Toxin Production Research Articles

Formation of the bacterial community as the basis of probiotic supplement for livestock

The paper presents the results of research on the creation of a community based on lactobacilli and bacilli with antimicrobial activity against pathogens of intestinal infections in farm animals. B. subtilis strain MP 2 and L. plantarum strain MP 5 included in the consortium are characterized by resistance to artificial gastric juice and bile salts, as well as the ability to form extracellular hydrolytic enzymes (xylanase, carboxymethylcellulase, avicelase, β-glucosidase, amylase, phytase, protease and lipase), antimicrobial (acids, bacteriocins, siderophores and exopolysaccharides) and antioxidant metabolites. B. subtilis strain MP 2 and L. plantarum strain MP 5 did not have hemolytic activity and did not carry genes responsible for toxin production, which indicates the potential safety of this bacteria. It was concluded that the developed consortium is promising for use as a probiotic for animal husbandry.

Evaluation of mycotoxins, mycobiota and toxigenic fungi in the traditional medicine Radix Dipsaci.

Medicinal herbs have been increasingly used for therapeutic purposes against a diverse range of human diseases worldwide. However, inevitable contaminants, including mycotoxins, in medicinal herbs can cause serious problems for humans despite their health benefits. The increasing consumption of medicinal plants has made their use a public health problem due to the lack of effective surveillance of the use, efficacy, toxicity, and quality of these natural products. Radix Dipsaci is commonly utilized in traditional Chinese medicine and is susceptible to contamination with mycotoxins. Here, we evaluated the mycotoxins, mycobiota and toxigenic fungi in the traditional medicine Radix Dipsaci. A total of 28 out of 63 Radix Dipsaci sample batches (44.4%) were found to contain mycotoxins. Among the positive samples, the contamination levels of AFB1, AFG1, AFG2, and OTA in the positive samples ranged from 0.52 to 32.13 μg/kg, 5.14 to 20.05 μg/kg, 1.52 to 2.33 μg/kg, and 1.81 to 19.43 μg/kg respectively, while the concentrations of ZEN and T-2 were found to range from 2.85 to 6.33 μg/kg and from 2.03 to 2.53 μg/kg, respectively. More than 60% of the contaminated samples were combined with multiple mycotoxins. Fungal diversity and community were altered in the Radix Dipsaci contaminated with various mycotoxins. The abundance of Aspergillus and Fusarium increased in the Radix Dipsaci contaminated with aflatoxins (AFs) and ZEN. A total of 95 strains of potentially toxigenic fungi were isolated from the Radix Dipsaci samples contaminated with mycotoxins, predominantly comprising Aspergillus (73.7%), Fusarium (20.0%), and Penicillium (6.3%). Through morphological identification, molecular identification, mycotoxin synthase gene identification and toxin production verification, we confirmed that AFB1 and AFG1 primarily derive from Aspergillus flavus, OTA primarily derives from Aspergillus westerdijkiae, ZEN primarily derives from Fusarium oxysporum, and T-2 primarily derives from Fusarium graminearum in Radix Dipsaci. These data can facilitate our comprehension of prevalent toxigenic fungal species and contamination levels in Chinese herbal medicine, thereby aiding the establishment of effective strategies for prevention, control, and degradation to mitigate the presence of fungi and mycotoxins in Chinese herbal medicine.

Transdisciplinary approaches for the study of cyanobacteria and cyanotoxins

Cyanobacteria, ancient aerobic and photoautotrophic prokaryotes, thrive in diverse ecosystems due to their extensive morphological and physiological adaptations. They play crucial roles in aquatic ecosystems as primary producers and resource providers but also pose significant ecological and health risks through blooms that produce harmful toxins, called cyanotoxins. The taxonomic affiliation of cyanobacteria has evolved from morphology-based methods to genomic analysis, which offers detailed structural and physiological insights that are essential for accurate taxonomic affiliation and monitoring. However, challenges posed by uncultured species have been extrapolated to the detection and quantification of cyanotoxins. Current advances in molecular biology and informatics improve the precision of monitoring and allow the analysis of groups of genes related to toxin production, providing crucial information for environmental biosafety and public health. Unfortunately, public genomic databases heavily underrepresent cyanobacteria, which limits the understanding of their diversity and metabolic capabilities. Despite the increasing availability of cyanobacterial genome sequences, research is still largely focused on a few model strains, narrowing the scope of genetic and metabolic studies. The challenges posed by cyanobacterial blooms and cyanotoxins necessitate improved molecular, cultivation, and polyphasic techniques for comprehensive classification and quantification, highlighting the need for advanced genomic approaches to better understand and manage cyanobacteria and toxins. This review explores the application of transdisciplinary approaches for the study of cyanobacteria and cyanotoxins focused on diversity analysis, population quantification, and cyanotoxin monitoring, emphasizing their genomic resources and their potential in the genomic mining of toxin-related genes.

Research progress of traditional Chinese medicine on the treatment of diarrhea by regulating intestinal microbiota and its metabolites based on renal-intestinal axis.

Intestinal microbiota and its metabolites are involved in many physiological processes of the human body and play a vital role in maintaining human health. The occurrence of kidney disease can cause intestinal microbiota imbalance, resulting in diarrhea. The change of intestinal microbiota and its metabolites content can aggravate renal function injury, which has a bidirectional regulating effect. The theory of renal-intestinal axis further clarified that the impaired renal function is related to the imbalance of intestinal microorganisms, and the impaired intestinal barrier is related to the accumulation of toxin products. Because of its unique therapeutic advantages, Traditional Chinese Medicine can treat diarrhea by enhancing the growth of beneficial bacteria, inhibiting pathogenic bacteria and immune regulation, and slow down the continuous deterioration of kidney disease. This paper focuses on the relationship between intestinal microbiota and its metabolites and diarrhea, the influence of Traditional Chinese Medicine on intestinal microbiota in the treatment of diarrhea, and the role of intestinal microbiota and its metabolites in the renal-intestinal axis. It provides a theoretical basis for Traditional Chinese Medicine to regulate intestinal microbiota and its metabolites based on the renal-intestinal axis theory to treat nephrology-induced diarrhea, and also provides a new idea and method for Traitional Chinese Medicine to treat nephrology-induced diarrhea.

Analysis of the properties of Komagataella phaffii CF-st401, a genetically modified producer of the sweet protein brazzein by using in silico methods

Currently, in order to reduce the consumption of mono- and disaccharides in diets, sweeteners are widely used. At the same time, none of the sweeteners approved for food industry usage matches the organoleptic properties of natural sugars. This circumstance was the basis for the development of technology for producing a new type of food ingredient with a sweet taste - brazzein using the producer strain Komagataella phaffii CF-st401. The purpose of the study was the application of in silico methods to assess the safety of K. phaffii CF-st401 genetically modified (GM) microorganism, which is the producer of the "Sweet protein Brazzein". Material and methods. The research object was the map of K. phaffii CF-st401 plasmid obtained by Biryuch LLC as a result of sequencing the plasmid of GM strain K. phaffii CF-st401, including: a synthetic nucleotide sequence similar to the sequence encoding the brazzein protein in the plant (Pentadiplandra brazzeana) and optimized for expression in the DNA of the recipient strain; the nucleotide sequence of plasmid M4794 from Saccharomyces cerevisiae and a linear fragment of the flanking DNA regions from K. phaffii used for integration into the genome of the recipient strain K. phaffii YIB Δleu2 VKPM Y-476, as well as amino acid sequence data of recombinant brazzein. By using bioinformatics methods (in silico), we investigated the DNA structure of the vector sequence of K. phaffii CF-st401, including the presence of operons responsible for toxin production, antibiotic resistance, and allergenicity. Results. As a result of the studies of K. phaffii CF-st401 vector plasmid, introduced into K. phaffii YIB Δleu2 VKPM Y-4761, it was shown that its regions responsible for the structure of the "Sweet protein Brazzein" coincide by more than 70% with elements of the brazzein P56552 reference protein from the plant P. brazzeana. The absence of selective markers and allergenicity in the vector plasmid was confirmed. Conclusion. The analysis of the structure of the DNA vector sequence of the K. phaffii CF-st401 GM strain confirmed the feasibility of using bioinformatics methods to predict the properties of technological microorganisms when assessing their safety for consumers.

Analysis of synthetic route and case correlation of methamphetamine crystals seized by ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) and Pearson correlation coefficient method.

Analysis of impurities in illicit drugs provides key information on the manufacture and distribution of methamphetamine (MA). An analysis of 22 specific target compounds in unlawful MA products was conducted using ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS). The peak area of the alkaloids was utilized as an important calculation data variable to determine the synthesis method. The results demonstrated that the application of target screening techniques for trace impurity components in samples can aid in determining the synthesis process routes for toxin production in 169 samples. These samples were initially categorized based on their synthesis modes, specifically synthesis routes N, E, and R. The correlation analysis of MA samples with known synthetic routes was carried out using principal coordinate analysis (PCoA) and the Pearson correlation coefficient method. Among the N and R synthesis routes, 15 and 7 groups with strong correlation were screened, respectively. Consistent with the observed scenario, the samples from the synthetic route E showed limited correlation with each other. The experiment provides a focused framework for the management of synthetic raw materials used for the production of drugs and the investigation of drug-related cases. This facilitates a holistic strategy to combat drug-related crimes, from manufacturing laboratory to end-user.

Comprehensive analyses of inhibition of the Chinese traditional food-packaging materials, Indocalamus latifolius leaves (Zongye) on Aspergillus flavus

Abstract Objectives The contamination of Aspergillus flavus and aflatoxins (AFs) is one of the most serious safety problems in food and feed. The discovery and application of plant-sourced antifungal agents are hot topics in mycotoxin control. In this study, we aim to develop efficient strategies to control A. flavus and subsequent contamination by AFs. Materials and Methods We focused on Zongye essential oil, which was extracted from Indocalamus latifolius leaves (Zongye, the traditional food-packaging materials). Its antifungal and antitoxin activities were observed by volatile test, and the significant morphological and ultrastructural variations were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses. The main components of Zongye essential oil were analyzed by gas chromatography-mass spectrometry (GC-MS). RNA sequencing (RNA-Seq) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses were used to reveal the inhibitory mechanism. Results Among them, No. 2 Zongye essential oil completely inhibited the growth and toxin production of A. flavus after 10 µL treatment. Under Zongye essential oil treatment, evidently morphological and ultrastructural variations were observed, such as hyphae shrinkage, partial distortion, and a decrease in conidia number. Longifolene and linalool were the major constituents of Zongye essential oil, accounting for 9.55% and 7.95%, respectively, and linalool had stronger inhibitory effects on fungal growth and mycotoxin biosynthesis than longifolene. Based on the experimental results, the antifungal mechanism was proposed: down-regulations of membrane proteins (AFLA_005560, AFLA_019420, and AFLA_084310, etc.) and conidial genes (fblC, steA, and abaA, etc.) inhibited fungal development, and anti-aflatoxigenic activity might be due to significant down-regulation of AF cluster genes and global regulators (AtfA and AtfB). Conclusion A novel antifungal agent, Zongye essential oil, was identified, and its inhibitory mechanism was comprehensively clarified, which is helpful to control AF contamination in an environmentally friendly way.

Atypical Haemolytic Uraemic Syndrome in an Infant with Ventricular Septal Defect: A Case Report

Atypical Haemolytic Uraemic Syndrome (aHUS) is a rare type of thrombotic microangiopathy that occurs without Shiga toxin producing bacteria. It is a condition related to complement regulation, which may be genetic or acquired. The complement system’s alternative pathway is commonly implicated, with around 6-10% of cases being caused by autoantibodies directed against factor H. While it typically affects children between 9 to 13-years-old, it can also occur in adults. Many patients do not have circulating Complement Factor H-related proteins 1 and 3 due to a homozygous deletion involving CFHR1 and CFHR3. Authors hereby report a case of a six-month-old female child who was diagnosed with Ventricular Septal Defect (VSD) at one and a half months of age. She presented with pneumonia and subsequently developed haemolytic anaemia with thrombocytopenia, oliguria, and acute kidney failure. She was diagnosed with Antifactor H antibody-mediated HUS. She was treated with plasma therapy, but the patient succumbed due to multiorgan dysfunction.

Comparative genomics analyses of fish pathogenic Streptococcus spp. isolated from tilapia and flounder

This comparative study was performed to analyze the whole genome sequences of three species of Streptococcus (S. agalactiae, S. iniae and S. parauberis) isolated from Tilapia and Flounder from six different countries (Brazil, China, Israel, South Korea, Taiwan and USA). The objectives were to compare the genomic features, virulence, and antibiotic resistance genes in the genome sequences of 11 isolates of Streptococcus spp. A total of 44 virulence genes were identified in the genomes of 11 strains. These genes are responsible for adherence, various enzyme production, immune evasion, immunoreactive antigen, and toxin production. Eight antibiotic resistance genes were identified in the eleven genome sequences of the Streptococcus sp. strains. All strains of S. agalactiae and S. iniae harbor macrolide resistance gene mreA. Although five secondary metabolites such as Arylpolyene (ary), Type III Polyketide synthases (T3PKS), RiPP-like peptide, linear azol(in)e-containing peptides (LAPs), RaS-RiPP antimicrobial compound, and T3PKS were detected in all 11 genomes; only T3PKS was common in all strains. Additionally, Cas cluster CAS-TypeIC and CAS-TypeIIA were identified among the ten strains of S. agalactiae and S. iniae. The findings indicated that the degree of pathogenicity of Streptococcus sp. remained closer regardless of origin, distribution and host. The results would be useful to understand the virulence factors of the Streptococcus sp. and the antibiotic resistance genes associated with their virulence in fish. Ann. Bangladesh Agric. (2023) 27 (2): 1-18

The Role of Microcin B17-Producing Escherichia Coli, Isolated from Patients with Inflammatory Bowel Disease on Rat Intestine

Background: Inflammatory Bowel Disease (IBD) is a chronic gastrointestinal disease, Crohn’s disease (CD) and ulcerative colitis (UC) are two distinct kinds of IBD. Recent researches have linked between Escherichia coli which produce the toxin microcin B17 (E. coli/+mB17) and the development of inflammatory bowel disease (IBD). Objective: This study aimed to demonstrate the relationship between these E. coli isolates and the emergence of IBD in an experimental animal models. Materials and Methods: Twenty female rats, five-weeks-old were divided in to four groups; first group, were orally given E. coli/+mB17 isolated from crohn’s patients stool, the second group rats were orally given E. coli/+mB17 isolated from patients stool with ulcerative colitis, in the third group were orally given E. coli non-producing microcin B17 (E. coli/-mB17) isolated from patients stool with IBD, and the last group received normal saline orally, as control. After fifteen weeks of administration, Blood was drawn for serological tests which include interlukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) and the rats sacrificed for evaluation of histological changes in their intestine. Results: All groups that were administrated bacteria, whether they were toxin producer or not, showed a decrease in their weights with a reduction of appetite. Serological changes observed included an increase in IL-10 level and an increase in TNF-α level in their sera. Regarding the histological examination, there were noticeable histological changes in the large intestines of all rats given bacterial suspension but not in small intestine. Conclusion: All E.coli isolates which were isolated from IBD patients stool (whether it was microcin B17 producing or not) were able to induce inflammatory responses and histological changes in the intestines of rats . Accordingly, other virulence factors (in addition to Microcin B17) may play role in the pathogenesis of IBD.

Identification and antibiotic sensitivity profile of potential bacterial toxin producers from some foods sold to students within Enugu state university of science and technology (ESUT) campus

Food borne diseases have become major public health burden and bacteria especially the toxin producers have been mostly implicated as among the most common causative agents. Thus, the need to screen and identify most frequent bacteria associated with ready to eat foods consumed within ESUT campus. The food samples were chicken, egg, tomato, vegetable salad and dried fish. A total of 25 samples were collected, with 5 for each sample .They were cultured for bacterial isolation using selective media prior to phenotypic characterization via morphology and biochemical analysis . The predominant isolates were further subjected to genotypic characterization using 16S rRNA sequencing. Each identified isolate of inoculum size of 1.5x 108cfu/ml was exposed in-vitro to analytical Gram+ve and Gram-ve antibiotics by disc diffusion method .The results revealed the presence of Staphylococcus aureus strain OPD001-1 in 5samples, Citrobacter werkmanii strain UMH18 in 3 samples, Salmonella enterica subsp. entericaserovar strain CFSAN027396 in 4 samples, Salmonella enterica subsp. entericaserovar typhimurium in 4 samples and Salmonella enteric strain 2011k-1440 in 4 samples and Salmonella enteric subsp. entericaserovar in 5 samples respectively. Their antibiotic susceptibility pattern differed. Gram positive Staphylococcus aureus was susceptible (>20mm) to 7 test antibiotics (Erythromycin, Levofloxacin, Cephalexin, Ciprofloxacin, Gentamicin, Ofloxacin and Clindamycin ) and resistant (<14mm) to 3 (Cetriaxone, Ampicillin and Cloxacillin). Citrobacter werkmanii and Salmonella enteric subsp. entericaserovar demonstrated similar susceptibility (>20mm) to 7 test Gram-ve antibiotics but resistant to Amoxicillin, Streptomycin and Gentamicin while Salmonella enterica strain 2011k-1440 and Salmonella enteric subsp. entericaserovar typhimurium were resistant (<14mm) to Nitrofurantion , Cetriaxone and Amoxicillin. Although these bacterial isolates can be inhibited by some antibiotics, their implications as potential toxin producers including the heat stable ones pose health problems to food consumers.

Infective Endocarditis by Clostridioides and Clostridium Species-A Narrative Review.

Bacteria of the genus Clostridium are anaerobic Gram-positive spore-forming bacilli that include more than 200 species. Some of them are known to cause invasive infections and diseases caused by the production of toxins. Some of the diseases that are mediated by toxins are colitis in patients with specific risk factors, such as previous administration of antimicrobials or foodborne botulism. Invasive diseases include bacteremia, infective endocarditis (IE), clostridial myonecrosis (gas gangrene), and other diseases that involve the destruction of soft tissue due to the local production of toxins. The present study aimed to review all cases of IE by Clostridioides and Clostridium species and describe the epidemiology, clinical characteristics, treatment, and outcomes of these infections. A narrative review was performed based on a search in PubMed and Scopus for studies published until 11 September 2023, providing such data of IE caused by Clostridioides and Clostridium species in humans. A total of 20 studies containing data for 21 patients were included. A prosthetic valve was present in 5 patients (23.8%). The aortic valve was the most commonly involved, followed by the mitral valve. Fever, sepsis, and embolic phenomena were the most common clinical presentations. Beta-lactams and metronidazole were the most commonly used antimicrobials. Surgery was performed in nine patients (45%). Mortality reached 33.3%. IE in multiple valves was associated with increased mortality. Despite the heterogeneous genetic and molecular characteristics that necessitate the taxonomic change of some of this genus's previous members, the clinical syndrome of IE caused by these bacteria seems to have similar characteristics.

Identification of the Enterotoxigenic Potential of Staphylococcus spp. from Raw Milk and Raw Milk Cheeses.

This study aimed to genotypic and phenotypic analyses of the enterotoxigenic potential of Staphylococcus spp. isolated from raw milk and raw milk cheeses. The presence of genes encoding staphylococcal enterotoxins (SEs), including the classical enterotoxins (sea-see), non-classical enterotoxins (seg-seu), exfoliative toxins (eta-etd) and toxic shock syndrome toxin-1 (tst-1) were investigated. Isolates positive for classical enterotoxin genes were then tested by SET-RPLA methods for toxin expression. Out of 75 Staphylococcus spp. (19 Staphylococcus aureus and 56 CoNS) isolates from raw milk (49/65.3%) and raw milk cheese samples (26/34.7%), the presence of enterotoxin genes was confirmed in 73 (97.3%) of them. Only one isolate from cheese sample (1.3%) was able to produce enterotoxin (SED). The presence of up to eight different genes encoding enterotoxins was determined simultaneously in the staphylococcal genome. The most common toxin gene combination was sek, eta present in fourteen isolates (18.7%). The tst-1 gene was present in each of the analyzed isolates from cheese samples (26/34.7%). Non-classical enterotoxins were much more frequently identified in the genome of staphylococcal isolates than classical SEs. The current research also showed that genes tagged in S. aureus were also identified in CoNS, and the total number of different genes detected in CoNS was seven times higher than in S. aureus. The obtained results indicate that, in many cases, the presence of a gene in Staphylococcus spp. is not synonymous with the ability of enterotoxins production. The differences in the number of isolates with genes encoding SEs and enterotoxin production may be mainly due to the limit of detection of the toxin production method used. This indicates the need to use high specificity and sensitivity methods for detecting enterotoxin in future studies.

Plasmid-encoded toxin defence mediates mutualistic microbial interactions

Gut environments harbour dense microbial ecosystems in which plasmids are widely distributed. Plasmids facilitate the exchange of genetic material among microorganisms while enabling the transfer of a diverse array of accessory functions. However, their precise impact on microbial community composition and function remains largely unexplored. Here we identify a prevalent bacterial toxin and a plasmid-encoded resistance mechanism that mediates the interaction between Lactobacilli and Enterococci. This plasmid is widespread across ecosystems, including the rumen and human gut microbiota. Biochemical characterization of the plasmid revealed a defence mechanism against reuterin, a toxin produced by various gut microbes, such as Limosilactobacillus reuteri. Using a targeted metabolomic approach, we find reuterin to be prevalent across rumen ecosystems with impacts on microbial community structure. Enterococcus strains carrying the protective plasmid were isolated and their interactions with L. reuteri, the toxin producer, were studied in vitro. Interestingly, we found that by conferring resistance against reuterin, the plasmid mediates metabolic exchange between the defending and the attacking microbial species, resulting in a beneficial relationship or mutualism. Hence, we reveal here an ecological role for a plasmid-coded defence system in mediating a beneficial interaction.

Effect of temperature on growth and yessotoxin production of Protoceratium reticulatum and Lingulodinium polyedra (Dinophyceae) isolates from the Portuguese coast (NE Atlantic)

The dinoflagellates Protoceratium reticulatum and Lingulodinium polyedra are potential yessotoxin (YTX) producers, which have been associated with blooms responsible for economic, social, and ecological impacts around the world. They occur in Iberian waters, but in this region, little is known of their ecophysiology and toxin profiles. This study investigated the growth and toxin production of two strains of each species, from the Portuguese coast, at 15 °C, 19 °C, and 23 °C. Growth curves showed higher growth rates at 19 °C, for both species. YTX and three analogs (homo YTX; 45-OH YTX; 45-OH homo YTX) were investigated by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), and the presence of other analogs was investigated by Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS). No evidence of toxin production was found in L. polyedra. By contrast, YTX and 45,55-diOH-YTX were detected in both strains of P. reticulatum. These results confirm P. reticulatum as a source of yessotoxins along the Portuguese coast and add to the observed high intraspecific variability on YTX production of both species, at a global scale.

Cell structure damage contributes to antifungal activity of sodium propylparaben against Trichothecium roseum

Trichothecium roseum is a type of fungus that causes pink rot in muskmelon after the melons are harvested. Pink rot leads to severe decay during storage and causes the production of toxins that can be harmful to human health. Sodium propylparaben (SPP, IUPAC name: sodium; 4-propoxycarbonylphenolate) is an antimicrobial preservative that can be used to treat the inedible parts of fruits in addition to food, medications, and packaging. In this study, the effectiveness of SPP in inhibiting T. roseum was tested, and the inhibition mechanism was investigated. The results show that SPP inhibited the growth and spore germination of T. roseum. The malondialdehyde (MDA) content, propidium iodide staining, alkaline phosphatase (AKP) activity, and calcofluor white (CFW) staining results show that SPP produced a disruption of the cell membrane and cell wall integrity of T. roseum. Scanning and transmission electron microscopy (SEM and TEM, respectively) results also indicate that SPP disrupted the cellular structure of T. roseum. Meanwhile, the large amounts of superoxide anion and hydrogen peroxide in T. roseum accumulated due to the effects of SPP on the activities of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase, and decreased catalase. In addition, SPP caused a significant reduction in the incidence rate and disease degree of muskmelon pink rot in vivo. In conclusion, SPP appears to be effective against T. roseum via disruption of the cell membrane and wall. SPP could be used to manage melon pink rot after fruit harvesting because of its disease inhibition effect in vivo.

Phage transcriptional regulator X (PtrX)-mediated augmentation of toxin production and virulence in Clostridioides difficile strain R20291

Clostridioides difficile is a Gram-positive, anaerobic, and spore-forming bacterial member of the human gut microbiome. The primary virulence factors of C. difficile are toxin A and toxin B. These toxins damage the cell cytoskeleton and cause various diseases, from diarrhea to severe pseudomembranous colitis. Evidence suggests that bacteriophages can regulate the expression of the pathogenicity locus (PaLoc) genes of C. difficile. We previously demonstrated that the genome of the C. difficile RT027 strain NCKUH-21 contains a prophage-like DNA sequence, which was found to be markedly similar to that of the φCD38–2 phage. In the present study, we investigated the mechanisms underlying the φNCKUH-21-mediated regulation of the pathogenicity and the PaLoc genes expression in the lysogenized C. difficile strain R20291. The carriage of φNCKUH-21 in R20291 cells substantially enhanced toxin production, bacterial motility, biofilm formation, and spore germination in vitro. Subsequent mouse studies revealed that the lysogenized R20291 strain caused a more severe infection than the wild-type strain. We screened three φNCKUH-21 genes encoding DNA-binding proteins to check their effects on PaLoc genes expression. The overexpression of NCKUH-21_03890, annotated as a transcriptional regulator (phage transcriptional regulator X, PtrX), considerably enhanced toxin production, biofilm formation, and bacterial motility of R20291. Transcriptome analysis further confirmed that the overexpression of ptrX led to the upregulation of the expression of toxin genes, flagellar genes, and csrA. In the ptrX-overexpressing R20291 strain, PtrX influenced the expression of flagellar genes and the sigma factor gene sigD, possibly through an increased flagellar phase ON configuration ratio.

Selection of Starter from Decaying Oranges and Pineapple for Bioethanol Production

Screening of yeasts for bioethanol production is an important aspect of industrial microbiology and biotechnology. Optimal yeast nutrition also requires availability of nutrients allowing for growth and ethanol production and ability to withstand the increasing concentration of the medium during fermentation. This study seeks to use appropriate methods to isolate, characterize and identify yeasts isolates with essential attributes for bioethanol production. Microbiological and Physicochemical assessment of the isolated strains was carried out on yeast maintenance media for the determination of ethanol, temperature and salt tolerance ability, growth at different pH and temperature, chloramphenicol and nalidixic acid test, determination of killer toxins production capacity of yeast, nitrate and carbon assimilation and sugar fermentation tests were carried out to select the best starter for production.
 Fifteen yeasts were isolated belonging to the genera Saccharomyces, Candida, Rhodotorula, Kluvyreomyces, Trichosporon, Pichia. S. cerevisae and K. marxianus showed efficient physico-chemical attributes. Proximate analysis of fruits juice showed a moisture content between 81.83 – 86.37%, crude protein 1.33 – 2.00%, Ash 0.73 – 0.83%, carbohydrate 11.87 – 15.67% and Ether 0.1 – 0.5%. The total sugar ranged from 1.83 – 13.17, equal mixture of oranges and pineapple juice showed a sugar content of 15 which reduced during fermentation while the alcohol content increased. Organisms were able to tolerate a percentage of 4-30% ethanol content, tolerating 15% sodium chloride which is an index of osmotolerance. At higher concentration, growth reduced, organisms produced catalase, reduced nitrate and showed a variation in utilization of different sugars.
 At the end of the screening, S. cerevisiae and K. marxianus showed the best attribute essential for bioethanol production and were chosen as starters. The ability of the selected yeast isolates to produce Killer toxins against E. coli showed a negative result. Antibiotic sensitivity test was carried out on the selected yeast isolates using chloramphenicol and nalidixic and the two organisms were resistant to the antibiotics tested.
 The ability of the organisms to grow in changing environmental conditions and ethanol tolerance are attributes essential for production while Saccharomyces cerevisiae showed the highest attribute followed by Kluveromyces marxinus. This study concluded that Saccharomyces cerevisiae can be employed as starter in the industry for the production of bioethanol and in the conversion of agricultural waste to wealth.

Effect of light intensities on the stress responses and toxin production of selected Microcystin producing Cyanobacteria

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Effects of polystyrene nanoplastics on growth and hemolysin production of microalgae Karlodinium veneficum

There are few studies on the effects of nanoplastics on growth and hemolysin production of harmful algal bloom species at present. In this study, Karlodinium veneficum was exposed to different concentrations (0, 5, 25, 50, 75 mg/L) of polystyrene nanoplastics (PS-NPs, 100 nm) for 96 h. The effects of PS-NPs on growth of K. veneficum were investigated by measuring algal cell abundance, growth inhibition rate (IR), total protein (TP), malondialdehyde (MDA), glutathione reductase (GSH), superoxide dismutase (SOD), ATPase activity (Na+/K+ ATPase and Ca2+/Mg2+ ATPase). Scanning electron microscope and transmission electron microscope (SEM and TEM) images of microalgae with or without nanoplastics were also observed. The effects of PS-NPs on hemolysin production of K. veneficum were studied by measuring the changes of hemolytic toxin production of K. veneficum exposed to PS-NPs on 1, 3, 5 and 7 days. High concentrations (50 and 75 mg/L) of PS-NPs seriously affected the growth of K. veneficum and different degrees of damage to cell morphology and ultrastructure were found. Excessive free radicals and other oxidants were produced in the cells, which disrupted the intracellular redox balance state and caused oxidative damage to the cells, and the basic activities such as photosynthesis and energy metabolism were weakened. The athletic ability of K. veneficum was decreased, but the ability to produce hemolysin was enhanced. It was suggested that the presence of nanoplastics in seawater may strengthen the threat of harmful algal bloom species to aquatic ecosystems and human health.