Correction: Additive Function of Vibrio vulnificus MARTX Vv and VvhA Cytolysins Promotes Rapid Growth and Epithelial Tissue Necrosis During Intestinal Infection.

[This corrects the article DOI: 10.1371/journal.ppat.1002581.].

Approach Study for Mass Balance of Pesticide Residues in Distillers' Stillage along with Distillate and Absence Verification of Pesticides in Distilled Spirits from Pilot-Scale of Distillation Column.

Herein, contaminants remaining in distillate and distillers' stillage were quantitatively measured after distillation. After rice bran powder was contaminated with 10 ppm of lead (Pb) and cadmium (Cd) or 0.02-1.27 ppm of five pesticides (terbufos, fenthion, iprobenfos, flutolanil, and ethoprophos) followed by fermentation, single-stage distillation was performed. In the obtained distillate, no Pb or Cd was found, as expected. However, when the pesticides were added as contaminants, trace-0.05 ppm of some pesticides were detected in the distillate, possibly due to the high vapor pressure (e.g., that of ethoprophos) and contamination amount (e.g., that of flutolanil, terbufos, and fenthion). In contrast, none of the contaminating pesticides were observed in the distilled spirits when a fermented liquefaction contaminated with 0.04-4 ppm of six pesticides (fenthion, terbufos, ethoprophos, iprobenfos, oxadiazon, and flutolanil) was distilled using a pilot-plant scale distillation column, indicating that the pesticides hardly migrate to the distilled spirits.

Vibrio vulnificus biotype 3 multifunctional autoprocessing RTX toxin is an adenylate cyclase toxin essential for virulence in mice.

Vibrio vulnificus is an environmental organism that causes both food-borne and wound infections with high morbidity and mortality in humans. The annual incidence and global distribution of infections associated with this pathogen are increasing with climate change. In the late 1990s, an outbreak of tilapia-associated wound infections in Israel was linked to a previously unrecognized variant of V. vulnificus designated biotype 3. The sudden emergence and clonality of the outbreak suggest that this strain may be a true newly emergent pathogen with novel virulence properties compared to those of other V. vulnificus strains. In a subcutaneous infection model to mimic wound infection, the multifunctional autoprocessing RTX (MARTX) toxin of biotype 3 strains was shown to be an essential virulence factor contributing to highly inflammatory skin wounds with severe damage affecting every tissue layer. We conducted a sequencing-based analysis of the MARTX toxin and found that biotype 3 MARTX toxin has an effector domain structure distinct from that of either biotype 1 or biotype 2. Of the two new domains identified, a domain similar to Pseudomonas aeruginosa ExoY was shown to confer adenylate cyclase activity on the MARTX toxin. This is the first demonstration that the biotype 3 MARTX toxin is essential for virulence and that the ExoY-like MARTX effector domain is a catalytically active adenylate cyclase.

Additive function of Vibrio vulnificus MARTX(Vv) and VvhA cytolysins promotes rapid growth and epithelial tissue necrosis during intestinal infection.

Vibrio vulnificus is a pathogen that causes both severe necrotizing wound infections and life-threatening food-borne infections. Food-borne infection is particularly lethal as the infection can progress rapidly to primary septicemia resulting in death from septic shock and multiorgan failure. In this study, we use both bioluminescence whole animal imaging and V. vulnificus bacterial colonization of orally infected mice to demonstrate that the secreted multifunctional-autoprocessing RTX toxin (MARTX(Vv)) and the cytolysin/hemolysin VvhA of clinical isolate CMCP6 have an important function in the gut to promote early in vivo growth and dissemination of this pathogen from the small intestine to other organs. Using histopathology, we find that both cytotoxins can cause villi disruption, epithelial necrosis, and inflammation in the mouse small intestine. A double mutant deleted of genes for both cytotoxins was essentially avirulent, did not cause intestinal epithelial tissue damage, and was cleared from infected mice by 36 hours by an effective immune response. Therefore, MARTX(Vv) and VvhA seem to play an additive role for pathogenesis of CMCP6 causing intestinal tissue damage and inflammation that then promotes dissemination of the infecting bacteria to the bloodstream and other organs. In the absence of these two secreted factors, we propose that this bacterium is unable to cause intestinal infection in humans.

Vibrio vulnificus rtxA1 gene recombination generates toxin variants with altered potency during intestinal infection.

Vibrio vulnificus is a food-borne bacterial pathogen associated with 1% of all food-related deaths, predominantly because of consumption of contaminated seafood. The ability of V. vulnificus to cause disease is linked to the production of a large cytotoxin called the "multifunctional-autoprocessing RTX" (MARTX(Vv)) toxin, a factor shown here to be an important virulence factor by the intragastric route of infection in mice. In this study, we examined genetic variation of the rtxA1 gene that encodes MARTX(Vv) in 40 V. vulnificus Biotype 1 strains and found four distinct variants of rtxA1 that encode toxins with different arrangements of effector domains. We provide evidence that these variants arose by recombination either with rtxA genes carried on plasmids or with the rtxA gene of Vibrio anguillarum. Contrary to expected results, the most common rtxA1 gene variant in clinical-type V. vulnificus encodes a toxin with reduced potency and is distinct from the toxin produced by strains isolated from market oysters. These results indicate that an important virulence factor of V. vulnificus is undergoing significant genetic rearrangement and may be subject to selection for reduced virulence in the environment. This finding would imply further that in the future on-going genetic variation of the MARTX(Vv) toxins could result in the emergence of novel strains with altered virulence in humans.

Correction: Anti­biofilm effects of sinomenine against Staphylococcus aureus.

[This corrects the article DOI: 10.1007/s10068-022-01174-0.].

Anti-biofilm effects of sinomenine against Staphylococcus aureus.

Staphylococcus aureus is a gram-positive foodborne pathogen capable of forming strong biofilms. This study identified that anti-biofilm natural compound against S. aureus. Sinomenine, a natural compound, showed significantly reduced biofilm formation (31.97-39.86%), but no effect on bacterial growth was observed. The dispersion of preformed biofilms was observed by confocal laser scanning microscopy (CLSM). qRT-PCR revealed that sinomenine treatment significantly up-regulated agrA by 3.8-fold and down-regulated icaA gene by 3.1-fold. These indicate that sinomenine treatment induces biofilm dispersal due to cell-cell adhesion, polysaccharide intercellular adhesin (PIA), and phenol-soluble modulin (PSM) peptides production. Our results suggest that sinomenine can be used as a promising agent for effectively controlling biofilm formation and dispersion, thereby making S. aureus more susceptible to the action of antimicrobial agents.

Characterization of the Bacterial Communities in Cichorium intybus According to Cultivation and Storage Conditions.

Chicory leaves (Cichorium intybus) are widely consumed due to their health benefits. They are mainly consumed raw or without adequate washing, which has led to an increase in food-borne illness. This study investigated the taxonomic composition and diversity of chicory leaves collected at different sampling times and sites. The potential pathogenic genera (Sphingomonas, Pseudomonas, Pantoea, Staphylococcus, Escherichia, and Bacillus) were identified on the chicory leaves. We also evaluated the effects of various storage conditions (enterohemorrhagic E. coli contamination, washing treatment, and temperature) on the chicory leaves' microbiota. These results provide an understanding of the microbiota in chicory and could be used to prevent food-borne illnesses.

Microbiota Analysis and Microbiological Hazard Assessment in Chinese Chive (Allium tuberosum Rottler) Depending on Retail Types.

Chinese chive (Allium tuberosum Rottler) has potential risks associated with pathogenic bacterial contamination as it is usually consumed raw. In this study, we investigated the microbiota of Chinese chives purchased from traditional markets and grocery stores in March (Spring) and June (Summer) 2017. Differences in bacterial diversity were observed, and the microbial composition varied across sampling times and sites. In June, potential pathogenic genera, such as Escherichia, Enterobacter, and Pantoea, accounted for a high proportion of the microbiota in samples purchased from the traditional market. A large number of pathogenic bacteria (Acinetobacter lwoffii, Bacillus cereus, Klebsiella pneumoniae, and Serratia marcescens) were detected in the June samples at a relatively high rate. In addition, the influence of the washing treatment on Chinese chive microbiota was analyzed. After storage at 26°C, the washing treatment accelerated the growth of enterohemorrhagic Escherichia coli (EHEC) because it caused dynamic shifts in Chinese chive indigenous microbiota. These results expand our knowledge of the microbiota in Chinese chives and provide data for the prediction and prevention of food-borne illnesses.

Effect of Fermentation on Cyanide and Ethyl Carbamate Contents in Cassava Flour and Evaluation of Their Mass Balance during Lab-Scale Continuous Distillation.

When cassava is used for the production of distilled spirits through fermentation and distillation, toxic hydrogen cyanide (HCN) is released from linamarin and carcinogenic ethyl carbamate is produced. Herein, cyanide and ethyl carbamate contents were monitored during the fermentation and lab-scale continuous distillation processes. Thereafter, mass balance and the influence of copper chips were evaluated. Results showed that 81.5% of cyanide was removed after fermentation. Use of copper chips completely prevented the migration of cyanide into the distilled spirits, while 88.3% of cyanide migrated from the fermented liquid in the absence of copper chips. Formation of ethyl carbamate was significantly promoted during distillation. Most of the produced ethyl carbamate (73.2%) was transferred into the distilled spirits in the absence of copper chips, only 9.6% of the ethyl carbamate was transferred when copper chips were used. Thus, copper chips effectively prevented the migration of cyanide and ethyl carbamate into the distilled spirts during continuous distillation.

The capability of catabolic utilization of N-acetylneuraminic acid, a sialic acid, is essential for Vibrio vulnificus pathogenesis.

N-acetylneuraminic acid (Neu5Ac, sialic acid) could provide a good substrate for enteropathogenic bacteria in the intestine, when the bacteria invade and colonize in human gut. In order to analyze the role of Neu5Ac catabolism in Vibrio vulnificus pathogenesis, a mutant with disruption of the nanA gene encoding Neu5Ac lyase was constructed by allelic exchanges. The nanA mutant was not able to utilize Neu5Ac as a sole carbon source and revealed an altered colony morphotype with reduced opacity in the presence of Neu5Ac. Compared to the wild type, the nanA mutant exhibited a low level of cytotoxicity toward INT-407 epithelial cells in vitro and reduced virulence in a mouse model. The disruption of nanA also resulted in a substantial decrease in histopathological damage in jejunum and colon tissues from the mouse intestine. These results indicated that NanA plays an important role in V. vulnificus pathogenesis. In addition, the nanA mutant was significantly diminished in growth with and adherence to INT-407 epithelial cells in vitro, and was defective for intestinal colonization, reflecting the impaired ability of the mutant to grow and survive with, persist in, and adhere to the intestine in vivo. Consequently, the combined results suggest that NanA and the capability of catabolic utilization of Neu5Ac contribute to V. vulnificus virulence by ensuring growth, adhesion, and survival during infection.

Leaf-associated microbiota on perilla (Perilla frutescens var. frutescens) cultivated in South Korea to detect the potential risk of food poisoning.

Perilla (Perilla frutescens) is a commonly consumed herb with various health benefits in Asia. However, the risks of food-borne illness owing to the presence of pathogens on perilla leaves have not been evaluated. In this study, we evaluated the microbiota of perilla leaves harvested in South Korea using Illumina MiSeq sequencing of the 16S rRNA gene. In total, 2,743,003 sequencing reads were obtained, and 92-437 operational taxonomic units were observed in all samples. Bacterial loads were quantified, and the diversity indices were compared. Differences in the microbiota among sampling times and regions were also investigated. Proteobacteria and Firmicutes were predominant phyla at both times. At the class level, the bacterial communities were composed primarily of Alphaproteobacteria, Bacilli, and Gammaproteobacteria. Diverse bacterial taxa, such as Bacillus, uncultured family Enterobacteriaceae, and Sphingomonas were detected, and the representative pathogenic species (i.e., Acinetobacter lwoffii, Klebsiella pneumoniae, and Staphylococcus aureus) were quantified by qRT-PCR. The results of the co-occurrence network analysis showed characteristics of bacterial taxa in the microbiome on perilla leaves and provided insights into the roles of correlations among diverse microbes, including potential pathogens. Based on these results, the potential risk of food-borne illness from consumption of perilla leaves may be higher in July than in April. In summary, the microbial compositions determined in this study can be used as a base data for food-safety management for prediction and prevention of future outbreaks.

Evidence that AphB, essential for the virulence of Vibrio vulnificus, is a global regulator.

The Vibrio vulnificus aphB mutant was significantly less virulent than the wild type and was impaired in motility and adherence to host cells. Microarray analysis revealed that AphB of V. vulnificus (AphB(Vv)) influences the expression of over 10% of the V. vulnificus genome. The combined results indicated that AphB(Vv) is a global regulator contributing to the pathogenesis of V. vulnificus.

Proteomic identification and characterization of Vibrio vulnificus proteins induced upon exposure to INT-407 intestinal epithelial cells.

Proteomic analysis led to identification of the proteins of Vibrio vulnificus that were induced upon exposure to INT-407 cells, and 7 of which belong to the functional categories such as amino acid transport/metabolism, nucleotide transport/metabolism, posttranslational modification/protein turnover/chaperones, and translation. Among the genes encoding the host-induced proteins, disruption of purH, trpD, tsaA, and groEL2 resulted in reduced cytotoxicity. The purH, trpD, and tsaA mutants showed impaired growth in the INT-407 lysate; however, the growth rate of the groEL2 mutant was not significantly changed, indicating that the possible roles of the host-induced proteins in the virulence of V. vulnificus are rather versatile.

Analysis of the Microbiota on Lettuce (Lactuca sativa L.) Cultivated in South Korea to Identify Foodborne Pathogens.

Lettuce (Lactuca sativa L.) is a major ingredient used in many food recipes in South Korea. Lettuce samples were collected during their maximum production period between April and July in order to investigate the microbiota of lettuce during different seasons. 16S rRNA gene-based sequencing was conducted using Illumina MiSeq, and real-time PCR was performed for quantification. The number of total bacterial was greater in lettuce collected in July than in that collected in April, albeit with reduced diversity. The bacterial compositions varied according to the site and season of sample collection. Potential pathogenic species such as Bacillus spp., Enterococcus casseliflavus, Klebsiella pneumoniae, and Pseudomonas aeruginosa showed season-specific differences. Results of the network co-occurrence analysis with core genera correlations showed characteristics of bacterial species in lettuce, and provided clues regarding the role of different microbes, including potential pathogens, in this microbiota. Although further studies are needed to determine the specific effects of regional and seasonal characteristics on the lettuce microbiota, our results imply that the 16S rRNA gene-based sequencing approach can be used to detect pathogenic bacteria in lettuce.

Characterization and evaluation of antimicrobial activity of actinonin against foodborne pathogens.

This study revealed the antimicrobial properties of actinonin against major foodborne pathogens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, Staphylococcus aureus, and Vibrio vulnificus. Among them, actinonin caused growth defect in S. Typhimurium and V. vulnificus. Minimal inhibitory concentration (MIC) values of actinonin were determined by broth microdilution methods. The MICs of actinonin were ≤0.768 µg/ml for S. Typhimurium and ≤0.192 µg/ml for V. vulnificus. Susceptibility to actinonin in both pathogens was measured by colony-forming ability and disc diffusion test. The results showed actinonin had antimicrobial activity against S. Typhimurium and V. vulnificus in a dose-dependent manner. The inhibitory effects on swarming motility were determined, and cytotoxicity of each pathogen against HeLa cells was decreased significantly by actinonin treatment. Furthermore, actinonin showed an antimicrobial efficacy in food models infected with these pathogens. These results demonstrate that actinonin is potentially an effective agent for food sanitization or preservation.

Increase in nitrite content and functionality of ethanolic extracts of Perilla frutescens following treatment with atmospheric pressure plasma.

This study investigated the effect of atmospheric pressure plasma (APP) treatment on nitrite content and functionality of plant extracts. Ethanolic extracts of Perilla frutescens (EEP) were prepared and treated with APP for 60min. Nitrite content increased from 0 to 45.8mg/l in EEP after APP treatment for 60min. Antimicrobial activity of EEP against Clostridium perfringens and Salmonella Typhimurium was increased by APP with no influence on antioxidative activity (p<0.05). Lyophilized EEP (LEEP) treated with APP for 60min contained 3.74mg/g nitrite. The control (LEEP without APP) contained no nitrite. The minimum inhibitory concentration (MIC) of LEEP for C. perfringens was 200µg/ml. The control did not inhibit C. perfringens growth between 25 and 1000µg/ml. MICs of LEEP and the control against S. Typhimurium were 25 and 50µg/ml, respectively. New nitrite sources with increased antimicrobial activity can be produced from natural plants by APP treatment.

Genome Sequence of Bacillus cereus FORC_021, a Food-Borne Pathogen Isolated from a Knife at a Sashimi Restaurant.

Bacillus cereus causes food-borne illness through contaminated foods; therefore, its pathogenicity and genome sequences have been analyzed in several studies. We sequenced and analyzed B. cereus strain FORC_021 isolated from a sashimi restaurant. The genome sequence consists of 5,373,294 bp with 35.36% GC contents, 5,350 predicted CDSs, 42 rRNA genes, and 107 tRNA genes. Based on in silico DNA-DNA hybridization values, B. cereus ATCC 14579T was closest to FORC_021 among the complete genome-sequenced strains. Three major enterotoxins were detected in FORC_021. Comparative genomic analysis of FORC_021 with ATCC 14579T revealed that FORC_021 harbored an additional genomic region encoding virulence factors, such as putative ADP-ribosylating toxin, spore germination protein, internalin, and sortase. Furthermore, in vitro cytotoxicity testing showed that FORC_021 exhibited a high level of cytotoxicity toward INT-407 human epithelial cells. This genomic information of FORC_021 will help us to understand its pathogenesis and assist in managing food contamination.

Vibrio vulnificus glycogen branching enzyme preferentially transfers very short chains: N1 domain determines the chain length transferred.

The glycogen branching enzyme from Vibrio vulnificus (VvGBE) transfers short side chains (DP 3-5) significantly greater than any other bacterial glycogen branching enzyme (GBE). To elucidate the role of the N-domain of VvGBE in the unique branching pattern, domain-truncated (N1 and N) and N1-domain-swapped (with VvGBE N1 replacing the counter part of Escherichia coli GBE) mutants were constructed. The truncation mutants synthesized branched products with a greatly reduced proportion of short chains. The swapping mutant exhibited a branching pattern of the short chain region similar to that of VvGBE. We conclude that the N1-domain of VvGBE has a crucial role in the determination of the branching pattern of glycogen.

Regulatory characteristics of the Vibrio vulnificus rtxHCA operon encoding a MARTX toxin.

Vibrio vulnificus MARTX encoded by rtxA, an open reading frame of the rtxHCA operon, is essential for virulence in vitro and in mice. In this study, a primer extension analysis revealed that transcription of the rtxHCA operon begins at a single site, and is under the direction of a single promoter, P( rtxHCA ). P( rtxHCA ) activity appeared at the beginning of growth and reached a maximum in mid-exponential phase. P( rtxHCA ) activity was induced by exposure to INT-407 cells, and the membrane fraction of INT-407 cells was the most effective for the induction.