Non-toxigenic Strains Research Articles

Detection and elimination of a novel non-toxigenic Clostridioides difficile strain from the microbiota of a mouse colony

ABSTRACT Clostridioides difficile is an enteric bacterial pathogen that can a cause nosocomial infection leading to debilitating colitis. The development of a murine model of C. difficile infection has led to fundamental discoveries in disease pathogenesis and the host immune response to infection. Recently, C. difficile endogenously present in the microbiota of mice has been reported and was found to complicate interpretation of mouse studies. Here, we report a novel C. difficile strain, named NTCD-035, isolated from the microbiota of our mouse colony. The presence of NTCD-035 in mice prior to challenge with a highly pathogenic C. difficile strain (VPI10463) led to significantly reduced disease severity. Phylogenetic characterization derived from whole genome sequencing and PCR ribotyping identified the isolate as a novel clade 1, ribotype 035 strain that lacks the pathogenicity locus required to produce toxins. Deficiency in toxin production along with sporulation capacity and secondary bile acid sensitivity was confirmed using in vitro assays. Inoculation of germ-free mice with NTCD-035 did not cause morbidity despite the strain readily colonizing the large intestine. Implementation of a culture-based screening procedure enabled the identification of mice harboring C. difficile in their microbiota, the establishment of a C. difficile-free mouse colony, and a monitoring system to prevent future contamination. Taken together, these data provide a framework for screening mice for endogenously harbored C. difficile and support clinical findings that demonstrate the therapeutic potential of non-toxigenic strains in preventing C. difficile associated disease. Abbreviations: PaLoc - Pathogenicity locus, CFUs - Colony forming units, TcdA - toxin-A, TcdB - toxin-B, CdtA - binary toxin A, CdtB - binary toxin B, CdtR - binary toxin R, NTCD - non-toxigenic C. difficile

Pheno- and Genotypical Features of Non-Toxigenic Strains of Cholera Vibrios of Different Origins, Isolated in the Territory of Russia

Aim. Analysis of the phenotypic characteristics and identification of peculiarities of the genotypic organization in non-toxigenic strains of cholera vibrios having different origin, isolated in Russia. Materials and methods. A sample of 548 non-toxigenic strains obtained using the author’s updated GIS “Cholera 1989–2014” was used. PCR genotyping was carried out in accordance with the patented “Method for the identification of non-toxigenic strains of cholera vibrio O1 serogroup using PCR to isolate genetic determinants.” Cluster analysis was performed applying the UPGMA method. The dendrogram was constructed using MEGA 5 software package.Results and discussion. Representative cultural-morphological, serological and biochemical properties of V. cholerae strains have been specified. The variability of the studied strains on the basis of phagolizability has been revealed. Unique phage-types not previously encountered in Russia have been identified. The population of non-toxigenic strains of cholera vibrio O139 serogroup is genetically homogeneous in contrast to V. cholerae O1 El Tor isolates and has identical PCR genotypes. The universality of the PCR genotyping by 14 target genes has been shown to differentiate the studied strains of V. cholerae O1 and O139, as well as to identify disparities among O139 strains isolated in different geographical regions of the country.

Rapid detection assay of toxigenic Clostridioides difficile through PathOC RightGene, a novel high-speed polymerase chain reaction device

Nucleic acid amplification tests for diagnosing Clostridioides difficile infections (CDI) are improving to become faster and more accurate. This study aimed to evaluate the accuracy of rapid detection of toxigenic C. difficile using the novel high-speed polymerase chain reaction (PCR) device, PathOC RightGene. These results were compared and evaluated with real-time PCR (qPCR) and enzyme immunoassays (EIA) kit. For this study, 102 C. difficile and 3 Clostridium species isolated from CDI patients were used. These C. difficile isolates were 85 toxigenic and 17 non-toxigenic strains. The results of qPCR served as a standard, and sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the PathOC Right Gene were 99.2%, 99.4%, 100%, 98.8%, and 99.3%, respectively. Turnaround time of qPCR and EIA was 85 and 30 minutes, whereas PathOC RightGene was only 25 minutes including DNA extraction. This novel high-speed PCR device detected toxigenic C. difficile rapidly and accurately.

Clostridioides difficile Strains in the Gut by Next-Generation Shotgun Sequencing: Innocent Bystander or Villain?

Background: Pathogenic Clostridioides difficile is the most common cause of nosocomial infections in the United States. However, the prevalence of C. difficile colonization in the general population is poorly understood. Objective: In this study, we sought to determine the presence and nature of various strains of Clostridioides difficile colonizing a representative sample of 121 asymptomatic adult volunteers from around the globe, consisting of 110 healthy and 11 stable Crohn’s patients. Methods: Next-generation sequencing was performed on fecal samples from 121 study participants. Stool samples were collected by patients utilizing a Zymo collection kit, which preserves bacterial DNA and RNA. Following collection, DNA was extracted, quantitated, and then normalized for downstream library fabrication utilizing shotgun methodology. Prepared and indexed libraries were subsequently pooled and sequenced on the Illumina NextSeq 550 System. Results: All 121 of 121 subjects (100%) were found to possess the bacterium Clostridioides difficile as identified by the NGS bioinformatics metagenomic pipeline. To visualize comparative abundances of Clostridioides difficile present in study participants, normalized read counts were highlighted (Fig. 1). Conclusions: NGS provides a unique opportunity to increase the resolution and identification of Clostridioides difficile compared to traditional categorizations, such as PCR ribotypes (ie, RT027), restriction endonuclease groups (BI), and North American pulsotypes (ie, NAP1). This is accomplished by its ability to differentiate species based on a nucleotides, while targeting entire bacterial genomes. Our approach for this study was to utilize a bioinformatics pipeline that would provide Clostridioides difficile strain-specific resolution when aligning to genomes in the NCBI (National Center for Biotechnology Information) database. In our representative sample of 121 volunteers, all (100%) possessed at least 1 Clostridioides difficile strain in their gut. Although it is recognized that some Clostridioides difficile strains are pathogenic, our findings suggest that nonpathogenic Clostridioides difficile strains make up an important component of the commensal gut microbiome and may perhaps play a protective role. Although symptomatic toxigenic CDI is a clear indication for therapy, Clostridioides difficile colonization with nontoxigenic strains is not believed to be a direct precursor for CDI. These findings demonstrate the need to be aware of the existence of numerous strains of Clostridioides difficile, and the relevance of sequencing prior to hospitalization or antibiotic treatment to help predict those at risk of CDI, and after treatment to be aware of any loss of what appear to be protective components of our microbiome.Funding: NoneDisclosures: Dr. Sabine Hazan reports that she is the founder and CEO of Ventura Clinical Trials and that she and her spouse receive salaries from the company. She also receives a salary from ProgenaBiome.

Analysis of the Zonula occludens Toxin Found in the Genome of the Chilean Non-toxigenic Vibrio parahaemolyticus Strain PMC53.7.

Vibrio parahaemolyticus non-toxigenic strains are responsible for about 10% of acute gastroenteritis associated with this species, suggesting they harbor unique virulence factors. Zonula occludens toxin (Zot), firstly described in Vibrio cholerae, is a secreted toxin that increases intestinal permeability. Recently, we identified Zot-encoding genes in the genomes of highly cytotoxic Chilean V. parahaemolyticus strains, including the non-toxigenic clinical strain PMC53.7. To gain insights into a possible role of Zot in V. parahaemolyticus, we analyzed whether it could be responsible for cytotoxicity. However, we observed a barely positive correlation between Caco-2 cell membrane damage and Zot mRNA expression during PMC53.7 infection and non-cytotoxicity induction in response to purified PMC53.7-Zot. Unusually, we observed a particular actin disturbance on cells infected with PMC53.7. Based on this observation, we decided to compare the sequence of PMC53.7-Zot with Zot of human pathogenic species such as V. cholerae, Campylobacter concisus, Neisseria meningitidis, and other V. parahaemolyticus strains, using computational tools. The PMC53.7-Zot was compared with other toxins and identified as an endotoxin with conserved motifs in the N-terminus and a variable C-terminal region and without FCIGRL peptide. Notably, the C-terminal diversity among Zots meant that not all of them could be identified as toxins. Structurally, PMC53.7-Zot was modeled as a transmembrane protein. Our results suggested that it has partial 3D structure similarity with V. cholerae-Zot. Probably, the PMC53.7-Zot would affect the actin cytoskeletal, but, in the absence of FCIGRL, the mechanisms of actions must be elucidated.

Genomic Variability of Pathogenicity Islands in Nontoxigenic Strains of Vibrio cholerae O1 Biotype El Tor

The results of comparative sequence analysis of pathogenicity islands (PIs), VPI-1 and VPI-2, in 43 nontoxigenic Vibrio cholerae strains are presented. It was demonstrated that these strains, differing from each other in the PI genome composition and structure, formed three genetically distinct groups (ctxA−tcpA+VPI-2+, ctxA−tcpA+VPI-2del+, and ctxA−tcpA−VPI-2del+) that differed in adaptive potential. It was found that the level of PI genomic variability caused by point mutations and deletions of different lengths was different. Nucleotide sequences of the VPI-1 structural and regulatory pathogenicity genes present in the chromosome of nontoxigenic ctxA−tcpA+ vibrios were mostly identical to that of the reference toxigenic strain. Only a small number of polymorphic sites (5) with single nucleotide substitutions in the acfB, асfС, tcpА, tcpF, and tcpH genes were identified. At the same time, high VPI-2 genomic instability, manifested in the formation of deletions of different lengths (31 131–49 986 kb) and the occurrence of multiple single nucleotide substitutions in all conserved genes, was observed. It was first demonstrated that the loss of VPI-2 DNA correlated with the appearance of multiple substitutions in its genes of utilization of amino sugars and sialic acids, located in the nan-nag region (57–274 substitutions), including the nanH gene. Twelve previously undescribed nanH allelic variants were identified. It seems likely that high genetic diversity of these genes that are important for the vibrio survival in different ecological niches is one of the reasons for high adaptive potential of V. cholerae O1 biotype El Tor сtxA−tcpA−VPI-2del+, ubiquitous in an aquatic environment. On the basis of sequence analysis of the nanH gene included in VPI-2, as well as six housekeeping genes, cluster analysis that determined the phylogenetic relationships of different groups of nontoxigenic strains with each other and with toxigenic strains was performed. The genetic closeness of nontoxigenic ctxA−tcpA+VPI-2+ and ctxA−tcpA+VPI-2del+ vibrios to the causative agent of cholera was discovered. The novel data obtained in the present study on the PI structure in nontoxigenic vibrios not only shed light on the molecular mechanisms of genetically diverse strains with different adaptive potentials. Nontoxigenic strains with the determined genome structure that do not require the removal of key virulence genes upon genome editing can be useful in creating a new generation of live cholera vaccines.

Antimicrobial Resistance Patterns in Clostridioides difficile Strains Isolated from Neonates in Germany.

Young children are frequently colonized with Clostridioides (C.) difficile. Depending on their resistance patterns, antibiotic treatment can facilitate gastrointestinal spreading in colonized individuals, potentially leading to transmission to others. C. difficile was isolated from stool samples from infants born in two hospitals in Göttingen and Darmstadt, Germany. All isolates were subjected to phenotypic antimicrobial resistance testing, PCR-based screening for toxin genes and mass spectrometry-based exclusion of ribotypes 027 and 176. Within an initial cohort of 324 neonates with a longitudinal survey of C. difficile, 137 strains were isolated from 48 individuals. Antimicrobial resistance was recorded against metronidazole in one (0.7%), erythromycin in 16 (11.7%) and moxifloxacin in 2 (1.5%) of the strains, whereas no resistance was observed against vancomycin (0.0%) or rifampicin (0.0%). Newly observed resistance against erythromycin in children with detection of previously completely sensitive isolates was reported for C. difficile isolates from 2 out of 48 children. In 20 children (42%), non-toxigenic strains were detected, and from 27 children (56%), toxigenic strains were isolated, while both toxigenic and non-toxigenic strains were recorded for 1 child (2%). Ribotypes 027 or 176 were not observed. In conclusion, the German C. difficile strains isolated from the children showed mild to moderate resistance with predominance of macrolide resistance, a substance class which is frequently applied in children. The observed switches to the dominance of macrolide-resistant isolates suggests likely selection of resistant C. difficile strains already in children.

Complete mitochondrial genome sequence of Afla-Guard®, commercially available non-toxigenic Aspergillus flavus

Afla-Guard® is a commercial non-toxigenic Aspergillus flavus strain used to decrease aflatoxin contamination level in field. Its mitochondrial genome was sequenced, showing that its length is 29,208 bp with typical configuration of Aspergillus mitochondrial genome. 17 SNPs and 27 INDELs were identified by comparing with previous A. flavus mitochondrial genome. Phylogenetic trees present that A. flavus of Afla-Guard® was clustered with the previous A. flavus mitochondrial genome.

A non-toxigenic Aspergillus flavus strain prevents the spreading of Fusarium verticillioides and fumonisins in maize

We evaluated Fusarium verticillioides and fumonisin contamination in maize samples after application of a non-aflatoxigenic Aspergillus flavus strain in the field. The sampling was performed 150 days after planting. The results showed a reduction in F. verticillioides frequency, as well as in fumonisin levels when samples were obtained from field areas treated with non-aflatoxigenic A. flavus strain. These results suggested a competition for substrate or space between fungi reducing the frequency of F. verticillioides and, consequently, fumonisin production.

Characterization of the genotype and the phenotype of nontoxigenic strains of Corynebacterium diphtheriae subsp. lausannense isolated in Russian residents

In 2018, a few sequencing studies were published revealing the existence of two monophyletic clusters within the C. diphtheriae species, meaning that this species can be divided into two subspecies: C. diphtheriae subsp. diphtheriae and C. diphtheriae subsp. lausannense. The objective of our study was to describe the genotype and the phenotype of 2 nontoxigenic C. diphtheriae strains isolated in Russia in 2017–2018, which were classified by us as C. diphtheriae subsp. lausannense based on the aggregated data yielded by a variety of techniques, including microbiological and molecular genetic techniques, as well as a bioinformatic search for subspecies-specific genes in the publicly available genomes of C. diphtheriae. The isolated strains had morphological and biochemical characteristics of C. diphtheriae. The strains were assigned to the MLST type ST199 included in the clonal complex associated with subsp. lausannense. PCR revealed that both analyzed strains of C. diphtheriae subsp. lausannense carried the ptsI gene encoding phosphoenolpyruvate-protein phosphotransferase and did not carry the narG gene encoding the synthesis of nitrate reductase subunits, whereas the strains of C. diphtheriae subsp. diphtheriae had the narG gene and did not have ptsI. We experimentally proved the ability of lausannense strains to ferment N-acetylglucosamine. Our findings expand the knowledge of the biological diversity of C. diphtheriae and indicate the need for estimating the spread of these microorganisms in Russia, as well as their pathogenic potential.

Characterization of the genotype and the phenotype of nontoxigenic strains of Corynebacterium diphtheriae subsp. lausannense isolated in Russian residents

В 2018 г. на основании полногеномных данных появились публикации о наличии двух монофилетических кластеров внутри вида C. diphtheriae, что позволяет дифференцировать этот вид на два подвида: C. diphtheriae subsp. diphtheriae и C. diphtheriae subsp. lausannense. Целью работы было описать генотип и фенотип двух нетоксигенных штаммов C. diphtheriae, выделенных в 2017–2018 гг. с профилактической целью, которые на основании совокупности результатов множества методов могут быть отнесены к C. diphtheriae subsp. lausannense. В исследовании использовали микробиологические и молекулярно-генетические методы, а также биоинформатический поиск подвид-специфичных генов в публично доступных геномах C. diphtheriae. Выделенные штаммы имели характерные для C. diphtheriae морфо-культуральные свойства и биохимическую характеристику. В МЛСТ штаммы принадлежали к сиквенс-типу ST199, входящему в клональный комплекс, ассоциированный с subsp. lausannense. С использованием ПЦР были показаны наличие ptsI (гена, кодирующего фосфоенолпируват-белок фосфотрансферазы) и отсутствие narG (гена, кодирующего синтез субъединиц нитратредуктазы) у двух исследуемых штаммов C. diphtheriae subsp. lausannense и противоположная картина — у штаммов C. diphtheriae subsp. diphtheriae. Была экспериментально подтверждена способность выделенных штаммов подвида lausannense сбраживать N-aцетилглюкозамин. Полученные результаты расширяют представления о биологическом разнообразии вида C. diphtheriae и свидетельствуют о необходимости дальнейших исследований по оценке распространенности этих микроорганизмов и изучению их патогенного потенциала.

Biological Control of Aflatoxin in Maize Grown in Serbia.

Aspergillus flavus is the main producer of aflatoxin B1, one of the most toxic contaminants of food and feed. With global warming, climate conditions have become favourable for aflatoxin contamination of agricultural products in several European countries, including Serbia. The infection of maize with A. flavus, and aflatoxin synthesis can be controlled and reduced by application of a biocontrol product based on non-toxigenic strains of A. flavus. Biological control relies on competition between atoxigenic and toxigenic strains. This is the most commonly used biological control mechanism of aflatoxin contamination in maize in countries where aflatoxins pose a significant threat. Mytoolbox Af01, a native atoxigenic A. flavus strain, was obtained from maize grown in Serbia and used to produce a biocontrol product that was applied in irrigated and non-irrigated Serbian fields during 2016 and 2017. The application of this biocontrol product reduced aflatoxin levels in maize kernels (51–83%). The biocontrol treatment had a highly significant effect of reducing total aflatoxin contamination by 73%. This study showed that aflatoxin contamination control in Serbian maize can be achieved through biological control methods using atoxigenic A. flavus strains.

Characterization of Circulating Clostridium difficile Strains, Host Response and Intestinal Microbiome in Hospitalized Children With Diarrhea.

Clostridium difficile is capable of causing severe enterocolitis in adults. The significance of toxin-producing C. difficile in children with diarrhea is unclear and practice differs on whether to institute treatment. We aimed to characterize the microbiome in relation to the presence of C. difficile and co-infection with other pathogens and to describe host response to infection. Participants were children with acute diarrhea, 0-16 years of age, from whom stool samples had been submitted to the hospital laboratory for routine microbiology/virology. Convenience sampling was used for 50 prospective and 150 retrospective samples. No participants were treated for C. difficile. Rates of culture positivity for C. difficile, presence of toxin and PCR-ribotype were compared between age groups. Presence of other potential pathogens, comorbidities and complications were recorded. Microbiotal diversity was measured by 16S profiling. Nineteen of 77 (25%) children <2 years of age and 13 of 119 (11%) children >2 years of age were C. difficile positive, of whom 10 (53%) and 9 (69%), respectively, carried toxigenic strains. Increased Shannon diversity was seen in children carrying C. difficile, with altered milieu. Presence of C. difficile was not associated with adverse clinical outcomes. In stools containing both Norovirus and C. difficile, there was increased relative abundance of verrucomicrobia. Children with diarrhea regularly carried toxigenic and non-toxigenic strains of C. difficile, demonstrating enhanced microbiotal diversity, and change in milieu, without apparent morbidity. This unexpected finding is contrary to that seen in adults with C. difficile disease.

A novel pre-CTX prophage in the Vibrio cholerae serogroup O139 strain

In Vibrio cholerae, the lysogenic bacteriophage CTXΦ carries the cholera toxin genes ctxAB, which can be transferred from toxigenic strains to nontoxigenic strains through infection and lysogenic conversion of CTXΦ. This phage also has the precursor genome which does not harbor ctxAB, named pre-CTXΦ. Based on the sequences of the transcriptional regulator-encoding gene rstR alleles in CTXΦ/pre-CTXΦ, multiple types of these prophages have been classified and identified in toxigenic and nontoxigenic V. cholerae strains. In this study, by combining the short-read and long-read sequencing approaches of next generation sequencing, we obtained the complete genome sequence of the studied V. cholerae toxigenic serogroup O139 strain and identified the CTXΦ and a pre-CTXΦ genome type encoding a novel rstR allele, pre-CTXZHJΦ. This pre-CTX prophage integrates into the small chromosome of the V. cholerae host strain and coexists with a typical CTXETΦ prophage present in the large chromosome, which is commonly present in the seventh pandemic serogroup O1 and toxigenic serogroup O139 strains. RstRZHJ could bind to the ig-2 region in the RstAB promotor in the pre-CTXZHJΦ genome, and could repress the expression of its own rstAB genes but could not repress rstAB expression in CTXETΦ and CTXclassΦ, suggesting that the V. cholerae strains carrying the pre-CTXZHJΦ prophage cannot prevent the infection of these epidemic CTXΦs, hence have the potentiality to become toxigenic by acquiring and lysogenic conversion of CTXΦs. Our study identified a novel pre-CTXΦ type, and presents the new evidence for the complexity and diversity of the CTXΦ/pre-CTXΦ family in V. cholerae.

Enhanced resistance of co-existing toxigenic and non-toxigenic Microcystis aeruginosa to pyrogallol compared with monostrains

Cyanobacteria species are sensitive to many plant allelochemicals, such as pyrogallol. However, little attention has been paid to the relative effects of these xenobiotics on co-occurring toxigenic and non-toxigenic cyanobacterial strains, despite their co-existence in blooms. Hence, the responses of one toxigenic (TS2) and two non-toxigenic (NS1, NS2) Microcystis aeruginosa strains to pyrogallol were tested under three conditions: mono-culture and co-cultured either directly or separately by dialysis membrane. The study showed that the inhibitory effects of pyrogallol on the growth and photosynthetic yield (Fv/Fm) of either toxigenic or non-toxigenic M. aeruginosa strains were lower in direct and dialysis co-culture conditions than those in mono-culture conditions. This result indicated that chemical-mediated reciprocal effects occur between the co-existing toxigenic and non-toxigenic strains. The toxigenic M. aeruginosa strain was more sensitive to pyrogallol than the non-toxigenic strains in both mono- and co-culture systems, though whether this outcome is due to the former's toxigenic status is unclear. Intracellular microcystin-LR (MC-LR) concentrations of the toxigenic strain decreased after pyrogallol addition in both mono- and co-culture systems, whereas extracellular MC-LR concentrations increased. This finding may reflect the cell damage of M. aeruginosa because of the pyrogallol. At the same initial number of cells, the extracellular MC-LR concentration released from the same amount of TS2 cells in mono-culture was slightly higher than that in dialysis co-culture conditions. Overall, this study shows that plant allelochemicals may have the potential to reduce bloom toxicity by reducing the proportion of toxigenic cyanobacterial strains, and the effects of co-existing strains must be considered when assessing the effects of plant allelochemicals on target strains.

Septicemia in an immunocompetent adult in the Czech Republic caused by Corynebacterium diphtheriae nontoxigenic strain biotype mitis: emergence of invasive cases in Western Europe

Septicemia in an immunocompetent adult in the Czech Republic caused by Corynebacterium diphtheriae nontoxigenic strain biotype mitis: emergence of invasive cases in Western Europe

Monitoring of Cyanobacteria in Water Using Spectrophotometry and First Derivative of Absorbance

Management of cyanobacteria blooms and their negative impact on human and ecosystem health requires effective tools for monitoring their concentration in water bodies. This research investigated the potential of derivative spectrophotometry in detection and monitoring of cyanobacteria using toxigenic and non-toxigenic strains of Microcystis aeruginosa. Microcystis aeruginosa was quantified in deionized water and surface water using traditional spectrophotometry and the first derivative of absorbance. The first derivative of absorbance was effective in improving the signal of traditional spectrophotometry; however, it was not adequate in differentiating between signal and noise at low concentrations. Savitzky-Golay coefficients for first derivative were used to smooth the derivative spectra and improve the correlation between concentration and noise at low concentrations. Derivative spectrophotometry improved the detection limit as much as eight times in deionized water and as much as four times in surface water. The lowest detection limit measured in surface water with traditional spectrophotometry was 392,982 cells/mL, and the Savitzky-Golay first derivative of absorbance was 90,231 cells/mL. The method provided herein provides a promising tool in real-time monitoring of cyanobacteria concentrations and spectrophotometry offers the ability to measure water quality parameters together with cyanobacteria concentrations.

Naphtho-Gamma-Pyrones Produced by Aspergillus tubingensis G131: New Source of Natural Nontoxic Antioxidants.

Seven naphtho-gamma-pyrones (NγPs), including asperpyrone E, aurasperone A, dianhydroaurasperone C, fonsecin, fonsecinone A, fonsecin B, and ustilaginoidin A, were isolated from Aspergillus tubingensis G131, a non-toxigenic strain. The radical scavenging activity of these NγPs was evaluated using ABTS assay. The Trolox equivalent antioxidant capacity on the seven isolated NγPs ranged from 2.4 to 14.6 μmol L−1. The toxicity and ability of the NγPs to prevent H2O2-mediated cell death were evaluated using normal/not cancerous cells (CHO cells). This cell-based assay showed that NγPs: (1) Are not toxic or weakly toxic towards cells and (2) are able to protect cells from oxidant injuries with an IC50 on H2O2-mediated cell death ranging from 2.25 to 1800 μmol mL−1. Our data show that A. tubingensis G131 strain is able to produce various NγPs possessing strong antioxidant activities and low toxicities, making this strain a good candidate for antioxidant applications in food and cosmetic industries.

Epidemiology and virulence-associated genes of Clostridioides difficile isolates and factors associated with toxin EIA results at a university hospital in Japan.

IntroductionClostridioides difficile is one of the most important nosocomial pathogens; however, reports regarding its clinical and molecular characteristics from Japan are scarce.AimsWe studied the multilocus sequence typing (MLST)-based epidemiology and virulence-associated genes of isolates and the clinical backgrounds of patients from whom the isolates had been recovered.Methods A total of 105 stool samples tested in a C. difficile toxin enzyme immune assay (EIA) were analysed at the University of Tokyo Hospital from March 2013 to July 2014. PCR for MLST and the virulence-associated genes tcdA, tcdB, cdtA, cdtB and tcdC was performed on C. difficile isolates meeting our inclusion criteria following retrospective review of medical records. EIA-positive and EIA-negative groups with toxigenic strains underwent clinical and molecular background comparison.Results The toxigenic strains ST17, ST81, ST2, ST54, ST8, ST3, ST37 and ST53 and the non-toxigenic strains ST109, ST15 and ST100 were frequently recovered. The prevalence rate of tcdA-negative ST81 and ST37, endemic in China and Korea, was higher (11.4%) than that reported in North America and Europe, and hypervirulent ST1(RT027) and ST11(RT078) strains that occur in North America and Europe were not recovered. The linkage between the EIA results and cdt A/B positivity, tcdC deletion, or tcdA variation was absent among toxigenic strains. Compared with the 38 EIA-negative cases, the 36 EIA-positive cases showed that the patients in EIA-positive cases were older and more frequently had chronic kidney disease, as well as a history of beta-lactam use and proton pump inhibitor therapy.Conclusion In Japan, the prevalence rates for tcdA-negative strains are high, whereas the cdtA/B-positive strains are rare. EIA positivity is linked to older age, chronic kidney disease and the use of beta-lactams and proton pump inhibitors.

Resilience of Biocontrol for Aflatoxin Minimization Strategies: Climate Change Abiotic Factors May Affect Control in Non-GM and GM-Maize Cultivars.

There has been significant interest in the development of formulations of non-toxigenic strains of Aspergillus flavus for control of toxigenic strains to reduce the aflatoxin B1 (AFB1) contamination of maize. In the future, climate change (CC) abiotic conditions of temperature (+2–4°C), CO2 (existing levels of 400 vs. 800–1,200 ppb), and drought stress will impact on the agronomy and control of pests and diseases. This study has examined (1) the effect of two-way interacting factors of water activity × temperature on colonization and AFB1 contamination of maize cobs of different ripening ages; (2) the effect of non-toxigenic strains of A. flavus (50:50 inoculum ratio) on relative control of toxigenic A. flavus and AFB1 contamination of ripening cobs; (3) post-harvest control of AFB1 by non-toxigenic strains of A. flavus in non-GM and isogenic GM maize cultivars using the same inoculum ratio; and (4) the impact of three-way interacting CC factors on relative control of AFB1 in maize cobs pre-harvest and in stored non-GM/GM cultivars. Pre-harvest colonization and AFB1 production by a toxigenic A. flavus strain was conserved at 37°C when compared with 30°C, at the three ripening stages of cob development examined: milk ripe (R3), dough (R4), and dent (R5). However, pre-harvest biocontrol with a non-toxigenic strain was only effective at the R3 and R4 stages and not at the R5 stage. This was supported by relative expression of the aflR regulatory biosynthetic gene in the different treatments. When exposed to three-way interacting CC factors for control of AFB1 pre-harvest, the non-toxigenic A. flavus strain was effective at R3 and £4 stages but not at the R5 stage. Post-harvest storage of non-GM and GM cultivars showed that control was achievable at 30°C, with slightly better control in GM-cultivars in terms of the overall inhibition of AFB1 production. However, in stored maize, the non-toxigenic strains of A. flavus had conserved biocontrol of AFB1 contamination, especially in the GM-maize cultivars under three-way interacting CC conditions (37°C × 1,000 ppm CO2 and drought stress). This was supported by the relative expression of the aflR gene in these treatments. This study suggests that the choice of the biocontrol strains, for pre- or post-harvest control, needs to take into account their resilience in CC-related abiotic conditions to ensure that control of AFB1 contamination can be conserved.