Characterization of the OCC Gene Cluster Required for the Production of Antifungal Compound Occidiofungion in Burkholderia Contaminans Strain MS14

Strain MS14, exhibiting antifungal activity, was classified to belong to Burkholderia contaminans. Occidiofungin produced by strain MS14 is an octapeptide dedicated to a broad range of antifungal activities of the bacterium. The 58.2-kb genomic fragment containing 18 open reading frames (ORFs), named occidiofungin (occ) gene cluster, is required for occidiofungin production. Putative proteins encoded by five nonribosomal peptide synthetase genes (occA – occE) of the gene cluster were predicted to contain the catalytic modules responsible for the biosynthesis of occidiofungin. Transcription of all the ORFs identified in the region except ORF1 and ORF16 was regulated by both ambR1 and ambR2, the LuxR-type regulatory genes located at the left border of the cluster. The functional ambR1 gene was essential for transcription of ambR2, and constitutive expression of ambR2 did not restore the phenotype of the mutant MS14GG44(ambR1::nptII). Sequence analysis revealed that the occ gene cluster shared high similarity (99% nucleotide coverage and 91% identity) to an uncharacterized DNA region of B. ambifaria strain AMMD. The gene cluster was not found in other Burkholderia strains available in GenBank (nucleotide coverage \u3c 24%). Analysis of G+C composition and prediction using “IslandPick” indicate that the occ gene cluster has possibly been horizontally transferred between bacteria. In addition, the absence of the gene cluster in clinical strains of Burkholderia indicates that occidiofungin is not required for potential human pathogenesis. The findings have provided insights into the development of antifungal medicines and agricultural fungicides based on occidiofungin

Characterization of the OCC Gene Cluster Required for the Production of Antifungal Compound Occidiofungion in Burkholderia Contaminans Strain MS14

Strain MS14, exhibiting antifungal activity, was classified to belong to Burkholderia contaminans. Occidiofungin produced by strain MS14 is an octapeptide dedicated to a broad range of antifungal activities of the bacterium. The 58.2-kb genomic fragment containing 18 open reading frames (ORFs), named occidiofungin (occ) gene cluster, is required for occidiofungin production. Putative proteins encoded by five nonribosomal peptide synthetase genes (occA – occE) of the gene cluster were predicted to contain the catalytic modules responsible for the biosynthesis of occidiofungin. Transcription of all the ORFs identified in the region except ORF1 and ORF16 was regulated by both ambR1 and ambR2, the LuxR-type regulatory genes located at the left border of the cluster. The functional ambR1 gene was essential for transcription of ambR2, and constitutive expression of ambR2 did not restore the phenotype of the mutant MS14GG44(ambR1::nptII). Sequence analysis revealed that the occ gene cluster shared high similarity (99% nucleotide coverage and 91% identity) to an uncharacterized DNA region of B. ambifaria strain AMMD. The gene cluster was not found in other Burkholderia strains available in GenBank (nucleotide coverage \u3c 24%). Analysis of G+C composition and prediction using “IslandPick” indicate that the occ gene cluster has possibly been horizontally transferred between bacteria. In addition, the absence of the gene cluster in clinical strains of Burkholderia indicates that occidiofungin is not required for potential human pathogenesis. The findings have provided insights into the development of antifungal medicines and agricultural fungicides based on occidiofungin

Secure and Fast Asynchronous Vertical Federated Learning via Cascaded Hybrid Optimization

Vertical Federated Learning (VFL) attracts increasing attention because it empowers multiple parties to jointly train a privacy-preserving model over vertically partitioned data. Recent research has shown that applying zeroth-order optimization (ZOO) has many advantages in building a practical VFL algorithm. However, a vital problem with the ZOO-based VFL is its slow convergence rate, which limits its application in handling modern large models. To address this problem, we propose a cascaded hybrid optimization method in VFL. In this method, the downstream models (clients) are trained with ZOO to protect privacy and ensure that no internal information is shared. Meanwhile, the upstream model (server) is updated with first-order optimization (FOO) locally, which significantly improves the convergence rate, making it feasible to train the large models without compromising privacy and security. We theoretically prove that our VFL framework converges faster than the ZOO-based VFL, as the convergence of our framework is not limited by the size of the server model, making it effective for training large models with the major part on the server. Extensive experiments demonstrate that our method achieves faster convergence than the ZOO-based VFL framework, while maintaining an equivalent level of privacy protection. Moreover, we show that the convergence of our VFL is comparable to the unsafe FOO-based VFL baseline. Additionally, we demonstrate that our method makes the training of a large model feasible.Comment: Under Revie

Survival of Salmonella enterica and shifts in the culturable mesophilic aerobic bacterial community as impacted by tomato wash water particulate size and chlorine treatment

Particulates of harvest debris are common in tomato packinghouse dump tanks, but their role in food safety is unclear. In this study we investigated the survival of Salmonella enterica and the shifts in relative abundance of culturable mesophilic aerobic bacteria (cMAB) as impacted by particulate size and interaction with chlorine treatment. Particulates suspended in grape tomato wash water spanned a wide size range, but the largest contribution came from particles of 3–20 μm. Filtration of wash water through 330 μm, applied after 100 mg/L free chlorine (FC) wash, reduced surviving cMAB by 98%. The combination of filtration (at 330 μm or smaller pore sizes) and chlorinated wash also altered the cMAB community, with the survivors shifting toward Gram-positive and spore producers (in both lab-simulated and industrial conditions). When tomatoes and harvest debris inoculated with differentially tagged Salmonella were washed in 100 mg/L FC for 1 min followed by filtration, only cells originating from harvest debris survived, with 85 and 93% of the surviving cells associated with particulates larger than 330 and 63 μm, respectively. This suggests that particulates suspended in wash water can protect Salmonella cells from chlorine action, and serve as a vector for cross-contamination

Internal Colonization of Salmonella enterica Serovar Typhimurium in Tomato Plants

Several Salmonella enterica outbreaks have been traced back to contaminated tomatoes. In this study, the internalization of S. enterica Typhimurium via tomato leaves was investigated as affected by surfactants and bacterial rdar morphotype, which was reported to be important for the environmental persistence and attachment of Salmonella to plants. Surfactants, especially Silwet L-77, promoted ingress and survival of S. enterica Typhimurium in tomato leaves. In each of two experiments, 84 tomato plants were inoculated two to four times before fruiting with GFP-labeled S. enterica Typhimurium strain MAE110 (with rdar morphotype) or MAE119 (without rdar). For each inoculation, single leaflets were dipped in 109 CFU/ml Salmonella suspension with Silwet L-77. Inoculated and adjacent leaflets were tested for Salmonella survival for 3 weeks after each inoculation. The surface and pulp of ripe fruits produced on these plants were also examined for Salmonella. Populations of both Salmonella strains in inoculated leaflets decreased during 2 weeks after inoculation but remained unchanged (at about 104 CFU/g) in week 3. Populations of MAE110 were significantly higher (P<0.05) than those of MAE119 from day 3 after inoculation. In the first year, nine fruits collected from one of the 42 MAE119 inoculated plants were positive for S. enterica Typhimurium. In the second year, Salmonella was detected in adjacent non-inoculated leaves of eight tomato plants (five inoculated with strain MAE110). The pulp of 12 fruits from two plants inoculated with MAE110 was Salmonella positive (about 106 CFU/g). Internalization was confirmed by fluorescence and confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can move inside tomato plants grown in natural field soil and colonize fruits at high levels without inducing any symptoms, except for a slight reduction in plant growth

Agricultural Practices Influence Salmonella Contamination and Survival in Pre-harvest Tomato Production

Between 2000 and 2010 the Eastern Shore of Virginia was implicated in four Salmonella outbreaks associated with tomato. Therefore, a multi-year study (2012–2015) was performed to investigate presumptive factors associated with the contamination of Salmonella within tomato fields at Virginia Tech’s Eastern Shore Agricultural Research and Extension Center. Factors including irrigation water sources (pond and well), type of soil amendment: fresh poultry litter (PL), PL ash, and a conventional fertilizer (triple superphosphate – TSP), and production practices: staked with plastic mulch (SP), staked without plastic mulch (SW), and non-staked without plastic mulch (NW), were evaluated by split-plot or complete-block design. All field experiments relied on naturally occurring Salmonella contamination, except one follow up experiment (worst-case scenario) which examined the potential for contamination in tomato fruits when Salmonella was applied through drip irrigation. Samples were collected from pond and well water; PL, PL ash, and TSP; and the rhizosphere, leaves, and fruits of tomato plants. Salmonella was quantified using a most probable number method and contamination ratios were calculated for each treatment. Salmonella serovar was determined by molecular serotyping. Salmonella populations varied significantly by year; however, similar trends were evident each year. Findings showed use of untreated pond water and raw PL amendment increased the likelihood of Salmonella detection in tomato plots. Salmonella Newport and Typhimurium were the most frequently detected serovars in pond water and PL amendment samples, respectively. Interestingly, while these factors increased the likelihood of Salmonella detection in tomato plots (rhizosphere and leaves), all tomato fruits sampled (n = 4800) from these plots were Salmonella negative. Contamination of tomato fruits was extremely low (&lt; 1%) even when tomato plots were artificially inoculated with an attenuated Salmonella Newport strain (104 CFU/mL). Furthermore, Salmonella was not detected in tomato plots irrigated using well water and amended with PL ash or TSP. Production practices also influenced the likelihood of Salmonella detection in tomato plots. Salmonella detection was higher in tomato leaf samples for NW plots, compared to SP and SW plots. This study provides evidence that attention to agricultural inputs and production practices may help reduce the likelihood of Salmonella contamination in tomato fields

Selection of aptamers targeted to food‐borne pathogenic bacteria Vibrio parahaemolyticus

Abstract Vibrio parahaemolyticus (Vp) is a common marine halophilic food‐borne pathogen, mainly found in seafood and food with a high salt content. Gastrointestinal reactions such as diarrhea, headache, vomiting, nausea, and abdominal cramps may occur after eating food infected with Vp. This study aimed to screen for high‐affinity aptamers that specifically recognize Vp. A high‐affinity modified aptamer screening kit was used to rapidly screen aptamers of the food‐borne Vp. The first round of screening involved release of target aptamers from the microspheres. The "false‐positive" aptamers were eliminated after specific binding to and elution of Vp in the second round. The second round of screening of the aptamers involved polymerase chain reaction (PCR), and the abundance of a sequence was determined using next‐generation sequencing. Nine high‐affinity aptamer sequences were obtained, and the first eight modified aptamer sequences were derived using a cloud‐based intelligent software of the American AM Biotech Co. Escherichia coli (E. coli) was used as a control, and aptamer ID 12 with the highest affinity for Vp was selected using real‐time PCR. According to the principle of color change caused by nano‐gold condensing under salt induction, Salmonella, Listeria monocytogenes (L. monocytogenes), and E. coli were used as counter‐screening bacteria, and the aptamer ID12 was combined with nano‐gold. The results showed that aptamer ID12 has strong specificity for Vp. Based on these findings, this study developed a simple, innovative, and rapid method for screening Vp aptamers

Cultivar was more influential than bacterial strain and other experimental factors in recovery of Escherichia coli O157:H7 populations from inoculated live Romaine lettuce plants

ABSTRACTThe varied choice of bacterial strain, plant cultivar, and method used to inoculate, retrieve, and enumerate Escherichia coli O157:H7 from live plants could affect comparability among studies evaluating lettuce–enterobacterial interactions. Cultivar, bacterial strain, incubation time, leaf side inoculated, and sample processing method were assessed for their influence in recovering and quantifying E. coli O157:H7 from live Romaine lettuce. Cultivar exerted the strongest effect on E. coli O157:H7 counts, which held up even when cultivar was considered in interactions with other factors. Recovery from the popularly grown green Romaine “Rio Bravo” was higher than from the red variety “Outredgeous.” Other modulating variables were incubation time, strain, and leaf side inoculated. Sample processing method was not significant. Incubation for 24 hours post-lettuce inoculation yielded greater counts than 48 hours, but was affected by lettuce cultivar, bacterial strain, and leaf side inoculated. Higher counts obtained for strain EDL933 compared to a lettuce outbreak strain 2705C emphasized the importance of selecting relevant strains for the system being studied. Inoculating the abaxial side of leaves gave higher counts than adaxial surface inoculation, although this factor interacted with strain and incubation period. Our findings highlight the importance of studying interactions between appropriate bacterial strains and plant cultivars for more relevant research results, and of standardizing inoculation and incubation procedures. The strong effect of cultivar exerted on the E. coli O157:H7-lettuce association supports the need to start reporting cultivar information for illness outbreaks to facilitate the identification and study of plant traits that impact food safety risk.IMPORTANCEThe contamination of Romaine lettuce with Escherichia coli O157:H7 has been linked to multiple foodborne disease outbreaks, but variability in the methods used to evaluate E. coli O157:H7 association with live lettuce plants complicates the comparability of different studies. In this study, various experimental variables and sample processing methods for recovering and quantifying E. coli O157:H7 from live Romaine lettuce were assessed. Cultivar was found to exert the strongest influence on E. coli O157:H7 retrieval from lettuce. Other modulating factors were bacterial incubation time on plants, strain, and leaf side inoculated, while sample processing method had no impact. Our findings highlight the importance of selecting relevant cultivars and strains, and of standardizing inoculation and incubation procedures, in these types of assessments. Moreover, results support the need to start reporting cultivars implicated in foodborne illness outbreaks to facilitate the identification and study of plant traits that impact food safety risk

Engineering the production of a conformational variant of occidiofungin that has enhanced inhibitory activity against fungal species

Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar activity. This invention is directed to compositions enriched for particular occidiofungin diastereomers/conformers, methods of making compositions enriched for particular diastereomers/conformers and microorganisms suitable for producing enriched compositions of particular diastereomers/conformers. Methods of treating fungal infections or plants infected by fungi are also provided.U