Quantitative detection and survival analysis of VBNC Salmonella Typhimurium in flour using droplet digital PCR and DNA-intercalating dyes.

The difficulty in detecting viable but non-culturable (VBNC) Salmonella by culture-dependent methods poses a risk to food safety. In our study, we applied a viability test to Salmonella following a lethal treatment and to flour samples inoculated with Salmonella to evaluate the effectiveness of viability polymerase chain reaction (PCR). Our findings revealed that the combination of both ddPCR and qPCR with those DNA-intercalating dyes could quantify viable cells at low concentrations when the plate counting method failed to detect them post-inactivation. Prolonged UV exposure did not induce cell membrane disruption, as confirmed with PMA-ddPCR, with insignificant differences in gene copies. However, samples exposed to DyeTox13 and DyeTox13 + EMA showed lower gene copy numbers, implying that enzymatic activity was decreased by UV exposure duration. In addition, temperature-dependent survival in flour revealed uniform decay rates and D values (time required for a 1 log reduction) of DNA in untreated samples across various temperatures. By contrast, different decay rates were observed with DNA-intercalating dyes (DyeTox13 and DyeTox13 + EMA), showing faster metabolic activity loss at higher temperatures in flour. The decay rates and D values, determined through plate counting and those DNA-intercalating dyes, indicated the potential presence of VBNC Salmonella. A strong correlation between DyeTox13 dyes and the plate counting method suggested DyeTox13 as a rapid alternative for detecting Salmonella in flour. The ddPCR with DNA-intercalating dyes could effectively evaluate Salmonella viability, facilitating more precise monitoring of VBNC in food. IMPORTANCE: Salmonella, a major foodborne pathogen, poses significant risks, particularly to vulnerable groups like infants, older people, and the immunocompromised. Accurate detection is vital for public health and food safety, given its potential to cause severe and life-threatening symptoms. Our study demonstrated digital polymerase chain reaction (ddPCR) with DNA-intercalating dyes for identifying the different physiological statuses of Salmonella. Also, the application of ddPCR with DNA-intercalating dyes offers quantification of viable cells post-disinfection as an alternative method in food. Utilizing ddPCR and DNA-intercalating dyes, we enhanced the detection of VBNC Salmonella, a form often undetectable by conventional methods. This innovative approach could significantly improve the precision and efficiency of detection for viable Salmonella. By providing deeper insights into its transmission potential, our method is a critical tool in preventing outbreaks and ensuring the safety of food products. This research contributes substantially to global efforts in controlling foodborne illnesses and safeguarding public health.

Spatial transcriptomic brain imaging reveals the effects of immunomodulation therapy on specific regional brain cells in a mouse dementia model.

Increasing evidence of brain-immune crosstalk raises expectations for the efficacy of novel immunotherapies in Alzheimer's disease (AD), but the lack of methods to examine brain tissues makes it difficult to evaluate therapeutics. Here, we investigated the changes in spatial transcriptomic signatures and brain cell types using the 10x Genomics Visium platform in immune-modulated AD models after various treatments. To proceed with an analysis suitable for barcode-based spatial transcriptomics, we first organized a workflow for segmentation of neuroanatomical regions, establishment of appropriate gene combinations, and comprehensive review of altered brain cell signatures. Ultimately, we investigated spatial transcriptomic changes following administration of immunomodulators, NK cell supplements and an anti-CD4 antibody, which ameliorated behavior impairment, and designated brain cells and regions showing probable associations with behavior changes. We provided the customized analytic pipeline into an application named STquantool. Thus, we anticipate that our approach can help researchers interpret the real action of drug candidates by simultaneously investigating the dynamics of all transcripts for the development of novel AD therapeutics.

Lumen air pressure regulated multifunctional microbiotas in membrane-aerated biofilm reactors for simultaneous nitrogen removal and antibiotic elimination from aquaculture wastewater.

In this study, two membrane-aerated biofilm reactors (MABRs) were constructed: one solely utilizing biofilm and another hybrid MABR (HMABR) incorporating both suspended-sludge and biofilm to treat low C/N aquaculture wastewater under varying lumen air pressure (LAP). Both HMABR and MABR demonstrated superior nitrogen removal than conventional aeration reactors. Reducing LAP from 10 kPa to 2 kPa could enhance denitrification processes without severely compromising nitrification, resulting in an increase in total inorganic nitrogen (TIN) removal from 50.2±3.1 % to 71.6±1.0 %. The HMABR exhibited better denitrification efficacy than MABR, underscoring its potential for advanced nitrogen removal applications. A decline in LAP led to decreased extracellular polymeric substance (EPS) production, which could potentially augment reactor performance by minimizing mass transfer resistance while maintaining microbial matrix stability and function. Gene-centric metagenomics analysis revealed decreasing LAP impacted nitrogen metabolic potentials and electron flow pathways. The enrichment of napAB at higher LAP and the presence of complete ammonia oxidation (Comammox) Nitrospira at lower LAP indicated aerobic denitrification and Comammox processes in nitrogen removal. Multifunctional microbial communities developed under LAP regulation, diversifying the mechanisms for simultaneous nitrification-denitrification. Increased denitrifying gene pool (narGHI, nirK, norB) and enzymatic activity at a low LAP can amplify denitrification by promoting denitrifying genes and electron flow towards denitrifying enzymes. Sulfamethoxazole (SMX) was simultaneously removed with efficiency up to 80.2 ± 3.7 %, mainly via biodegradation, while antibiotic resistome and mobilome were propagated. Collectively, these findings could improve our understanding of nitrogen and antibiotic removal mechanisms under LAP regulation, offering valuable insights for the effective design and operation of MABR systems in aquaculture wastewater treatment.

Psychometric properties and measurement invariance of the short form of grit scale in Korean adolescents.

This study aimed to identify the factor structure of the Korean version of the Short Grit Scale (Grit-S) and examine its cross-sectional and longitudinal measurement invariance (MI). Data from the Korean Children and Youth Panel Survey 2018 were analyzed, which included two cohorts, comprising 2,327 and 2,325 fourth-year elementary and first-year middle school students, respectively. It was found that the two-factor model fit the data well for the elementary and middle school samples. The results of the cross-sectional MI tests across genders indicated that the full threshold and loading invariance were also supported for the elementary school sample, and the partial threshold and loading invariance were supported for the middle school sample. The analyses of the longitudinal MI revealed that the partial threshold and loading invariance were supported for both samples. The reliability analysis revealed satisfactory McDonald's Omega values for both samples at each time point and moderate stability coefficients over time. Based on these findings, it was concluded that the Korean version of the Grit-S demonstrated satisfactory psychometric properties and exhibited MI across gender and time in Korean adolescents.

Novel use of ferrous iron/peroxymonosulfate for high-performance seawater desalination pretreatment under harmful algal blooms.

Marine harmful algae bloom (HAB) is a growing threat to desalination plants worldwide. This work proposes ferrous iron/peroxymonosulfate (Fe2+/PMS) as a novel pretreatment technology for seawater reverse osmosis (SWRO) under HAB. Herein, Fe2+/PMS achieved a significantly higher reduction of negative charge of algae-laden seawater as compared to conventional coagulation (i.e., coagulant is Fe3+), which thereby facilitated improved flocculation to remove algal cells, turbidity and algal organics matters (AOMs), and marine Ca2+ (∼430 mg/L) could partially contribute to the enhanced coagulation performance. A new understanding of the improved coagulation efficiency achieved with Fe2+/PMS in seawater has been proposed as compared to freshwater: seawater matrix (e.g., 504 mM Cl-) was demonstrated to significantly enhance the generation of high-valent iron (FeO2+) as the main reactive intermediate instead of the long-recognized Fe3+ and free radicals, as revealed by methyl phenyl sulfoxide (PMSO) probe, radicals scavenging analysis and electron spin resonance (ESR) spectra. This new mechanism is expected to provide valuable insights for the development of more novel oxidative seawater treatment technologies. Of note, while trade-off between particles and AOMs played an important role in membrane fouling reduction by different dosages of Fe2+/PMS, Fe2+/PMS with an optimal dosage of 0.1 mM/0.05 mM achieved an unprecedentedly higher reduction (95.26%) of modified fouling index (MFI) as compared to conventional coagulation (13.28%-42.36% with 0.1-0.2 mM of Fe3+). Optical-photothermal infrared spectromicroscopy with sub-micron spatial resolution was employed to analyze membrane foulants for the first time, and Fe2+/PMS was found to mainly cause reduced cake layer resistance, which was attributed to the collectively reduced concentration of algae cells, micro-particles with sizes from 2 to 10 µm, humic substances and biopolymers. Moreover, Fe2+/PMS resulted in lower dissolved Fe3+ (<0.027 mg/L) in ultrafiltration (UF) permeate, which would make it more reliable for SWRO operation as compared to conventional coagulation. When energy-intensive dissolved air flotation (DAF) was employed to withstand HAB, Fe2+/PMS outperformed it and was instrumental in achieving reduced MFI with 56.4% lower operational cost. In this context, Fe2+/PMS would facilitate a high-performance and low-cost pretreatment technology for seawater desalination plants under HAB.

Spatiotemporal characterization of glial cell activation in an Alzheimer's disease model by spatially resolved transcriptomics.

The molecular changes that occur with the progression of Alzheimer's disease (AD) are well known, but an understanding of the spatiotemporal heterogeneity of changes in the brain is lacking. Here, we investigated the spatially resolved transcriptome in a 5XFAD AD model at different ages to understand regional changes at the molecular level. Spatially resolved transcriptomic data were obtained from 5XFAD AD models and age-matched control mice. Differentially expressed genes were identified using spots clustered by anatomical structures. Gene signatures of activation of microglia and astrocytes were calculated and mapped on the spatially resolved transcriptomic data. We identified early alterations in the white matter (WM) of the AD model before the definite accumulation of amyloid plaques in the gray matter (GM). Changes in the early stage of the disease involved primarily glial cell activation in the WM, whereas the changes in the later stage of pathology were prominent in the GM. We confirmed that disease-associated microglia (DAM) and astrocyte (DAA) signatures also showed initial changes in WM and that activation spreads to GM. Trajectory inference using microglial gene sets revealed the subdivision of DAMs with different spatial patterns. Taken together, these results help to understand the spatiotemporal changes associated with reactive glial cells as a major pathophysiological characteristic of AD. The heterogeneous spatial molecular changes apply to identifying diagnostic and therapeutic targets caused by amyloid accumulation in AD.

Unveiling the impact of short-term polyethylene microplastics exposure on metabolomics and gut microbiota in earthworms (Eudrilus euganiae).

Microplastics (MPs) pose a significant environmental concern, particularly for terrestrial fauna. In this study, earthworms were used as a model organism to investigate the ecotoxicological effects of short-term exposure to virgin MPs on changes in metabolome and gut microbiota. High-throughput untargeted metabolomics showed significant internal reactions in the earthworms' metabolic processes due to MPs exposure, even when no visible stress signs, such as changes in growth or mortality rates, were present. Earthworms exposed to different concentrations of polyethylene (PE) MP exhibited significant disruption in 39 and 199 molecular features related to energy and lipid metabolism, anti-inflammatory, cell signaling, and membrane integrity. The activities of enzymes and transport proteins in earthworms were dysregulated when exposed to PE. Changes in the gut microbiota's community structure and complexity were observed in response to PE MPs exposure. Despite the relative stability in alpha-diversity and relative abundance, shifts in beta-diversity and network analysis in the PE-exposed group were indicative of an adaptive response to MPs. Earthworms exhibited resilience or adaptation in response to MPs exposure, potentially maintaining their functionality. This study provides preliminary insights into the impact of MPs on soil invertebrates like earthworms and highlights the need for further exploration of long-term effects and underlying molecular mechanisms.

Multichannel Microfluidic Platform for Temporal-Spatial Investigation of Niche Roles of Pseudomonas aeruginosa and Escherichia coli within a Dual-Species Biofilm.

In natural or man-made environments, microorganisms exist predominantly as biofilms forming surface-associated bacterial communities embedded in extracellular polymeric substances (EPSs). Often, biofilm reactors used for endpoint and disruptive analyses of biofilm are not suitable for periodic observation of biofilm formation and development. In this study, a microfluidic device designed with multiple channels and a gradient generator was used for high-throughput analysis and real-time monitoring of dual-species biofilm formation and development. We compared the structural parameters of monospecies and dual-species biofilms containing Pseudomonas aeruginosa (expressing mCherry) and Escherichia coli (expressing green fluorescent protein [GFP]) to understand the interactions in the biofilm. The rate of biovolume increase of each species in monospecies biofilm (2.7 × 105 µm3) was higher than those in a dual-species biofilm (9.68 × 104 µm3); however, synergism was still observed in the dual-species biofilm due to overall increases in biovolume for both species. Synergism was also observed in a dual-species biofilm, where P. aeruginosa forms a "blanket" over E. coli, providing a physical barrier against shear stress in the environment. The microfluidic chip was useful for monitoring the dual-species biofilm in the microenvironment, indicating that different species in a multispecies biofilm exhibit different niches for the survival of the biofilm community. Finally, we demonstrated that the nucleic acids can be extracted from the dual-species biofilm in situ after biofilm imaging analysis. In addition, gene expression supported that the activation and suppression of different quorum sensing genes resulted in the different phenotype seen in the biofilm. This study showed that the integration of microfluidic device with microscopy analysis and molecular techniques could be a promising tool for studying biofilm structure and gene quantification and expression simultaneously. IMPORTANCE In natural or man-made environments, microorganisms exist predominantly as biofilms forming surface-associated bacterial communities embedded in extracellular polymeric substances (EPSs). Often, biofilm reactors used for endpoint and disruptive analyses of biofilm are not suitable for periodic observation of biofilm formation and development. Here, we demonstrate that a microfluidic device with multiple channels and a gradient generator can be useful for high-throughput analysis and real-time monitoring of dual-species biofilm formation and development. Our study revealed synergism in the dual-species biofilm, where P. aeruginosa forms a "blanket" over E. coli, providing a physical barrier against shear stress in the environment. Furthermore, different species in a multispecies biofilm exhibit different niches for the survival of the biofilm community. This study showed that the integration of microfluidic device with microscopy analysis and molecular techniques could be a promising tool for studying biofilm structure and gene quantification and expression simultaneously.

spSeudoMap: cell type mapping of spatial transcriptomics using unmatched single-cell RNA-seq data.

Since many single-cell RNA-seq (scRNA-seq) data are obtained after cell sorting, such as when investigating immune cells, tracking cellular landscape by integrating single-cell data with spatial transcriptomic data is limited due to cell type and cell composition mismatch between the two datasets. We developed a method, spSeudoMap, which utilizes sorted scRNA-seq data to create virtual cell mixtures that closely mimic the gene expression of spatial data and trains a domain adaptation model for predicting spatial cell compositions. The method was applied in brain and breast cancer tissues and accurately predicted the topography of cell subpopulations. spSeudoMap may help clarify the roles of a few, but crucial cell types.

Different types of land use influence soil physiochemical properties, the abundance of nitrifying bacteria, and microbial interactions in tropical urban soil.

Anthropogenic activities have led to unexpected changes in microbial community composition and structure, resulting in an interruption of soil ecological roles in urban environments. We questioned the impact of the different land use (e.g., agricultural, industrial, recreational, coastal, and residential areas) on the distribution of nitrifying bacteria and microbial interaction in tropical soil. The dominant nitrifying bacteria were ammonia-oxidizing archaea (AOA) in tropical soils up to 107 copies/g of soil, while the abundance of ammonia-oxidizing bacteria (AOB) was significantly higher in agricultural soil only. Comammox (CMX) was ubiquitous up to 105 copies/g of tropical soil, indicating that CMX might share ecological niches with AOA and considerably contribute to nitrification in urban areas. The most abundant phylum is Actinobacteria, accounting for 27-34 % relative abundance among most land-use types, but Proteobacteria was observed as the most prevalent phylum in agricultural soil. The physicochemical properties (e.g., soil pH and nutrient contents) of different types of land use influenced microbial richness and diversities associated with nitrogen cycling. Multivariate analysis disclosed that agricultural soils were distinct from other land uses because of the concentrations of nutrients and heavy metals and the abundance of microorganisms associated with nitrogen cycles. Also, the microbial co-occurrence network revealed that agricultural soils were a highly interconnected network of the microbial community. In this study, C: N ratio might have a significant impact on ecological networks and the abundance of nitrogen-related taxa, which could influence microbial interactions and complexity in tropical soils. Thus, the impact of anthropogenic land use induced changes in microbial composition and diversity, co-occurrence network, and nitrifying bacteria, leading to potential transformation in ecological services of tropical soils and nitrogen cycling in urban environments.

Reliability and feasibility of visual grading systems and quantitative indexes on [99mTc]Tc-DPD imaging for cardiac amyloidosis.

We aimed to evaluate the reliability and feasibility of visual grading systems and various quantitative indexes of [99mTc]Tc-DPD imaging for cardiac amyloidosis (CA). Patients who underwent [99mTc]Tc-DPD imaging with suspicion of CA were enrolled. On the planar image, myocardial uptake was visually graded using Perugini's and Dorbala's methods (PS and DS). As [99mTc]Tc-DPD indexes, heart-to-whole body ratio (H/WB) and heart-to-contralateral lung ratio (H/CL) were measured on planar image. SUVmax, SUVmean, total myocardial uptake (TMU), and C-index were measured on SPECT/CT. Inter-observer agreement of the indexes and their association with visual grading and clinical factors were evaluated. A total of 152 [99mTc]Tc-DPD images, of which 18 were positive, were analyzed. Inter-observer agreement was high for both DS (κ = 0.95) and PS (κ = 0.96). However, DS showed a higher correlation with quantitative indexes than PS. Inter-observer agreement was also high for SPECT/CT indexes, particularly SUVmax. SUVmax was significantly different between different DS groups (P = 0.014-0.036), and showed excellent correlations with H/WB and H/CL (r = 0.898 and 0.910). SUVmax also showed significant differences between normal, AL, and ATTR pathology (P = 0.022-0.037), and a significant correlation with extracellular volume on cardiac MRI (r = 0.772, P < 0.001). DS is a visual grading system for CA that is more significantly matched with quantitative indexes than PS. SUVmax is a reliable quantitative index on SPECT/CT, with a high inter-observer agreement, correlations with the visual grade, and potential association with cardiac MRI findings.

Acute exposure to microplastics induces metabolic disturbances and gut dysbiosis in adult zebrafish (Danio rerio).

There is limited knowledge of the ecotoxicological impacts of MPs at the environmentally relevant concentration on freshwater animals, even though numerous studies have demonstrated the toxic effects of MPs on living organisms. In this study, zebrafish (Danio rerio) was used as a model organism to investigate the ecotoxicological effects of acute exposure of virgin MPs on changes in metabolome and gut microbiota. High-throughput untargeted metabolomics using liquid chromatography with tandem mass spectrometry (LC-MS/MS) provided comprehensive insights into the metabolic responses of zebrafish exposed to PE (polyethylene) and PES (polyester) MPs. Statistical analysis of metabolomics data indicated that 39 and 27 metabolites, such as lysophosphatidylcholine, phosphocholine, phosphatidylserine, triglyceride, glycosphingolipid, psychosine, 8-amino-7-oxononanoate, cholesterol fatty acid ester, phosphatidylinositol, n-Triacontanol, were significantly altered in PE- and PES-exposed zebrafish, respectively. Furthermore, the enrichment pathway analysis unveiled the synthesis of the structural and functional lipids, signaling molecules, fatty alcohol metabolism, and amino acid metabolism, which was considerably perturbated in MPs-exposed zebrafish. In addition, high-throughput DNA sequencing was conducted to examine changes in gut microbiota in the MPs-treated zebrafish. The MPs exposure increased in the relative abundance of Fusobacteria and Proteobacteria, while the relative abundance of Firmicutes declined in MPs-treated zebrafish. Also, microbial diversity and linear discriminant analyses indicated microbiota dysbiosis, metabolomic dysregulation, and oxidative stress. Taken together, the acute exposure of MPs at environmentally relevant concentrations could disrupt the metabolic interaction via the microbiota-gut-liver-brain relationship, implying gastrointestinal and neurological/immune disorders in zebrafish.

Changes in physiological states of Salmonella Typhimurium measured by qPCR with PMA and DyeTox13 Green Azide after pasteurization and UV treatment.

Diarrheal diseases caused by Salmonella pose a major threat to public health, and assessment of bacterial viability is critical in determining the safety of food and drinking water after disinfection. Viability PCR could overcome the limitations of traditional culture-dependent methods for a more accurate assessment of the viability of a microbial sample. In this study, the physiological changes in Salmonella Typhimurium induced by pasteurization and UV treatment were evaluated using a culture-based method, RT-qPCR, and viability PCR. The plate count results showed no culturable S. Typhimurium after the pasteurization and UV treatments, while viability PCR with propidium monoazide (PMA) and DyeTox13-qPCR indicated that the membrane integrity of S. Typhimurium remained intact with no metabolic activity. The RT-qPCR results demonstrated that invasion protein (invA) was detectable in UV-treated cells even though the log2-fold change ranged from - 2.13 to - 5.53 for PMA treatment. However, the catalytic activity gene purE was under the detection limit after UV treatment, indicating that most Salmonella entered metabolically inactive status after UV disinfection. Also, viability PCRs were tested with artificially contaminated eggs to determine physiological status on actual food matrices. DyeTox13-qPCR methods showed that most Salmonella lost their metabolic activity but retained membrane integrity after UV disinfection. RT-qPCR may not determine the physiological status of Salmonella after UV disinfection because mRNA could be detectable in UV-treated cells depending on the choice of target gene. Viability PCR demonstrated potential for rapid and specific detection of pathogens with physiological states such as membrane integrity and metabolic activity.Key Points• Membrane integrity of Salmonella remained intact with no metabolic activity after UV.• mRNA could be detectable in UV-treated cells depending on the choice of target gene.• Viability PCR could rapidly detect specific pathogens with their physiological states.

CellDART: cell type inference by domain adaptation of single-cell and spatial transcriptomic data.

Deciphering the cellular composition in genome-wide spatially resolved transcriptomic data is a critical task to clarify the spatial context of cells in a tissue. In this study, we developed a method, CellDART, which estimates the spatial distribution of cells defined by single-cell level data using domain adaptation of neural networks and applied it to the spatial mapping of human lung tissue. The neural network that predicts the cell proportion in a pseudospot, a virtual mixture of cells from single-cell data, is translated to decompose the cell types in each spatial barcoded region. First, CellDART was applied to a mouse brain and a human dorsolateral prefrontal cortex tissue to identify cell types with a layer-specific spatial distribution. Overall, the proposed approach showed more stable and higher accuracy with short execution time compared to other computational methods to predict the spatial location of excitatory neurons. CellDART was capable of decomposing cellular proportion in mouse hippocampus Slide-seq data. Furthermore, CellDART elucidated the cell type predominance defined by the human lung cell atlas across the lung tissue compartments and it corresponded to the known prevalent cell types. CellDART is expected to help to elucidate the spatial heterogeneity of cells and their close interactions in various tissues.

Acute exposure effects of tetracycline, ampicillin, sulfamethoxazole, and their mixture on nutrient removal and microbial communities in the activated sludge of air-scouring and reciprocation membrane bioreactors.

The fate of antibiotics, their effects on non-target species, and the spread of antibiotic resistance in wastewater treatment systems have been of concern in recent years. Despite its importance, the effects of these antibiotics on biological nutrient removal in WWTPs have not been completely elucidated. To evaluate the effects of antimicrobial compounds on nutrient removal performance and microbiome, batch experiments were performed using activated sludge samples taken from two distinct membrane bioreactor systems (reciprocation MBR vs. air-scouring MBR). We exposed the activated sludge to 0 mg/L, 0.1 mg/L, and 1.0 mg/L of tetracycline (TET), ampicillin (AMP), sulfamethoxazole (SUL), and their mixture. The mixture of antibiotics significantly decreased ammonia removal efficiency in the reciprocation MBR (rMBR) and air-scouring MBR (AS MBR) by 5% and 12%, respectively. A significant reduction (p < 0.05) in the amoA-AOB gene was observed in AS MBR, while this gene remained unaffected in the rMBR. Interestingly, the gene abundance of amoA from comammox Nitrospira increased from 2.8 × 108 gene copies per gram sludge (0 mg/L) to 5.0 × 108 gene copies per gram sludge (1.0 mg/L) in the setup with antibiotics in the mixture. Correlation analysis of the relative abundance of prevalent taxa and antibiotic concentrations showed that the microbial communities of the AS MBR were more susceptible to TET and MXD antibiotics than the rMBR microbiome.

Assessment of physiological responses of bacteria to chlorine and UV disinfection using a plate count method, flow cytometry and viability PCR.

AIMS: This study aimed to investigate the physiological responses of two gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two gram-positive bacteria (Enterococcus faecalis and Bacillus sphaericus) to ultraviolet (UV) and chlorine disinfection. METHODS AND RESULTS: Bacterial inactivation by UV and chlorine disinfection were evaluated with a plate count method for culturability, FCM and PMA-qPCR for membrane integrity and DyeTox13-qPCR for enzymatic activity, respectively. Both UV and chorine disinfection caused complete loss of culturability while membrane integrity remained intact after UV disinfection. Both DyeTox13-qPCR and PMA-qPCR showed high ΔCt values up to 8.9 after chlorine disinfection, indicating that both methods were able to distinguish non-treated from chlorine-treated cells. Although PMA-qPCR could not differentiate membrane integrity of cells on UV exposure, DyeTox13-qPCR showed significant differences in ΔCt values of 5.05 and 10.4 for gram-negative (E. coli) and gram-positive (Enterococcus) bacteria, respectively. However, DyeTox13-qPCR for gram-negative bacteria displayed relatively small differences in ΔCt values compared with gram-positive bacteria. CONCLUSION: UV and chlorine disinfection led to changes in physiological state of gram-negative and gram-positive bacteria. Particularly, UV disinfection could induce active but non-culturable (ABNC) for gram-negative bacteria and dormant cell for gram-positive bacteria where intact cells no longer showed the enzymatic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: UV and chlorine are commonly used to disinfect water, food and fomites to inactivate pathogenic bacteria. However, a viable but non-culturable (VBNC) state of bacteria induced by disinfection may underestimate the health risks because of the potential resuscitation of VBNC cells. This study highlighted that bacteria could undergo different physiological (ABNC or dormant) states during UV and chlorine disinfection. In addition, viability PCR techniques could provide insight into the changes in physiological states during disinfection processes.

Debromination of TetraBromoBisphenol-A (TBBPA) depicting the metabolic versatility of Dehalococcoides.

TetraBromoBisphenol-A (TBBPA) is a widely used brominated flame retardant and an emerging contaminant that has amassed significant environmental impacts. Though there are a few studies that report the bioremediation of TBBPA, there is no direct evidence to suggest a metabolic use of TBBPA as the sole electron acceptor, which offers an advantage in the complete and energy-efficient process of debromination under anaerobic conditions. In this study, Dehalococcoides mccartyi strain CG1 was identified to be capable of utilizing TBBPA as the sole electron acceptor at its maximum soluble concentrations (7.3 µM) coupled with cell growth. A previously characterized reductive dehalogenase (RDase), PcbA1, and six other RDases of strain CG1 were detected during TBBPA debromination via transcriptional and proteomic analyses. Furthermore, as a commonly co-contaminated brominated flame retardant of TBBPA, penta-BDEs were debrominated synchronously with TBBPA by strain CG1. This study provides deeper insights into the versatile dehalogenation capabilities of D. mccartyi strain CG1 and its role in in situ remediations of persistent organic pollutants in the environment.

Limited implication of initial bone scintigraphy on long-term condylar bone change in temporomandibular disorders-Comparison with cone beam computed tomography at 1 year.

BACKGROUND: The current diagnostic criteria for temporomandibular disorders (TMD) do not require imaging for the diagnosis of degenerative joint disease (DJD) of the temporomandibular joint (TMJ) condyle, and there is a lack of data investigating the effectiveness of imaging modalities in predicting long-term TMJ DJD prognosis. OBJECTIVES: To verify the association between initial bone scintigraphy results and long-term DJD bone changes occurring in the TMJ condyle on cone beam computed tomography (CBCT). METHODS: Initial bone scintigraphy, panoramic radiography and CBCT results were analysed in relation to long-term (12 months) TMJ DJD bone change on CBCTs in 55 TMD patients (110 joints). Clinical and radiographic indices were statistically analysed among three groups (improved, no change, and worsened) based on long-term TMJ DJD prognosis calculated by destructive change index (DCI). RESULTS: Neither the uptake ratio nor visual assessment results from initial bone scintigraphy showed a significant difference according to long-term condylar bone change groups. The cut-off value of bone scintigraphy uptake ratio was 2.53 for long-term worsening of TMJ DJD. Worsening of TMJ DJD was significantly associated with the diagnosis based on panoramic radiography (p = .011) and CBCT (p < .001). Initial DCI (ß = -.291, p = .046) had a significant association with long-term worsening of TMJ DJD. CONCLUSION: Initial bone scintigraphy results did not show sufficiently close associations with long-term TMJ DJD prognosis. This should be considered in the selection process of imaging modalities for TMJ DJD patients. Future studies are needed to develop prognostic indices that comprise both clinical and imaging contents for improved predictive ability.

Rapid quantification of fecal indicator bacteria in water using the most probable number - loop-mediated isothermal amplification (MPN-LAMP) approach on a polymethyl methacrylate (PMMA) microchip.

Fecal contamination of water and its associated pathogens are a major public health concern in both developing and industrialized areas. Fecal indicator bacteria (FIB) are commonly used to assess microbial water quality, but they require a relatively long period of incubation time. Currently, molecular techniques have been applied to rapidly detect FIB. However, these molecular techniques require expensive and sophisticated equipment. In this study, we developed a rapid on-chip gene quantification method based on loop-mediated isothermal amplification (LAMP) PCR. The LAMP assays can measure the target genes of the fecal indicator bacteria (FIB), including E. coli and Enterococcus spp, using the most probable number (MPN) approach. The colorimetric LAMP assay allows for naked-eye observation of the PCR reaction as few as 4 gene copies / well. When the reaction ends, MPN measurement of positive outcomes on the white-based PMMA (polymethacrylic acid) microchips provides the concentrations of the target genes of FIB with a confidence interval. We validated the feasibility of the MPN-LAMP approach by obtaining a strong correlation between the results of the MPN estimations and the qPCR analysis. Moreover, the MPN-LAMP approach was used to quantify the FIB in different environmental water collected from the freshwater reservoirs, beach, agriculture farm, and sewage. Our research demonstrates that the MPN- LAMP method enables us to easily and quickly quantifying FIB genes isolated from the environment without expensive qPCR instruments.

Effects of monochloramine on culturability, viability and persistence of Pseudomonas putida and tap water mixed bacterial community.

Bacterial biofilms are able to persist in drinking water distribution systems (DWDS) even if disinfectants such as monochloramine are used to inhibit bacterial colonization and biofilm formation. While studies have determined the monochloramine concentrations required to inhibit bacterial biofilms, not much is known about how bacterial biofilms develop resistance towards monochloramine. This study covers the development of resistance to monochloramine in both single species and mixed bacterial biofilms. Through culturability tests and flow cytometry, exposing bacterial biofilms to monochloramine disinfection using a sub-lethal concentration (1.5 mg/L Cl2, experimentally determined) was sufficient to cause an increase of the monochloramine's inhibitory concentrations by as much as two times than what is initially required to inhibit biofilm growth. Through persister cultures and 16S rRNA next generation sequencing (NGS) studies, mixed bacterial biofilms experienced to monochloramine exposure resulted in more bacterial genera becoming persistent and resistant towards monochloramine. Through this study, bacterial genera that were persistent towards monochloramine were suggested to share common traits including the ability (1) to readily enter a persister or viable but non-culturable (VBNC) state and (2) to form biofilms primarily comprising proteinaceous extra-polymeric substances (EPS). Both of these traits also suggested that selected bacterial genera tended to be more persistent to monochloramine and produce EPS. This study advances our understanding of bacterial biofilm resistance towards monochloramine and showed the importance of maintaining monochloramine concentrations in DWDS to prevent the development of bacterial resistance towards monochloramine. KEY POINTS: • Monochloramine-resistant biofilm was developed after sub-lethal disinfection. • Mixed-species culture experienced monochloramine showed more persistence to monochloramine. • Ability to enter a persister/VBNC state is a common trait of persistent bacteria genera.