Microorganisms — Open Access Microbiology Journal

Klebsiella pneumoniae is a prominent etiological agent of healthcare associated infections (HAIs). In this context, multidrug-resistant and biofilm-producing bacteria are of special public health concern due to the difficulties associated with treatment of human infections and eradication from hospital environments. Here, in order to study the impact of medical devices-associated materials on the biofilm dynamics, we performed biofilm phenotypic analyses through a classic and a new scanning electron microscopy (SEM) technique for three multidrug-resistant K. pneumoniae isolates growing on polystyrene and silicone. We also applied whole-genome sequencing (WGS) to search for genetic clues underlying biofilm phenotypic differences. We found major differences in the extracellular polymeric substances (EPS) content among the three strains, which were further corroborated by in-depth EPS composition analysis. WGS analysis revealed a high nucleotide similarity within the core-genome, but relevant differences in the accessory genome that may account for the detected biofilm phenotypic dissimilarities, such as genes already associated with biofilm formation in other pathogenic bacteria (e.g., genes coding haemogglutinins and haemolysins). These data reinforce that the research efforts to defeat bacterial biofilms should take into account that their dynamics may be contingent on the medical devices-associated materials. Full article

Biofilm-associated infections pose a complex problem to the medical community, in that residence within the protection of a biofilm affords pathogens greatly increased tolerances to antibiotics and antimicrobials, as well as protection from the host immune response. This results in highly recalcitrant, chronic infections and high rates of morbidity and mortality. Since as much as 80% of human bacterial infections are biofilm-associated, many researchers have begun investigating therapies that specifically target the biofilm architecture, thereby dispersing the microbial cells into their more vulnerable, planktonic mode of life. This review addresses the current state of research into medical biofilm dispersal. We focus on three major classes of dispersal agents: enzymes (including proteases, deoxyribonucleases, and glycoside hydrolases), antibiofilm peptides, and dispersal molecules (including dispersal signals, anti-matrix molecules, and sequestration molecules). Throughout our discussion, we provide detailed lists and summaries of some of the most prominent and extensively researched dispersal agents that have shown promise against the biofilms of clinically relevant pathogens, and we catalog which specific microorganisms they have been shown to be effective against. Lastly, we discuss some of the main hurdles to development of biofilm dispersal agents, and contemplate what needs to be done to overcome them. Full article

Analysis of 49 strains of Fusarium langsethiae originating from northern Europe (Russia, Finland, Sweden, UK, Norway, and Latvia) revealed the presence of spontaneous auxotrophic mutants that reflect natural intraspecific diversity. Our investigations detected that 49.0% of F. langsethiae strains were auxotrophic mutants for biotin, and 8.2% of the strains required thiamine as a growth factor. They failed to grow on vitamin-free media. For both prototrophic and auxotrophic strains, no growth defect was observed in rich organic media. Without essential vitamins, a significant reduction in the growth of the auxotrophic strains results in a decrease of the formation of T-2 toxin and diacetoxyscirpenol. In addition, all analysed F. langsethiae strains were distinguished into two subgroups based on PCR product sizes. According to our results, 26 and 23 strains of F. langsethiae belong to subgroups I and II respectively. We determined that the deletion in the intergenic spacer (IGS) region of the rDNA of F. langsethiae belonging to subgroup II is linked with temperature sensitivity and causes a decrease in strain growth at 30 °C. Four thiamine auxotrophic strains were found in subgroup I, while 21 biotin auxotrophic strains were detected in subgroups II. To the best of our knowledge, the spontaneous mutations in F. langsethiae observed in the present work have not been previously reported. Full article

Coccidioidomycosis is a fungal infection caused by Coccidioides posadasii and Coccidioides immitis. It is estimated that 150,000 new infections occur in the United States each year. The incidence of this infection continues to rise in endemic regions. There is an urgent need for the development of better therapeutic drugs and a vaccine against coccidioidomycosis. This review discusses the features of host innate and adaptive immune responses to Coccidioides infection. The focus is on the recent advances in the immune response and host-pathogen interactions, including the recognition of spherules by the host and defining the signal pathways that guide the development of the adaptive T-cell response to Coccidioides infection. Also discussed is an update on progress in developing a vaccine against these fungal pathogens. Full article

Food-borne pathogens are a serious human health concern worldwide, and the emergence of antibiotic-resistant food pathogens has further confounded this problem. Once-highly-efficacious antibiotics are gradually becoming ineffective against many important pathogens, resulting in severe treatment crises. Among several reasons for the development and spread of antimicrobial resistance, their overuse in animal food production systems for purposes other than treatment of infections is prominent. Many pathogens of animals are zoonotic, and therefore any development of resistance in pathogens associated with food animals can spread to humans through the food chain. Human infections by antibiotic-resistant pathogens such as Campylobacter spp., Salmonella spp., Escherichia coli and Staphylococcus aureus are increasing. Considering the human health risk due to emerging antibiotic resistance in food animal–associated bacteria, many countries have banned the use of antibiotic growth promoters and the application in animals of antibiotics critically important in human medicine. Concerted global efforts are necessary to minimize the use of antimicrobials in food animals in order to control the development of antibiotic resistance in these systems and their spread to humans via food and water. Full article

Filamentous fungi of the Ascomycota phylum are known to contain a family of conserved conidiation regulating proteins with distinctive velvet domains. In Aspergilli, this velvet family includes four proteins, VeA, VelB, VelC and VosA, and is involved in conidiation and secondary metabolism along with a global regulator LaeA. In A. terreus, the overexpression of LaeA has been observed to increase the biogenesis of the pharmaceutically-important secondary metabolite, lovastatin, while the role of the velvet family has not been studied. The secondary metabolism and conidiation of A. terreus have also been observed to be increased by butyrolactone I in a quorum-sensing manner. An enlightenment of the interplay of these regulators will give potential advancement to the industrial use of this fungus, as well as in resolving the pathogenic features. In this study, the Aspergillus terreus MUCL 38669 transcriptome was strand-specifically sequenced to enable an in-depth gene expression analysis to further investigate the transcriptional role of butyrolactone I in these processes. The sequenced transcriptome revealed intriguing properties of the velvet family transcripts, including the regulator laeA, and uncovered the velC gene in A. terreus. The reliability refining microarray gene expression analysis disclosed a positive regulatory role for butyrolactone I in laeA expression, as well as an influence on the expression of the canonical conidiation-regulating genes under submerged culture. All of this supports the suggested regulative role of butyrolactone I in A. terreus secondary metabolism, as well as conidiation.
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Excessive intake of purine-rich foods elevates serum levels of uric acid. Animal and fish meats contain high amounts of inosine and its related purines, and the reduction of taking those purines is crucial for the improvement of serum uric acid levels. We previously showed that Lactobacillus gasseri PA-3 (PA-3) incorporates adenosine and its related purines and that oral treatment with PA-3 reduced adenosine absorption in rats. This study investigated whether PA-3 also incorporates IMP (inosine 5′-monophosphate), inosine, and hypoxanthine, and whether it reduces their absorption in rats. PA-3 was incubated in vitro with radioisotope (RI)-labeled IMP, inosine, and hypoxanthine, and the incorporation of these compounds by PA-3 was evaluated. In addition, rats were orally administered PA-3 along with RI-labeled inosine 5′-monophosphate, inosine, or hypoxanthine, and the ability of PA-3 to attenuate the absorption of these purines was determined. PA-3 incorporated all three purines and displayed greater proliferation in the presence than in the absence of these purines. Oral administration of PA-3 to rats reduced the absorption of IMP, inosine, and hypoxanthine. These results indicate that PA-3 reduces the absorption of purines contained in foods and it is expected that PA-3 contributes attenuation of the excessive intake of dietary purines. Full article

Background is provided on biofilms, including their formation, tolerance mechanisms, structure, and morphology within the context of chronic wounds. The features of biofilms in chronic wounds are discussed in detail, as is the impact of biofilm on wound chronicity. Difficulties associated with the use of standard susceptibility tests (minimum inhibitory concentrations or MICs) to determine appropriate treatment regimens for, or develop new treatments for use in, chronic wounds are discussed, with alternate test methods specific to biofilms being recommended. Animal models appropriate for evaluating biofilm treatments are also described. Current and potential future therapies for treatment of biofilm-containing chronic wounds, including probiotic therapy, virulence attenuation, biofilm phenotype expression attenuation, immune response suppression, and aggressive debridement combined with antimicrobial dressings, are described. Full article

Rhodoferax antarcticus is an Antarctic purple nonsulfur bacterium and the only characterized anoxygenic phototroph that grows best below 20 °C. We present here a high-quality draft genome of Rfx. antarcticus strain ANT.BR^T, isolated from an Antarctic microbial mat. The circular chromosome (3.8 Mbp) of Rfx. antarcticus has a 59.1% guanine + cytosine (GC) content and contains 4036 open reading frames. In addition, the bacterium contains a sizable plasmid (198.6 kbp, 48.4% GC with 226 open reading frames) that comprises about 5% of the total genetic content. Surprisingly, genes encoding light-harvesting complexes 1 and 3 (LH1 and LH3), but not light-harvesting complex 2 (LH2), were identified in the photosynthesis gene cluster of the Rfx. antarcticus genome, a feature that is unique among purple phototrophs. Consistent with physiological studies that showed a strong capacity for nitrogen fixation in Rfx. antarcticus, a nitrogen fixation gene cluster encoding a molybdenum-type nitrogenase was present, but no alternative nitrogenases were identified despite the cold-active phenotype of this phototroph. Genes encoding two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase were present in the Rfx. antarcticus genome, a feature that likely provides autotrophic flexibility under varying environmental conditions. Lastly, genes for assembly of both type IV pili and flagella are present, with the latter showing an unusual degree of clustering. This report represents the first genomic analysis of a psychrophilic anoxygenic phototroph and provides a glimpse of the genetic basis for maintaining a phototrophic lifestyle in a permanently cold, yet highly variable, environment. Full article

Robust colony formation by Bacillus subtilis is recognized as one of the sessile, multicellular lifestyles of this bacterium. Numerous pathways and genes are responsible for the architecturally complex colony structure development. Cells in the biofilm colony secrete extracellular polysaccharides (EPS) and protein components (TasA and the hydrophobin BslA) that hold them together and provide a protective hydrophobic shield. Cells also secrete surfactin with antimicrobial as well as surface tension reducing properties that aid cells to colonize the solid surface. Depending on the environmental conditions, these secreted components of the colony biofilm can also promote the flagellum-independent surface spreading of B. subtilis, called sliding. In this study, we emphasize the influence of Ca²⁺ in the medium on colony expansion of B. subtilis. Interestingly, the availability of Ca²⁺ has no major impact on the induction of complex colony morphology. However, in the absence of this divalent ion, peripheral cells of the colony expand radially at later stages of development, causing colony size to increase. We demonstrate that the secreted extracellular compounds, EPS, BslA, and surfactin facilitate colony expansion after biofilm maturation. We propose that Ca²⁺ hinders biofilm colony expansion by modifying the amphiphilic properties of surfactin. Full article

The gastrointestinal (GI) tract performs key functions that regulate the relationship between the host and the microbiota. Research has shown numerous benefits of probiotic intake in the modulation of immune responses and human metabolic processes. However, unfavorable attention has been paid to temporal changes of the microbial composition and diversity of the GI tract. This study aimed to investigate the effects of yogurt consumption on the GI microbiome bacteria community composition, structure and diversity during and after a short-term period (42 days). We used a multi-approach combining classical fingerprinting techniques (T-RFLPs), Sanger analyses and Illumina MiSeq 16S rRNA gene amplicon sequencing to elucidate bacterial communities and Lactobacilli and Bifidobacteria populations within healthy adults that consume high doses of yogurt daily. Results indicated that overall GI microbial community and diversity was method-dependent, yet we found individual specific changes in bacterial composition and structure in healthy subjects that consumed high doses of yogurt throughout the study. Full article

Microbial biofilm systems are defined as interface-associated microorganisms embedded into a self-produced matrix. The extracellular matrix represents a continuous challenge in terms of characterization and analysis. The tools applied in more detailed studies comprise extraction/chemical analysis, molecular characterization, and visualisation using various techniques. Imaging by laser microscopy became a standard tool for biofilm analysis, and, in combination with fluorescently labelled lectins, the glycoconjugates of the matrix can be assessed. By employing this approach a wide range of pure culture biofilms from different habitats were examined using the commercially available lectins. From the results, a binary barcode pattern of lectin binding can be generated. Furthermore, the results can be fine-tuned and transferred into a heat map according to signal intensity. The lectin barcode approach is suggested as a useful tool for investigating the biofilm matrix characteristics and dynamics at various levels, e.g. bacterial cell surfaces, adhesive footprints, individual microcolonies, and the gross biofilm or bio-aggregate. Hence fluorescence lectin bar-coding (FLBC) serves as a basis for a subsequent tailor-made fluorescence lectin-binding analysis (FLBA) of a particular biofilm. So far, the lectin approach represents the only tool for in situ characterization of the glycoconjugate makeup in biofilm systems. Furthermore, lectin staining lends itself to other fluorescence techniques in order to correlate it with cellular biofilm constituents in general and glycoconjugate producers in particular. Full article

Due to advancements in sequencing technology, sequence data production is no longer a constraint in the field of microbiology and has made it possible to study uncultured microbes or whole environments using metagenomics. However, these new technologies introduce different biases in metagenomic sequencing, affecting the nucleotide distribution of resulting sequence reads. Here, we illustrate such biases using two methods. One is based on phylogenetic heatmaps (PGHMs), a novel approach for compact visualization of sequence composition differences between two groups of sequences containing the same phylogenetic groups. This method is well suited for finding noise and biases when comparing metagenomics samples. We apply PGHMs to detect noise and bias in the data produced with different DNA extraction protocols, different sequencing platforms and different experimental frameworks. In parallel, we use principal component analysis displaying different clustering of sequences from each sample to support our findings and illustrate the utility of PGHMs. We considered contributions of the read length and GC-content variation and observed that in most cases biases were generally due to the GC-content of the reads. Full article

Magnaporthe oryzae is the causal agent of rice blast disease, the most important infection of rice worldwide. Half the world’s population depends on rice for its primary caloric intake and, as such, rice blast poses a serious threat to food security. The stages of M. oryzae infection are well defined, with the formation of an appressorium, a cell type that allows penetration of the plant cuticle, particularly well studied. However, many of the key pathways and genes involved in this disease stage are yet to be identified. In this study, I have used network-extracted ontologies (NeXOs), hierarchical structures inferred from RNA-Seq data, to identify pathways involved in appressorium development, which in turn highlights novel genes with potential roles in this process. This study illustrates the use of NeXOs for pathway identification from large-scale genomics data and also identifies novel genes with potential roles in disease. The methods presented here will be useful to study disease processes in other pathogenic species and these data represent predictions of novel targets for intervention in M. oryzae. Full article

Lactic acid bacteria (LAB) form a major component of gut microbiota and are often used as probiotics for fermented foods, such as yoghurt. In this study, we aimed to evaluate immunomodulatory activity of LAB, especially that of Lactobacillus bulgaricus ME-552 (ME552) and Streptococcus thermophilus ME-553 (ME553). In vivo/in vitro assay was performed in order to investigate their effects on T cell function. After oral administration of ME553 to C57BL/6 mice, the amount of both interferon γ (IFN-γ) and interleukin 17 (IL-17) produced by cluster of differentiation (CD) 4⁺ T cells from Peyer’s patches (PPs) were significantly enhanced. On the other hand, ME552 only up-regulated the production of IL-17 from PP cells. The extent of induction for IFN-γ production differed between ME552 and ME553. These results suggest that LAB modulate T cell effector functions and mucosal immunity. Full article

This review addresses specific regulatory mechanisms involved in the host immune response to fungal organisms. We focus on key cells and regulatory pathways involved in these responses, including a brief overview of their broader function preceding a discussion of their specific relevance to fungal disease. Important cell types discussed include dendritic cells and regulatory T cells, with a focus on specific studies relating to their effects on immune responses to fungi. We highlight the interleukin-10, programmed cell death 1, and cytotoxic T lymphocyte-associated protein 4 signaling pathways and emphasize interrelationships between these pathways and the regulatory functions of dendritic cells and regulatory T cells. Throughout our discussion, we identify selected studies best illustrating the role of these cells and pathways in response to specific fungal pathogens to provide a contextual understanding of the tightly-controlled network of regulatory mechanisms critical to determining the outcome of exposure to fungal pathogens. Lastly, we discuss two unique phenomena relating to immunoregulation, protective tolerance and immune reactivation inflammatory syndrome. These two clinically-relevant conditions provide perspective as to the range of immunoregulatory mechanisms active in response to fungi. Full article

The consumption of fresh nopal cactus juice is widely popular among health-conscious consumers in Mexico. The juice is prepared from fresh cladodes that have only been rinsed with tap water and are not subjected to a pasteurization or terminal bacterial reduction process. The aim of this study was to evaluate the microbial quality of commercially available fresh juices (n = 162) made with nopal in Texcoco, State of Mexico, during the summer and spring season. Standard microbiological methods, the PCR technique and the serological method were used for isolation and identification of bacteria. All samples contained total coliforms and 91% were positive for Escherichia coli. Although total coliforms and E. coli were detected throughout the study, their populations were significantly lower (p < 0.05) in winter and spring, respectively. Citrobacter youngae was found in 20% of the samples, an unidentified species of Citrobacter in 10%, C. freundii and Proteus mirabilis in 3%, and Salmonella Javiana in 1%. The presence of these microorganisms, especially Salmonella, in the nopal juices is unacceptable due to its health significance. The information generated in this study is relevant for human health risk assessment associated with the consumption of unpasteurized nopal juices and potential interventions to minimize pathogen contamination. Full article

Extremophiles are organisms able to thrive in extreme environmental conditions and some of them show the ability to survive high doses of heavy metals thanks to defensive mechanisms provided by primary and secondary metabolic products, i.e., extremolytes, lipids, and extremozymes. This is why there is a growing scientific and industrial interest in the use of thermophilic bacteria in a host of tasks, from the environmental detoxification of heavy metal to industrial activities, such as bio-machining and bio-metallurgy. In this work Thermus thermophilus was challenged against increasing Pb²⁺ concentrations spanning from 0 to 300 ppm in order to ascertain the sensitiveness of this bacteria to the Pb environmental pollution and to give an insight on its heavy metal resistance mechanisms. Analysis of growth parameters, enzyme activities, protein profiles, and lipid membrane modifications were carried out. In addition, genotyping analysis of bacteria grown in the presence of Pb²⁺, using random amplified polymorphic DNA-PCR and DNA melting evaluation, were also performed. A better knowledge of the response of thermophilic bacteria to the different pollutants, as heavy metals, is necessary for optimizing their use in remediation or decontamination processes. Full article

A global model of the lactic fermentation step of gowé was developed by assembling blocks hosting models for bacterial growth, lactic acid production, and the drop of pH during fermentation. Commercial strains of Lactobacillus brevis and of Lactobacillus plantarum were used; their growth was modeled using Rosso’s primary model and the gamma concept as a secondary model. The optimum values of pH and temperature were 8.3 ± 0.3, 44.6 ± 1.2 °C and 8.3 ± 0.3, 3.2 ± 37.1 °C with μ_max values of 1.8 ± 0.2 and 1.4 ± 0.1 for L. brevis and L. plantarum respectively. The minimum inhibitory concentration of undissociated lactic acid was 23.7 mM and 35.6 mM for L. brevis and L. plantarum, respectively. The yield of lactic acid was five times higher for L. plantarum than for L. brevis, with a yield of glucose conversion to lactic acid close to 2.0 for the former and 0.8 for the latter. A model was developed to predict the pH drop during gowé fermentation. The global model was partially validated during manufacturing of gowé. The global model could be a tool to aid in the choice of suitable starters and to determine the conditions for the use of the starter. Full article