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The effect of plasma-activated water (PAW) generated with a dielectric barrier discharge diffusor (DBDD) system on microbial load and organoleptic quality of cucamelons was investigated and compared to the established sanitizer, sodium hypochlorite (NaOCl). Pathogenic serotypes of Escherichia coli, Salmonella enterica, and Listeria monocytogenes were inoculated onto the surface of cucamelons (6.5 log CFU g-1) and into the wash water (6 log CFU mL-1). PAW treatment involved 2 min in situ with water activated at 1,500 Hz and 120 V and air as the feed gas; NaOCl treatment was a wash with 100 ppm total chlorine; control treatment was a wash with tap water. PAW treatment produced a 3-log CFU g-1 reduction of pathogens on the cucamelon surface without negatively impacting quality or shelf life. NaOCl treatment reduced the pathogenic bacteria on the cucamelon surface by 3 to 4 log CFU g-1; however, this treatment also reduced fruit shelf life and quality. Both systems reduced 6-log CFU mL-1 pathogens in the wash water to below detectable limits. The critical role of superoxide anion radical (·O2-) in the antimicrobial power of DBDD-PAW was demonstrated through a Tiron scavenger assay, and chemistry modeling confirmed that ·O2- generation readily occurs in DBDD-PAW generated with the employed settings. Modeling of the physical forces produced during plasma treatment showed that bacteria likely experience strong local electric fields and polarization. We hypothesize that these physical effects synergize with reactive chemical species to produce the acute antimicrobial activity seen with the in situ PAW system. IMPORTANCE Plasma-activated water (PAW) is an emerging sanitizer in the fresh food industry, where food safety must be achieved without a thermal kill step. Here, we demonstrate PAW generated in situ to be a competitive sanitizer technology, providing a significant reduction of pathogenic and spoilage microorganisms while maintaining the quality and shelf life of the produce item. Our experimental results are supported by modeling of the plasma chemistry and applied physical forces, which show that the system can generate highly reactive ·O2- and strong electric fields that combine to produce potent antimicrobial power. In situ PAW has promise in industrial applications as it requires only low power (12 W), tap water, and air. Moreover, it does not produce toxic by-products or hazardous effluent waste, making it a sustainable solution for fresh food safety.

The ability of Candida albicans to switch its morphology from yeast to filaments, known as polymorphism, may bias the methods used in microbial quantification. Here, we compared the quantification methods [cell/mL, colony forming units (CFU)/mL, and the number of nuclei estimated by viability polymerase chain reaction (vPCR)] of three strains of C. albicans (one reference strain and two clinical isolates) grown as yeast, filaments, and biofilms. Metabolic activity (XTT assay) was also used for biofilms. Comparisons between the methods were evaluated by agreement analyses [Intraclass and Concordance Correlation Coefficients (ICC and CCC, respectively) and Bland-Altman Plot] and Pearson Correlation (α = 0.05). Principal Component Analysis (PCA) was employed to visualize the similarities and differences between the methods. Results demonstrated a lack of agreement between all methods irrespective of fungal morphology/growth, even when a strong correlation was observed. Bland-Altman plot also demonstrated proportional bias between all methods for all morphologies/growth, except between CFU/mL X vPCR for yeasts and biofilms. For all morphologies, the correlation between the methods were strong, but without linear relationship between them, except for yeast where vPCR showed weak correlation with cells/mL and CFU/mL. XTT moderately correlated with CFU/mL and vPCR and weakly correlated with cells/mL. For all morphologies/growth, PCA showed that CFU/mL was similar to cells/mL and vPCR was distinct from them, but for biofilms vPCR became more similar to CFU/mL and cells/mL while XTT was the most distinct method. As conclusions, our investigation demonstrated that CFU/mL underestimated cells/mL, while vPCR overestimated both cells/mL and CFU/mL, and that the methods had poor agreement and lack of linear relationship, irrespective of C. albicans morphology/growth.1

Background: One of the most common preventive methods of Surgical Site Infection (SSI) is preoperative skin preparation. Selecting the skin antiseptic before surgery is an important step that can reduce SSI risk. Aim: The present study was performed with aim to compare the effect of 7.5% povidone-iodine (PVP-I) and 70% alcohol versus 0.2% chlorhexidine (CHG) and 70% alcohol on the microbial count of the surgical site in the abdominal surgery. Method: This double-blind randomized clinical trial study was conducted between March 2017 and July 2018 at the educational-therapeutic centers of Iran University of Medical Sciences. The patients aged ≥18 years who underwent elective abdominal surgery were randomly assigned into two groups to have their skin cleaned before surgery with CHG-alcohol or PVP-I-alcohol. Also, before skin prep, after the primary prep and after the secondary prep, microbial cultures were taken. Data analysis was performed using SPSS (version 16) and Chi-square, Fisher's exact, Kolmogorov-Smirnov, Wilcoxon and U-Mann-Whitney tests. P0.05). Implications for Practice: This study did not demonstrate an overall superiority of 2% CHG over 7.5% PVP-I skin preparation solution or vice versa. Both groups can be used to prepare patients' skin before abdominal surgery due to the affordability conditions and availability.

Background: Water quality and safety are fundamental to human development and well-being. Therefore, the purpose of this study was to determine the bacteriological and mineral content of water in Ho, the capital city of the Volta Region of Ghana. Methods: Sachet, bottled, and tap water were sampled from January to February in 2019 due to the high rate of consumption and their presumed quality which were taken at random from five different locations throughout the municipality. Water quality assessment protocols were utilized to ascertain the bacteriological as well as mineral contents of the samples, whilst ANOVA was used to determine statistical difference and significance at p

In assessing food microbial safety, the presence of Escherichia coli is a critical indicator of fecal contamination. However, conventional detection methods require the isolation of bacterial macrocolonies for biochemical or genetic characterization, which takes a few days and is labor-intensive. In this study, we show that the real-time object detection and classification algorithm You Only Look Once version 4 (YOLOv4) can accurately identify the presence of E. coli at the microcolony stage after a 3-h cultivation. Integrating with phase-contrast microscopic imaging, YOLOv4 discriminated E. coli from seven other common foodborne bacterial species with an average precision of 94%. This approach also enabled the rapid quantification of E. coli concentrations over 3 orders of magnitude with an R2 of 0.995. For romaine lettuce spiked with E. coli (10 to 103 CFU/g), the trained YOLOv4 detector had a false-negative rate of less than 10%. This approach accelerates analysis and avoids manual result determination, which has the potential to be applied as a rapid and user-friendly bacterial sensing approach in food industries. IMPORTANCE A simple, cost-effective, and rapid method is desired to identify potential pathogen contamination in food products and thus prevent foodborne illnesses and outbreaks. This study combined artificial intelligence (AI) and optical imaging to detect bacteria at the microcolony stage within 3 h of inoculation. This approach eliminates the need for time-consuming culture-based colony isolation and resource-intensive molecular approaches for bacterial identification. The approach developed in this study is broadly applicable for the identification of diverse bacterial species. In addition, this approach can be implemented in resource-limited areas, as it does not require expensive instruments and significantly trained human resources. This AI-assisted detection not only achieves high accuracy in bacterial classification but also provides the potential for automated bacterial detection, reducing labor workloads in food industries, environmental monitoring, and clinical settings.

Background: Water quality and safety are fundamental to human development and wellbeing. Therefore, the purpose of this study was to determine the bacteriological and mineral content of water in Ho, the capital city of the Volta Region of Ghana. Methods: Sachet, bottled, and tap water were sampled from January to February in 2019 due to the high rate of consumption and their presumed quality which were taken at random from five different locations throughout the municipality. Water quality assessment protocols were utilized to ascertain the bacteriological as well as mineral contents of the samples, whilst ANOVA was used to determine statistical difference and significance at p<0.05. Results: The maximum Heterotrophic Plate Count for tap water was 9.95±0.64x105 Colony Forming Unit (CFU)/ml, for sachet water was 7.46±0.09x106 CFU/ml, and for bottled water was 1.10±0.56x105 CFU/ml, all obtained on nutrient agar. For MacConkey agar, maximum growth was 2.94±0.03x106, 9.42±1.67x106, and 2.31±0.77x105 CFU/ml for tap, sachet, and bottled water, respectively. The Xylose Lysine Deoxycholate Agar indicated maximum growth of 1.84±0.34x10³, 5.72±0.06x106, and 5.50±2.12x104 CFU/ml for tap, sachet, and bottled water, respectively. The physical parameters such as pH, turbidity, color, and conductivity were within the recommended limits set by the Ghana Standards Authority. However, tap water recorded the highest turbidity, bottled water recorded the highest and least pH and turbidity, respectively. Moreover, the mineral analysis revealed high levels of phosphate (PO43-), chloride (C-), and sodium (Na) in bottled water, and total iron (Fe) was relatively high in several tap and sachet water samples, the latter item also recorded the highest for ammonia (NH3). Conclusions: Overall, the tap, sachet, and bottled water samples exhibited varied levels of microbial, and mineral contents whilst the physical parameters were relatively within the recommended levels. The sachet and tap water were the least wholesome in comparison with the bottled water samples. [ABSTRACT FROM AUTHOR]

Copyright of China Fiber Inspection is the property of China Fiber Inspection and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Food Science & Biotechnology is the property of Journal of Food Science & Biotechnology Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Small RNAs (sRNAs) are known to regulate pathogenic plant-microbe interactions. Emerging evidence from the study of these model systems suggests that microRNAs (miRNAs) can be translocated between microbes and plants to facilitate symbiosis. The roles of sRNAs in mutualistic mycorrhizal fungal interactions, however, are largely unknown. In this study, we characterized miRNAs encoded by the ectomycorrhizal fungus Pisolithus microcarpus and investigated their expression during mutualistic interaction with Eucalyptus grandis. Using sRNA sequencing data and in situ miRNA detection, a novel fungal miRNA, Pmic_miR-8, was found to be transported into E. grandis roots after interaction with P. microcarpus Further characterization experiments demonstrate that inhibition of Pmic_miR-8 negatively impacts the maintenance of mycorrhizal roots in E. grandis, while supplementation of Pmic_miR-8 led to deeper integration of the fungus into plant tissues. Target prediction and experimental testing suggest that Pmic_miR-8 may target the host NB-ARC domain containing transcripts, suggesting a potential role for this miRNA in subverting host signaling to stabilize the symbiotic interaction. Altogether, we provide evidence of previously undescribed cross-kingdom sRNA transfer from ectomycorrhizal fungi to plant roots, shedding light onto the involvement of miRNAs during the developmental process of mutualistic symbioses.

Understanding the molecular determinants underlying the interaction between the leaf and human pathogenic bacteria is key to provide the foundation to develop science-based strategies to prevent or decrease the pathogen contamination of leafy greens. In this study, we conducted a dual RNA-sequencing analysis to simultaneously define changes in the transcriptomic profiles of the plant and the bacterium when they come in contact. We used an economically relevant vegetable crop, lettuce (Lactuca sativa L. cultivar Salinas), and a model plant, Arabidopsis thaliana Col-0, as well as two pathogenic bacterial strains that cause disease outbreaks associated with fresh produce, Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium 14028s (STm 14028s). We observed commonalities and specificities in the modulation of biological processes between Arabidopsis and lettuce and between O157:H7 and STm 14028s during early stages of the interaction. We detected a larger alteration of gene expression at the whole transcriptome level in lettuce and Arabidopsis at 24 h post inoculation with STm 14028s compared to that with O157:H7. In addition, bacterial transcriptomic adjustments were substantially larger in Arabidopsis than in lettuce. Bacterial transcriptome was affected at a larger extent in the first 4 h compared to the subsequent 20 h after inoculation. Overall, we gained valuable knowledge about the responses and counter-responses of both bacterial pathogen and plant host when these bacteria are residing in the leaf intercellular space. These findings and the public genomic resources generated in this study are valuable for additional data mining.

Background: There is currently no specific requirement for operator’s attires performing amniocentesis, especially for repetitive amniocenteses. We investigated the contamination status on the surface of operators with or without long-sleeved surgical gowns during repetitive amniocenteses to examine whether they are different. Methods: In a prospective cohort study, consecutive samples were collected from the surface of amniocentesis operators with short-sleeved scrubs (bare arms) or with long-sleeved surgical gowns on 4 sites (including the left and right proximal medial forearm, and the left and right ventral wrist). There were 7 time spots during each sampling period. Results: The highest colony count among all plates was 65 colony forming units (cfu)/plate. We observed 85.3% (382/448) and 65.1% (255/392) plates with 0 colony count from operators with bare arms and in surgical gowns, respectively. Moreover, the bacterial load of the group with surgical gowns had an increasing trend with time while the other group showed a contrary trend. Conclusions: All colony counts observed were within the hygiene specification of the National Health Commission of China. Instead of attires, other measures with high-quality evidence including surveillance of hand hygiene and control of indoor air bacterial load should be given more attention.

The high incidence of demineralization around orthodontic brackets has led to the development of preventive measures. Incorporation of antibacterial or remineralizing agents into orthodontic adhesives is an attractive method. This single-center, split-mouth, randomized controlled clinical trial was conducted to assess the effect of a modified composite containing TiO2 nanoparticles on the Streptococcus mutans population and to prevent demineralization around orthodontic brackets. Each participant was assigned a random sequence (AB or BA). During the bonding session, the control lateral incisor was bonded with a conventional composite and the contralateral incisor was bonded with a composite containing nano TiO2 particles (1%weight). The eligibility criteria included the presence of S. mutans in the dental plaque and absence of active caries, fractures or cracks. The S. mutans count in the dental plaque immediately around the brackets was evaluated at baseline and 1, 3, and 6 months after bonding. The specificity of the colonies was determined by PCR. The DIAGNOdent score was assessed at baseline and re-assessed every month up to the sixth month. Salivary samples were collected at T0, T1, and T3 to assess the amount of Ti released from the composite. The cytotoxicity of the modified composites was evaluated using an MTT assay. Participants, examiners, and data analyzers were blinded to the test and intervention groups. Forty-two patients ranging from 12 to 25 years were enrolled in this study. The amount of Ti released into saliva was insignificant and far below the toxic level. There was no significant difference between the S. mutans counts of the studied tooth S. mutans counts at any time point evaluated. DIAGNOdent scores on both sides increased significantly after the first month. However, this increase was higher on the test side (p < 0.001), and a significant difference of 2.6 scores remained throughout the study period. No severe adverse events were observed. Orthodontic composites containing TiO2 nanoparticles may prevent demineralization induced around brackets during orthodontic treatment. However, the antibacterial effects were not statistically significant. Registration: The protocol was registered with the IRCT.ir (IRCT20140215016582N6). [ABSTRACT FROM AUTHOR]

The Greenland Ice Sheet harbours a wealth of microbial life, yet the total biomass stored or exported from its surface to downstream environments is unconstrained. Here, we quantify microbial abundance and cellular biomass flux within the near-surface weathering crust photic zone of the western sector of the ice sheet. Using groundwater techniques, we demonstrate that interstitial water flow is slow (~10-2 m d-1), while flow cytometry enumeration reveals this pathway delivers 5 × 108 cells m-2 d-1 to supraglacial streams, equivalent to a carbon flux up to 250 g km-2 d-1. We infer that cellular carbon accumulation in the weathering crust exceeds fluvial export, promoting biomass sequestration, enhanced carbon cycling, and biological albedo reduction. We estimate that up to 37 kg km-2 of cellular carbon is flushed from the weathering crust environment of the western Greenland Ice Sheet each summer, providing an appreciable flux to support heterotrophs and methanogenesis at the bed.

Staphylococcus aureus colonizes patients with atopic dermatitis (AD) and exacerbates disease by promoting inflammation. The present study investigated the safety and mechanisms of action of Staphylococcus hominis A9 (ShA9), a bacterium isolated from healthy human skin, as a topical therapy for AD. ShA9 killed S. aureus on the skin of mice and inhibited expression of a toxin from S. aureus (psmα) that promotes inflammation. A first-in-human, phase 1, double-blinded, randomized 1-week trial of topical ShA9 or vehicle on the forearm skin of 54 adults with S. aureus-positive AD (NCT03151148) met its primary endpoint of safety, and participants receiving ShA9 had fewer adverse events associated with AD. Eczema severity was not significantly different when evaluated in all participants treated with ShA9 but a significant decrease in S. aureus and increased ShA9 DNA were seen and met secondary endpoints. Some S. aureus strains on participants were not directly killed by ShA9, but expression of mRNA for psmα was inhibited in all strains. Improvement in local eczema severity was suggested by post-hoc analysis of participants with S. aureus directly killed by ShA9. These observations demonstrate the safety and potential benefits of bacteriotherapy for AD.

Background: Canned foods may be contaminated with microbes and primarily with spore-forming bacteria. This study was designed to give information about microbial load of canned foods imported through Ibrahim Khalil International Border, Iraq. Methods: Total of 119 samples of canned foods comprising 35 poultry meats, 40 fishes, and 44 tomato pastes were collected from Ibrahim Khalil International Border. Using conventional protocols, samples were evaluated for total plate counts (aerobic and anaerobic microorganisms), spoilage pathogenic, and coliform organisms. The obtained results were analysed by One-Way Analysis of Variance (ANOVA) suing GraphPad Prism (V.5.01). Results: The total aerobic plate counts at 37 °C incubation were 1.30±0.2 log Colony Forming Unit (CFU)/g in canned meats, 1.32±0.3 log CFU/g for fishes, and tomato paste accounts for 2.11±0.5 log CFU/g. On the other hand, the counts of anaerobic plate were 0.95±0.2 log CFU/g in meat samples, 1.08±0.2 log CFU/g for fishes, and tomatoes were scored at 0.95±0.2 log CFU/g. Bacillus subtilis, B. coagulans, Clostridium perfringens, and Klebsiella spp. were recovered from some of the canned samples. Conclusion: Canned tomatoes and fishes relatively had more microorganisms than the poultry meat products. These data suggested that poor hygiene standards in the processing line may result in microbial control loss. DOI: 10.18502/jfqhc.10.1.11989

Background: Fermented foods are gaining interest because of their ability to improve health, as well as their good taste, and the desire of many to eat them. This investigation aimed to enhance the chemical and physical properties, sensory evaluation, and shelf life of fermented orange juice by adding liquid hibiscus leave (Hibiscus sabdariffa) and potato peel (Solanum tuberosum) extracts. Methods: The extracts of hibiscus leaves and potato peel were added to fermented orange juice, and the samples were divided into five parts. Two parts with different concentrations of hibiscus leave extracts (5%, FHL5 and 10%, FHL10), and the other two parts with different concentrations of potato peel extracts (5%, FPP5 and 10%, FPP10). The Fifth part was fermented orange juice without any additives (PC). Then, they were estimated for the acidity, total solid solution, viscosity, carbohydrates, vitamin C, antioxidants profile, color, total microbial count, and sensory evaluation during storage time at 4±2 °C. Statistical analysis was done by IBM SPSS version 25.0 software. Results: The results showed no significant mean difference (p>0.05) between samples in acidity. FHL10 registered the highest values in total solid solution, vitamin C, antioxidants profile, and sensory evaluation, while FPP10 registered the highest values in viscosity and total microbial count. Also, FPP10 registered the highest values in overall acceptability (8.85±0.24, 8.78±0.26, 8.71±0.26, and 8.78±0.26 at zero, 7th, 14th, and 21st days of storage time, respectively) followed by FPP5 with no significant mean difference values (p>0.05). Conclusion: The extracts of hibiscus leaves and potato peel can improve acceptability and shelf life of fermented orange juice summarized in the enhancement vitamin content, antioxidants profile, color, and sensory properties. DOI: 10.18502/jfqhc.10.1.11988

Netherton syndrome (NS) is a monogenic skin disease resulting from loss of function of lymphoepithelial Kazal-type-related protease inhibitor (LEKTI-1). In this study we examine if bacteria residing on the skin are influenced by the loss of LEKTI-1 and if interaction between this human gene and resident bacteria contributes to skin disease. Shotgun sequencing of the skin microbiome demonstrates that lesional skin of NS subjects is dominated by Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). Isolates of either species from NS subjects are able to induce skin inflammation and barrier damage on mice. These microbes promote skin inflammation in the setting of LEKTI-1 deficiency due to excess proteolytic activity promoted by S. aureus phenol-soluble modulin α as well as increased bacterial proteases staphopain A and B from S. aureus or EcpA from S. epidermidis. These findings demonstrate the critical need for maintaining homeostasis of host and microbial proteases to prevent a human skin disease.

The healthy cornea is remarkably resistant to infection, quickly clearing deliberately inoculated bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. Contrasting with the adjacent conjunctiva and other body surfaces, it also lacks a resident viable bacterial microbiome. Corneal resistance to microbes depends on intrinsic defenses involving tear fluid and the corneal epithelium. Dry eye, an ocular surface disease associated with discomfort and inflammation, can alter tear fluid composition and volume, and impact epithelial integrity. We previously showed that experimentally-induced dry eye (EDE) in mice does not increase corneal susceptibility to P. aeruginosa infection. Here, we explored if EDE alters corneal resistance to bacterial colonization. EDE was established in mice using scopolamine injections and dehumidified air-flow, and verified by phenol-red thread testing after 5 and 10 days. As expected, EDE corneas showed increased fluorescein staining versus controls consistent with compromised epithelial barrier function. Confocal imaging using mT/mG knock-in mice with red-fluorescent membranes revealed no other obvious morphological differences between EDE corneas and controls for epithelium, stroma, and endothelium. EDE corneas were imaged ex vivo and compared to controls after alkyne-functionalized D-alanine labeling of metabolically-active colonizing bacteria, or by FISH using a universal 16S rRNA gene probe. Both methods revealed very few viable bacteria on EDE corneas after 5 or 10 days (median of 0, upper quartile of ≤ 1 bacteria per field of view for each group [9-12 eyes per group]) similar to control corneas. Furthermore, there was no obvious difference in abundance of conjunctival bacteria, which included previously reported filamentous forms. Thus, despite reduced tear flow and apparent compromise to corneal barrier function (fluorescein staining), EDE murine corneas continue to resist bacterial colonization and maintain the absence of a resident viable bacterial microbiome.

Gene-drive systems in diploid organisms bias the inheritance of one allele over another. CRISPR-based gene-drive expresses a guide RNA (gRNA) into the genome at the site where the gRNA directs Cas9-mediated cleavage. In the presence of Cas9, the gRNA cassette and any linked cargo sequences are copied via homology-directed repair (HDR) onto the homologous chromosome. Here, we develop an analogous CRISPR-based gene-drive system for the bacterium Escherichia coli that efficiently copies a gRNA cassette and adjacent cargo flanked with sequences homologous to the targeted gRNA/Cas9 cleavage site. This "pro-active" genetic system (Pro-AG) functionally inactivates an antibiotic resistance marker on a high copy number plasmid with ~ 100-fold greater efficiency than control CRISPR-based methods, suggesting an amplifying positive feedback loop due to increasing gRNA dosage. Pro-AG can likewise effectively edit large plasmids or single-copy genomic targets or introduce functional genes, foreshadowing potential applications to biotechnology or biomedicine.

The nutritional and sensory quality of different flour blends for baked products has been investigated. However, there is paucity of information on the quality of the conventional all-purpose flour which is expected to be chiefly of wheat origin and thus very expensive. Comparative analysis of the microbial, proximate and sensory quality of bread produced from all-purpose flour and blends of locally made flour was undertaken. Composite breads were produced from all-purpose flour (APF) 100%, wheat flour (WF) 100%, potato flour (PF) 100% and WF:PF blend (50%:50%). The microbial, proximate and sensory qualities, as well as the shelf life of the bread samples were determined. Data was analyzed using SPSS software. The bacterial and fungal counts were less than the maximum of 100 cfu/g recommended by the Standard Organization of Nigeria. The moisture and ash content of the PF bread was significantly higher (p < 0.05) than the rest of the flour blends. Among the flour blends, APF recorded the lowest fiber, ash and protein content. There was no significant difference (p > 0.05) in the mean sensory scores of APF bread and WF:PF bread with respect to taste and overall acceptability. PF and WF:PF bread had the highest shelf life of 8 and 7 days respectively. The nutritional composition of the bread produced from 100% WF and 50%:50% WF:PF blends were higher than that produced using the conventional APF. Thus, PF could be blended with WF to improve the nutritional quality and minimize cost of bread and other baked products.

Abstract As one of the most widely used assays in biological research, an enumeration of the bacterial cell colonies is an important but time-consuming and labor-intensive process. To speed up the colony counting, a machine learning method is presented for counting the colony forming units (CFUs), which is referred to as CFUCounter. This cell-counting program processes digital images and segments bacterial colonies. The algorithm combines unsupervised machine learning, iterative adaptive thresholding, and local-minima-based watershed segmentation to enable an accurate and robust cell counting. Compared to a manual counting method, CFUCounter supports color-based CFU classification, allows plates containing heterologous colonies to be counted individually, and demonstrates overall performance (slope 0.996, SD 0.013, 95%CI: 0.97–1.02, p value

Background: Fermented foods are gaining interest because of their ability to improve health, as well as their good taste, and the desire of many to eat them. This investigation aimed to enhance the chemical and physical properties, sensory evaluation, and shelf life of fermented orange juice by adding liquid hibiscus leave (Hibiscus sabdariffa) and potato peel (Solanum tuberosum) extracts. Methods: The extracts of hibiscus leaves and potato peel were added to fermented orange juice, and the samples were divided into five parts. Two parts with different concentrations of hibiscus leave extracts (5%, FHL5 and 10%, FHL10), and the other two parts with different concentrations of potato peel extracts (5%, FPP5 and 10%, FPP10). The Fifth part was fermented orange juice without any additives (PC). Then, they were estimated for the acidity, total solid solution, viscosity, carbohydrates, vitamin C, antioxidants profile, color, total microbial count, and sensory evaluation during storage time at 4±2 °C. Statistical analysis was done by IBM SPSS version 25.0 software. Results: The results showed no significant mean difference (p>0.05) between samples in acidity. FHL10 registered the highest values in total solid solution, vitamin C, antioxidants profile, and sensory evaluation, while FPP10 registered the highest values in viscosity and total microbial count. Also, FPP10 registered the highest values in overall acceptability (8.85±0.24, 8.78±0.26, 8.71±0.26, and 8.78±0.26 at zero, 7th, 14th, and 21st days of storage time, respectively) followed by FPP5 with no significant mean difference values (p>0.05). Conclusion: The extracts of hibiscus leaves and potato peel can improve acceptability and shelf life of fermented orange juice summarized in the enhancement vitamin content, antioxidants profile, color, and sensory properties. [ABSTRACT FROM AUTHOR]

Microbially Induced Calcite Precipitation (MICP) is a bio-mediated cementation process that can improve the engineering properties of granular soils through the precipitation of calcite. The process is made possible by soil microorganisms containing urease enzymes, which hydrolyze urea and enable carbonate ions to become available for precipitation. While most researchers have injected non-native ureolytic bacteria to complete bio-cementation, enrichment of native ureolytic microorganisms may enable reductions in process treatment costs and environmental impacts. In this study, a large-scale bio-cementation experiment involving two 1.7-meter diameter tanks and a complementary soil column experiment were performed to investigate biogeochemical differences between bio-cementation mediated by either native or augmented (Sporosarcina pasteurii) ureolytic microorganisms. Although post-treatment distributions of calcite and engineering properties were similar between approaches, the results of this study suggest that significant differences in ureolysis rates and related precipitation rates between native and augmented microbial communities may influence the temporal progression and spatial distribution of bio-cementation, solution biogeochemical changes, and precipitate microstructure. The role of urea hydrolysis in enabling calcite precipitation through sustained super-saturation following treatment injections is explored.

Candida albicans is an opportunistic human fungal pathogen that is able to cause both mucosal and systemic infections. It is also a frequent human commensal, where it is typically found inhabiting multiple niches including the gastrointestinal tract. One of the most remarkable features of C. albicans biology is its ability to undergo heritable and reversible switching between different phenotypic states, a phenomenon known as phenotypic switching. This is best exemplified by the white-opaque switch, in which cells undergo epigenetic transitions between two alternative cellular states. Here, we describe assays to quantify the frequency of switching between states, as well as methods to help identify cells in different phenotypic states. We also describe the use of environmental cues that can induce switching into either the white or opaque state. Finally, we introduce the use of mNeonGreen and mScarlet fluorescent proteins that have been optimized for use in C. albicans and which outperform commonly used fluorescent proteins for both fluorescence microscopy and flow cytometry. © 2019 by John Wiley & Sons, Inc.

Lifelong infection of the gastric mucosa by Helicobacter pylori can lead to peptic ulcers and gastric cancer. However, how the bacteria maintain chronic colonization in the face of constant mucus and epithelial cell turnover in the stomach is unclear. Here, we present a new model of how H. pylori establish and persist in stomach, which involves the colonization of a specialized microenvironment, or microniche, deep in the gastric glands. Using quantitative three-dimensional (3D) confocal microscopy and passive CLARITY technique (PACT), which renders tissues optically transparent, we analyzed intact stomachs from mice infected with a mixture of isogenic, fluorescent H. pylori strains with unprecedented spatial resolution. We discovered that a small number of bacterial founders initially establish colonies deep in the gastric glands and then expand to colonize adjacent glands, forming clonal population islands that persist over time. Gland-associated populations do not intermix with free-swimming bacteria in the surface mucus, and they compete for space and prevent newcomers from establishing in the stomach. Furthermore, bacterial mutants deficient in gland colonization are outcompeted by wild-type (WT) bacteria. Finally, we found that host factors such as the age at infection and T-cell responses control bacterial density within the glands. Collectively, our results demonstrate that microniches in the gastric glands house a persistent H. pylori reservoir, which we propose replenishes the more transient bacterial populations in the superficial mucosa.

Microbial food safety of raw or minimally processed fresh produce is a significant challenge. The current sanitation processes are effective for inactivation of bacteria in wash water but have limited efficacy (5-log reduction) in the presence of relatively high organic content (chemical oxygen demand of 500 mg/liter), demonstrating a potential to address a significant unmet need to improve fresh produce sanitation. The particle-based sanitizer had no significant effect on the quality of fresh lettuce.IMPORTANCE The limitation of current sanitation processes for inactivation of microbes on the surfaces of fresh produce is due to nonspecific consumption of sanitizers by reactions with the food matrix and complexity of surface chemistries and structural features of produce surfaces. This study demonstrates a novel approach to enhance sanitation effectiveness of fresh produce using a particle-based sanitizer. The particle-based sanitizer concept provides localized high concentration delivery of chlorine to the surfaces of fresh produce and enables more than 5 logs of inactivation of inoculated bacteria on fresh produce surfaces without significant changes in produce quality. The results of this study illustrate the potential of this approach to address the unmet need for improving sanitation of fresh produce. Further validation of this approach using a scaled-up produce washing system will enable commercialization of this novel concept.

PurposeContact lens wear carries a risk of complications, including corneal infection. Solving these complications has been hindered by limitations of existing animal models. Here, we report development of a new murine model of contact lens wear.MethodsC57BL/6 mice were fitted with custom-made silicone-hydrogel contact lenses with or without prior inoculation with Pseudomonas aeruginosa (PAO1-GFP). Contralateral eyes served as controls. Corneas were monitored for pathology, and examined ex vivo using high-magnification, time-lapse imaging. Fluorescent reporter mice allowed visualization of host cell membranes and immune cells. Lens-colonizing bacteria were detected by viable counts and FISH. Direct-colony PCR was used for bacterial identification.ResultsWithout deliberate inoculation, lens-wearing corneas remained free of visible pathology, and retained a clarity similar to non-lens wearing controls. CD11c-YFP reporter mice revealed altered numbers, and distribution, of CD11c-positive cells in lens-wearing corneas after 24 h. Worn lenses showed bacterial colonization, primarily by known conjunctival or skin commensals. Corneal epithelial cells showed vacuolization during lens wear, and after 5 days, cells with phagocyte morphology appeared in the stroma that actively migrated over resident keratocytes that showed altered morphology. Immunofluorescence confirmed stromal Ly6G-positive cells after 5 days of lens wear, but not in MyD88 or IL-1R gene-knockout mice. P. aeruginosa-contaminated lenses caused infectious pathology in most mice from 1 to 13 days.ConclusionsThis murine model of contact lens wear appears to faithfully mimic events occurring during human lens wear, and could be valuable for experiments, not possible in humans, that help solve the pathogenesis of lens-related complications.

Ground beef makes up more than half of the beef consumed in the U.S. market. Although numerous studies have been conducted on microbial safety and shelf life of ground beef limited work has been done using a culture-independent approach. While past studies have allowed for the evaluation of a few organisms of interest, there is limited work on the microbial community associated with fresh ground beef. In order to have a more complete picture of the microbial ecology of the product, a culture-independent approach utilizing 16S rRNA gene amplicon sequencing was used. The objectives of this study were to characterize the fresh ground beef microbiome and the effect that antimicrobial interventions and antioxidants, applied to beef trim before grinding, and product storage have on community composition using 16S rRNA gene amplicon sequencing. Beef trimmings were treated with antimicrobials and an antioxidant. Samples were ground, loafed, and overwrapped before being packaged in modified-atmosphere packaging. Samples were in dark storage for 21 days followed by five days in retail display. Periodically during storage, samples were collected for microbiological analysis and DNA isolation. Due to low microbial biomass, only 52 of 210 samples were included in the final analysis. These samples represented two antimicrobial treatments (peroxyacetic acid, and a sulfuric acid and sodium sulfate blend) and a control, from day-15 of dark storage and day-5 of retail display. As sample age increased, so did the number of raw reads (P < 0.001) and aerobic plate counts (P < 0.001), which were correlated (r = 0.94, P = 0.017). Across all samples, lactic acid bacteria were most abundant followed by Enterobacteriaceae; several rare taxa were also identified (namely Geobacillus, Thermus, and Sporosarcina). Antimicrobial treatment altered the bacterial alpha (P < 0.001) and beta (P = 0.001) diversity, while storage day altered alpha (P = 0.001) diversity. Enterobacteriaceae relative abundance differed (P < 0.05) among treatments and was highest in control samples. In addition to confirming previously described dominant microbial differences in culture-dependent results, these data identified genera not typically associated with ground beef and allowed for study of shifts in the entire microbiome and not just a subset of indicator organisms.

As one of the most widely used assays in biological research, an enumeration of the bacterial cell colonies is an important but time-consuming and labor-intensive process. To speed up the colony counting, a machine learning method is presented for counting the colony forming units (CFUs), which is referred to as CFUCounter. This cell-counting program processes digital images and segments bacterial colonies. The algorithm combines unsupervised machine learning, iterative adaptive thresholding, and local-minima-based watershed segmentation to enable an accurate and robust cell counting. Compared to a manual counting method, CFUCounter supports color-based CFU classification, allows plates containing heterologous colonies to be counted individually, and demonstrates overall performance (slope 0.996, SD 0.013, 95%CI: 0.97–1.02, p value < 1e-11, r = 0.999) indistinguishable from the gold standard of point-and-click counting. This CFUCounter application is open-source and easy to use as a unique addition to the arsenal of colony-counting tools. [ABSTRACT FROM AUTHOR]

Background: Kalahari truffle (Kalaharituber pfeilii) is found in the Kalahari desert and nearby regions (Africa). This study assessed the microbiological quality and safety, mycotoxins, and heavy metals contents of raw Kalahari truffle sold in Namibia. Methods: Batches of Kalahari truffles were purchased from informal markets and different vendors in Namibia. Total aerobic, coliform, yeast, and moulds counts, and Salmonella were assessed. Also, some mycotoxins and heavy metals were determined. Data were analyzed using SPSS Statistics Software, Version 25. Results: Total aerobic count of unwashed truffles ranged from 4.4 to 7.3 log Colony Forming Unit (CFU)/g. Total coliform counts detected in truffles were 6.0 log CFU/g. Salmonella was not detected. Doxynivalenol was the most prevalent mycotoxin. Fumonisin B1 levels ranged from 17.4 to 142.1 µg/kg. Ochratoxin A levels in unwashed truffles ranged from 0.1 to 48.5 µg/kg. Total aflatoxin levels were 26.3 to 27.5 µg/kg, while zearalenone levels ranged from 45.0 to 9,680 µg/kg. The iron content was up to 746.72 mg/kg. Cadmium and zinc were detected in the studied samples, but mercury and nickel were no detectable in any samples. Conclusion: The studied truffle samples were safe in terms of Salmonella, mercury, and nickel. However, some of the detected microorganisms, mycotoxins, and heavy metals in underprized Kalahari truffles may impair the safety, shelf life, and human health. Thus, they should be subjected to appropriate processing before consumption. DOI: 10.18502/jfqhc.9.1.9687

Listeriolysin O (LLO) is a cholesterol-dependent cytolysin that mediates escape of Listeria monocytogenes from a phagosome, enabling growth of the bacteria in the host cell cytosol. LLO contains a PEST-like sequence that prevents it from killing infected cells, but the mechanism involved is unknown. We found that the LLO PEST-like sequence was necessary to mediate removal of LLO from the interior face of the plasma membrane, where it coalesces into discrete puncta. LLO interacts with Ap2a2, an adaptor protein involved in endocytosis, via its PEST-like sequence, and Ap2a2-dependent endocytosis is required to prevent LLO-induced cytotoxicity. An unrelated PEST-like sequence from a human G protein-coupled receptor (GPCR), which also interacts with Ap2a2, could functionally complement the PEST-like sequence in L. monocytogenes LLO. These data revealed that LLO co-opts the host endocytosis machinery to protect the integrity of the host plasma membrane during L. monocytogenes infection.

Sporulation in Bacillus subtilis is a paradigm of bacterial development, which involves the interaction between a larger mother cell and a smaller forespore. The mother cell and the forespore activate different genetic programs, leading to the production of sporulation-specific proteins. A critical gap in our understanding of sporulation is how vegetative proteins, made before sporulation initiation, contribute to spore formation. Here we present a system, spatiotemporally regulated proteolysis (STRP), which enables the rapid, developmentally regulated degradation of target proteins, thereby providing a suitable method to dissect the cell- and developmental stage-specific role of vegetative proteins. STRP has been used to dissect the role of two major vegetative sigma factors, σH and σA , during sporulation. The results suggest that σH is only required in predivisional cells, where it is essential for sporulation initiation, but that it is dispensable during subsequent steps of spore formation. However, evidence has been provided that σA plays different roles in the mother cell, where it replenishes housekeeping functions, and in the forespore, where it plays an unexpected role in promoting spore germination and outgrowth. Altogether, the results demonstrate that STRP has the potential to provide a comprehensive molecular dissection of every stage of sporulation, germination and outgrowth.

Reports of the test accuracy of the urinalysis for diagnosing urinary tract infections (UTIs) in young febrile infants have been variable. We evaluated the test characteristics of the urinalysis for diagnosing UTIs, with and without associated bacteremia, in young febrile infants. We performed a planned secondary analysis of data from a prospective study of febrile infants ≤60 days old at 26 emergency departments in the Pediatric Emergency Care Applied Research Network. We evaluated the test characteristics of the urinalysis for diagnosing UTIs, with and without associated bacteremia, by using 2 definitions of UTI: growth of ≥50 000 or ≥10 000 colony-forming units (CFUs) per mL of a uropathogen. We defined a positive urinalysis by the presence of any leukocyte esterase, nitrite, or pyuria (>5 white blood cells per high-power field). Of 4147 infants analyzed, 289 (7.0%) had UTIs with colony counts ≥50 000 CFUs/mL, including 27 (9.3%) with bacteremia. For these UTIs, a positive urinalysis exhibited sensitivities of 0.94 (95% confidence interval [CI]: 0.91-0.97), regardless of bacteremia; 1.00 (95% CI: 0.87-1.00) with bacteremia; and 0.94 (95% CI: 0.90-0.96) without bacteremia. Specificity was 0.91 (95% CI: 0.90-0.91) in all groups. For UTIs with colony counts ≥10 000 CFUs/mL, the sensitivity of the urinalysis was 0.87 (95% CI: 0.83-0.90), and specificity was 0.91 (95% CI: 0.90-0.92). The urinalysis is highly sensitive and specific for diagnosing UTIs, especially with ≥50 000 CFUs/mL, in febrile infants ≤60 days old, and particularly for UTIs with associated bacteremia.

Microbiological water quality is usually assessed by the identification of Escherichia coli (E. coli), a fecal indicator. The hydrogen sulfide (H2S) test is an inexpensive, easy-to-use, and portable alternative field-based water quality test. Our study evaluated the H2S test's effectiveness as a water quality indicator for diarrhea risk. Field workers collected stored drinking water samples for H2S analysis and detection of E. coli by membrane filtration and measured caregiver-reported diarrhea among children < 5 years in the same households 1 month later. We assessed the association between the H2S test (incubated for 24 hours and 48 hours) and diarrhea prevalence, with 2-day and 7-day symptom recall periods (N = 1,348). We determined the sensitivity, specificity, and positive and negative predictive value (PPV, NPV) of the H2S test compared with E. coli (N = 525). Controlling for potentially confounding covariates, H2S-positive water (at 24 or 48 hours) was not associated with 2-day diarrhea prevalence (24-hour prevalence ratio [PR] = 1.03, 95% confidence interval [CI]: 0.63-1.69; 48-hour PR = 0.89, 95% CI: 0.58-1.38) or 7-day diarrhea prevalence (24-hour PR = 1.17, 95% CI: 0.76-1.78; 48-hour PR = 1.21, 95% CI: 0.81-1.80). The sensitivity, PPV, and NPV of the H2S test was significantly higher when the H2S test was incubated for 48 versus 24 hours whereas specificity showed the opposite trend. H2S test sensitivity, PPV, and NPV increased with increasing E. coli levels, consistent with previous evidence that the H2S test is a useful water quality tool in high-contamination settings. However, our results suggest that the H2S test is not an effective indicator for waterborne diarrhea.

Growth rates are an important tool in microbiology because they provide high throughput fitness measurements. The release of GrowthRates, a program that uses the output of plate reader files to automatically calculate growth rates, has facilitated experimental procedures in many areas. However, many sources of variation within replicate growth rate data exist and can decrease data reliability. We have developed a new statistical package, CompareGrowthRates (CGR), to enhance the program GrowthRates and accurately measure variation in growth rate data sets. We define a metric, Variability-score (V-score), that can help determine if variation within a data set might result in false interpretations. CGR also uses the bootstrap method to determine the fraction of bootstrap replicates in which a strain will grow the fastest. We illustrate the usage of CGR with growth rate data sets similar to those in Mira, Meza, et al. (Adaptive landscapes of resistance genes change as antibiotic concentrations change. Mol Biol Evol. 32(10): 2707-2715). These statistical methods are compatible with the analytic methods described in Growth Rates Made Easy and can be used with any set of growth rate output from GrowthRates.

Abstract Background In resource-limited settings, sputum smear conversion is used to document treatment response. Many People living with HIV (PLHIV) are smear-negative at baseline. The Xpert MTB/RIF test can indirectly measure bacterial load through cycle threshold (ct) values. This study aimed to determine if baseline Xpert MTB/RIF could predict time to culture negativity in PLHIV with newly diagnosed TB. Methods A subset of 138 PLHIV from the ‘SOUTH’ study on outcomes related to TB and antiretroviral drug concentrations were included. Bacterial load was estimated by Mycobacterium Growth Indicator Tubes (MGIT) culture time-to-positivity (TTP) and Lowenstein Jensen (LJ) colony counts. Changes in TTP and colony counts were analyzed with Poisson Generalised Estimating Equations (GEE) and multilevel ordered logistic regression models, respectively, while time to culture negativity analysed with Cox proportional hazard models. ROC curves were used to explore the accuracy of the ct value in predicting culture negativity. Results A total of 81 patients (58.7%) were males, median age 34 (IQR 29 ̶ 40) years, median CD4 cell count of 180 (IQR 68 ̶ 345) cells/μL and 77.5% were ART naive. The median baseline ct value was 25.1 (IQR 21.0 ̶ 30.1). A unit Increase in the ct value was associated with a 5% (IRR = 1.05 95% CI 1.04 ̶ 1.06) and 3% (IRR = 1.03 95% CI 1.03 ̶ 1.04) increase in TTP at week 2 and 4 respectively. With LJ culture, a patient’s colony grade was reduced by 0.86 times (0R = 0.86 95% CI 0.74 ̶ 0.97) at week 2 and 0.84 times (OR = 0.84 95% CI 0.79 ̶ 0.95 P = 0.002) at week 4 for every unit increase in the baseline ct value. There was a 3% higher likelihood of earlier conversion to negativity for every unit increase in the ct value. A ct cut point ≥28 best predicted culture negativity at week 4 with a sensitivity of 91. 7% & specificity 53.7% while a cut point ≥23 best predicted culture negativity at week 8. Conclusion Baseline Xpert MTB/RIF ct values predict sputum conversion in PLHIV on anti-TB treatment. Surrogate biomarkers for sputum conversion in PLHIV are still a research priority.

Bacterial plant pathogens often encounter reactive oxygen species (ROS) during host invasion. In foliar bacterial pathogens, multiple regulatory proteins are involved in the sensing of oxidative stress and the activation of the expression of antioxidant genes. However, it is unclear whether xylem-limited bacteria, such as Xylella fastidiosa, experience oxidative stress during the colonization of plants. Examination of the X. fastidiosa genome uncovered only one homologue of oxidative stress regulatory proteins, OxyR. Here, a knockout mutation in the X. fastidiosa oxyR gene was constructed; the resulting strain was significantly more sensitive to hydrogen peroxide (H2 O2 ) relative to the wild-type. In addition, during early stages of grapevine infection, the survival rate was 1000-fold lower for the oxyR mutant than for the wild-type. This supports the hypothesis that grapevine xylem represents an oxidative environment and that X. fastidiosa must overcome this challenge to achieve maximal xylem colonization. Finally, the oxyR mutant exhibited reduced surface attachment and cell-cell aggregation and was defective in biofilm maturation, suggesting that ROS could be a potential environmental cue stimulating biofilm development during the early stages of host colonization.

PurposeTo determine whether patients who had a positive repeated culture was predictive of worse clinical outcome than those who achieved microbiological cure at 6 days in the Mycotic Ulcer Treatment Trial I (MUTT-I).DesignSecondary analysis from a multicenter, double-masked, randomized clinical trial.Methodssetting: Multiple hospital sites of the Aravind Eye Care System, India.Study populationPatients with culture-positive filamentous fungal ulcers and visual acuity of 20/40 to 20/400 reexamined 6 days after initiation of treatment.InterventionCorneal scraping and cultures were obtained from study participants at day 6 after enrollment.Main outcome measuresWe assessed 3-month best spectacle-corrected visual acuity (BSCVA), 3-month infiltrate/scar size, corneal perforation, and re-epithelialization rates stratified by culture positivity at day 6.ResultsOf the 323 patients with smear-positive ulcers enrolled in MUTT-I, 299 (92.6%) were scraped and cultured 6 days after enrollment. Repeat culture positivity was 31% (92/299). Among patients who tested positive at enrollment, those with positive 6-day cultures had significantly worse 3-month BSCVA (0.39 logMAR; 95% confidence interval [CI]: 0.24-0.44; P < .001), had larger 3-month scar size (0.39 mm; 95% CI: 0.06-0.73; P = .02), were more likely to perforate or require therapeutic penetrating keratoplasty (odds ratio: 6.27; 95% CI: 2.73-14.40; P < .001), and were slower to re-epithelialize (hazard ratio: 0.33; 95% CI: 0.21-0.50; P < .001) than those with a negative 6-day culture result.ConclusionsEarly microbiological cure on culture is a predictor of clinical response to treatment.

Neutrophils are the first line of defense against bacterial infections, and the generation of reactive oxygen species is a key part of their arsenal. Pathogens use detoxification systems to avoid the bactericidal effects of reactive oxygen species. Here we demonstrate that the Gram-negative pathogen Pseudomonas aeruginosa is susceptible to reactive oxygen species but actively blocks the reactive oxygen species burst using two type III secreted effector proteins, ExoS and ExoT. ExoS ADP-ribosylates Ras and prevents it from interacting with and activating phosphoinositol-3-kinase (PI3K), which is required to stimulate the phagocytic NADPH-oxidase that generates reactive oxygen species. ExoT also affects PI3K signaling via its ADP-ribosyltransferase activity but does not act directly on Ras. A non-ribosylatable version of Ras restores reactive oxygen species production and results in increased bacterial killing. These findings demonstrate that subversion of the host innate immune response requires ExoS-mediated ADP-ribosylation of Ras in neutrophils.

Based upon knowledge of the hydrolytic profile of major β-lactamases found in Gram-negative bacteria, we tested the efficacy of the combination of ceftazidime-avibactam (CAZ-AVI) with aztreonam (ATM) against carbapenem-resistant enteric bacteria possessing metallo-β-lactamases (MBLs). Disk diffusion and agar-based antimicrobial susceptibility testing were initially performed to determine the in vitro efficacy of a unique combination of CAZ-AVI and ATM against 21 representative Enterobacteriaceae isolates with a complex molecular background that included blaIMP, blaNDM, blaOXA-48, blaCTX-M, blaAmpC, and combinations thereof. Time-kill assays were conducted, and the in vivo efficacy of this combination was assessed in a murine neutropenic thigh infection model. By disk diffusion assay, all 21 isolates were resistant to CAZ-AVI alone, and 19/21 were resistant to ATM. The in vitro activity of CAZ-AVI in combination with ATM against diverse Enterobacteriaceae possessing MBLs was demonstrated in 17/21 isolates, where the zone of inhibition was ≥21 mm. All isolates demonstrated a reduction in CAZ-AVI agar dilution MICs with the addition of ATM. At 2 h, time-kill assays demonstrated a ≥4-log10-CFU decrease for all groups that had CAZ-AVI with ATM (8 μg/ml) added, compared to the group treated with CAZ-AVI alone. In the murine neutropenic thigh infection model, an almost 4-log10-CFU reduction was noted at 24 h for CAZ-AVI (32 mg/kg every 8 h [q8h]) plus ATM (32 mg/kg q8h) versus CAZ-AVI (32 mg/kg q8h) alone. The data presented herein require us to carefully consider this new therapeutic combination to treat infections caused by MBL-producing Enterobacteriaceae.

This study developed RNA-based predictive models describing the survival of Vibrio parahaemolyticus in Eastern oysters (Crassostrea virginica) during storage at 0, 4, and 10°C. Postharvested oysters were inoculated with a cocktail of five V. parahaemolyticus strains and were then stored at 0, 4, and 10°C for 21 or 11 days. A real-time reverse transcription-PCR (RT-PCR) assay targeting expression of the tlh gene was used to evaluate the number of surviving V. parahaemolyticus cells, which was then used to establish primary molecular models (MMs). Before construction of the MMs, consistent expression levels of the tlh gene at 0, 4, and 10°C were confirmed, and this gene was used to monitor the survival of the total V. parahaemolyticus cells. In addition, the tdh and trh genes were used for monitoring the survival of virulent V. parahaemolyticus Traditional models (TMs) were built based on data collected using a plate counting method. From the MMs, V. parahaemolyticus populations had decreased 0.493, 0.362, and 0.238 log10 CFU/g by the end of storage at 0, 4, and 10°C, respectively. Rates of reduction of V. parahaemolyticus shown in the TMs were 2.109, 1.579, and 0.894 log10 CFU/g for storage at 0, 4, and 10°C, respectively. Bacterial inactivation rates (IRs) estimated with the TMs (-0.245, -0.152, and -0.121 log10 CFU/day, respectively) were higher than those estimated with the MMs (-0.134, -0.0887, and -0.0732 log10 CFU/day, respectively) for storage at 0, 4, and 10°C. Higher viable V. parahaemolyticus numbers were predicted using the MMs than using the TMs. On the basis of this study, RNA-based predictive MMs are the more accurate and reliable models and can prevent false-negative results compared to TMs.IMPORTANCE One important method for validating postharvest techniques and for monitoring the behavior of V. parahaemolyticus is to establish predictive models. Unfortunately, previous predictive models established based on plate counting methods or on DNA-based PCR can underestimate or overestimate the number of surviving cells. This study developed and validated RNA-based molecular predictive models to describe the survival of V. parahaemolyticus in oysters during low-temperature storage (0, 4, and 10°C). The RNA-based predictive models show the advantage of being able to count all of the culturable, nonculturable, and stressed cells. By using primers targeting the tlh gene and pathogenesis-associated genes (tdh and trh), real-time RT-PCR can evaluate the total surviving V. parahaemolyticus population as well as differentiate the pathogenic ones from the total population. Reliable and accurate predictive models are very important for conducting risk assessment and management of pathogens in food.

Accurately assessing the microbiological safety of water sources is essential to reduce waterborne fecal exposures and track progress toward global targets of safe water access. Sanitary inspections are a recommended tool to assess water safety. We collected 1,684 water samples from 902 shallow tubewells in rural Bangladesh and conducted sanitary surveys to assess whether sanitary risk scores could predict water quality, as measured by Escherichia coli. We detected E. coli in 41% of tubewells, mostly at low concentrations. Based on sanitary scores, 31% of wells were low risk, 45% medium risk, and 25% high or very high risk. Older wells had higher risk scores. Escherichia coli levels were higher in wells where the platform was cracked or broken (Δlog10 = 0.09, 0.00-0.18) or undercut by erosion (Δlog10 = 0.13, 0.01-0.24). However, the positive predictive value of these risk factors for E. coli presence was low (< 50%). Latrine presence within 10 m was not associated with water quality during the wet season but was associated with less frequent E. coli detection during the dry season (relative risk = 0.72, 0.59-0.88). Sanitary scores were not associated with E. coli presence or concentration. These findings indicate that observed characteristics of a tubewell, as measured by sanitary inspections in their current form, do not sufficiently characterize microbiological water quality, as measured by E. coli. Assessments of local groundwater and geological conditions and improved water quality indicators may reveal more clear relationships. Our findings also suggest that the dominant contamination route for shallow groundwater sources is short-circuiting at the wellhead rather than subsurface transport.

The microbiome can promote or disrupt human health by influencing both adaptive and innate immune functions. We tested whether bacteria that normally reside on human skin participate in host defense by killing Staphylococcus aureus, a pathogen commonly found in patients with atopic dermatitis (AD) and an important factor that exacerbates this disease. High-throughput screening for antimicrobial activity against S. aureus was performed on isolates of coagulase-negative Staphylococcus (CoNS) collected from the skin of healthy and AD subjects. CoNS strains with antimicrobial activity were common on the normal population but rare on AD subjects. A low frequency of strains with antimicrobial activity correlated with colonization by S. aureus The antimicrobial activity was identified as previously unknown antimicrobial peptides (AMPs) produced by CoNS species including Staphylococcus epidermidis and Staphylococcus hominis These AMPs were strain-specific, highly potent, selectively killed S. aureus, and synergized with the human AMP LL-37. Application of these CoNS strains to mice confirmed their defense function in vivo relative to application of nonactive strains. Strikingly, reintroduction of antimicrobial CoNS strains to human subjects with AD decreased colonization by S. aureus These findings show how commensal skin bacteria protect against pathogens and demonstrate how dysbiosis of the skin microbiome can lead to disease.

The current drug regimens for treating tuberculosis are lengthy and onerous, and hence complicated by poor adherence leading to drug resistance and disease relapse. Previously, using an output-driven optimization platform and an in vitro macrophage model of Mycobacterium tuberculosis infection, we identified several experimental drug regimens among billions of possible drug-dose combinations that outperform the current standard regimen. Here we use this platform to optimize the in vivo drug doses of two of these regimens in a mouse model of pulmonary tuberculosis. The experimental regimens kill M. tuberculosis much more rapidly than the standard regimen and reduce treatment time to relapse-free cure by 75%. Thus, these regimens have the potential to provide a markedly shorter course of treatment for tuberculosis in humans. As these regimens omit isoniazid, rifampicin, fluoroquinolones and injectable aminoglycosides, they would be suitable for treating many cases of multidrug and extensively drug-resistant tuberculosis.

A zygomycete fungus, Choanephora cucurbitarum is a plant pathogen that causes blossom rot in cucurbits and other plants. Here we report the genome sequence of Choanephora cucurbitarum KUS-F28377 isolated from squash. The assembled genome has a size of 29.1 Mbp and 11,977 protein-coding genes. The genome analysis indicated that C. cucurbitarum may employ a plant pathogenic mechanism similar to that of bacterial plant pathogens. The genome contained 11 genes with a Streptomyces subtilisin inhibitor-like domain, which plays an important role in the defense against plant immunity. This domain has been found only in bacterial genomes. Carbohydrate active enzyme analysis detected 312 CAZymes in this genome where carbohydrate esterase family 6, rarely found in dikaryotic fungal genomes, was comparatively enriched. The comparative genome analysis showed that the genes related to sexual communication such as the biosynthesis of β-carotene and trisporic acid were conserved and diverged during the evolution of zygomycete genomes. Overall, these findings will help us to understand how zygomycetes are associated with plants.

pyrE (STM3733) encodes orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10), the fifth enzyme of the de novo pyrimidine biosynthetic pathway. We identified a ΔpyrE mutant as under selection in screening of a Salmonella mutant library in 4-day old chicks. Here, we confirm that a ΔpyrE mutant colonizes 4-day old chicks poorly in competitive infection with isogenic wild type, and that the ability of this mutant to colonize chicks could be restored by providing a copy of pyrE in trans. We further show that our ΔpyrE mutant grows poorly in nutrient poor conditions in vitro, and that the ability of this mutant to grow is restored, both in vitro and in chicks, when precursors to the pyrimidine salvage pathway were provided. This finding suggests that the environment in the chick intestine during our infections lacks sufficient precursors of the pyrimidine salvage pathway to support Salmonella growth. Finally, we show that the colonization defect of a ΔpyrE mutant during infection occurs in to chicks, but not in CBA/J mice or ligated ileal loops in calves. Our data suggest that de novo pyrimidine synthesis is necessary for colonization of Salmonella Typhimurium in the chick, and that the salvage pathway is not used in this niche.

Infections with Gram-negative pathogens pose a serious threat to public health. This scenario is exacerbated by increases in antibiotic resistance and the limited availability of vaccines and therapeutic tools to combat these infections. Here, we report an immunization approach that targets siderophores, which are small molecules exported by enteric Gram-negative pathogens to acquire iron, an essential nutrient, in the host. Because siderophores are nonimmunogenic, we designed and synthesized conjugates of a native siderophore and the immunogenic carrier protein cholera toxin subunit B (CTB). Mice immunized with the CTB-siderophore conjugate developed anti-siderophore antibodies in the gut mucosa, and when mice were infected with the enteric pathogen Salmonella, they exhibited reduced intestinal colonization and reduced systemic dissemination of the pathogen. Moreover, analysis of the gut microbiota revealed that reduction of Salmonella colonization in the inflamed gut was accompanied by expansion of Lactobacillus spp., which are beneficial commensal organisms that thrive in similar locales as Enterobacteriaceae. Collectively, our results demonstrate that anti-siderophore antibodies inhibit Salmonella colonization. Because siderophore-mediated iron acquisition is a virulence trait shared by many bacterial and fungal pathogens, blocking microbial iron acquisition by siderophore-based immunization or other siderophore-targeted approaches may represent a novel strategy to prevent and ameliorate a broad range of infections.

Aspergillus fumigatus is an opportunistic fungal pathogen that invades pulmonary epithelial cells and vascular endothelial cells by inducing its own endocytosis, but the mechanism by which this process occurs is poorly understood. Here, we show that the thaumatin-like protein CalA is expressed on the surface of the A. fumigatus cell wall, where it mediates invasion of epithelial and endothelial cells. CalA induces endocytosis in part by interacting with integrin α5β1 on host cells. In corticosteroid-treated mice, a ΔcalA deletion mutant has significantly attenuated virulence relative to the wild-type strain, as manifested by prolonged survival, reduced pulmonary fungal burden and decreased pulmonary invasion. Pretreatment with an anti-CalA antibody improves survival of mice with invasive pulmonary aspergillosis, demonstrating the potential of CalA as an immunotherapeutic target. Thus, A. fumigatus CalA is an invasin that interacts with integrin α5β1 on host cells, induces endocytosis and enhances virulence.

This study aimed to examine the effect of Zataria multiflora Boiss. mouthwash on the microbial load of the oral cavity in patients under mechanical ventilation. This randomized controlled trial was conducted in 2019 on patients under mechanical ventilation. Sampling was performed using the consecutive method. Using a block randomization method, 90 patients were allocated to three equal groups of 30 to receive mouth care using chlorhexidine, Z. multiflora, or normal saline. Mouthwash was used three times a day for a week. Eight hours before and after the intervention, sterile samples of saliva were collected and cultured in the laboratory. Chi-square, Fisher's exact test, paired t and McNemar’s tests, and analysis of variance were used to analyze the data. After the interventions, a significant difference in the microbial load was found between the three groups (P < 0.021). The Tukey post hoc test showed a significant difference between the group treated with Z. multiflora and those who received chlorhexidine (P value = 0.016). The frequency of patients with positive cultures was lower in the group treated with Z. multiflora and the differences were statistically significant in terms of Acinetobacter (P = 0.01) and Klebsiella pneumonia (P = 0.02). Z. multiflora mouthwash was effective in decreasing the microbial load of the oral cavity. This mouthwash can be used to reduce the microbial load of the oral cavity in patients under mechanical ventilation and reduce the risk of ventilator-associated pneumonia.