Dormant (resting) cyst formation (encystment) in unicellular eukaryotes is the process of a large-scale digestion of vegetative cell structures and reconstruction into the dormant form, which is performed by cell signaling pathways accompanied by up- or down-regulation of protein expression, and by posttranslational modification such as phosphorylation. In this review, the author describes the morphogenetic events during encystment of Colpoda and the early molecular events in the Ca2+/calmodulin-triggered signaling pathways for encystment, based mainly on our research results of the past 10 years; especially, the author discusses the role of c-AMP dependently phosphorylated proteins (ribosomal P0 protein, ribosomal S5 protein, Rieske iron-sulfur protein, actin and histone H4) and encystment-dependently upregulated (EF-1α-HSP60, actin-related protein) and downregulated proteins (ATP synthase β-chain). In addition, the roles of AMPK, a key molecule in the signaling pathways leading to Colpoda encystment, and differentially expressed genes and proteins during encystment of other ciliates are discussed.

Plastic is omnipresent in the oceans and serves as a surface for biofilm-forming microorganisms. Plastic debris comprises different polymers, which may influence microbial colonization; here, we evaluated whether polymer type affects bacterial biofilm formation. Quantifying the biofilm on polyethylene (PE), polypropylene (PP) or polystyrene (PS) pellets by six marine bacterial strains (Vibrio,Pseudoalteromonas,Phaeobacter) demonstrated that each strain had a unique colonization behavior with either a preference for PS or PP over the other polymer types or no preference for a specific plastic type. PE, PP and PS pellets were exposed to natural seawater microbiota using free-living or total communities as inoculum. Microbial assembly as determined by 16S rRNA (V4) amplicon sequencing was affected by the composition of the initial inoculum and also by the plastic type. Known polymer and hydrocarbon degraders such as Paraglaciecola, Oleibacter and Hydrogenophaga were found in the plastic biofilms. Thus, on a community level, bacterial colonization on plastic is influenced by the microorganisms as well as the polymer type, and also individual strains can demonstrate polymer-specific colonization.

Elizabethkingia are found to cause severe neonatal meningitis, nosocomial pneumonia, endocarditis and bacteremia. However, there are few studies on Elizabethkingia genus by comparative genomic analysis. In this study, three species of Elizabethkingia were found: E. meningoseptica, E. anophelis and E. miricola. Resistance genes and associated proteins of seven classes of antibiotics including beta-lactams, aminoglycosides, macrolides, tetracyclines, quinolones, sulfonamides and glycopeptides, as well as multidrug resistance efflux pumps were identified from 20 clinical isolates of Elizabethkingia by whole-genome sequence. Genotype and phenotype displayed a good consistency in beta-lactams, aminoglycosides and glycopeptides, while contradictions exhibited in tetracyclines, quinolones and sulfonamides. Virulence factors and associated genes such as hsp60 (htpB), exopolysaccharide (EPS) (galE/pgi), Mg2+ transport (mgtB/mgtE) and catalase (katA/katG) existed in all clinical and reference strains. The functional analysis of the clusters of orthologous groups indicated that 'metabolism' occupied the largest part in core genome, 'information storage and processing' was the largest group in both accessory genome and unique genome. Abundant mobile elements were identified in E. meningoseptica and E. anophelis. The most significant finding in our study was that a single clone of E. anophelis had been circulating within diversities of departments in a clinical setting for nearly 18 months.

Azospirillum brasilense is a non-photosynthetic member of the family Rhodospirillaceae. Some strains of this bacterium are reported to produce bacterioruberin type of carotenoids, which are generally produced by halophilic or psychrophilic bacteria. Since no other member of Rhodospirillaceae produces bacterioruberin type of carotenoids, we investigated the presence of genes involved in bacterioruberin and spirilloxanthin biosynthetic pathways in A. brasilense Cd. Although genes encoding the spirilloxanthin pathway were absent, homologs of several genes (crtC and crtF) involved in the biosynthesis of bacterioruberins were present in the genome of A. brasilense Cd. However, the homolog of CruF responsible for the final step in bacterioruberin biosynthesis could not be found. We also characterized the carotenoids of A. brasilense Cd using thin-layer chromatography, high-performance liquid chromatography, absorption spectra and high-resolution mass spectrometry (HRMS). Resolution of the methanol extract of carotenoids in ultra-performance liquid chromatography showed nine peaks, out of which six peaks showed absorption spectra characteristic of carotenoids. HRMS of each peak produced 1-14 fragments with different m/z values. Two of these fragments were identified as 19'-hydroxyfucoxanthinol and 8'-apoalloxanthinal, which are the carotenoids found in aquatic microalgae.

Probiotics is widespreadly used nowadays. However, the safety issue with the use of live probiotics is still a matter of contention. In recent years, an expanding body of evidence supports the beneficial role of heat-killed probiotics in the maintenance of systemic health, whereas the role of these heat-killed bacteria on periodontal health remains unclear. This study aimed to evaluate the effects of heat-killed probiotics on periodontal pathogen virulence and associated mechanisms. We demonstrated that heat-killed Lactobacillus acidophilus was able to coaggregate with Fusobacterium nucleatum, the bridging bacteria of oral biofilm, and inhibit the adhesion and invasion of F. nucleatum, leading to a subsequent elimination of pro-inflammatory cytokine production in oral epithelial cells. This coaggregation further caused a suppression of the virulence gene fap2 expression in F. nucleatum. Therefore, heat-killed L. acidophilus might downregulate the pro-inflammatory cytokine expression in epithelial cells via coaggregation with F. nucleatum and suppression of F. nucleatum fap2 expression, which was the first demonstration that heat-killed probiotics modulate periodontal disease pathogenesis via coaggregation. Collectively, this finding provides new evidence that heat-killed probiotics might exert beneficial effects to periodontal health by coaggregating with periodontal pathogens and modulating their virulence.

In recent years, the relationship between type 2 diabetes (T2D) and gut microbiota has attracted much interest. Dendrobium officinale is a valuable traditional Chinese medicine (TCM) with anti-T2D potential, while its action mechanism remains to be further studied. This study was designed to investigate the modulation effects of D. officinale on gut microbiota of T2D model mice to provide clues to its pharmacology by high-throughput sequencing techniques. It was found that D. officinale supplement could significantly reduce the fasting blood glucose levels of T2D mice. Dendrobium officinale supplement could modulate the composition of gut microbiota and increase the relative abundances of key bacterial taxa associated with T2D development, including Akkermansia and Parabacteroides. Compared with placebo group mice, several Kyoto Encyclopedia of Gene and Genomes pathways associated with T2D altered in the D. officinale treated group. These findings indicated the modulation of D. officinale on gut microbiota of T2D mice, which provide potential pharmacological implications.

The study aimed to evaluate qualitatively and quantitatively the antimicrobial capacity of 10 potential probiotic Lactobacillus strains against model enteropathogens and spoilage microorganisms. The probiotic strains (live and heat-killed forms) were also assessed for their ability to inhibit adhesion of selected pathogens to Caco-2 cells. The largest inhibition zones (the diffusion method) were connected with the usage of whole bacteria cultures (WBC), also high and moderate with cell-free supernatant (CFS) and the lowest with cell-free neutralized supernatant (CNS). The highest antagonistic activity of Lactobacillus strains was observed against L. monocytogenes strains, moderate activity against Salmonella, Shigella, Escherichia coli, Pseudomonas and, the lowest against S.aureus, Bacillus and Enterococcus. The inhibition of adhesion to Caco-2 cells was very high in the case of E. coli, Salmonella and L. monocytogenes, and moderate in the case of S.aureus. On average, the inhibition effect was higher when pathogenic bacteria were treated by WBC, than heat-killed Lactobacillus. Although, in most samples, the effect was not significantly different (P> 0.05). The strains Lb. brevis O24 and Lb. rhamnosus K3 showed the biggest overall antimicrobial properties, and were most effective in adherence inhibition of investigated indicator strains. These bacteria or their metabolites can be used for the production of various foods or pharmaceutical products.

Bacillus thuringiensis has been widely used as a biological control agent against insect pests. Additionally, nematicidal strains have been under investigation. In this report, 310 native strains of B. thuringiensis against Caenorhabditis elegans were tested. Only the LBIT-596 and LBIT-107 strains showed significant mortality. LC50s of spore-crystal complexes were estimated at 37.18 and 31.89 μg/mL for LBIT-596 and LBIT-107 strains, respectively, while LC50s of partially purified crystals was estimated at 23.76 and 20.25 μg/mL for LBIT-596 and LBIT-107, respectively. The flagellin gene sequence and plasmid patterns indicated that LBIT-596 and LBIT-107 are not related to each other. Sequences from internal regions of a cry5B and a cyt1A genes were found in the LBIT-596 strain, while a cry21A, a cry14A and a cyt1A genes were found in the LBIT-107 strain. Genome sequence of the LBIT-107 strain showed new cry genes, along with other virulence factors, hence, total nematicidal activity of the LBIT-107 strain may be the result of a multifactorial effect. The highlight of this contribution is that translocation of spore-crystal suspensions of LBIT-107 into tomato plants inoculated at their rhizosphere decreased up to 90% the number of galls of Meloidogyne incognita, perhaps the most important nematode pest in the world.

Many people spend most of their time indoors, thereby exposing themselves to indoor environmental microbial communities that might interact with the human microbiota. These potential interactions have only been considered for personal identification; however, accumulating evidence indicates that these microbial interactions are potentially implicated with the identification of human interactions and location-specific factors including time and seasonal variations in the microbial community. To augment the potential of metagenomics-based forensic tools, we compared the composition of microbial communities in blood spot surfaces from healthy adults placed in different environments, such as in the bathroom of a female single-person household and on a laboratory, which were sampled across seasons and time points. The laboratory samples showed more changes in the bacterial community over time owing to the higher number of individuals using the laboratory, whereas the microbial communities in the bathroom samples remained relatively stable over time. Moreover, the two locations could be distinguished according to their specific bacterial community compositions. Variations were also observed related to changes in temperature and humidity, allowing for prediction of season-based microbial community. These findings offer a new perspective regarding the use of microbial community analysis in forensic science.

This study aimed to evaluate the Cronobacter spp. strains isolated on the American continent and characterized using multi-locus sequence typing (MLST) available in the PubMLST database and current literature. From 465 Cronobacter spp. strains, the majority (n = 267, 57.4%) was from North America, mainly from USA (n = 234) and 198 (42.6%) were from South America, mainly from Brazil (n = 196). A total of 232 (49.9%) were isolated from foods, 102 (21.9%) from environmental, 87 (18.7%) from clinical, 27 (5.8%) from PIF, one from water (0.2%) and 16 (3.5%) from unknown sources. A total of five species were represented: Cronobacter sakazakii (374, 80.4%), Cronobacter malonaticus (41, 8.8%), Cronobacter dublinensis (29, 6.2%), Cronobacter turicensis (16, 3.5%) and Cronobacter muytjensii (5, 1.1%). The strains with complete MLST profile (n = 345) were assigned to 98 STs, a ratio of 3.5 strain by ST found and the calculated Simpson`s index was 0.93. The strains showed a high diversity and after eBURST analysis, 30 STs (n = 189) formed 12 single and/or double-locus variant clonal complexes (CC). A total of 38 STs (38.7%) were associated with clinical cases of infection, including well established C. sakazakii CC 1, 4, 8 and 83; C. malonaticus ST60, 307, 394 and 440; and C. sakazakii ST 12 and 494.

Antimicrobial materials are tools used to reduce the transmission of infectious microorganisms. Photo-illuminated titania (TiO2) is a known antimicrobial material. Used as a coating on door handles and similar surfaces, it may reduce viability and colonization by pathogens and limit their spread. We tested the survival of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Saccharomyces cerevisiae on a nano-structured TiO2-based thin film, called 'NsARC', and on stainless steel under a variety of light wavelengths and intensities. There was significantly less survival (P <0.001) of all the organisms tested on NsARC compared to inert uncoated stainless steel under all conditions. NsARC was active in the dark and possible mechanisms for this are suggested. NsARC inhibited biofilm formation as confirmed by scanning electron microscopy. These results suggest that NsARC can be used as a self-cleaning and self-sterilizing antimicrobial surface coating for the prevention and reduction in the spread of potentially infectious microbes.

Salmonellosis is an important worldwide food-borne disease. Increasing resistance to Salmonella spp. has been reported in recent years, and now the prevalence of multidrug-resistant Salmonella spp. is a worldwide problem. This necessitates alternative approaches like phage therapy. This study aimed to isolate bacteriophages specific for Salmonella enterica serovar Paratyphi B and S. enterica serovar Typhimurium isolated from different sources (chicken meat, beef and eggshells). The antibiotic resistance profiles of the bacteria were determined by phenotypic and genotypic methods. The prevalence of extended-spectrum β-lactamase genes was examined by polymerase chain reaction. In total, 75% of the isolated Salmonella strains were resistant to tetracycline, whereas 70% of them were resistant to azithromycin. All of the isolates from beef were resistant to nalidixic acid. The most common extended-spectrum β-lactamase genes among the isolates were blaSHV (15%) followed by blaTEM (10%) and blaCTX (5%). Two specific bacteriophages were isolated and characterized. The host range for vB_SparS-ui was Salmonella Paratyphi B, S. enterica serovar Paratyphi A and S. enterica, while that for vB_StyS-sam phage was Salmonella Typhimurium and S. enterica serovar Enteritidis. The characteristics of the isolated phages indicate that they are proper candidates to be used to control some foodstuff contaminations and also phage therapy of infected animals.

The gut microbiota and metabolome could play a role in primary biliary cholangitis (PBC) progression. We aimed to assess fecal microbiota and fecal short-chain fatty acids (SCFAs) in PBC according to fibrosis. In a cross-sectional study of 23 PBC patients, fecal microbiota and SCFAs were determined using 16S rRNA sequencing and nuclear magnetic resonance spectroscopy, respectively. Fecal acetate and SCFAs were higher in advanced fibrosis. Advanced fibrosis microbiota exhibited decreased alpha diversity, increased Weisella and a distinct community composition. SCFAs correlated with individual taxa in non-advanced fibrosis. Fecal microbiota and SCFAs correspond to fibrosis in PBC.

BACKGROUND: Food allergy has been a significant public health issue with growing severity, prevalence and limited treatments. The neutrophil-activating protein A subunit (NapA) of Helicobacter pylori has been shown to have therapeutic potential in allergic diseases. METHODS: The NapA expression efficiency of recombinant Lactococcus lactis(L.lactis) were determined. The effects of recombinant bacterium on food allergy in Balb/c mice were also investigated. RESULTS: NapA were delivered and expressed efficiently via L. lactis. The engineered bacterium ameliorated food allergy symptoms (acute diarrhea and intestinal inflammation) and decreased serum histamine levels. In addition, the secretion of OVA-specific IgG2a, IFN-γ was promoted and the level of IL-4, OVA-specific IgE was restrained. CONCLUSIONS: The recombinant strain may attenuate food allergy in mice through immune regulatory effect, which may be a promising approach for preventing or treating food allergy.

LcrF is the master regulator that positively regulates the Ysc type III secretion system (T3SS) in Yersinia and shares a high similarity with the DNA-binding domain of the T3SS master regulator ExsA in Pseudomonas aeruginosa. Based on these features, bioinformatics analysis has predicted a putative LcrF-binding site in its target promoters. Here, we experimentally characterized its binding motif. An adenine-rich LcrF-binding region in the lcrG promoter sequence, a typical regulatory target of LcrF, was first confirmed. To obtain detailed information, this binding region was cloned into a synthetized promoter and mutations in this region were further constructed. We demonstrated that the 5'-AAAAA-n5-GnCT-3' sequence is required for LcrF regulation and this motif is strictly located 4-bp upstream of a noncanonical promoter, in which the -35 and -10 elements are separated by a 21-bp spacer. Consistently, the putative binding motif was found in promoters of nine T3SS related operons or genes positively regulated by LcrF. Transcriptome analysis further confirmed that LcrF specifically activates T3SS genes in Yersinia. Collectively, our data suggest that LcrF has evolved to be a specific T3SS activator with a stringent sequence requirement for transcriptional regulation.

It is widely acknowledged that having experience conducting research is invaluable for undergraduate science students. Most undergraduate research is undertaken by students in a mentor's laboratory, but this limits the number of opportunities for students, as each laboratory can only take on a certain number of undergraduate researchers each semester. Additionally, it is also widely acknowledged that it is difficult for teachers to meet research goals while providing the best possible coursework for undergraduate students. Both of these bottlenecks can be circumvented via Classroom Undergraduate Research Experiences (CUREs), which integrate research into the curricula of structured undergraduate classes. Students enrolled in classes that include CUREs conduct research to address open-ended questions as part of their coursework. In this commentary, I describe the many ways in which CUREs are helpful for students and teachers, as well as considerations for designing successful CUREs. I provide several examples of CUREs from Microbial Physiology laboratory classes and Genomics classes that I have taught. Results from these CUREs have been successfully integrated into many peer-reviewed publications in which the students are co-authors, which has been a boon both to students' post-baccalaureate opportunities, as well as my research agenda.

Russula griseocarnosa is one of the uncultivable important mycorrhizal edible fungi. Currently, there is a limited insight into the dynamic composition of the microbial communities associated with Russula. Here, the microbiota in the root and mycorrhizosphere from Russula-Fagaceae nature areas of Fujian province were identified by Illumina MiSeq high-throughput sequencing. First, we compared three types of fungal communities associated with Russula-Fagaceae root mycelia-running stage (stage-1), Russula sporocarping stage-2 (stage-2) and Russula-free Fagaceae root (stage-3). Fungal diversity negatively correlated with Russula. Russula, Tomentella and Lactarius were core EcM in Fagaceae roots. A total of eight genera, including Boletus, are likely a positive indicator of Russula sporocarp production in Russula-Fagaceae roots, while Tomentella and Elaphomyces for Russula symbiosis. Secondly, analysis of fungal and bacterial communities within rhizosphere soils from the three stages revealed six genera, including Dacryobolus and Acidocella, as possible indicator species associated with sporocarping in Russula. Elaphomyces, Tomentella, Sorangium, Acidicaldus, Acidobacterium and Haliangium occurred more frequently in the Russula rhizosphere. Furthermore, operational taxonomic unit (OTU) network analysis showed a positive correlation between Russula,Tomentella, Elaphomyces and Sorangium. Overall, our results revealed a relationship between micro-community and Russula, which may provide a new strategy for improving Russula symbiosis and sporocarp production.

Several fungi have been shown to harbor microorganisms that regulate the key components of fungal metabolism. We explored the symbiotic association of an endophyte, Aspergillus terreus, which led to the isolation of a yeast, Meyerozyma caribbica, as its symbiont. An axenic fungal culture, free of the symbiont, was developed to study the effect of this association on the endophytic fungus. The symbiotic yeast partner was found to play an important role in the adaptation of A. terreus to thermal as well as osmotic stress. Under these stress conditions, the symbiont enhanced the production of lovastatin and the growth of the host fungus. The symbiotic yeast was found to induce the expression of the global regulator gene, the key genes involved in the lovastatin biosynthetic pathway as well as those involved in general growth and development, under stress conditions, in the fungal partner. Analysis by PCR and fluorescent in situ hybridization microscopy indicated that the yeast may be present inside the hyphae of the fungus. However, a direct method like transmission electron microscopy may help to better understand the dynamics of this association, including the distribution of the yeast cells in/on the fungal hyphae and spores.

T-2 is a common mycotoxin contaminating cereal crops. Chronic consumption of food contaminated with T-2 toxin can lead to death, so simple and accurate detection methods in food and feed are necessary. In this paper, we establish a highly sensitive and accurate method for detecting T-2 toxin using AlphaLISA. The system consists of acceptor beads labeled with T-2-bovine serum albumin (BSA), streptavidin-labeled donor beads and biotinylated T-2 antibodies. T-2 in the sample matrix competes with T-2-BSA for antibodies. Adding biotinylated antibodies to the test well followed by T-2 and T-2-BSA acceptor beads yielded a detection range of 0.03-500 ng/mL. The half-maximal inhibitory concentration was 2.28 ng/mL and the coefficient of variation was <10%. In addition, this method had no cross-reaction with other related mycotoxins. This optimized method for extracting T-2 from food and feed samples achieved a recovery rate of approximately 90% in T-2 concentrations as low as 1 ng/mL, better than the performance of a commercial ELISA kit. This competitive AlphaLISA method offers high sensitivity, good specificity, good repeatability and simple operation for detecting T-2 toxin in food and feed.

The study aimed to evaluate the probiotic and safety properties of lactic acid bacterial (LAB) strains isolated from the gut microbiota of honey bee Apis mellifera L., since this source remains a promising reservoir of microbial diversity. A total of five bacterial isolates were molecularly identified using 16S rRNA gene sequencing as Enterococcus faecalis-HBE1, Lactobacillus brevis-HBE2, Enterococcus faecalis-HBE3, Enterococcus faecalis-HBE4 and Lactobacillus casei-HBE5. Gut tolerance conditions (low pH and bile salt) were evaluated. Exopolysaccharides (EPS) production, hemolytic, antioxidant activity, resistance toward antibiotics and technological characteristics (starter activity, pH and proteolysis) were examined. The five isolates showed a high survival rate (>95%), under gastrointestinal tract conditions indicating excellent potential for application as probiotics. The isolates showed no hemolytic activities and good acidification rates in the range of pH 4.6-4.98 after incubation at 37°C for 24h. The isolates exhibited promising proteolytic activity as well as DPPH radical scavenging activity in the range of 16.52-59.39%. All the tested isolates had the capability to produce exopolysaccharides except Lactobacillus casei-HBE5. These results put forward that lactic acid bacterial strain isolated from honey bee workers can be considered as promising candidates for future applications as starter cultures and could constitute new potential probiotics for the production of functional dietary products promoting health benefits.

The Qaidam Basin is the most extensive (120 000 km2) basin on the Qinghai-Tibet Plataea (QTP). Recent studies have shown that environmental selection and dispersal limitation influence the soil fungal community significantly in a large-scale distance. However, less is known about large-scale soil fungal community assemblages and its response to the elevation gradient in the high-elevation basin ecosystems. We studied fungal assemblages using Illumina sequencing of the ITS1 region from 35 sites of the Qaidam Basin. As the increase of elevation, fungal species richness and Chao1 index also increased. The Ascomycota was the most abundant phylum (more than 70% of total sequences), and six of the 10 most abundance fungal family was detected in all 35 soil samples. The key factors influencing the soil fungal community composition in the Qaidam Basin were environmental filtering (soil properties and climate factors). The Mantel test showed no significant relationship between geographic distance and community similarity (r = 0.05; p = 0.81). The absence of the distance effect might be caused by lacking dispersal limitation for the soil fungal community.

Classical Bordetella species are primarily isolated from animals and humans causing asymptomatic infection to lethal pneumonia. However, isolation of these bacteria from any extra-host environmental niche has not been reported so far. Here, we have characterized the genomic plasticity and antibody response of Bordetella bronchiseptica strain HT200, isolated from a thermal spring. Genomic ANI value and SNPs-based phylogenetic tree suggest a divergent evolution of strain HT200 from a human-adapted lineage of B. bronchiseptica. Growth and survivability assay showed strain HT200 retained viability for more than 5 weeks in the filter-sterilized spring water. In addition, genes or loci encoding the Bordetella virulence factors such as DNT, ACT and LPS O-antigen were absent in strain HT200, while genes encoding other virulence factors were highly divergent. Phenotypically, strain HT200 was non-hemolytic and showed weak hemagglutination activity, but was able to colonize in the respiratory organs of mice. Further, both infection and vaccination with strain HT200 induced protective antibody response in mouse against challenge infection with virulent B. bronchiseptica strain RB50. In addition, genome of strain HT200 (DSM 26023) showed presence of accessory genes and operons encoding predicted metabolic functions pertinent to the ecological conditions of the thermal spring.