Direct or DNA Extraction-Free Amplification and Quantification of Foodborne Pathogens.

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

A Comprehensive Guide to Quality Assessment and Data Submission for Genomic Surveillance of Enteric Pathogens.

This document outlines the steps necessary to assemble and submit the standard data package required for contributing to the global genomic surveillance of enteric pathogens. Although targeted to GenomeTrakr laboratories and collaborators, these protocols are broadly applicable for enteric pathogens collected for different purposes. There are five protocols included in this chapter: (1) quality control (QC) assessment for the genome sequence data, (2) validation for the contextual data, (3) data submission for the standard pathogen package or Pathogen Data Object Model (DOM) to the public repository, (4) viewing and querying data at NCBI, and (5) data curation for maintaining relevance of public data. The data are available through one of the International Nucleotide Sequence Database Consortium (INSDC) members, with the National Center for Biotechnology Information (NCBI) being the primary focus of this document. NCBI Pathogen Detection is a custom dashboard at NCBI that provides easy access to pathogen data plus results for a standard suite of automated cluster and genotyping analyses important for informing public health and regulatory decision-making.

Identifying Putative Biomarkers of Foodborne Pathogens Using a Metabolomic Approach.

Metabolomics is the study of low molecular weight biochemical molecules (typically <1500 Da) in a defined biological organism or system. In case of food systems, the term "food metabolomics" is often used. Food metabolomics has been widely explored and applied in various fields including food analysis, food intake, food traceability, and food safety. Food safety applications focusing on the identification of pathogen-specific biomarkers have been promising. This chapter describes a nontargeted metabolite profiling workflow using gas chromatography coupled with mass spectrometry (GC-MS) for characterizing three globally important foodborne pathogens, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica, from selective enrichment liquid culture media. The workflow involves a detailed description of food spiking experiments followed by procedures for the extraction of polar metabolites from media, the analysis of the extracts using GC-MS, and finally chemometric data analysis using univariate and multivariate statistical tools to identify potential pathogen-specific biomarkers.

Effects of reductive soil disinfestation on potential pathogens and antibiotic resistance genes in soil.

Reductive soil disinfestation (RSD) is commonly employed for soil remediation in greenhouse cultivation. However, its influence on antibiotic resistance genes (ARGs) in soil remains uncertain. This study investigated the dynamic changes in soil communities, potential bacterial pathogens, and ARG profiles under various organic material treatments during RSD, including distillers' grains, potato peel, peanut vine, and peanut vine combined with charcoal. Results revealed that applying diverse organic materials in RSD significantly altered bacterial community composition and diminished the relative abundance of potential bacterial pathogens (P < 0.05). The relative abundance of high-risk ARGs decreased by 10.7%-30.6% after RSD treatments, the main decreased ARG subtypes were AAC(3)_Via, dfrA1, ErmB, lnuB, aadA. Actinobacteria was the primary host of ARGs and was suppressed by RSD. Soil physicochemical properties, such as total nitrogen, soil pH, total carbon, were crucial factors affecting ARG profiles. Our findings demonstrated that RSD treatment inhibited pathogenic bacteria and could be an option for reducing high-risk ARG proliferation in soil.

An electrochemical sensor based on 2D Zn-MOFs and 2D C-Ti3C2Tx composite materials for rapid and direct detection of various foodborne pathogens.

Rapid screening for foodborne pathogens is crucial for food safety. A rapid and one-step electrochemical sensor has been developed for the detection of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Salmonella typhimurium (S. typhimurium). Through the construction of aptamer/two-dimensional carboxylated Ti3C2Tx (2D C-Ti3C2Tx)/two-dimensional Zn-MOF (2D Zn-MOF) composites, the recognition elements, signal tags, and signal amplifiers are integrated on the electrode surface. Pathogens are selectively captured using the aptamer, which increases the impedance of the electrode surface，leads to a decrease in the 2D Zn-MOF current. Bacteria can be rapidly quantified using a one-step detection method and the replacement of aptamers. The detection limits for E. coli, S. aureus, and S. typhimurium are 6, 5, and 5 CFU·mL-1, respectively. The sensor demonstrated reliable detection capabilities in real-sample testing. Therefore, the one-step sensor based on the 2D Zn-MOF and 2D C-Ti3C2Tx has significant application value in the detection of foodborne pathogens.

Carvacrol-loaded chia mucilage nanocapsules as sanitizer to control Salmonella, Escherichia coli and Listeria monocytogenes in green cabbage.

Cabbage is susceptible to various microbiological risks, frequently serving as a vehicle for pathogenic bacteria, mainly Salmonella and Escherichia coli. Therefore, ensuring the safety of this vegetable is essential to reduce the risk of foodborne illnesses. Traditional sanitization using chlorinated water, although effective, raises concerns due to the production of potentially carcinogenic compounds, and this method is banned in some countries. In recent years, alternative sanitizing methods have been developed using essential oils (EOs). However, EOs present high volatility, limited solubility in water, and strong odor and taste. This study introduces an innovative approach to overcome these disadvantages by employing carvacrol encapsulated into chia mucilage nanocapsules (CMNC), prepared through high-energy homogenization. Encapsulating carvacrol in chia mucilage nanocapsules helps to mask its strong sensory characteristics, making it more suitable and acceptable for use in food applications. The antimicrobial efficacy of CMNC (1.67 mg/mL), carvacrol emulsion (CE: 10.6 mg/mL), and chlorine solution (CS: 200 ppm) was evaluated against Salmonella, E. coli, and Listeria monocytogenes. CMNC decreased Salmonella to levels below the detection limit of the technique (< 2 log CFU/g), reduced 3.5 log CFU/g of E. coli, and 2.5 log CFU/g of L. monocytogenes. These results are similar to or better than those obtained with CS. In addition, sanitizing cabbage with CMNC preserved the firmness and color of the samples, important aspects for consumer acceptance. This innovative approach is promising for increasing the food safety of cabbage, while mitigating the potential drawbacks associated with traditional sanitization methods.

Field-scale gene flow of fungicide resistance in Pyrenophora teres f. teres and the effect of selection pressure on the population structure.

The effectiveness of fungicides to control foliar fungal crop diseases is being diminished by the increasing spread of resistances to fungicides. One approach that may help to maintain efficacy is remediation of resistant populations by sensitive ones. However, the success of such approaches can be compromised by re-incursion of resistance through aerial spore dispersal; although, knowledge of localized gene flow is lacking. Here, we report on a replicated mark-release-recapture field experiment with several treatments set up to study spore-dispersal-mediated gene flow of a mutated allele that confers demethylase inhibitor resistance in Pyrenophora teres f. teres (Ptt). Artificial inoculation of the host, barley (Hordeum vulgare), was successful across the 12-ha trial, where the introduced sensitive- and resistant-populations were, respectively, 6- and 13-fold the DNA concentration of the native Ptt population. Subsequent disease pressure remained low which hampered spread of the epidemic to such extent that gene flow was not detected at, or beyond 2.5 m from source points. In the absence of gene flow, plots were assessed for treatment effects; fungicide applied to populations that contained 14.3% of allele mutation increased in frequency to 24.5%, whereas sensitive populations had no change in structure. Untreated controls of native Ptt population remained genetically stable, yet untreated controls that were inoculated with sensitive Ptt had half the resistance frequency of the native population structure. The trial demonstrates the potential for management to remediate fungicide resistant pathogen populations, where localized gene flow is minimal; to safeguard chemical crop protection into the future.

Strain-level multidrug-resistant pathogenic bacteria in urban wastewater treatment plants: Transmission, source tracking and evolution.

Wastewater treatment plants (WWTPs) serve as reservoirs for various pathogens and play a pivotal role in safeguarding environmental safety and public health by mitigating pathogen release. Pathogenic bacteria, known for their potential to cause fatal infections, present a significant and emerging threat to global health and remain poorly understood regarding their origins and transmission in the environment. Using metagenomic approaches, we identified a total of 299 pathogens from three full-scale WWTPs. We comprehensively elucidated the occurrence, dissemination, and source tracking of the pathogens across the WWTPs, addressing deficiencies in traditional detection strategies. While indicator pathogens in current wastewater treatment systems such as Escherichia coli are effectively removed, specific drug-resistant pathogens, including Pseudomonas aeruginosa, Pseudomonas putida, and Aeromonas caviae, persist throughout the treatment process, challenging complete eradication efforts. The anoxic section plays a predominant role in controlling abundance but significantly contributes to downstream pathogen diversity. Additionally, evolution throughout the treatment process enhances pathogen diversity, except for upstream transmission, such as A. caviae str. WP8-S18-ESBL-04 and P. aeruginosa PAO1. Our findings highlight the necessity of expanding current biomonitoring indicators for wastewater treatment to optimize treatment strategies and mitigate the potential health risks posed by emerging pathogens. By addressing these research priorities, we can effectively mitigate risks and safeguard environmental safety and public health.

POD-like nanozyme constructed from perspective of charge transfer engineering for biosensing of magnetic separation treated Listeria monocytogenes.

For foodborne pathogens pose a serious threat to public health, a magnetic separation strategy and a nanozyme-based biosensor are proposed for biosensing of Listeria monocytogenes (L. monocytogenes). In this work, doripenem is selected as a recognized molecule for the modification of magnetic beads to capture L.monocytogenes in food and environmental samples. Furthermore, the POD-like MXene-Hemin-Au is constructed from perspective of charge transfer engineering which provides a vivid example to rational design of nanozymes. Finally, the captured L.monocytogenes is labeled with MXene-Hemin-Au@mAb, forming the sandwich complexes for quantitative determination. The current signals that generated by the complexes exhibit a good linear relationship with a limit of detection of 2.3 × 101 CFU/mL. The biosensor shows a satisfactory applicability in real samples with recoveries of 91.19% to 102.98%. Overall, the biosensor with integrated magnetic separation strategy presents a potential approach for high sensitivity biosensing of foodborne pathogens.

Comparative Evaluation of Antimicrobial Efficacy of Silver Nanoparticles Infused with Azadirachta indica Extract and Chlorhexidine Against Red-complex Pathogens.

AIM: The present study aimed to evaluate the antimicrobial efficacy of silver nanoparticles infused with Azadirachta indica extract and chlorhexidine against red-complex periopathogens. MATERIALS AND METHODS: Neem leaf extraction was done followed by standardization to the synthesis of neem-infused silver nanoparticles and fractionation of compounds done by using thin layer chromatography to separate the mixture of neem leaf extract. Characterization of neem-infused silver nanoparticles was done by scanning electron microscopy and UV-Visible spectroscopy. The compound identified in neem-infused silver nanoparticles was gedunin which was confirmed by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. Determination of antibacterial activity done by disc diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) methods. Group I-99% ethanolic extract, group II-neem-infused silver nanoparticles (NAgNPs), group III-chlorhexidine. RESULTS: The relative inhibitory zone value for Tannerella forsythia (180) in neem-infused silver nanoparticles (group II) was greater when compared with other periopathogens Porphyromonas gingivalis (133) and Treponema denticola (160) than 99% ethanolic extract (group I), chlorhexidine (group III). Neem-infused silver nanoparticles (group III) showed superior antimicrobial activity against T. forsythia (19.3 ± 31.1547) and T. denticola (18±0) when compared with P. gingivalis (17.6 ± 0.5774). On evaluating MIC and minimum bacterial concentrations, P. gingivalis is more resistant than other pathogens in neem-infused silver nanoparticles (group III). CONCLUSION: Neem-infused silver nanoparticles exhibited superior antibacterial activity as compared with gold-standard chlorhexidine against red-complex periodontal pathogens. For MIC and MBC all the three periopathogens were effective but P. gingivalis was more resistant. CLINICAL SIGNIFICANCE: Antibiotics are effective against many drug-resistant bacteria. As a ready-made medicine, they can be used to treat many infections. Silver nanoparticles in drug delivery systems generally increase solubility, stability, and biodistribution, thereby increasing their effectiveness. Green synthesis using plant extracts as precursors to synthesize nanoparticles has proven to be environmentally non-hazardous combined with remarkably improved efficacy against bacterial and viral diseases. So neem-infused silver nanoparticles can be utilized as a drug delivery system. Hence, it can be used as a potential antibacterial ingredient in formulations for periodontal use like mouthwashes and gels for local drug delivery. How to cite this article: Krishnappan S, Ravindran S, Balu P, et al. Comparative Evaluation of Antimicrobial Efficacy of Silver Nanoparticles Infused with Azadirachta indica extract and Chlorhexidine Against Red-Complex Pathogens. J Contemp Dent Pract 2024;25(6):547-553.

Advances in nucleic acid aptamer-based detection of respiratory virus and bacteria: a mini review.

Respiratory pathogens infecting the human respiratory system are characterized by their diversity, high infectivity, rapid transmission, and acute onset. Traditional detection methods are time-consuming, have low sensitivity, and lack specificity, failing to meet the needs of rapid clinical diagnosis. Nucleic acid aptamers, as an emerging and innovative detection technology, offer novel solutions with high specificity, affinity, and broad target applicability, making them particularly promising for respiratory pathogen detection. This review highlights the progress in the research and application of nucleic acid aptamers for detecting respiratory pathogens, discussing their selection, application, potential in clinical diagnosis, and future development. Notably, these aptamers can significantly enhance the sensitivity and specificity of detection when combined with detection techniques such as fluorescence, colorimetry and electrochemistry. This review offers new insights into how aptamers can address the limitations of traditional diagnostic methods and advance clinical diagnostics. It also highlights key challenges and future research directions for the clinical application of nucleic acid aptamers.

Frequently Touched Sites in the Intensive Care Unit Environment Returning 100 Colony-Forming Units per Surface Area Sampled Are Associated With Increased Risk of Major Bacterial Pathogen Detection.

BACKGROUND: A threshold for surface hygiene has not been defined for the healthcare arena. We aimed to identify the magnitude of bacterial contamination of frequently touched sites in the intensive care unit (ICU) environment that could be used to guide quality improvement initiatives. METHODS: Nineteen patients in a mixed ICU environment (providing care for medical and surgical patients) were followed from admission for 72 hours in 2010. Baseline cultures of frequently touched environmental sites were obtained at time zero following active decontamination and at 12, 24, 48, and 72 hours without further disinfection. We tested for an association of environmental reservoirs returning ≥ 100 colony-forming units (CFU) per surface area sampled with major bacterial pathogen detection. RESULTS: There were 446 ICU room, day, and reservoir combinations sampled from 19 patients. There were pathogens detected in 40% (79/199) of samples with ≥ 100 CFU vs. 14% (35/247) of samples returning < 100 CFU. The relative risk was 2.80 (95% CI: 1.97-3.98, P <0.0001). The odds ratio adjusted for time in hours was 3.11 (95% CI: 1.84-5.34, P < 0.0001). CONCLUSIONS: Frequently touched ICU environmental sites returning ≥ 100 CFU are associated with major bacterial pathogen detection. This threshold for surface hygiene can be used to ensure compliance with ICU environmental cleaning protocols and to guide quality improvement initiatives.

Mucosal infection with Tsukamurella species following nasal septum procedure: a rare case report.

Introduction and importance: Tsukamurella species are rare, aerobic, gram-positive bacteria known to cause infections, primarily in immunocompromised individuals. This case report presents a rare instance of a mucosal infection caused by Tsukamurella species following a nasal septum procedure in an immunocompetent patient. Case presentation: A 51-year-old man with a history of multiple hereditary exostosis, allergic rhinitis, and recent nasal fracture repair presented with persistent fevers and low back pain. Postoperatively, he developed sinus pain and small oral lesions, initially treated with antibiotics for presumed sinusitis. Despite treatment, his fever persisted, leading to an emergency department visit. Laboratory tests indicated sepsis, but a CT scan of the sinuses showed no sinusitis. Despite broad-spectrum antibiotics, the patient's fever continued. On admission day 9, nasal endoscopy and culture identified Tsukamurella species. The patient was treated with augmentin, fluconazole, and levofloxacin, leading to the resolution of symptoms and discharge with ongoing treatment. Clinical discussion: Tsukamurella species are uncommon pathogens that are often associated with bacteremia in immunocompromised individuals. This case highlights the diagnostic challenges and the importance of considering unusual pathogens in postprocedural infections, even in immunocompetent patients. Accurate identification and appropriate management are critical in improving outcomes for patients with Tsukamurella infections. Conclusion: This case underscores the need for vigilance in diagnosing rare infections like Tsukamurella, even in immunocompetent individuals. The successful resolution with combination therapy highlights the importance of appropriate antibiotic selection in managing such infections.

Cryptophytes as potential source of natural antimicrobials for food preservation.

Cryptophytes are a promising source of bioactive compounds that have not been fully explored. This research investigated the antimicrobial activity of total phenolic compounds (TPC) and exopolysaccharides (EPS) extracted from several cryptophytes against a range of harmful foodborne bacteria and fungi. To measure the minimum inhibitory concentration (MIC) value, the broth microdilution method was used. In the antibacterial evaluation of TPC, the MIC ranged between 31.25 and 500 µg/mL, while for the antifungal activity test, it varied from 31.25 to 125 µg/mL. In the antibacterial activity test of EPS, the MIC values ranged from 125 to 1,000 µg/mL, whereas in the antifungal susceptibility test, it ranged between 62.5 and 1,000 µg/mL. The most resistant pathogen against TPC was Escherichia coli, while Campylobacter jejuni was the most susceptible. In the case of EPS, the most resistant pathogen was Salmonella Typhimurium, while Aspergillus versicolor exhibited the highest susceptibility. Overall, in terms of antimicrobial activity, TPC was more effective than EPS. Finally, the tolerance level (TL) for TPC and EPS was ≤4 in all tested samples, indicating their bactericidal/fungicidal mechanism of action. In conclusion, TPC and EPS isolated from cryptophytes demonstrated remarkable antimicrobial properties and ability to fully eradicate pathogens, and could be considered as natural preservatives in the food industry.

First report of Sclerotinia minor causing soft rot of Scabiosa atropurpurea in California.

In May of 2019, Scabiosa atropurpurea samples with brown discoloration, soft rot of the crown and lower stem, with presence of white mycelium and black sclerotia (Supp. Fig. 1A, B) were collected from a 0.10 ha open field diversified cut flower production in San Luis Obispo County, CA. Approximately 30 to 40% of the scabiosa crop planted in a quarter of the field, exhibited symptoms. Symptomatic crowns and lower stems from five plants were surface disinfested by rinsing in 0.1% Tween 20, soaking in 70% ethanol for 30 s, 1% sodium hypochlorite for 2 min and sterile water. Disinfested tissue was placed in 1/10 potato dextrose agar (PDA) and incubated at 20°C (12 h photoperiod). Resulting colonies (n = 5) formed abundant white mycelia, with black sclerotia formed on the outer edge of the plates after two weeks (Suppl. Fig. 1C). Sclerotia (n = 50) had an average size of 1.6 (± 0.19) mm in diameter. Morphological identification resulted in Sclerotinia sp. (Hao et al., 2003). The pathogen was further identified by DNA extraction of two hyphal tipped isolates, followed by amplification and sequencing of the rDNA internal transcribed spacer (ITS) region, ITS1/ITS4 (White et al. 1990), calmodulin (CaM), CAL-228F/CAL-737R (Carbone and Kohn, 1999), and DNA replication licensing factor Mcm7 Mcm7-709for/Mcm7-1348rev (Schmitt et al., 2009). NCBI BLAST searches with consensus sequences for each maker revealed 99 to 100% identity with S. minor ex-types for all loci (Supp. Table 1). A maximum parsimony multilocus phylogenetic analysis clustered Californian isolates with reference strains of S. minor (Supp. Fig. 1F). Sequences were deposited in GenBank (Supp. Table 1). Pathogenicity tests were conducted with isolate CS435, which was transferred onto PDA plates and incubated at 20°C for one week. Inoculum consisted of CS435 infested PDA plugs (1 cm3). In the greenhouse, the experiment was set as a complete randomized design and observed for six weeks. Fourteen-week-old scabiosa 'Merlot Red' grown in 3.78 L pots (n = 6), were inoculated by wounding plants at 0.5 cm above the crowns with a 1 mm probe. Inoculum was placed directly on top of the wound and was secured with parafilm. Negative control plants (n = 6) were wounded as above and inoculated with PDA plugs. In experiment 1(19.4 (± 3) °C, RH 46.9), 83% of plants exhibited yellowing of the lower leaves and wilting at one week post inoculation (wpi). Symptoms progressed over time until wilting, major leaf and stem necrosis, was observed in all inoculated plants (Supp. Fig 1E, D). Plant mortality incidence at five wpi was 83%. Pathogen signs including white mycelia and black sclerotia were also observed. In experiment 2 (20.0 (± 10) °C, RH 39.6), 66% of the total plants were symptomatic at five wpi: 33% exhibited yellowing of the lower leaves and wilting, and 33% of plants died. Disease did not develop in non-inoculated plants in either experiment. S. minor was successfully reisolated from surface disinfested tissue of at least 50% (Exp. 1) and 100% (Exp. 2) of inoculated plants, yielding identical sequences to those of the inoculated isolate. S. minor is a soil-borne pathogen that infects tomato, lettuce, brassica, and sunflower crops in California. This study stablishes S. minor as a new soil-borne pathogen of Scabiosa in California. Soil-borne pathogens are a recurrent issue in the cut flower industry, characterizing new pathogens in these crops can inform crop advisors and disease diagnostician to improve disease management in the ornamental industry.

The applicability of the SERS technique in food contamination testing - The detailed spectroscopic, chemometric, genetic, and comparative analysis of food-borne Cronobacter spp. strains.

Microorganisms assigned as Cronobacter are Gram-negative, facultatively anaerobic, bacteria widely distributed in nature, home environments, and hospitals. They can also be detected in foods, milk powder, and powdered infant formula (PIF). Additionally, as an opportunistic pathogen, Cronobacter may cause serious infections, sometimes leading to the death of neonates and infants. Thus, it is essential to test food products for the presence of Cronobacter spp. The currently used standard described in ISO 22964:2017 is a laborious method that could be easily replaced by surface-enhanced Raman scattering (SERS). Here, we demonstrate that SERS allows the identification of food-borne bacteria belonging to Cronobacter spp. based on their SERS spectra. For this purpose, twenty-six Cronobacter strains from different food samples were analyzed. Additionally, it was shown that it is possible to differentiate them from other closely related pathogens such as Salmonella enterica subsp. enterica, Escherichia coli, or Enterobacter spp. The SERS results were supported by principal component analysis (PCA), as well as and sequencing of 16S rRNA, rpoB and fusA genes. Last but not least, it was demonstrated that the cells of Cronobacter sakazakii may be easily separated from PIF using an appropriate filter, microfluidic chip, and dielectrophoresis (DEP) technique.

Optimization of loop-mediated isothermal amplification assay for sunflower mildew disease detection.

Loop-Mediated Isothermal Amplification (LAMP) represents a valuable technique for DNA/RNA detection, known for its exceptional sensitivity, specificity, speed, accuracy, and affordability. This study focused on optimizing a LAMP-based method to detect early signs of Plasmopara halstedii, the casual pathogen of sunflower downy mildew, a severe threat to sunflower crops. Specifically, a set of six LAMP primers (two outer, two inner, and two loop) were designed from P. halstedii genomic DNA, targeting the ribosomal Large Subunit (LSU). These primers were verified by in silico analysis and experimental validation using both target and non-target species' DNAs. Optimizations encompassing reaction conditions (temperature, time) and component concentrations (magnesium, Bst DNA polymerase, primers, and dNTP) were determined. Validation of these optimizations was performed by agarose gel electrophoresis. Furthermore, various colorimetric chemicals (Neutral Red, Hydroxynaphthol Blue, SYBR Safe, Thiazole Green) were evaluated to facilitate method analysis, and the real-time analysis has been optimized, presenting multiple approaches for detecting sunflower downy mildew using the LAMP technique. The analytical sensitivity of the method was confirmed by detecting P. halstedii DNA concentrations as low as 0.5 pg/µl. This pioneering study, establishing P. halstedii detection through the LAMP method, stands as unique in its field. The precision, robustness, and practicality of the LAMP protocol make it an ideal choice for studies focusing on sunflower mildew, emphasizing its recommended use due to its operational ease and reliability.

Municipal-treated wastewater as a practical alternative to conventional rice irrigation: Effects on antibiotic resistance genes, virulence factors and human bacterial pathogens in soil, and responses of rice grain quality.

Reuse of municipal-treated wastewater for agricultural irrigation is becoming increasingly prevalent due to growing demand and decline in freshwater supplies. However, the microbial contamination profile, including antibiotic resistance genes (ARGs), virulence factors (VFs), and human bacterial pathogens (HBPs) in agricultural soil irrigated with municipal-treated wastewater for paddy cultivation, was unknown. Here, metagenomic analysis was applied to provide a systematic insight into the resistome, VFs and HBPs in paddy soils irrigated with municipal-treated wastewater. The obtained results revealed that the residual antibiotics in municipal-treated wastewater has an impact on the antibiotic resistome by increasing both the total number and abundance of ARGs. Furthermore, it was found that sul1 could serve as a potential risk indicator for assessing ARG contamination. VFs, core HBP abundance, and dangerous pathogens remain unaffected by municipal-treated wastewater irrigation for paddy. The good coexistence patterns of ARGs-HBPs and ARGs-VFs demonstrated the presence of resistant pathogenic bacteria. The network analysis revealed that ARGs-bearing Legionella pneumophila, Mycobacterium marinum, Bordetella pertussis, Staphylococcus aureus, and Pseudomonas aeruginosa might be ranked as high-risk HBPs. Additionally, our investigation also demonstrated that reuse of municipal-treated wastewater for agricultural irrigation had no detrimental effects on rice plant growth and grain quality. This study was the first to investigate the response of VFs and HBPs in paddy soil under long-term municipal-treated wastewater irrigation. The obtained results provide a scientific basis for the safe application of municipal-treated wastewater.

Screening potential biomarkers for acute respiratory infectious diseases from antipyretics, antiviral, and antibiotics in wastewater.

Since the onset of COVID-19, respiratory diseases have emerged as a focal concern within the field of public health. This study aims to reveal the prevalence of acute respiratory infectious diseases by screening antipyretic, antiviral, and antibiotic biomarkers through wastewater analysis. Samples were collected over a seven-day period each year in 2022, 2023, and 2024 from a northern city in China, assessing the concentrations of two antipyretics (paracetamol and ibuprofen), one antiviral drug (oseltamivir), eleven antibiotics, and three pathogens (influenza A, influenza B, and Mycoplasma pneumoniae). The usage of most antipyretics and antibiotics was higher in 2023 and 2024, primarily due to the outbreak of COVID-19 in 2023 and the prevalence of influenza A, influenza B, and Mycoplasma pneumoniae in 2024. The prevalence assessed using antipyretics (2.68 %) and pathogens (2.70 %) demonstrated a high degree of consistency, whereas the prevalence estimated using antibiotics and antiviral drugs was only 0.53 % and 0.36 %, respectively. Antibiotics are generally used to treat a broad spectrum of bacterial infections rather than targeting a specific pathogen, so their presence in wastewater may not directly reflect the prevalence of a particular disease. In contrast, antipyretics and specific pathogens exhibit a stronger correlation, suggesting that they may serve as more reliable biomarkers than antiviral and antibiotic drugs. The research findings offer alternative biomarkers, such as antipyretics, aside from pathogens, for the assessment of acute respiratory infectious diseases.