Microorganisms

16 pages, 853 KiB

Open AccessArticle

Fusarium Species and Mycotoxins Contaminating Veterinary Diets for Dogs and Cats

by Natalia Witaszak, Łukasz Stępień, Jan Bocianowski and Agnieszka Waśkiewicz

Microorganisms 2019, 7(1), 26; https://doi.org/10.3390/microorganisms7010026 - 21 Jan 2019

Cited by 16 | Viewed by 6821

Veterinary diets are intended for diseased animals and may contain cereal grains, mainly maize and/or wheat. These, in turn, are often infected with pathogens of the Fusarium genus, which are able to produce numerous harmful mycotoxins. Forty-two samples of veterinary diets for dogs [...] Read more.

Veterinary diets are intended for diseased animals and may contain cereal grains, mainly maize and/or wheat. These, in turn, are often infected with pathogens of the Fusarium genus, which are able to produce numerous harmful mycotoxins. Forty-two samples of veterinary diets for dogs and cats were analyzed for the presence of Fusarium species and mycotoxins. Species were identified using molecular methods and the ergosterol and mycotoxins (fumonisin B₁, deoxynivalenol, nivalenol and zearalenone) were quantified using HPLC methods. Two Fusarium species were identified: Fusarium proliferatum and Fusarium verticillioides. The highest concentrations of fumonisin B₁, deoxynivalenol, nivalenol and zearalenone were 74.83, 2318.05, 190.90, and 45.84 ng/g, respectively. Only 9.5% of the samples were free from Fusarium mycotoxins. The acceptable limits of mycotoxin content in animal feed, specified by the EU regulations, were not exceeded in any of the samples tested. The mean mycotoxin content in veterinary diets for cats was lower than for dogs. Thus, it is recommended that veterinary diets are examined, since the mycotoxin contamination pose additional risk to animal health. The knowledge on Fusarium occurrence in veterinary diets is scarce and as far as we are aware this is the first report concerning the occurrence of Fusarium spp. and their important secondary metabolites—mycotoxins—in different types of veterinary diets for companion animals in Poland. Full article

(This article belongs to the Section Food Microbiology)

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15 pages, 2275 KiB

Open AccessArticle

Into the Thermus Mobilome: Presence, Diversity and Recent Activities of Insertion Sequences Across Thermus spp.

by Alba Blesa, Mercedes Sánchez, Eva Sacristán-Horcajada, Sandra González-de la Fuente, Ramón Peiró and José Berenguer

Microorganisms 2019, 7(1), 25; https://doi.org/10.3390/microorganisms7010025 - 21 Jan 2019

Cited by 15 | Viewed by 4505

Abstract

A high level of transposon-mediated genome rearrangement is a common trait among microorganisms isolated from thermal environments, probably contributing to the extraordinary genomic plasticity and horizontal gene transfer (HGT) observed in these habitats. In this work, active and inactive insertion sequences (ISs) spanning [...] Read more.

A high level of transposon-mediated genome rearrangement is a common trait among microorganisms isolated from thermal environments, probably contributing to the extraordinary genomic plasticity and horizontal gene transfer (HGT) observed in these habitats. In this work, active and inactive insertion sequences (ISs) spanning the sequenced members of the genus Thermus were characterized, with special emphasis on three T. thermophilus strains: HB27, HB8, and NAR1. A large number of full ISs and fragments derived from different IS families were found, concentrating within megaplasmids present in most isolates. Potentially active ISs were identified through analysis of transposase integrity, and domestication-related transposition events of ISTth7 were identified in laboratory-adapted HB27 derivatives. Many partial copies of ISs appeared throughout the genome, which may serve as specific targets for homologous recombination contributing to genome rearrangement. Moreover, recruitment of IS1000 32 bp segments as spacers for CRISPR sequence was identified, pointing to the adaptability of these elements in the biology of these thermophiles. Further knowledge about the activity and functional diversity of ISs in this genus may contribute to the generation of engineered transposons as new genetic tools, and enrich our understanding of the outstanding plasticity shown by these thermophiles. Full article

(This article belongs to the Special Issue Thermophiles and Thermozymes)

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8 pages, 3118 KiB

Open AccessArticle

Ganoderma boninense Disease of Oil Palm to Significantly Reduce Production After 2050 in Sumatra if Projected Climate Change Occurs

by R Russell M Paterson

Microorganisms 2019, 7(1), 24; https://doi.org/10.3390/microorganisms7010024 - 19 Jan 2019

Cited by 52 | Viewed by 6382

Abstract

Palm oil is a valuable crop. This relates to the high economic return from sales of the commodity, where Indonesia is the major producer in the world and the island of Sumatra is the most important region for palm oil production in the [...] Read more.

Palm oil is a valuable crop. This relates to the high economic return from sales of the commodity, where Indonesia is the major producer in the world and the island of Sumatra is the most important region for palm oil production in the country. The island can be considered as a model for other oil palm growing regions in SE Asia. The area in Sumatra with a suitable climate for growing oil palm will decrease in size due to projected climate change as demonstrated specifically herein. The more unsuitable climate will lead to concomitant increases in basal stem rot (BSR) by Ganoderma boninense, as previously predicted, which is of major concern to sustainability in SE Asia. A novel approach is described herein, whereby (a) a determination of suitable climate for growing oil palm in Sumatra and (b) deductions to determine future BSR levels on the island were undertaken. The unsuitability of the climate for oil palm is predicted to increase dramatically after 2050 when BSR is predicted to increase to very high levels on most parts of the island. This is likely to make palm oil production unsustainable at some stage between 2050 and 2100. North Sumatra may be more sustainable than the other areas considered in Sumatra. These effects of projected climate change require amelioration before the high levels of BSR and the unsuitable climate for oil palm are realized. Full article

(This article belongs to the Section Environmental Microbiology)

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10 pages, 1213 KiB

Open AccessArticle

Geographic Distribution of Avirulence Genes of the Rice Blast Fungus Magnaporthe oryzae in the Philippines

by Ana Liza C. Lopez, Tapani Yli-Matilla and Christian Joseph R. Cumagun

Microorganisms 2019, 7(1), 23; https://doi.org/10.3390/microorganisms7010023 - 19 Jan 2019

Cited by 10 | Viewed by 5798

Abstract

A total of 131 contemporary and 33 reference isolates representing a number of multi-locus genotypes of Magnaporthe oryzae were subjected to a PCR test to detect the presence/absence of avirulence (Avr) genes. Results revealed that the more frequently occurring genes were [...] Read more.

A total of 131 contemporary and 33 reference isolates representing a number of multi-locus genotypes of Magnaporthe oryzae were subjected to a PCR test to detect the presence/absence of avirulence (Avr) genes. Results revealed that the more frequently occurring genes were Avr-Pik (81.50%), Avr-Pita (64.16%) and Avr-Pii (47.98%), whereas the less frequently occurring genes were Avr-Pizt (19.08%) and Avr-Pia (5.20%). It was also laid out that the presence of Avr genes in M. oryzae is strongly associated with agroecosystems where the complementary resistant (R) genes exist. No significant association, however, was noted on the functional Avr genes and the major geographic locations. Furthermore, it was identified that the upland varieties locally known as “Milagrosa” and “Waray” contained all the R genes complementary to the Avr genes tested. Full article

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14 pages, 2308 KiB

Open AccessArticle

Identification, Characterization, and Formulation of a Novel Carbapenemase Intended to Prevent Antibiotic-Mediated Gut Dysbiosis

by Sheila Connelly, Todd Parsley, Hui Ge and Michael Kaleko

Microorganisms 2019, 7(1), 22; https://doi.org/10.3390/microorganisms7010022 - 16 Jan 2019

Cited by 5 | Viewed by 4255

Abstract

Antibiotics can damage the gut microbiome leading to opportunistic infections and the emergence of antibiotic resistance. Microbiome protection via antibiotic inactivation in the gastrointestinal (GI) tract represents a strategy to limit antibiotic exposure of the colonic microbiota. Proof of concept for this approach [...] Read more.

Antibiotics can damage the gut microbiome leading to opportunistic infections and the emergence of antibiotic resistance. Microbiome protection via antibiotic inactivation in the gastrointestinal (GI) tract represents a strategy to limit antibiotic exposure of the colonic microbiota. Proof of concept for this approach was achieved with an orally-administered beta-lactamase enzyme, SYN-004 (ribaxamase), that was demonstrated to degrade ceftriaxone excreted into the GI tract and protect the gut microbiome from antibiotic-mediated dysbiosis. Ribaxamase efficiently degrades penicillin and cephalosporin beta-lactam antibiotics, but is not active against carbapenems. To expand this microbiome protection strategy to include all classes of beta-lactams, three distinct carbapenemases were evaluated for manufacturability, antibiotic degradation spectrum, and stability in human intestinal fluid. E. coli production strains were generated for P2A, a novel metallo-enzyme isolated from B. cereus, New Delhi metallo-beta-lactamase (NDM), and Klebsiella pneumoniae carbapenemase (KPC). While all three enzymes effectively inactivated a broad range of antibiotics, including penicillins, most cephalosporins, and carbapenems in vitro, only P2A retained biological activity when incubated with human chyme. As functional stability in the intestinal tract is a key requirement for an orally-delivered enzyme, P2A was chosen as a potential clinical candidate. An enteric formulation of P2A was developed, called SYN-006, that was inert under high acid conditions, with enzyme dissolution occurring at pH > 5.5. SYN-006 has the potential to expand microbiome protection via antibiotic inactivation to include all classes of beta-lactam antibiotics. Full article

(This article belongs to the Special Issue Gastrointestinal Microbiota Impacts Human Health and Disease)

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2 pages, 160 KiB

Open AccessEditorial

Editorial for the Special Issue: Human Pathogenic Filamentous Fungi from Food/Water and Mycotoxins from Water

by R. Russell M. Paterson

Microorganisms 2019, 7(1), 21; https://doi.org/10.3390/microorganisms7010021 - 16 Jan 2019

Cited by 1 | Viewed by 2664

Abstract

This special issue was conceived due to the success of the book by Paterson and Lima [...] Full article

(This article belongs to the Special Issue Human Pathogenic Filamentous Fungi from Food/Water and Mycotoxins from Water)

14 pages, 1032 KiB

Open AccessReview

Role of Oral Microbiota in Cancer Development

by Tomasz M. Karpiński

Microorganisms 2019, 7(1), 20; https://doi.org/10.3390/microorganisms7010020 - 13 Jan 2019

Cited by 237 | Viewed by 16333

Abstract

Nowadays cancer is the second main cause of death in the world. The most known bacterial carcinogen is Helicobacter pylori. Pathogens that can have an impact on cancer development in the gastrointestinal tract are also found in the oral cavity. Some specific [...] Read more.

Nowadays cancer is the second main cause of death in the world. The most known bacterial carcinogen is Helicobacter pylori. Pathogens that can have an impact on cancer development in the gastrointestinal tract are also found in the oral cavity. Some specific species have been identified that correlate strongly with oral cancer, such as Streptococcus sp., Peptostreptococcus sp., Prevotella sp., Fusobacterium sp., Porphyromonas gingivalis, and Capnocytophaga gingivalis. Many works have also shown that the oral periopathogens Fusobacterium nucleatum and Porphyromonas gingivalis play an important role in the development of colorectal and pancreatic cancer. Three mechanisms of action have been suggested in regard to the role of oral microbiota in the pathogenesis of cancer. The first is bacterial stimulation of chronic inflammation. Inflammatory mediators produced in this process cause or facilitate cell proliferation, mutagenesis, oncogene activation, and angiogenesis. The second mechanism attributed to bacteria that may influence the pathogenesis of cancers by affecting cell proliferation is the activation of NF-κB and inhibition of cellular apoptosis. In the third mechanism, bacteria produce some substances that act in a carcinogenic manner. This review presents potentially oncogenic oral bacteria and possible mechanisms of their action on the carcinogenesis of human cells. Full article

(This article belongs to the Special Issue Oral Microbiota in Health and Disease)

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14 pages, 1667 KiB

Open AccessReview

Microbial Fermentation of Dietary Protein: An Important Factor in Diet–Microbe–Host Interaction

by Natalie E. Diether and Benjamin P. Willing

Microorganisms 2019, 7(1), 19; https://doi.org/10.3390/microorganisms7010019 - 13 Jan 2019

Cited by 306 | Viewed by 21060

Abstract

Protein fermentation by gut microbiota contributes significantly to the metabolite pool in the large intestine and may contribute to host amino acid balance. However, we have a limited understanding of the role that proteolytic metabolites have, both in the gut and in systemic [...] Read more.

Protein fermentation by gut microbiota contributes significantly to the metabolite pool in the large intestine and may contribute to host amino acid balance. However, we have a limited understanding of the role that proteolytic metabolites have, both in the gut and in systemic circulation. A review of recent studies paired with findings from previous culture-based experiments suggests an important role for microbial protein fermentation in altering the gut microbiota and generating a diverse range of bioactive molecules which exert wide-ranging host effects. These metabolic products have been shown to increase inflammatory response, tissue permeability, and colitis severity in the gut. They are also implicated in the development of metabolic disease, including obesity, diabetes, and non-alcoholic fatty liver disease (NAFLD). Specific products of proteolytic fermentation such as hydrogen sulfide, ammonia, and p-Cresol may also contribute to the development of colorectal cancer. These findings are in conflict with other studies showing that tryptophan metabolites may improve gut barrier function and attenuate severity in a multiple sclerosis model. Further research examining proteolytic fermentation in the gut may be key to our understanding of how microbial and host metabolism interact affecting health. Full article

(This article belongs to the Special Issue Gastrointestinal Microbiota Impacts Human Health and Disease)

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9 pages, 819 KiB

Open AccessArticle

Failure of Staphylococcus aureus to Acquire Direct and Cross Tolerance after Habituation to Cinnamon Essential Oil

by Xiaoqiu Song, Yue Sun, Qian Zhang, Xiaobo Yang, Feng Zheng, Shoukui He and Yifei Wang

Microorganisms 2019, 7(1), 18; https://doi.org/10.3390/microorganisms7010018 - 11 Jan 2019

Cited by 12 | Viewed by 4000

Abstract

Utilization of sublethal concentrations of cinnamon essential oil (CEO) for food preservation has been proposed. However, exposure to stressful, sublethal growth conditions may induce bacterial tolerance to homologous or heterologous stressing agents. Hence, the ability of CEO to stimulate bacterial stress response was [...] Read more.

Utilization of sublethal concentrations of cinnamon essential oil (CEO) for food preservation has been proposed. However, exposure to stressful, sublethal growth conditions may induce bacterial tolerance to homologous or heterologous stressing agents. Hence, the ability of CEO to stimulate bacterial stress response was evaluated in the current work. Staphylococcus aureus was exposed to 1/4 and 1/2 of the minimum inhibitory concentration (MIC, 500 μL/L) of CEO for 18 h. It was found that overnight habituation to CEO failed to induce direct tolerance and cross-tolerance to lactic acid (pH 4.5), NaCl (10 g/100 mL) and high temperature (45 °C) in S. aureus. Likewise, S. aureus cells subjected to successive habituation with increasing amounts (1/16 MIC to 2× MIC) of CEO developed no direct tolerance. Taken together, CEO has no inductive effect on the acquisition of stress tolerance in S. aureus. Full article

(This article belongs to the Section Food Microbiology)

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20 pages, 2307 KiB

Open AccessArticle

Socioeconomic Status and the Gut Microbiome: A TwinsUK Cohort Study

by Ruth C. E. Bowyer, Matthew A. Jackson, Caroline I. Le Roy, Mary Ni Lochlainn, Tim D. Spector, Jennifer B. Dowd and Claire J. Steves

Microorganisms 2019, 7(1), 17; https://doi.org/10.3390/microorganisms7010017 - 11 Jan 2019

Cited by 92 | Viewed by 10336

Abstract

Socioeconomic inequalities in health and mortality are well established, but the biological mechanisms underlying these associations are less understood. In parallel, the gut microbiome is emerging as a potentially important determinant of human health, but little is known about its broader environmental and [...] Read more.

Socioeconomic inequalities in health and mortality are well established, but the biological mechanisms underlying these associations are less understood. In parallel, the gut microbiome is emerging as a potentially important determinant of human health, but little is known about its broader environmental and social determinants. We test the association between gut microbiota composition and individual- and area-level socioeconomic factors in a well-characterized twin cohort. In this study, 1672 healthy volunteers from twin registry TwinsUK had data available for at least one socioeconomic measure, existing fecal 16S rRNA microbiota data, and all considered co-variables. Associations with socioeconomic status (SES) were robust to adjustment for known health correlates of the microbiome; conversely, these health-microbiome associations partially attenuated with adjustment for SES. Twins discordant for IMD (Index of Multiple Deprivation) were shown to significantly differ by measures of compositional dissimilarity, with suggestion the greater the difference in twin pair IMD, the greater the dissimilarity of their microbiota. Future research should explore how SES might influence the composition of the gut microbiota and its potential role as a mediator of differences associated with SES. Full article

(This article belongs to the Special Issue Gut Microbiota Diversity Relates to Lifestyle)

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8 pages, 224 KiB

Open AccessEditorial

Do Antimicrobial Proteins Contribute to Overcoming the Hidden Antifungal Crisis at the Dawn of a Post-Antibiotic Era?

by László Galgóczy and Florentine Marx

Microorganisms 2019, 7(1), 16; https://doi.org/10.3390/microorganisms7010016 - 11 Jan 2019

Cited by 8 | Viewed by 3226

Abstract

The incidence of fungal infections has been grossly underestimated in the past decades as a consequence of poor identification techniques and a lack of regular epidemiologic surveys in low- and middle-income countries [...] Full article

(This article belongs to the Special Issue Antimicrobial Proteins in Filamentous Fungi)

16 pages, 767 KiB

Open AccessReview

Complement Activation Contributes to the Pathophysiology of Shiga Toxin-Associated Hemolytic Uremic Syndrome

by Simona Buelli, Carlamaria Zoja, Giuseppe Remuzzi and Marina Morigi

Microorganisms 2019, 7(1), 15; https://doi.org/10.3390/microorganisms7010015 - 10 Jan 2019

Cited by 23 | Viewed by 7204

Abstract

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections have become a threat to public health globally because of the severe illnesses that they can trigger, such as hemorrhagic colitis and the post-diarrheal hemolytic uremic syndrome (HUS), characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute [...] Read more.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections have become a threat to public health globally because of the severe illnesses that they can trigger, such as hemorrhagic colitis and the post-diarrheal hemolytic uremic syndrome (HUS), characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney failure. Glomerular endothelial cells are primary targets of Stx which, after binding to its specific receptor globotriaosylceramide, upregulates proinflammatory proteins involved both in the recruitment and adhesion of leukocytes and thrombus formation at the site of endothelial injury. In this review, we discuss the role of complement activation in promoting glomerular microvascular dysfunction, providing evidence from experimental models and patients with STEC-HUS. Within the glomerulus, an important target for Stx-induced complement activation is the podocyte, a cell type that is in close contact with endothelial cells and participates in maintaining the filtration barrier. Recently, podocyte injury and loss have been indicated as potential risk factors for long-term renal sequelae in patients with STEC-HUS. Therapeutic approaches targeting the complement system, that may be useful options for patients with STEC-HUS, will also be discussed. Full article

(This article belongs to the Special Issue Pathogenesis of Enterohaemorrhagic Escherichia coli)

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22 pages, 876 KiB

Open AccessReview

What is the Healthy Gut Microbiota Composition? A Changing Ecosystem across Age, Environment, Diet, and Diseases

by Emanuele Rinninella, Pauline Raoul, Marco Cintoni, Francesco Franceschi, Giacinto Abele Donato Miggiano, Antonio Gasbarrini and Maria Cristina Mele

Microorganisms 2019, 7(1), 14; https://doi.org/10.3390/microorganisms7010014 - 10 Jan 2019

Cited by 1960 | Viewed by 84103

Abstract

Each individual is provided with a unique gut microbiota profile that plays many specific functions in host nutrient metabolism, maintenance of structural integrity of the gut mucosal barrier, immunomodulation, and protection against pathogens. Gut microbiota are composed of different bacteria species taxonomically classified [...] Read more.

Each individual is provided with a unique gut microbiota profile that plays many specific functions in host nutrient metabolism, maintenance of structural integrity of the gut mucosal barrier, immunomodulation, and protection against pathogens. Gut microbiota are composed of different bacteria species taxonomically classified by genus, family, order, and phyla. Each human’s gut microbiota are shaped in early life as their composition depends on infant transitions (birth gestational date, type of delivery, methods of milk feeding, weaning period) and external factors such as antibiotic use. These personal and healthy core native microbiota remain relatively stable in adulthood but differ between individuals due to enterotypes, body mass index (BMI) level, exercise frequency, lifestyle, and cultural and dietary habits. Accordingly, there is not a unique optimal gut microbiota composition since it is different for each individual. However, a healthy host–microorganism balance must be respected in order to optimally perform metabolic and immune functions and prevent disease development. This review will provide an overview of the studies that focus on gut microbiota balances in the same individual and between individuals and highlight the close mutualistic relationship between gut microbiota variations and diseases. Indeed, dysbiosis of gut microbiota is associated not only with intestinal disorders but also with numerous extra-intestinal diseases such as metabolic and neurological disorders. Understanding the cause or consequence of these gut microbiota balances in health and disease and how to maintain or restore a healthy gut microbiota composition should be useful in developing promising therapeutic interventions. Full article

(This article belongs to the Special Issue Gastrointestinal Microbiota Impacts Human Health and Disease)

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5 pages, 152 KiB

Open AccessEditorial

Acknowledgement to Reviewers of Microorganisms in 2018

by Microorganisms Editorial Office

Microorganisms 2019, 7(1), 13; https://doi.org/10.3390/microorganisms7010013 - 9 Jan 2019

Viewed by 3144

Abstract

Rigorous peer-review is the corner-stone of high-quality academic publishing [...] Full article

12 pages, 3492 KiB

Open AccessArticle

SNP and SCAR Markers for Specific Discrimination of Antler-Shaped Ganoderma lucidum

by O-Chul Kwon, Chang-Soo Lee and Young-Jin Park

Microorganisms 2019, 7(1), 12; https://doi.org/10.3390/microorganisms7010012 - 9 Jan 2019

Cited by 7 | Viewed by 4557

Abstract

In this study we identified single nucleotide polymorphism (SNP) and sequence characteristic amplification region (SCAR) markers for specific identification of antler-shaped Ganoderma lucidum strains. When the partial mitochondrial SSU rDNA gene sequence of various antler- and kidney-shaped G. lucidum strains were analyzed and [...] Read more.

In this study we identified single nucleotide polymorphism (SNP) and sequence characteristic amplification region (SCAR) markers for specific identification of antler-shaped Ganoderma lucidum strains. When the partial mitochondrial SSU rDNA gene sequence of various antler- and kidney-shaped G. lucidum strains were analyzed and aligned, an SNP was found only in the antler-shaped G. lucidum strain at position 456 bp. In addition, this SNP of antler-shaped strains was digested by HinfI restriction enzyme. We further analyzed the polymorphism of various G. lucidum strains by random amplified polymorphic DNA (RAPD) analysis. In RAPD analysis, we isolated and sequenced a fragment, specific for antler-shaped G. lucidum strains. Based on this specific fragment sequence, two sets of specific primer pairs for antler-shaped G. lucidum strains were designed. PCR analysis revealed that two specific bands were observed only from antler-shaped strains. These two molecular markers will be helpful for identification of morphological characteristics of G. lucidum. Full article

(This article belongs to the Section Molecular Microbiology and Immunology)

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11 pages, 1729 KiB

Open AccessArticle

An Alternative Platform for Protein Expression Using an Innate Whole Expression Module from Metagenomic DNA

by Dae-Eun Cheong, So-Youn Park, Ho-Dong Lim and Geun-Joong Kim

Microorganisms 2019, 7(1), 9; https://doi.org/10.3390/microorganisms7010009 - 8 Jan 2019

Cited by 3 | Viewed by 4090

Abstract

Many integrated gene clusters beyond a single genetic element are commonly trapped as the result of promoter traps in (meta)genomic DNA libraries. Generally, a single element, which is mainly the promoter, is deduced from the resulting gene clusters and employed to construct a [...] Read more.

Many integrated gene clusters beyond a single genetic element are commonly trapped as the result of promoter traps in (meta)genomic DNA libraries. Generally, a single element, which is mainly the promoter, is deduced from the resulting gene clusters and employed to construct a new expression vector. However, expression patterns of target proteins under the incorporated promoter are often inconsistent with those shown in clones harboring plasmids with gene clusters. These results suggest that the integrated set of gene clusters with diverse cis- and trans-acting elements is evolutionarily tuned as a complete set for gene expression, and is an expression module with all the components for the expression of a nested open reading frame (ORF). This possibility is further supported by truncation and/or serial deletion analysis of this module in which the expression of the nested ORF is highly fluctuated or reduced frequently, despite being supported by plentiful cis-acting elements in the spanning regions around the ORF such as the promoter, ribosome binding site (RBS), terminator, and 3′-/5′-UTRs for gene expression. Here, we examined whether an innate module with a naturally overexpressed gene could be considered as a scaffold for an expression system. For a proof-of-principle study, we mined a complete expression module with an innately overexpressed ORF in E. coli from a metagenomics DNA library, and incorporated it into a vector that had no regulatory element for expressing the insert. We obtained successful expression of several inserts such as MBP, GFPuv, β-glucosidase, and esterase using this simple construct without tuning and codon optimization of the target insert. Full article

(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)

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14 pages, 2166 KiB

Open AccessArticle

Expression and Purification of Chemokine MIP-3α (CCL20) through a Calmodulin-Fusion Protein System

by Gopal Ramamourthy, Mauricio Arias, Leonard T. Nguyen, Hiroaki Ishida and Hans J. Vogel

Microorganisms 2019, 7(1), 8; https://doi.org/10.3390/microorganisms7010008 - 8 Jan 2019

Cited by 9 | Viewed by 5308

Abstract

Human macrophage inflammatory protein 3α (MIP-3α), also known as CCL20, is a 70 amino acid chemokine that selectively binds and activates chemokine receptor 6 (CCR6). This chemokine is responsible for inducing the migration of immature dendritic cells, effector, or memory T-cells, and B-cells. [...] Read more.

Human macrophage inflammatory protein 3α (MIP-3α), also known as CCL20, is a 70 amino acid chemokine that selectively binds and activates chemokine receptor 6 (CCR6). This chemokine is responsible for inducing the migration of immature dendritic cells, effector, or memory T-cells, and B-cells. Moreover, the MIP-3α protein has been shown to display direct antimicrobial, antiviral and antiprotozoal activities. Because of the potential therapeutic uses of this protein, the efficient production of MIP-3α is of great interest. However, bacterial recombinant production of the MIP-3α protein has been limited by the toxicity of this extremely basic protein (pI 9.7) toward prokaryotic cells, and by solubility problems during expression and purification. In an attempt to overcome these issues, we have investigated the bacterial recombinant expression of MIP-3α by using several common expression and fusion tags, including 6× histidine (His), small ubiquitin modifier protein (SUMO), thioredoxin (TRX), ketosteroid isomerase (KSI), and maltose binding protein (MBP). We have also evaluated a recently introduced calmodulin (CaM)-tag that has been used for the effective expression of many basic antimicrobial peptides (AMPs). Here, we show that the CaM fusion tag system effectively expressed soluble MIP-3α in the cytoplasm of Escherichia coli with good yields. Rapid purification was facilitated by the His-tag that was integrated in the CaM-fusion protein system. Multidimensional nuclear magnetic resonance (NMR) studies demonstrated that the recombinant protein was properly folded, with the correct formation of disulfide bonds. In addition, the recombinant MIP-3α had antibacterial activity, and was shown to inhibit the formation of Pseudomonas aeruginosa biofilms. Full article

(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)

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14 pages, 4783 KiB

Open AccessArticle

Exploitation of Three Non-Conventional Yeast Species in the Brewing Process

by Laura Canonico, Edoardo Galli, Enrico Ciani, Francesca Comitini and Maurizio Ciani

Microorganisms 2019, 7(1), 11; https://doi.org/10.3390/microorganisms7010011 - 8 Jan 2019

Cited by 56 | Viewed by 5842

Abstract

Consumers require high-quality beers with specific enhanced flavor profiles and non-conventional yeasts could represent a large source of bioflavoring diversity to obtain new beer styles. In this work, we investigated the use of three different non-conventional yeasts belonging to Lachancea thermotolerans, Wickerhamomyces [...] Read more.

Consumers require high-quality beers with specific enhanced flavor profiles and non-conventional yeasts could represent a large source of bioflavoring diversity to obtain new beer styles. In this work, we investigated the use of three different non-conventional yeasts belonging to Lachancea thermotolerans, Wickerhamomyces anomalus, and Zygotorulaspora florentina species in pure and mixed fermentation with the Saccharomyces cerevisiae commercial starter US-05. All three non-conventional yeasts were competitive in co-cultures with the S. cerevisiae, and they dominated fermentations with 1:20 ratio (S. cerevisiae/non-conventional yeasts ratios). Pure non-conventional yeasts and co-cultures affected significantly the beer aroma. A general reduction in acetaldehyde content in all mixed fermentations was found. L. thermotolerans and Z. florentina in mixed and W. anomalus in pure cultures increased higher alcohols. L. thermotolerans led to a large reduction in pH value, producing, in pure culture, a large amount of lactic acid (1.83 g/L) while showing an enhancement of ethyl butyrate and ethyl acetate in all pure and mixed fermentations. W. anomalus decreased the main aroma compounds in comparison with the S. cerevisiae but showed a significant increase in ethyl butyrate and ethyl acetate. Beers produced with Z. florentina were characterized by an increase in the isoamyl acetate and α-terpineol content. Full article

(This article belongs to the Special Issue Non-conventional Yeasts: Genomics and Biotechnology)

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19 pages, 2595 KiB

Open AccessArticle

Salinity and Temperature Influence Growth and Pigment Production in the Marine-Derived Fungal Strain Talaromyces albobiverticillius 30548

by Mekala Venkatachalam, Léa Gérard, Cathie Milhau, Francesco Vinale, Laurent Dufossé and Mireille Fouillaud

Microorganisms 2019, 7(1), 10; https://doi.org/10.3390/microorganisms7010010 - 8 Jan 2019

Cited by 32 | Viewed by 6965

Abstract

Marine-derived fungi that inhabit severe changing environments have gained increasing interest for their ability to produce structurally unique natural products. Fungi belonging to the Talaromyces and the close Penicillium genera are among the most promising microbes for bioactive compound production, including colored metabolites. [...] Read more.

Marine-derived fungi that inhabit severe changing environments have gained increasing interest for their ability to produce structurally unique natural products. Fungi belonging to the Talaromyces and the close Penicillium genera are among the most promising microbes for bioactive compound production, including colored metabolites. Coupling pigment producing capability with bioactive effectiveness would be a valuable challenge in some specific fields such as dyeing, cosmeceutical, or food industries. In this sense, Talaromyces albobiverticillius 30548, a red pigment producing strain, has been isolated from the marine environment of Reunion Island, Indian Ocean. In this research, we analyzed the effect of temperatures (21–27 °C) and salinity levels (0–9%) on fungal growth and pigment production. Maximum pigment yield was obtained in non-salted media, when cultured at 27 °C after 10 days of submerged fermentation in PDB. However, maximum dry biomass production was achieved at stressed condition with 9% sea salts concentrated media at the same temperature. The results indicate that salinity of the culture media positively influences the growth of the biomass. Inversely, pigment production decreases with increase in salinity over 6%. Color coordinates of secreted pigments were expressed in CIELAB color system. The hue angles (h°) ranged from red to yellow colors. This indicated that the color distribution of fungal pigments depends on the salinity in the culture media. This study emphasizes the impact of abiotic stress (salt and temperature) on the growth and metabolome of marine-derived fungal strains. Full article

(This article belongs to the Section Microbial Biotechnology)

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11 pages, 970 KiB

Open AccessReview

Exposure to Aspergillus in Home and Healthcare Facilities’ Water Environments: Focus on Biofilms

by Malcolm Richardson and Riina Rautemaa-Richardson

Microorganisms 2019, 7(1), 7; https://doi.org/10.3390/microorganisms7010007 - 5 Jan 2019

Cited by 29 | Viewed by 5566

Abstract

Aspergillus conida are ubiquitous in the environment, including freshwater, water for bathing, and in drinking water. Vulnerable patients and those suffering from allergic diseases are susceptible to aspergillosis. Avoidance of Aspergillus is of paramount importance. Potential outbreaks of aspergillosis in hospital facilities have [...] Read more.

Aspergillus conida are ubiquitous in the environment, including freshwater, water for bathing, and in drinking water. Vulnerable patients and those suffering from allergic diseases are susceptible to aspergillosis. Avoidance of Aspergillus is of paramount importance. Potential outbreaks of aspergillosis in hospital facilities have been described where the water supply has been implicated. Little is known regarding the risk of exposure to Aspergillus in water. How does Aspergillus survive in water? This review explores the biofilm state of Aspergillus growth based on recent literature and suggests that biofilms are responsible for the persistence of Aspergillus in domestic and healthcare facilities’ water supplies. Full article

(This article belongs to the Special Issue Human Pathogenic Filamentous Fungi from Food/Water and Mycotoxins from Water)

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2 pages, 149 KiB

Open AccessEditorial

Special Issue: Gut Bacteria-Mucus Interaction

by Nathalie Juge

Microorganisms 2019, 7(1), 6; https://doi.org/10.3390/microorganisms7010006 - 4 Jan 2019

Cited by 7 | Viewed by 3828

Abstract

The mucus layer covering the gastrointestinal tract plays a critical role in maintaining a homeostatic relationship with our gut microbiota. [...] Full article

(This article belongs to the Special Issue Gut Bacteria-Mucus Interaction)

10 pages, 1240 KiB

Open AccessReview

Structure and Synthesis of Antifungal Disulfide β-Strand Proteins from Filamentous Fungi

by Györgyi Váradi, Gábor K. Tóth and Gyula Batta

Microorganisms 2019, 7(1), 5; https://doi.org/10.3390/microorganisms7010005 - 27 Dec 2018

Cited by 13 | Viewed by 4553

Abstract

The discovery and understanding of the mode of action of new antimicrobial agents is extremely urgent, since fungal infections cause 1.5 million deaths annually. Antifungal peptides and proteins represent a significant group of compounds that are able to kill pathogenic fungi. Based on [...] Read more.

The discovery and understanding of the mode of action of new antimicrobial agents is extremely urgent, since fungal infections cause 1.5 million deaths annually. Antifungal peptides and proteins represent a significant group of compounds that are able to kill pathogenic fungi. Based on phylogenetic analyses the ascomycetous, cysteine-rich antifungal proteins can be divided into three different groups: Penicillium chrysogenum antifungal protein (PAF), Neosartorya fischeri antifungal protein 2 (NFAP2) and “bubble-proteins” (BP) produced, for example, by P. brevicompactum. They all dominantly have β-strand secondary structures that are stabilized by several disulfide bonds. The PAF group (AFP antifungal protein from Aspergillus giganteus, PAF and PAFB from P. chrysogenum, Neosartorya fischeri antifungal protein (NFAP)) is the best characterized with their common β-barrel tertiary structure. These proteins and variants can efficiently be obtained either from fungi production or by recombinant expression. However, chemical synthesis may be a complementary aid for preparing unusual modifications, e.g., the incorporation of non-coded amino acids, fluorophores, or even unnatural disulfide bonds. Synthetic variants up to ca. 6–7 kDa can also be put to good use for corroborating structure determination. A short overview of the structural peculiarities of antifungal β-strand disulfide bridged proteins will be given. Here, we describe the structural propensities of some known antifungal proteins from filamentous fungi which can also be prepared with modern synthetic chemistry methods. Full article

(This article belongs to the Special Issue Antimicrobial Proteins in Filamentous Fungi)

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20 pages, 2853 KiB

Open AccessArticle

The Microbial Communities of Leaves and Roots Associated with Turtle Grass (Thalassia testudinum) and Manatee Grass (Syringodium filliforme) are Distinct from Seawater and Sediment Communities, but Are Similar between Species and Sampling Sites

by Kelly Ugarelli, Peeter Laas and Ulrich Stingl

Microorganisms 2019, 7(1), 4; https://doi.org/10.3390/microorganisms7010004 - 26 Dec 2018

Cited by 50 | Viewed by 7470

Abstract

Seagrasses are vital members of coastal systems, which provide several important ecosystem services such as improvement of water quality, shoreline protection, and serving as shelter, food, and nursery to many species, including economically important fish. They also act as a major carbon sink [...] Read more.

Seagrasses are vital members of coastal systems, which provide several important ecosystem services such as improvement of water quality, shoreline protection, and serving as shelter, food, and nursery to many species, including economically important fish. They also act as a major carbon sink and supply copious amounts of oxygen to the ocean. A decline in seagrasses has been observed worldwide, partly due to climate change, direct and indirect human activities, diseases, and increased sulfide concentrations in the coastal porewaters. Several studies have shown a symbiotic relationship between seagrasses and their microbiome. For instance, the sulfur, nitrogen, and carbon cycles are important biochemical pathways that seem to be linked between the plant and its microbiome. The microbiome presumably also plays a key role in the health of the plant, for example in oxidizing phyto-toxic sulfide into non-toxic sulfate, or by providing protection for seagrasses from pathogens. Two of the most abundant seagrasses in Florida include Thalassia testudinum (turtle grass) and Syringodium filliforme (manatee grass), yet there is little data on the composition of the microbiome of these two genera. In this study, the microbial composition of the phyllosphere and rhizosphere of Thalassia testudinum and Syringodium filiforme were compared to water and sediment controls using amplicon sequencing of the V4 region of the 16S rRNA gene. The microbial composition of the leaves, roots, seawater, and sediment differ from one another, but are similar between the two species of seagrasses. Full article

(This article belongs to the Section Environmental Microbiology)

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17 pages, 2103 KiB

Open AccessArticle

Employment of L. paracasei K5 as a Novel Potentially Probiotic Freeze-Dried Starter for Feta-Type Cheese Production

by Antonia Terpou, Ioanna Mantzourani, Alex Galanis, Maria Kanellaki, Eugenia Bezirtzoglou, Argyro Bekatorou, Athanasios A. Koutinas and Stavros Plessas

Microorganisms 2019, 7(1), 3; https://doi.org/10.3390/microorganisms7010003 - 26 Dec 2018

Cited by 23 | Viewed by 4588

Abstract

In the present study, a novel potentially probiotic Lactobacillus paracasei strain, previously isolated from dairy products, was evaluated as a starter culture of Feta-type cheese production. Targeting industrial applications, the starter culture was applied as a ready-to-use freeze-dried culture that was either free [...] Read more.

In the present study, a novel potentially probiotic Lactobacillus paracasei strain, previously isolated from dairy products, was evaluated as a starter culture of Feta-type cheese production. Targeting industrial applications, the starter culture was applied as a ready-to-use freeze-dried culture that was either free or immobilized. The immobilized biocatalyst composed of Lactobacillus paracasei K5 cells absorbed within delignified wheat bran prebiotic carrier. All produced cheeses were compared with cheese manufactured by renin enzyme. Several parameters that affect acceptability, quality and shelf-life of Feta-type cheese were investigated, including microbial populations, physicochemical characteristics and cheese volatiles through 90 days of ripening and storage. Survival of L. paracasei K5 remained in high levels (≥6.0 log cfu/g) after the 90th day of cheese production, as recorded by combining microbiological enumeration and strain-specific multiplex PCR analysis. The use of the freeze-dried novel starter culture (free or immobilized) enhanced the aromatic profile of Feta-type cheeses. Finally, the use of the potentially synbiotic immobilized biocatalyst further improved aromatic characteristics of produced cheese and decrease of possible spoilage or pathogenic microorganisms. These findings indicate the potential industrial use of freeze-dried L. paracasei K5 as starter culture for the production of good-quality functional Feta-type cheese. Full article

(This article belongs to the Special Issue Probiotics: From Quality Assessment to Microbial Ecology)

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7 pages, 375 KiB

Open AccessCommunication

Susceptibility of Virulent Yersinia pestis Bacteria to Predator Bacteria in the Lungs of Mice

by Riccardo Russo, Irina Kolesnikova, Thomas Kim, Shilpi Gupta, Androulla Pericleous, Daniel E. Kadouri and Nancy D. Connell

Microorganisms 2019, 7(1), 2; https://doi.org/10.3390/microorganisms7010002 - 21 Dec 2018

Cited by 21 | Viewed by 5575

Abstract

Multi-drug resistant bacterial infections are a serious threat to global public health. Changes in treatment modalities and prudent use of antibiotics can assist in reducing the threat, but new approaches are also required for untreatable cases. The use of predatory bacteria, such as [...] Read more.

Multi-drug resistant bacterial infections are a serious threat to global public health. Changes in treatment modalities and prudent use of antibiotics can assist in reducing the threat, but new approaches are also required for untreatable cases. The use of predatory bacteria, such as Bdellovibrio bacteriovorus, is among the novel approaches being considered as possible therapeutics for antibiotic resistant and/or unidentified bacterial infections. Previous studies have examined the feasibility of using predatory bacteria to reduce colony-forming units (CFUs) in the lungs of rats exposed to lethal doses of Klebsiella pneumoniae; here we apply the approach to the Tier 1 select agent Yersinia pestis, and show that three doses of B. bacteriovorus introduced every six hours reduces the number of CFUs of Y. pestis in the lungs of inoculated mice by 86% after 24 h of infection. These experiments further demonstrate that predatory bacteria may serve to combat Gram negative bacterial infections, including those considered potential bioweapon agents, in the future. Full article

(This article belongs to the Special Issue Multidrug-Resistant Pathogens)

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34 pages, 3678 KiB

Open AccessReview

Characterised Flavin-Dependent Two-Component Monooxygenases from the CAM Plasmid of Pseudomonas putida ATCC 17453 (NCIMB 10007): ketolactonases by Another Name

by Andrew Willetts

Microorganisms 2019, 7(1), 1; https://doi.org/10.3390/microorganisms7010001 - 20 Dec 2018

Cited by 11 | Viewed by 4093

Abstract

The CAM plasmid-coded isoenzymic diketocamphane monooxygenases induced in Pseudomonas putida ATCC 17453 (NCIMB 10007) by growth of the bacterium on the bicyclic monoterpene (rac)-camphor are notable both for their interesting history, and their strategic importance in chemoenzymatic syntheses. Originally named ‘ketolactonase—an [...] Read more.

The CAM plasmid-coded isoenzymic diketocamphane monooxygenases induced in Pseudomonas putida ATCC 17453 (NCIMB 10007) by growth of the bacterium on the bicyclic monoterpene (rac)-camphor are notable both for their interesting history, and their strategic importance in chemoenzymatic syntheses. Originally named ‘ketolactonase—an enzyme system for cyclic lactonization’ because of its characterised mode of action, (+)-camphor-induced 2,5-diketocamphane 1,2-monooxygenase was the first example of a Baeyer-Villiger monooxygenase activity to be confirmed in vitro. Both this enzyme and the enantiocomplementary (−)-camphor-induced 3,6-diketocamphane 1,6-monooxygenase were mistakenly classified and studied as coenzyme-containing flavoproteins for nearly 40 years before being correctly recognised and reinvestigated as FMN-dependent two-component monooxygenases. As has subsequently become evident, both the nature and number of flavin reductases able to supply the requisite reduced flavin co-substrate for the monooxygenases changes progressively throughout the different phases of camphor-dependent growth. Highly purified preparations of the enantiocomplementary monooxygenases have been exploited successfully for undertaking both nucleophilic and electrophilic biooxidations generating various enantiopure lactones and sulfoxides of value as chiral synthons and auxiliaries, respectively. In this review the chequered history, current functional understanding, and scope and value as biocatalysts of the diketocamphane monooxygenases are discussed. Full article

(This article belongs to the Section Microbial Biotechnology)

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