome and transcriptome before and after administration of the anti-TNF agent Infliximab (IFX) and investigate their potential to predict patient response to IFX at baseline. Mucosal biopsy samples from 20 IBD patients and nine healthy controls (HC) were examined for differences in microbiota composition (16S rRNA gene sequencing) and mucosal gene expression (RT-qPCR) at baseline and upon completion of IFX treatment, accordingly, via an in silico pipeline. Significant differences in microbiota composition were found between the IBD and HC groups. Several bacterial genera, which were found only in IBD patients and not HC, had their populations dramatically reduced after anti-TNF treatment regardless of response. Alpha and beta diversity metrics showed significant differences between our study groups. Correlation analysis revealed six microbial genera associated with differential expression of inflammation-associated genes in IFX treatment responders at baseline. This study shows that IFX treatment has a notable impact on both the gut microbial composition and the inflamed tissue transcriptome in IBD patients. Importantly, our results identify enterotypes that correlate with transcriptome changes and help differentiate IFX responders versus non-responders at baseline, suggesting that, in combination, these signatures can be an effective tool to predict anti-TNF response. Full article
(This article belongs to the Special Issue Mycobacteria Infections and Autoimmune Diseases)
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<p>Overall design of the study. This protocol allowed us to showcase IBD vs healthy controls dysbiosis, to highlight the influence of Infliximab on microbiota composition and identify response-related microbial and transcriptional biomarkers.</p> Full article ">Figure 2
<p>(<b>A</b>) Microbiota composition changes at phylum level among healthy controls, Crohn’s disease and ulcerative colitis patients. (<b>B</b>) α-diversity (quantification of biodiversity) differences in the 3 groups. (<b>C</b>) β-diversity (qualitative enterotype differences) of the 3 groups.</p> Full article ">Figure 3
<p><b>(</b><b>A</b>) Relative abundance changes of the microbial genera among healthy controls (control), Crohn’s disease (CD) and ulcerative colitis (UC) patients. (<b>B</b>) LEfSe analysis showing microbial genera associated with the 3 groups. (<b>C</b>) Venn diagram depicting the microbial genera constantly present (core microbiome) in the samples of the 3 groups versus those found exclusively in HC, CD and UC.</p> Full article ">Figure 4
<p>(<b>A</b>) Microbiota composition at phylum level among Crohn’s disease patients before treatment (CD_PRE) and after treatment in non-responders (CD_NONR) and responders (CD_R). (<b>B</b>) α-diversity (quantification of biodiversity) differences of the 3 groups. (<b>C</b>) β-diversity (qualitative enterotype differences) of the 3 groups.</p> Full article ">Figure 5
<p>(<b>A</b>) Relative abundance of microbial genera among Crohn’s disease patients before treatment (CD_PRE) and after treatment in non-responders (CD_NONR) and responders (CD_R). (<b>B</b>) LEfSe analysis showcasing microbial genera associated with the sample groups. (<b>C</b>) Venn Diagram depicting the microbial genera constantly present (core microbiome) in the samples of the 3 groups versus those found only in responders, non-responders and at baseline (before initiation of treatment).</p> Full article ">Figure 6
<p>Microbiome analysis of Crohn’s disease samples before treatment of which we know the response outcome: response (CD_PRE_R) and non-response (CD_PRE_NONR). (<b>A</b>) β-diversity (qualitive enterotype differences). (<b>B</b>) Relative abundance changes of the microbial genera (<b>C</b>) LEfSe analysis showcasing microbial genera associated with the sample groups. (<b>D</b>) Venn diagram depicting the microbial genera constantly present (core microbiome) in the samples of the 2 groups.</p> Full article ">Figure 7
<p>Overview of the genera with differential abundance between responders and non-responders and how these correlate with specific differentially expressed genes. IL18, CCR3, CXCL8, TLR6, TLR9 and TNFSF14 are upregulated and CCR4 is downregulated in both groups (2/2 genera abundant in responders and 3/4 abundant in non-responders) and appear to meet the criteria to be characterised as biomarkers for prediction of response to IFX therapy.</p> Full article ">
19 pages, 4737 KiB  
Article
Genome-Scale Metabolic Model Reconstruction and in Silico Investigations of Methane Metabolism in Methylosinus trichosporium OB3b
by Sanzhar Naizabekov and Eun Yeol Lee
Microorganisms 2020, 8(3), 437; https://doi.org/10.3390/microorganisms8030437 - 20 Mar 2020
Cited by 24 | Viewed by 6018
Abstract
Methylosinus trichosporium OB3b is an obligate aerobic methane-utilizing alpha-proteobacterium. Since its isolation, M. trichosporium OB3b has been established as a model organism to study methane metabolism in type II methanotrophs. M. trichosporium OB3b utilizes soluble and particulate methane monooxygenase (sMMO and pMMO respectively) [...] Read more.
Methylosinus trichosporium OB3b is an obligate aerobic methane-utilizing alpha-proteobacterium. Since its isolation, M. trichosporium OB3b has been established as a model organism to study methane metabolism in type II methanotrophs. M. trichosporium OB3b utilizes soluble and particulate methane monooxygenase (sMMO and pMMO respectively) for methane oxidation. While the source of electrons is known for sMMO, there is less consensus regarding electron donor to pMMO. To investigate this and other questions regarding methane metabolism, the genome-scale metabolic model for M. trichosporium OB3b (model ID: iMsOB3b) was reconstructed. The model accurately predicted oxygen: methane molar uptake ratios and specific growth rates on nitrate-supplemented medium with methane as carbon and energy source. The redox-arm mechanism which links methane oxidation with complex I of electron transport chain has been found to be the most optimal mode of electron transfer. The model was also qualitatively validated on ammonium-supplemented medium indicating its potential to accurately predict methane metabolism in different environmental conditions. Finally, in silico investigations regarding flux distribution in central carbon metabolism of M. trichosporium OB3b were performed. Overall, iMsOB3b can be used as an organism-specific knowledgebase and a platform for hypothesis-driven theoretical investigations of methane metabolism. Full article
(This article belongs to the Special Issue Genome-Scale Modeling of Microorganisms in the Real World)
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<p>Modes of electron transfer to pMMO in iMsOB3b model developed in this study. The dotted lines represent possible electron flow in 3 different modes of electron transfer. The green dotted lines refer to electron flow in redox-arm mode. The blue dotted lines refer to electron flow in uphill electron transfer mode. The red dotted lines represent electron flow during direct coupling mode. pMMO refers to particulate methane monooxygenase, q refers to ubiquinone, cytC refers to cytochrome C, NADH11 refers to ubiquinone oxidoreductase, UQCYOR_2p refers to ubiquinol-cytochrome-c reductase, CYO_2p refers to cytochrome C oxidase, H4MTP refers to tetrahydromethanopterin pathway.</p> Full article ">Figure 2
<p>Nitrogen assimilation and fixation pathways implemented in iMsOB3b. NO3t7pp refers to nitrate transporter via nitrite antiport, q refers to ubiquinone. NO3R1 refers to NADP-dependent nitrate reductase, NO3Ras refers to ubiquinol-dependent nitrate reductase, NITRIR2x refers to NAD-dependent nitrite reductase, NIT_mc refers to nitrogenase, and redfdx refers to reduced ferredoxin.</p> Full article ">Figure 3
<p>Parameter fitting. The grey dotted lines refer to literature values for oxygen:methane molar consumption ratio ranges on high-copper nitrate medium. (<b>a</b>) Comparison of predicted O<sub>2</sub>/CH<sub>4</sub> molar ratios between 3 modes of electron transfer to pMMO, with no additional parameter fitting. (<b>b</b>) For redox-arm mode, the number of protons necessary for the synthesis of 1 mol ATP was iterated. There was no change in the predicted O<sub>2</sub>/CH<sub>4</sub> molar ratio with all values fitting into experimental range. (<b>c</b>) For direct-coupling mode, a part of the flux was constrained to go via PMMOipp. Despite a slight improvement in the predicted O<sub>2</sub>/CH<sub>4</sub> molar ratio, this value is still below experimentally determined range. (<b>d</b>) For uphill electron transfer mode, the reverse flux via ubiquinol-cytochrome c reductase was fixed at different values. The predicted O<sub>2</sub>/CH<sub>4</sub> molar ratios felt into experimental values when reverse reaction flux was in 0.20–1.20 mmol gDCW<sup>−1</sup> h<sup>−1</sup> range.</p> Full article ">Figure 4
<p>Formaldehyde and formate oxidation and assimilation pathways as implemented in iMsOB3b. The continuous lines represent a single reaction, while the dashed lines represent a series of reaction. ALDD1 refers to non-specific aldehyde dehydrogenase, FALDHp refers to formaldehyde dehydrogenase, 5-10-MTHF refers to 5-10-methylenetetrahydrofolate.</p> Full article ">Figure 5
<p>The flux distribution maps between the serine cycle, EMC pathway and TCA cycle. The maps were drawn with the help of Escher software [<a href="#B52-microorganisms-08-00437" class="html-bibr">52</a>]. The circles refer to metabolites, the arrows refer to reactions, and numbers refer to flux values in mmol gDCW<sup>−1</sup> h<sup>−1</sup>. (<b>a</b>) Reference map for metabolite names, carbon and energy balance. CRN refers to crotonyl-CoA, 3HB refers to 3-hydroxybutyrate, AACOA refers to acetoacetyl-CoA, PEP refers to phosphoenolpyruvate, OAA refers to oxaloacetate, AKG refers to alpha-ketoglutarate, FUM refers to fumarate, MAL refers to malate; <span class="html-italic">S</span>- and <span class="html-italic">R</span>-MMCOA refer to <span class="html-italic">S</span>- and <span class="html-italic">R</span>-methylmalonyl-CoA respectively; and PPCOA refers to propionyl-CoA. (<b>b</b>) Unconstrained flux distribution map. No acetyl-CoA production flux via pyruvate is recorded. (<b>c</b>) Flux distribution when 1/3 of acetyl-CoA is forced to be produced from pyruvate. <a href="#microorganisms-08-00437-f005" class="html-fig">Figure 5</a>D,E,F refers to reversible TCA flux distribution maps: (<b>d</b>) 2-oxoglutarate is allowed to be produced from succinyl-CoA via reversible 2-oxoglutarate dehydrogenase reaction; (<b>e</b>) 2-oxoglutarate is allowed to be produced from succinyl-CoA via forward 2-oxoacid ferredoxin synthase reaction only; (<b>f</b>) both irreversible 2-oxoglutarate reaction and 2-oxoacid ferredoxin synthase reactions are allowed to carry flux.</p> Full article ">Figure 5 Cont.
<p>The flux distribution maps between the serine cycle, EMC pathway and TCA cycle. The maps were drawn with the help of Escher software [<a href="#B52-microorganisms-08-00437" class="html-bibr">52</a>]. The circles refer to metabolites, the arrows refer to reactions, and numbers refer to flux values in mmol gDCW<sup>−1</sup> h<sup>−1</sup>. (<b>a</b>) Reference map for metabolite names, carbon and energy balance. CRN refers to crotonyl-CoA, 3HB refers to 3-hydroxybutyrate, AACOA refers to acetoacetyl-CoA, PEP refers to phosphoenolpyruvate, OAA refers to oxaloacetate, AKG refers to alpha-ketoglutarate, FUM refers to fumarate, MAL refers to malate; <span class="html-italic">S</span>- and <span class="html-italic">R</span>-MMCOA refer to <span class="html-italic">S</span>- and <span class="html-italic">R</span>-methylmalonyl-CoA respectively; and PPCOA refers to propionyl-CoA. (<b>b</b>) Unconstrained flux distribution map. No acetyl-CoA production flux via pyruvate is recorded. (<b>c</b>) Flux distribution when 1/3 of acetyl-CoA is forced to be produced from pyruvate. <a href="#microorganisms-08-00437-f005" class="html-fig">Figure 5</a>D,E,F refers to reversible TCA flux distribution maps: (<b>d</b>) 2-oxoglutarate is allowed to be produced from succinyl-CoA via reversible 2-oxoglutarate dehydrogenase reaction; (<b>e</b>) 2-oxoglutarate is allowed to be produced from succinyl-CoA via forward 2-oxoacid ferredoxin synthase reaction only; (<b>f</b>) both irreversible 2-oxoglutarate reaction and 2-oxoacid ferredoxin synthase reactions are allowed to carry flux.</p> Full article ">Figure 5 Cont.
<p>The flux distribution maps between the serine cycle, EMC pathway and TCA cycle. The maps were drawn with the help of Escher software [<a href="#B52-microorganisms-08-00437" class="html-bibr">52</a>]. The circles refer to metabolites, the arrows refer to reactions, and numbers refer to flux values in mmol gDCW<sup>−1</sup> h<sup>−1</sup>. (<b>a</b>) Reference map for metabolite names, carbon and energy balance. CRN refers to crotonyl-CoA, 3HB refers to 3-hydroxybutyrate, AACOA refers to acetoacetyl-CoA, PEP refers to phosphoenolpyruvate, OAA refers to oxaloacetate, AKG refers to alpha-ketoglutarate, FUM refers to fumarate, MAL refers to malate; <span class="html-italic">S</span>- and <span class="html-italic">R</span>-MMCOA refer to <span class="html-italic">S</span>- and <span class="html-italic">R</span>-methylmalonyl-CoA respectively; and PPCOA refers to propionyl-CoA. (<b>b</b>) Unconstrained flux distribution map. No acetyl-CoA production flux via pyruvate is recorded. (<b>c</b>) Flux distribution when 1/3 of acetyl-CoA is forced to be produced from pyruvate. <a href="#microorganisms-08-00437-f005" class="html-fig">Figure 5</a>D,E,F refers to reversible TCA flux distribution maps: (<b>d</b>) 2-oxoglutarate is allowed to be produced from succinyl-CoA via reversible 2-oxoglutarate dehydrogenase reaction; (<b>e</b>) 2-oxoglutarate is allowed to be produced from succinyl-CoA via forward 2-oxoacid ferredoxin synthase reaction only; (<b>f</b>) both irreversible 2-oxoglutarate reaction and 2-oxoacid ferredoxin synthase reactions are allowed to carry flux.</p> Full article ">Figure 5 Cont.
<p>The flux distribution maps between the serine cycle, EMC pathway and TCA cycle. The maps were drawn with the help of Escher software [<a href="#B52-microorganisms-08-00437" class="html-bibr">52</a>]. The circles refer to metabolites, the arrows refer to reactions, and numbers refer to flux values in mmol gDCW<sup>−1</sup> h<sup>−1</sup>. (<b>a</b>) Reference map for metabolite names, carbon and energy balance. CRN refers to crotonyl-CoA, 3HB refers to 3-hydroxybutyrate, AACOA refers to acetoacetyl-CoA, PEP refers to phosphoenolpyruvate, OAA refers to oxaloacetate, AKG refers to alpha-ketoglutarate, FUM refers to fumarate, MAL refers to malate; <span class="html-italic">S</span>- and <span class="html-italic">R</span>-MMCOA refer to <span class="html-italic">S</span>- and <span class="html-italic">R</span>-methylmalonyl-CoA respectively; and PPCOA refers to propionyl-CoA. (<b>b</b>) Unconstrained flux distribution map. No acetyl-CoA production flux via pyruvate is recorded. (<b>c</b>) Flux distribution when 1/3 of acetyl-CoA is forced to be produced from pyruvate. <a href="#microorganisms-08-00437-f005" class="html-fig">Figure 5</a>D,E,F refers to reversible TCA flux distribution maps: (<b>d</b>) 2-oxoglutarate is allowed to be produced from succinyl-CoA via reversible 2-oxoglutarate dehydrogenase reaction; (<b>e</b>) 2-oxoglutarate is allowed to be produced from succinyl-CoA via forward 2-oxoacid ferredoxin synthase reaction only; (<b>f</b>) both irreversible 2-oxoglutarate reaction and 2-oxoacid ferredoxin synthase reactions are allowed to carry flux.</p> Full article ">
14 pages, 2568 KiB  
Article
Application of an O-Linked Glycosylation System in Yersinia enterocolitica Serotype O:9 to Generate a New Candidate Vaccine against Brucella abortus
by Jing Huang, Chao Pan, Peng Sun, Erling Feng, Jun Wu, Li Zhu and Hengliang Wang
Microorganisms 2020, 8(3), 436; https://doi.org/10.3390/microorganisms8030436 - 20 Mar 2020
Cited by 19 | Viewed by 3569
Abstract
Brucellosis is a major zoonotic public health threat worldwide, causing veterinary morbidity and major economic losses in endemic regions. However, no efficacious brucellosis vaccine is yet available, and live attenuated vaccines commonly used in animals can cause human infection. N- and O [...] Read more.
Brucellosis is a major zoonotic public health threat worldwide, causing veterinary morbidity and major economic losses in endemic regions. However, no efficacious brucellosis vaccine is yet available, and live attenuated vaccines commonly used in animals can cause human infection. N- and O-linked glycosylation systems have been successfully developed and exploited for the production of successful bioconjugate vaccines. Here, we applied an O-linked glycosylation system to a low-pathogenicity bacterium, Yersinia enterocolitica serotype O:9 (Y. enterocolitica O:9), which has repeating units of O-antigen polysaccharide (OPS) identical to that of Brucella abortus (B. abortus), to develop a bioconjugate vaccine against Brucella. The glycoprotein we produced was recognized by both anti-B. abortus and anti-Y. enterocolitica O:9 monoclonal antibodies. Three doses of bioconjugate vaccine-elicited B. abortus OPS-specific serum IgG in mice, significantly reducing bacterial loads in the spleen following infection with the B. abortus hypovirulent smooth strain A19. This candidate vaccine mitigated B. abortus infection and prevented severe tissue damage, thereby protecting against lethal challenge with A19. Overall, the results indicated that the bioconjugate vaccine elicited a strong immune response and provided significant protection against brucellosis. The described vaccine preparation strategy is safe and avoids large-scale culture of the highly pathogenic B. abortus. Full article
(This article belongs to the Special Issue Infectious Diseases, New Approaches to Old Problems)
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<p>Analysis of glycosylated recombinant cholera toxin B subunit (rCTB) and CTB-OPS<sub>Ba</sub> expressed in YeO9_52212. (<b>A</b>) YeO9_52212 was transformed with pET-CTB4573H or pET-pglL-CTB4573H and then induced with IPTG. A wild type strain was treated in the same way (negative control). Coomassie Blue staining (left) and western blotting (right) were performed. (<b>B</b>) The CTB-OPS<sub>Ba</sub> (C-OPS<sub>Ba</sub>) glycoprotein was purified from strain YeO9_52212 co-expressing PglL and rCTB. Samples were separated by 12% SDS-PAGE and analyzed by Coomassie Blue staining or western blotting using anti-His, anti-<span class="html-italic">B. abortus</span> (anti-Ba) or anti-<span class="html-italic">Y. enterocolitica</span> O:9 (anti-Ye) antibodies. (<b>C</b>) Coomassie Blue staining after native gel electrophoresis of C-OPS<sub>Ba</sub>.</p> Full article ">Figure 2
<p>IgG responses against <span class="html-italic">B. abortus</span> A19 lipopolysaccharide (LPS). (<b>A</b>) IgG titers against A19 LPS were measured in sera from PBS-, OPS<sub>Ba</sub>-, OPS<sub>Ba</sub>+Al-, C-OPS<sub>Ba</sub>-, and C-OPS<sub>Ba</sub>+Al-vaccinated mice. (<b>B</b>) IgG subclass titers (IgG1, IgG2a, IgG2b, and IgG3) against A19 LPS were measured in sera from PBS-, OPS<sub>Ba</sub>-, and C-OPS<sub>Ba</sub>-vaccinated mice. Each value represents the mean ± standard deviation of log<sub>2</sub>-transformed titers in the sera of individual mice (shown as data points on the graphs) from each group (<span class="html-italic">n</span> = 10). The unpaired <span class="html-italic">t</span>-test was used to evaluate differences between IgG titers (**, <span class="html-italic">p</span> &lt;0.01; ****, <span class="html-italic">p</span> &lt;0.0001; ns, no statistically significant difference).</p> Full article ">Figure 3
<p>Immune responses of mice following non-lethal <span class="html-italic">B. abortus</span> A19 infection. Immunized mice were infected intraperitoneally with 1.03 × 10<sup>7</sup> CFU of A19 on the 14th day following the third immunization. As a control, another group of naive mice was injected intraperitoneally with normal saline. (<b>A</b>) After infection, the sera of mice in each group were collected on the 1st, 3rd, 5th, and 7th day and the TNF-α levels were measured. The unpaired <span class="html-italic">t</span>-test was used to evaluate differences between TNF-α levels at different time points. Each value represents the mean ± standard deviation (<span class="html-italic">n</span> = 3). (<b>B</b>) On the 7th day post-infection, mouse spleens were collected and weighed and the bacterial loads were measured. Each value represents the mean ± standard deviation of spleen weight or log<sub>10</sub>-transformed bacterial loads (CFU/spleen) of individual mice (shown as data points on the graphs) from each group (<span class="html-italic">n</span> = 5 per group). The unpaired <span class="html-italic">t</span>-test was used to evaluate differences between spleen weights or bacterial loads (***, <span class="html-italic">p</span> &lt;0.001; ****, <span class="html-italic">p</span> &lt;0.0001; ns, no statistically significant difference). (<b>C</b>) The livers and spleens of infected mice and normal mice (Control) were fixed with 4% paraformaldehyde, paraffin sectioned, and then stained with hematoxylin and eosin. The yellow boxes in the top panels represent the field of view in the corresponding figures below, which were magnified four times. Green arrows indicate multinucleated giant cells and blue arrows indicate hyperplastic nodules.</p> Full article ">Figure 4
<p>Survival of BALB/c mice after challenge with a lethal dose of <span class="html-italic">B. abortus</span> A19. Mice were challenged 2 weeks after final immunization by intraperitoneal injection with different doses of A19 and survival was monitored. (<b>A</b>) Schematic diagram of the challenge experiment. (<b>B</b>) Mice were immunized with PBS, OPS<sub>Ba</sub>, OPS<sub>Ba</sub>+Al, C-OPS<sub>Ba</sub> or C-OPS<sub>Ba</sub>+Al and challenged with ~1.54 × 10<sup>8</sup> CFU/mouse (3 × LD<sub>50</sub>) of A19 (<span class="html-italic">n</span> = 10). (<b>C</b>) Mice were immunized with C-OPS<sub>Ba</sub>, C-OPS<sub>Ba</sub>+Al or PBS then challenged with approximately 2.51 × 10<sup>8</sup> CFU/mouse (5 × LD<sub>50</sub>) of A19 (<span class="html-italic">n</span> = 10).</p> Full article ">
10 pages, 1687 KiB  
Communication
OXA-48 Carbapenemase in Klebsiella pneumoniae Sequence Type 307 in Ecuador
by José E. Villacís, Jorge A. Reyes, Hugo G. Castelán-Sánchez, Sonia Dávila-Ramos, Miguel Angel Lazo, Ahmad Wali, Luis A. Bodero, Yadira Toapanta, Cristina Naranjo, Lorena Montero, Josefina Campos, Marcelo G. Galas and Mónica C. Gestal
Microorganisms 2020, 8(3), 435; https://doi.org/10.3390/microorganisms8030435 - 19 Mar 2020
Cited by 7 | Viewed by 4814
Abstract
Antibiotic resistance is on the rise, leading to an increase in morbidity and mortality due to infectious diseases. Klebsiella pneumoniae is a Gram-negative bacterium that causes bronchopneumonia, abscesses, urinary tract infection, osteomyelitis, and a wide variety of infections. The ubiquity of this microorganism [...] Read more.
Antibiotic resistance is on the rise, leading to an increase in morbidity and mortality due to infectious diseases. Klebsiella pneumoniae is a Gram-negative bacterium that causes bronchopneumonia, abscesses, urinary tract infection, osteomyelitis, and a wide variety of infections. The ubiquity of this microorganism confounds with the great increase in antibiotic resistance and have bred great concern worldwide. K. pneumoniae sequence type (ST) 307 is a widespread emerging clone associated with hospital-acquired infections, although sporadic community infections have also been reported. The aim of our study is to describe the first case of Klebsiella pneumoniae (ST) 307 harboring the blaOXA-48-like gene in Ecuador. We characterized a new plasmid that carry OXA-48 and could be the source of future outbreaks. The strain was recovered from a patient with cancer previously admitted in a Ukrainian hospital, suggesting that this mechanism of resistance could be imported. These findings highlight the importance of programs based on active molecular surveillance for the intercontinental spread of multidrug-resistant microorganisms with emergent carbapenemases. Full article
(This article belongs to the Special Issue Infectious Diseases, New Approaches to Old Problems)
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<p>From center to outside, the circular map of plasmid pOXA-48 reference CP034283.1 outside in blue plasmid, pOXA-48 from Ecuador, intrinsic curvature, stacking energy, position preference, CDS+, CDS-, global inverted repeats, GC content, GC skew, and percent AT.</p> Full article ">Figure 2
<p>Plasmid OXA-48 of <span class="html-italic">K. pneumoniae</span> of Ecuador. Whole sequence shows a high degree of identity with plasmids from Czech Republic.</p> Full article ">
40 pages, 1853 KiB  
Review
An Overview of Potential Oleaginous Microorganisms and Their Role in Biodiesel and Omega-3 Fatty Acid-Based Industries
by Alok Patel, Dimitra Karageorgou, Emma Rova, Petros Katapodis, Ulrika Rova, Paul Christakopoulos and Leonidas Matsakas
Microorganisms 2020, 8(3), 434; https://doi.org/10.3390/microorganisms8030434 - 19 Mar 2020
Cited by 195 | Viewed by 16058
Abstract
Microorganisms are known to be natural oil producers in their cellular compartments. Microorganisms that accumulate more than 20% w/w of lipids on a cell dry weight basis are considered as oleaginous microorganisms. These are capable of synthesizing vast majority of fatty acids from [...] Read more.
Microorganisms are known to be natural oil producers in their cellular compartments. Microorganisms that accumulate more than 20% w/w of lipids on a cell dry weight basis are considered as oleaginous microorganisms. These are capable of synthesizing vast majority of fatty acids from short hydrocarbonated chain (C6) to long hydrocarbonated chain (C36), which may be saturated (SFA), monounsaturated (MUFA), or polyunsaturated fatty acids (PUFA), depending on the presence and number of double bonds in hydrocarbonated chains. Depending on the fatty acid profile, the oils obtained from oleaginous microorganisms are utilized as feedstock for either biodiesel production or as nutraceuticals. Mainly microalgae, bacteria, and yeasts are involved in the production of biodiesel, whereas thraustochytrids, fungi, and some of the microalgae are well known to be producers of very long-chain PUFA (omega-3 fatty acids). In this review article, the type of oleaginous microorganisms and their expertise in the field of biodiesel or omega-3 fatty acids, advances in metabolic engineering tools for enhanced lipid accumulation, upstream and downstream processing of lipids, including purification of biodiesel and concentration of omega-3 fatty acids are reviewed. Full article
(This article belongs to the Special Issue Yeast and Fungal Metabolites)
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<p>On the basis of the fatty acid profiles, oleaginous microorganisms can be used for biodiesel production or nutraceuticals. Some oleaginous microorganisms such as microalgae, yeast, fungi, and bacteria are rich in saturated and monounsaturated fatty acids in their oils, while some of them are a good source of polyunsaturated fatty acids such as thraustochytrids and microalgae.</p> Full article ">Figure 2
<p>Role of oleaginous microorganisms to combat the problems of greenhouse gas emissions and improving air quality by using biodiesel in vehicles; likewise production of polyunsaturated fatty acids to fulfill the ever-rising global demand of omega-3 fatty acids and replace the use of fish oil that have become a persistent problem for the global aquatic ecosystem.</p> Full article ">Figure 3
<p>(<b>A</b>) <span class="html-italic">De-novo</span> fatty acid synthesis in oleaginous microorganisms (adapted from [<a href="#B13-microorganisms-08-00434" class="html-bibr">13</a>,<a href="#B16-microorganisms-08-00434" class="html-bibr">16</a>,<a href="#B18-microorganisms-08-00434" class="html-bibr">18</a>,<a href="#B221-microorganisms-08-00434" class="html-bibr">221</a>,<a href="#B243-microorganisms-08-00434" class="html-bibr">243</a>,<a href="#B244-microorganisms-08-00434" class="html-bibr">244</a>]), and enzymes involved in lipid accumulation. AC, aconitase; ACC, acetyl-CoA carboxylase; ACL, ATP-citrate lyase; ACP, acyl carrier protein; FAS, fatty acid synthetase; ICDH, iso-citrate dehydrogenase; MD, malate dehydrogenase (cytoplasmic); PD, pyruvate dehydrogenase; PAP, phosphatidic acid phosphohydrolase; DGAT; diacylglycerol acyltransferase; FAS: fatty acid synthase. (<b>B</b>) Biosynthesis pathway of omega-3 and -6 fatty acids (EPA and DHA) from parent fatty acids (LA and ALA) through a series of desaturation and elongation reactions [<a href="#B17-microorganisms-08-00434" class="html-bibr">17</a>,<a href="#B161-microorganisms-08-00434" class="html-bibr">161</a>,<a href="#B245-microorganisms-08-00434" class="html-bibr">245</a>,<a href="#B246-microorganisms-08-00434" class="html-bibr">246</a>].</p> Full article ">
22 pages, 7207 KiB  
Article
Molecular Ecological Network Complexity Drives Stand Resilience of Soil Bacteria to Mining Disturbances among Typical Damaged Ecosystems in China
by Jing Ma, Yongqiang Lu, Fu Chen, Xiaoxiao Li, Dong Xiao and Hui Wang
Microorganisms 2020, 8(3), 433; https://doi.org/10.3390/microorganisms8030433 - 19 Mar 2020
Cited by 19 | Viewed by 4228
Abstract
Understanding the interactions of soil microbial species and how they responded to disturbances are essential to ecological restoration and resilience in the semihumid and semiarid damaged mining areas. Information on this, however, remains unobvious and deficiently comprehended. In this study, based on the [...] Read more.
Understanding the interactions of soil microbial species and how they responded to disturbances are essential to ecological restoration and resilience in the semihumid and semiarid damaged mining areas. Information on this, however, remains unobvious and deficiently comprehended. In this study, based on the high throughput sequence and molecular ecology network analysis, we have investigated the bacterial distribution in disturbed mining areas across three provinces in China, and constructed molecular ecological networks to reveal the interactions of soil bacterial communities in diverse locations. Bacterial community diversity and composition were classified measurably between semihumid and semiarid damaged mining sites. Additionally, we distinguished key microbial populations across these mining areas, which belonged to Proteobacteria, Acidobacteria, Actinobacteria, and Chloroflexi. Moreover, the network modules were significantly associated with some environmental factors (e.g., annual average temperature, electrical conductivity value, and available phosphorus value). The study showed that network interactions were completely different across the different mining areas. The keystone species in different mining areas suggested that selected microbial communities, through natural successional processes, were able to resist the corresponding environment. Moreover, the results of trait-based module significances showed that several environmental factors were significantly correlated with some keystone species, such as OTU_8126 (Acidobacteria), OTU_8175 (Burkholderiales), and OTU_129 (Chloroflexi). Our study also implied that the complex network of microbial interaction might drive the stand resilience of soil bacteria in the semihumid and semiarid disturbed mining areas. Full article
(This article belongs to the Section Environmental Microbiology)
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<p>Location of the study area.</p> Full article ">Figure 2
<p>Bacterial categories (<b>a</b>–<b>d</b>) in soil samples across Peibei (PB), Zoucheng (ZC), Yangquan (YQ), and Datong (DT) mining areas.</p> Full article ">Figure 3
<p>Principal component analysis (PCA; (<b>a</b>)) and non-metric multidimensional scaling (NMDS; (<b>b</b>)) analysis results of soil bacteria at the phylum level in mining areas.</p> Full article ">Figure 4
<p>Overview of the networks in different mining areas, with node sizes being proportional to node degrees. A red link means a negative correlation and a blue link means a positive correlation.</p> Full article ">Figure 5
<p>Z–P plot showing the keystone species in the different mining area networks. Different symbols with special colors represent different networks as follows: black star for the PB network, red circle for the ZC network, blue upward-facing triangles for the YQ network, and rose downward-facing triangles for the DT network. The module hubs and connectors are labeled with phylogenetic affiliations (<span class="html-italic">Acido—Acidobacteria, Actino—Actinobacteria, Bacteroi—Bacteroidetes, Chloro—Chloroflexi</span>, and <span class="html-italic">Proteo—Proteobacteria</span>. *2 means that there are 2 connectors or 2 module hubs belong to that phylum).</p> Full article ">Figure 6
<p>Network graph with module structure produced by the fast-greedy modularity optimization method. Each node corresponds to a microbial population. Arable numbers such as 1, 2, 3, and 4 stands for the module number. A red link indicates a negative correlation, and a blue link indicates a positive correlation. (<b>a</b>–<b>d</b>) stand for the PB, ZC, YQ, and DT network, respectively.</p> Full article ">Figure 7
<p>Correlations and heatmap of module eigengenes of the four networks (<b>a</b>). Correlations between the signal intensity of a module and each soil characteristic for the four networks (<b>b</b>).</p> Full article ">Figure 7 Cont.
<p>Correlations and heatmap of module eigengenes of the four networks (<b>a</b>). Correlations between the signal intensity of a module and each soil characteristic for the four networks (<b>b</b>).</p> Full article ">Figure 8
<p>Correspondence analysis (CCA; (<b>a</b>)) and variation partition analysis (VPA; (<b>b</b>)) plots indicate the relationships between the bacterial community and soil properties.</p> Full article ">
14 pages, 2120 KiB  
Article
Small Sample Stress: Probing Oxygen-Deprived Ammonia-Oxidizing Bacteria with Raman Spectroscopy In Vivo
by Ann-Kathrin Kniggendorf, Regina Nogueira, Somayeh Nasiri Bahmanabad, Andreas Pommerening-Röser and Bernhard Wilhelm Roth
Microorganisms 2020, 8(3), 432; https://doi.org/10.3390/microorganisms8030432 - 19 Mar 2020
Cited by 9 | Viewed by 3159
Abstract
The stress response of ammonia-oxidizing bacteria (AOB) to oxygen deprivation limits AOB growth and leads to different nitrification pathways that cause the release of greenhouse gases. Measuring the stress response of AOB has proven to be a challenge due to the low growth [...] Read more.
The stress response of ammonia-oxidizing bacteria (AOB) to oxygen deprivation limits AOB growth and leads to different nitrification pathways that cause the release of greenhouse gases. Measuring the stress response of AOB has proven to be a challenge due to the low growth rates of stressed AOB, making the sample volumes required to monitor the internal stress response of AOB prohibitive to repeated analysis. In a proof-of-concept study, confocal Raman microscopy with excitation resonant to the heme c moiety of cytochrome c was used to compare the cytochrome c content and activity of stressed and unstressed Nitrosomonas europaea (Nm 50), Nitrosomonas eutropha (Nm 57), Nitrosospira briensis (Nsp 10), and Nitrosospira sp. (Nsp 02) in vivo. Each analysis required no more than 1000 individual cells per sampling; thus, the monitoring of cultures with low cell concentrations was possible. The identified spectral marker delivered reproducible results within the signal-to-noise ratio of the underlying Raman spectra. Cytochrome c content was found to be elevated in oxygen-deprived and previously oxygen-deprived samples. In addition, cells with predominantly ferrous cytochrome c content were found in deprived Nitrosomonas eutropha and Nitrosospira samples, which may be indicative of ongoing electron storage at the time of measurement. Full article
(This article belongs to the Special Issue Ecology, Diversity and Functions of Ammonia-Oxidizing Bacteria)
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<p>Cytochrome-c-resonant Raman spectra of <span class="html-italic">Nitrosomonas europaea</span> (Nm 50) (<b>a</b>,<b>b</b>), <span class="html-italic">Nitrosomonas eutropha</span> (Nm 57) (<b>c</b>,<b>d</b>), <span class="html-italic">Nitrosospira</span> sp. (Nsp 02) (<b>e</b>,<b>f</b>), and <span class="html-italic">Nitrosospira briensis</span> (Nsp 10) (<b>g</b>,<b>h</b>) as recorded in vivo from individual bacteria cells in well-aerated (<b>a</b>,<b>c</b>,<b>e</b>,<b>g</b>) and oxygen-deprived (<b>b</b>,<b>d</b>,<b>f</b>,<b>h</b>) cultures. Black spectra represent a maximal relRI (most ferrous cytochrome c content of the cell), and red spectra a minimal relRI (least ferrous) for the respective culture. Please note the different intensity ranges.</p> Full article ">Figure 2
<p>relRI sorted in ascending order of 100 random cells each from four different batches of Nm 50 grown non-simultaneously from two different stock cultures (C03, C46) under optimal growth conditions. The underlying Raman spectra were recorded with three different excitation times (1.0 s, 1.5 s, 2.0 s).</p> Full article ">Figure 3
<p>Oxygenation history (<b>a</b>) and relative Raman line intensities (relRI) sorted in ascending order of 1000 random cells each (<b>b</b>) of <span class="html-italic">Nitrosomonas europaea</span> (Nm 50) (black) and <span class="html-italic">Nitrosomonas eutropha</span> (Nm 57) (red) grown under well-aerated (solid) or oxygen-deprived (dashed) conditions.</p> Full article ">Figure 4
<p>Oxygenation history (<b>a</b>) and relative Raman line intensities (relRI) sorted in ascending order of 1000 random cells each (<b>b</b>) of <span class="html-italic">Nitrosospira</span> sp. (Nsp 02) (black) and <span class="html-italic">Nitrosospira briensis</span> (Nsp 10) (red) grown under well-aerated (solid) or oxygen-deprived (dashed) conditions.</p> Full article ">
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