Journal of Applied Microbiology

Antimicrobial resistance (AMR) is a critical One Health challenge at the interface of agriculture, the environment and human health. In Ireland, cattle production systems generate large volumes of manure and slurry, which are routinely applied to land. However, data on the prevalence and environmental persistence of clinically important resistance phenotypes remain limited. This study investigated the occurrence, characterisation and persistence of extended-spectrum {beta}-lactamase (ESBL) and carbapenemase-producing Enterobacterales (CPE), as well as fluoroquinolone-resistant bacteria on Irish cattle farms. A total of 450 pooled bovine faecal samples were collected from farms across 25 counties, screened using selective culture, antimicrobial susceptibility testing, and MALDI-TOF MS identification. Carbapenemase activity in presumptive CPE isolates was confirmed using phenotypic and biochemical assays, including MALDI-TOF MBT STAR analysis to demonstrate carbapenem hydrolysis. In parallel, a controlled persistence study evaluated the survival of phenotypically resistant ESBL and CPE bacteria in bovine slurry under simulated seasonal conditions. Overall, 29.6% of farms were positive for ESBL and/or CPE, with ESBL only resistance most prevalent (13.7%), followed by combined ESBL and CPE phenotypes (7.5%). Fluoroquinolone resistance was frequently detected and commonly co-occurred with {beta}-lactam resistance, indicating multidrug resistance. Escherichia coli was the dominant carrier of both ESBL and carbapenemase phenotypes. Persistence experiments demonstrated sustained phenotypic resistance in slurry for up to nine months under simulated winter conditions and up to three months during simulated summer conditions, with resistance becoming undetectable following extended verification. These findings demonstrate that clinically important AMR phenotypes are embedded within Irish cattle production systems and can persist in agricultural waste streams, highlighting slurry as a significant environmental reservoir with implications for environmental dissemination and One Health surveillance.

Urinary tract infections (UTIs) are frequently caused by Escherichia coli and are increasingly associated with antimicrobial resistance, highlighting the need for alternative therapies. Although phage therapy is a promising approach, the rational selection of phages and phage cocktails remains challenging, particularly when detailed genomic information on the infecting strain is unavailable. In this study, we isolated 18 phages from environmental samples collected in Sherbrooke, Quebec, Canada. Genome sequencing identified 15 pure lytic phages representing diverse taxonomic groups and revealed prophage-derived contaminants in three isolates, underscoring the importance of genomic validation during phage discovery. Host range was determined using efficiency-of-plating (EOP) assays against UTI-associated E. coli isolates. Based on host-range overlap and cumulative EOP values, we developed a phenotype-based framework for phage cocktail design and classified combinations as redundant or complementary. Complementary cocktails composed of phages with distinct host ranges and high infection effectiveness consistently outperformed redundant combinations in time-kill assays, demonstrating that host-range diversity and lytic effectiveness are independent properties that should be considered separately during cocktail design. To evaluate cocktail performance under physiologically relevant conditions, the most effective combinations were tested in artificial urine. Both cocktails exhibited reduced activity compared with standard laboratory media, indicating that environmental conditions strongly influence phage-host interactions. Together, our results demonstrate that host-range overlap and cumulative infection effectiveness provide a practical framework for identifying effective phage combinations in the absence of detailed bacterial genomic information and may facilitate the rational development of phage therapies for UTIs.

Background: Bovine respiratory disease (BRD) is a major cause of antimicrobial use in cattle, raising concerns about antimicrobial resistance (AMR). This systematic review estimated pooled AMR proportions in BRD pathogens: Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni, and assessed temporal and geographic trends in resistance. Methods: Five antimicrobials commonly used for BRD treatment were evaluated: penicillin G (PEN), tetracycline (TET), florfenicol (FLO), tulathromycin (TUL), and enrofloxacin (ENR). Following PRISMA guidelines, 1,097 records were screened and 24 studies met inclusion criteria. Study quality and risk of bias were assessed, and random-effects meta-analyses estimated pooled proportions of resistant isolates by antimicrobial, continent and period. Results: A total of 13,949 bacterial isolates were analysed including P. multocida (5,615), M. haemolytica (5,585), and H. somni (2,749). TET resistance was highest across all pathogens (12-30%), followed by TUL (6-11%), PEN (4-12%), FLO (0.5-1.6%) and ENR (0.2-1.1%). TET resistance in M. haemolytica and P. multocida, and FLO resistance in P. multocida were significantly more frequent in North America than in Europe (p[≤]0.05). TUL resistance increased significantly in P. multocida after 2013 (p=0.038), with similar non significant trends in M. haemolytica and H. somni. FLO resistance decreased significantly in H. somni after 2013 (p=0.013). Egger's test identified significant funnel plot asymmetry for ENR resistance in H. somni and PEN resistance in M. haemolytica, suggesting possible small study effects. Conclusion: These results offer insights into distribution of phenotypic resistance across pathogens and regions, informing BRD antimicrobial treatment guidelines and One-Health antimicrobial stewardship aimed at preserving the effectiveness of medically important antimicrobials.

Co-administration of phages and antibiotics has been proposed as a therapeutic approach against antibiotic-resistant bacteria. The relationship, however, between antibiotic resistance and phage resistance in clinical isolates is unclear. Here, we examine associations between phage and antibiotic resistance profiles across a panel of Pseudomonas aeruginosa clinical isolates from the Centers for Disease Control (CDC) and Food and Drug Administration (FDA) Antimicrobial Resistance Isolate (ARI) Bank comprising 55 clinical strains with full genome sequences and antibiotic susceptibility testing (AST) data for 11 clinically relevant antibiotics. As phages in this study, we use three well-characterized, morphologically distinct phages, OMKO1, Luz19, and PAML31-1. We screen for phage resistance using a growth suppression assay, then conduct statistical analysis against antibiotic MIC (Minimum Inhibitory Concentration) data provided by the CDC to define association patterns across this dataset. We find multiple significant susceptibility correlations between pairs of antibiotics and phages, and a positive overall association between average phage resistance and antibiotic resistance across the 55 strains, even controlling for phylogenetic associations (=0.358, p<0.005). We conclude that phage and antibiotic resistance are positively associated across this clinical isolate collection, suggesting that the two resistance phenotypes are not independent in P. aeruginosa. These findings have implications for the development of phage-antibiotic cocktails.

Objective Heme-dependent metronidazole (MTZ) resistance has emerged in Clostridioides difficile and is primarily mediated by a T-to-G single-nucleotide polymorphism (SNP) in the promoter of nimB (PnimBG). We aimed to develop a CRISPR-Cas12a-based assay for rapid detection of the PnimBG mutation associated with MTZ resistance. Methods A series of primers and CRISPR RNAs (crRNAs) were designed and optimized to identify the most effective combination for discriminating between PnimBG (MTZ-resistant) and PnimBT (MTZ-susceptible) alleles. Detection was evaluated by visual fluorescence using blue-light and UV imaging systems, as well as by quantitative fluorescence measurements. Results The CRISPR-Cas12a assay accurately differentiated PnimBG and PnimBT alleles in C. difficile. In a collection of clinical isolates, the assay correctly identified all 11 MTZ-susceptible isolates carrying the PnimBT allele and all 10 MTZ-resistant isolates carrying the PnimBG allele. Fluorescence signals were readily detected both visually and quantitatively. Furthermore, colony PCR using crude colony lysates yielded results comparable to those obtained with purified genomic DNA, enabling rapid colony-based detection without conventional DNA extraction. Conclusions The CRISPR-Cas12a assay enables rapid molecular detection of the PnimBG mutation associated with heme-dependent MTZ resistance in C. difficile. This approach could complement conventional susceptibility testing for MTZ resistance detection.

Antimicrobial resistance is a global One Health threat connecting antimicrobial use in human health, animal husbandry, and companion animal health with concern for persistence in environmental reservoirs. Complicating this threat is the gap that exists in our understanding of the intersection of antimicrobial resistance among humans and our companion and food production animals. This study applies a visual and statistical approach to further understand antimicrobial resistance characteristics and relatedness of Escherichia coli found in humans, dogs and cattle. The primary study collection included third-generation cephem resistant (3GCR) E. coli isolates recovered from human, dog, and cattle gastrointestinal specimens and secondarily human and dog isolates recovered from infection sources, primarily urinary tract infections. We previously reported on the transferable gene targets encoding for 3GCR in colonizing isolates and here further identified the familial-like clonal group of each resistant isolate using a novel clonotype diagnostic (septatyping). Together, this approach identified distinct genetic signatures among these 3GCR isolates, many of which are peculiar to their respective host species. We found that the overlap measured by the correlation in abundance of shared clonotypes of resistant E. coli among hosts revealed statistically greater similarity between dogs and humans than between cattle and humans. Yet, the reservoir of AMR genes was highest in cattle and secondarily dogs. While these findings demonstrate that clonotypes of 3GCR E. coli in humans, dogs and cattle display significant host specificity, transient colonization from a host enriched with resistant strains may still pose considerable risk of horizontal transfer of resistance genes between hosts living in proximity to each other.

GeneCapture is developing a novel, point-of-care, rapid AST technique that, when fully automated, can accomplish AST within 2 hours directly from a raw sample, representing a significant advancement in diagnostic capabilities. For this study, a panel of uropathogen isolates was collected from clinical specimens positive for urinary tract infection from various laboratories in the southern US. A selection of these isolates were then spiked into fresh clean-catch urine samples to produce mock samples covering a variety of antibiotic responses. Testing was performed on 20 strains of Escherichia coli, the cause of over 80% of UTIs, and 6 strains each of Klebsiella pneumoniae and Proteus mirabilis, the next most common causes. Each strain was tested against the susceptible, intermediate and resistant (S-I-R) breakpoints of up to 5 appropriate antibiotics including Amoxicillin-Clavulanate, Cefpodoxime, Ciprofloxacin, Nitrofurantoin and Trimethoprim-Sulfamethoxazole. The new method uses a phenotypic assessment of bacterial growth, comparing responses from bacteria treated with antibiotics at S-I-R breakpoint concentrations to untreated controls as the metric for determining susceptibility. Exposure to the drugs for only 90 minutes gave a Categorical Agreement of 98% to gold standard culture results across the resulting 247 bacteria/antibiotic pairs. Testing on separate days of nine of these strains in triplicate gave a reproducibility of 97%. The sequential steps of this novel AST - sample processing, growth, labeling, and counting - can readily be performed on an automated cartridge that is currently under development. The presented work, which took place in a laboratory setting, demonstrates the efficacy of the assay.

Coastal waters often experience fecal contamination from human (e.g., septic systems, wastewater treatment catchments and outfalls) and non-human (e.g., wildlife, domesticated pets, and livestock) origins, posing human health risks through contaminated fisheries and recreational waters. Quantitative PCR (qPCR) methods can identify and quantify fecal origins for their management by targeting Bacteroides, a dominant group of gastrointestinal bacteria with high host specificity. This study identified major contributors and drivers of fecal contamination to Mobile Bay, Alabama, an urbanized estuary in the southern United States. We utilized a multi-species qPCR source tracking approach targeting total Bacteroides and human-, pig-, dog-, cow-, and fowl-associated Bacteroides. Water samples and environmental parameters were collected over one year from 13 sites that represent a range of potential fecal inputs common to coastal waters. Human-associated feces were a consistent major contributor to the system (positive in 82% of samples), with localized inputs from pig (wild or domestic) and dog. Freshwater discharge and nitrogen constituents were good predictors of Bacteroides concentrations in this system, while areas with impervious surfaces and developed land cover may be contributing elevated fecal indicator levels. The multi-species qPCR source tracking approach described in this study can be implemented in other watersheds to support the development of better detection, identification, and fecal management strategies for community health of coastal areas.

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is one the most common cause of post-surgical infections in Malawi and poses a significant public health challenge due to resistance to commonly used antibiotics. We isolated and evaluated the stability and lytic activity of a MRSA-specific bacteriophage (Staphylophage) from Mzuzu Central hospital (MZCH) sewage against MRSA strains isolated from surgical wards. MethodsA prospective in vitro experimental study was conducted using 56 pus swabs collected from post-surgical patients at Mzuzu Central Hospital (MZCH), Malawi. Standard microbiological methods and VITEK analysis were used for bacterial isolation, identification and antimicrobial susceptibility testing of Staphylococcus aureus and MRSA according to the 2024 EUCAST guidelines. A lytic Staphylophage was isolated and purified from sewage wastewater using filtration, enrichment, and double-layer agar techniques. The purified Staphylophage was evaluated for its lytic activity through host range testing and stability under ultraviolet (UV) exposure (up to 60 minutes), different pH levels (2-12), and temperatures (4{degrees}C, -20{degrees}C, and -80{degrees}C). ResultsOut of 56 bacterial isolates, 18 (32.1%) were S. aureus, of which 11 (61.1%) were MRSA. The isolated Staphylophage showed lytic activity against 87% of Staphylococcus species tested. The phage remained stable at temperatures between -80{degrees}C and 4{degrees}C and across a pH range of 2- 12. It also retained viability after UV exposure for up to 15 minutes, although a reduction in phage titre was observed after 30 minutes. ConclusionsA lytic Staphylophage isolated from hospital sewage at MZCH demonstrated activity against Staphylococcus species, including MRSA strains from post-surgical wound infections. These findings illustrate the feasibility of generating candidate phages for alternative therapeutic approaches in a central hospital setting in Malawi.

Abstract Background: Soil microbiomes play critical roles in plant health and resilience, yet it is still poorly understood how disruptions in local microbial communities, such as during soil management, impact these ecological functions. In this study, we investigated how compost addition, a widely used soil management practice, and microbiome origin influence microbiome-mediated plant resistance to insects. We combined surveys of working organic farms across New York with controlled laboratory experiments, amplicon sequencing, plant-insect bioassays, and mass spectrometry. Results: Compost use was associated with shifts in the relative abundance of 20 fungal and 2 bacterial genera across ten working farms. However, no consistent changes in bacterial or fungal diversity or community structure in fields with compost were observed across the farms. Under controlled conditions using a common compost source, compost increased rhizosphere microbial diversity and significantly altered rhizosphere community structure, including the relative abundance of 6 fungal and 41 bacterial genera. Despite these substantial community-level changes, compost did not affect populations of green peach aphids (Myzus persicae) on kale (Brassica oleracea). Instead, aphid populations and plant salicylic acid concentrations were more strongly associated with microbiome origin, demonstrating that compost can substantially restructure soil and rhizosphere microbial communities without corresponding changes in microbiome-mediated plant resistance to insects. Conclusions: Our findings demonstrate that microbial diversity and compositional responses to management are not necessarily reliable indicators of all microbiome functions. More broadly, this work highlights the importance of local soil microbiome variation in shaping plant-insect interactions and emphasizes the need to link community-level responses to direct functional measurements when evaluating microbiome responses to agricultural management.

Background: Indwelling urinary catheters can cause pain, urethral irritation and trauma and provide surfaces for microbial colonisation and biofilm formation. The LubriShield(R) Foley catheter incorporates a covalently bonded, superhydrophilic antifouling hydrogel designed to reduce surface friction and limit biofilm accumulation without releasing an antimicrobial agent. This study evaluated its safety and clinical performance compared with an uncoated silicone Foley catheter, including usability and patient comfort, and explored catheter-associated microbial colonisation and biofilm formation. Methods: This prospective, randomised, single-blind, single-centre controlled clinical investigation enrolled 33 adults requiring indwelling urinary catheterisation for 72 hours to 14 days. Participants received either the LubriShield(R) Foley catheter (n = 16) or an uncoated Sterimed all-silicone Foley catheter (n = 17). The primary endpoint was safety based on adverse-event reporting; the prespecified safety criterion was the absence of a serious adverse device effect in the LubriShield(R) group. Secondary endpoints included investigator-rated ease and functionality of catheter insertion and removal and participant-reported pain and irritation/discomfort assessed using 0-10 numerical rating scales. Urine microbiology, catheter colonisation and catheter-level biofilm measurements were exploratory endpoints. Analyses were performed in the as-treated population. Numerical rating scale outcomes were compared using two-sided Mann-Whitney U tests; exploratory biofilm analyses used permutation tests with bootstrap confidence intervals. Results: No serious adverse events or serious adverse device effects occurred. Nine non-serious adverse events were reported and resolved completely. At least one adverse event occurred in 4/16 (25.0%) participants in the LubriShield(R) group and 4/17 (23.5%) participants in the control group. The prespecified safety criterion, defined as the absence of a SADE in the LubriShield(R) group, was met. LubriShield(R) was rated more favourably for ease of insertion (p = 0.0096), functionality during insertion (p = 0.0051), ease of removal (p = 0.0037) and functionality during removal (p = 0.0085). Participants reported significantly less pain and irritation/discomfort during insertion, while the catheter was indwelling and during removal (all p < 0.05). Outcomes 7 {+/-} 1 days after catheter removal were similar between groups. Absolute biofilm biomass and bacterial and fungal loads did not differ significantly. In a post hoc exploratory normalised analysis, the biofilm-to-bacterial-load ratio was lower with LubriShield(R) (median 0.052 vs 0.080; permutation p = 0.043), although the bootstrap 95% confidence interval for the between-group difference included zero. Conclusions: The LubriShield(R) Foley catheter demonstrated a favourable short-term safety profile and was associated with improved investigator-rated usability and participant-reported comfort compared with an uncoated silicone Foley catheter. A post hoc exploratory analysis was compatible with reduced biofilm accumulation relative to bacterial load, but this finding is hypothesis-generating only and requires confirmation in larger, adequately powered studies with prespecified biofilm endpoints.

This study developed a streamlined, sensitive and rapid method for co-detection of low concentrations of Salmonella enterica, Shigella flexneri, Listeria monocytogenes, and pathogenic Escherichia coli from soil. First, autoclaved soil from Iowa with serial dilutions of bacteria to confirm individual species recovery rates on selective agar by qPCR under varying primary enrichment times was used. All pathogens were detected by qPCR of colonies recovered from replicate sterile soils spiked with 10^1 or greater bacteria within 5 hours of enrichment. Shiga toxin expressing E. coli (STEC) and L. monocytogenes were consistently co-recovered in autoclaved and natural soil, pathogen spiking concentration, and secondary enrichment time. S.enterica was consistently recovered at autoclaved and natural concentrations, while S. flexneri detection rates fluctuated. Twenty-three soil samples from Kenyan neighborhoods, with prior evidence of contamination, was compared to qPCR detection at each protocol stage: processing, primary enrichment, secondary enrichment, and selective colony isolation. Detection rates substantially increased for S. enterica, STEC and other pathogenic E. coli after enrichment, with optimal detection rates occurring after secondary enrichment. Testing colonies from selective agar resulted in high false negative rates, possibly reflecting competition from native soil microbiota that was absent in spiked sterile soil experiment. Evaluation of the method with other environmental sample types yielded consistent detection patterns. Evidence suggests a protocol including 24-hour primary enrichment, followed by 24-hour secondary enrichment, and qPCR testing of soil DNA at primary and secondary steps is a reproducible and more expedient method for co-detecting common Enterobacteriaceae and L. monocytogenes pathogens in soil.

An increasing number of researchers, educators, and at-home growers are turning to hydroponics as an alternative method of food production, diversifying and strengthening the food supply chain. This has led to a substantial increase in the amount of academic work involving hydroponic production of plants for a variety of purposes, including but not limited to a more controlled and customizable environment than conventional production methods, utilizing hydroponics as a method of wastewater treatment, and investigating the vertical production of plants on a much smaller footprint than traditional commercial-scale production. Because so many unique stakeholders are interested in hydroponics for a variety of reasons, academic studies use a wide variety of hydroponic systems with varying levels of technology and automation, making comparisons across studies and systems challenging. A few institutions have sophisticated plant growth and research facilities, like those used by commercial companies, but these systems are often prohibitively expensive, costing upwards of $10,000 USD for a very limited amount of growing space for experimental trials. To enable more researchers, educators, and citizen scientists to conduct experiments and easily compare their results, this study details three pilot-scale hydroponic production systems (Kratky, NFT, and DWC), utilizing entirely off-the-shelf components, for under $5,000 USD each, that are capable of maintaining the desired consistent temperature and relative humidity levels, and have space for 216, 108 (6 irrigation loops), and 64 (16 irrigation bins) plants, respectively. The material lists and prices are provided along with assembly instructions and general guidelines for each hydroponic system. The testing and optimization process of environmental conditions within each growth system is also detailed, with suggested customizations for potential experimental setups. The authors developed these systems during their Masters and Doctoral degree programs, enabling improved hydroponic research at the University of Illinois Urbana-Champaign. They hope that other aspiring hydroponic researchers can benefit from this work in the development and operation of their hydroponic systems, increasing research efficiency and collaboration opportunities in this field of study.

Pyrroloquinoline quinone (PQQ) is known to promote plant growth, yet how the rhizosphere microbiome varies with PQQ concentration remains poorly understood. We applied metagenomic sequencing to characterize the taxonomic composition and functional potential of the tobacco rhizosphere microbiome across PQQ treatments at 0.5, 1 and 2 mol/L plus an untreated control. The rhizosphere microbiome was dominated by Pseudomonadota and Actinomycetota and differed among treatments in a concentration-associated, non-monotonic manner. The most pronounced differences were observed at 1 mol/L, where alpha diversity showed dimension-specific variation and beta-diversity analysis resolved the most distinct community profile. Taxonomic and functional profiles varied concurrently, with Streptomyces, Sphingomonas, Rhodanobacter and Lysobacter, together with functions related to biosynthesis, energy metabolism, colonization and environmental sensing, showing higher relative abundance at this concentration. No broad-spectrum antimicrobial effect was detected at the community level, and antibiotic resistance gene abundance showed no significant differences among treatments after correction for multiple testing. These findings provide a basis for further evaluation of PQQ in modulating the rhizosphere microbiome in continuous cropping systems.

Purpose: Vancomycin-resistant Gram-positive organisms represent a potential therapeutic challenge in endophthalmitis. To evaluate alternative treatment options, the FDA-approved oxazolidinones linezolid (LZD) and tedizolid (TZD) were tested against a collection of Gram-positive bacterial endophthalmitis isolates. Methods: Minimum inhibitory concentrations (MICs) were determined using epsilometers (Etest strips) for coagulase negative staphylococci (CoNS, n=20), Staphylococcus aureus (n=18), Bacillus species (n=10), Enterococcus species (n=5), and Streptococcus pneumoniae (n=1). Available CLSI and EUCAST breakpoints were applied to determine susceptibility. Results: TZD exhibited a >5-fold reduction in median MIC compared with LZD across all organisms tested (p<0.0001). MIC90 values for CoNS were 2.0 /ml for LZD and 0.25 /ml for TZD. For S. aureus, MIC90 values were 1.5 /ml (LZD) and 0.19 /ml (TZD). For Bacillus spp., MIC90 values were 1.0 /ml (LZD) and 0.25 /ml (TZD). Among Enterococcus species, the MIC range was 0.5-4.0 /ml (LZD) and 0.064-0.75 /ml (TDZ). The single S. pneumoniae strain had a MIC of 0.5 for LZD and 0.064 for TZD. All isolates were susceptible to TZD. One Enterococcus isolate was non-susceptible to LZD; the remaining isolates were susceptible. Conclusions: LZD and TZD demonstrated in vitro activity against Gram-positive bacterial endophthalmitis isolates, with TZD displaying consistently lower MICs than LZD. These findings suggest that oxazolidinones may represent potential alternatives when vancomycin cannot be used, including infections caused by vancomycin-resistant Gram-positive organisms. Given their excellent oral bioavailability, favorable safety profile, and reported anti-virulence effects in certain pathogens, further evaluation of oxazolidinones for the treatment of intraocular infections is warranted.

Gray leaf spot can cause severe turfgrass damage, particularly in intensively managed sports fields, while disease management remains challenging due to the rapid development of symptoms under favorable environmental conditions. Early detection of Pyricularia oryzae could therefore provide valuable information for anticipating disease development and supporting management decisions. This study aimed to develop a digital PCR (dPCR) molecular tool to support the monitoring and management of gray leaf spot disease in stadium turfgrass. The assay showed high sensitivity, consistently detecting P. oryzae when infected tissue represented only 0.05% of a mixture with healthy turfgrass, and enabled pathogen detection before symptom appearance under both controlled and field conditions. Different matrices were also investigated across 15 stadiums to identify the most relevant material for disease monitoring. Roots showed the highest prevalence of P. oryzae detection, including during asymptomatic periods, highlighting their potential as a relevant matrix for pathogen surveillance. In a stadium monitored several months after a gray leaf spot outbreak, P. oryzae DNA was still detected in roots from 27 of the 32 sampled zones despite the absence of visible symptoms. Overall, these results demonstrate the potential of dPCR for the early detection and monitoring of P. oryzae in stadium turfgrass, particularly through root sampling during periods when environmental conditions are not favorable for gray leaf spot development, and a spatio-temporal surveillance at stadium scale.

Nanoplastics (NPs) are widespread environmental contaminants, yet most toxicity studies rely on short-term, single-dose exposures that poorly reflect daily human exposure. The kidney, particularly the proximal tubule, is a likely site of repeated NP exposure given its role in filtration and reabsorption, but chronic exposure effects are largely unexplored. Here, a long-term in vitro model was developed in which human proximal tubule (HK-2) cells were continuously exposed to 20 nm or 100 nm carboxylated polystyrene NPs (100 {micro}g/mL) over 12 days. Continuous 20 nm NP exposure caused progressive loss of viability whereas 100 nm NPs left viable cell numbers largely unaffected. RNA-sequencing showed 20 nm NPs drove extensive, time-dependent transcriptional dysregulation (1,548 differentially expressed genes at day 6, rising to 4,431 at day 9), far exceeding the minimal alterations in gene expression (35 genes) caused by 100 nm NPs. Cell-cycle analysis and pathway enrichment revealed KRAS pathway activation and S-phase arrest in 20 nm-treated cells, with ANGPTL4 the most upregulated gene. Validation with qRT-PCR confirmed increased ANGPTL4, HMOX1 and FN1 expression, genes implicated in renal fibrosis and chronic kidney disease. These findings show that continuous, size-dependent NP exposure elicits progressive cytotoxic and transcriptional responses in kidney proximal tubule cells and indicate ANGPTL4 as a candidate early biomarker of NP-induced renal stress.

Antibiotic resistance genes (ARGs) travel from cities into rivers and coastal waters through wastewater treatment plants. Monitoring them normally requires metagenomic sequencing, which is sufficiently costly that most utilities can sample only occasionally. Weather and location data are freely available on a daily basis for virtually any location worldwide, making them attractive predictors for identifying where limited sequencing resources should be prioritized. However, whether such models generalize to previously unseen cities remains largely untested, as most published studies train and evaluate models within the same catchments, thereby assessing interpolation rather than geographic transferability. To address this gap, we paired 235 wastewater metagenomes collected from five European cities with 23 abiotic predictors spanning geospatial, meteorological, hydrological, radiative, and temporal domains. Model performance was evaluated using leave-one-group-out (LOGO) cross-validation, in which all samples from one city were withheld for testing while the remaining cities were used for training. CatBoost achieved a median LOGO ROC-AUC of 0.929 and a median F1-score of 0.750. Using freely available environmental reanalysis predictors (meteorological, hydrological, radiative, geospatial, and temporal) - without any metagenomic sequencing - CatBoost achieved a mean ROC-AUC of 0.722, recovering 78% of the predictive performance of the full omics-integrated model. Removing latitude and longitude reduced ROC-AUC by only 0.003, whereas replacing random cross-validation with city-wise validation reduced ROC-AUC by 0.052. Predictive performance varied across ARG classes, ranging from a ROC-AUC of 0.981 for {beta}-lactam resistance genes to 0.762 for glycopeptide resistance genes. These findings demonstrate that freely available environmental reanalysis predictors - spanning meteorological, hydrological, radiative, geospatial, and temporal domains - recover 78% of the predictive signal for antibiotic resistance gene occurrence in urban wastewater, allowing scarce sequencing capacity to be directed to the catchments where it changes a decision.

Biological filtration in newly established aquaria depends on the establishment of nitrifying microorganisms that prevent accumulation of toxic ammonia and nitrite. Many aquarists use commercial nitrifying supplements to accelerate biofilter establishment and reduce ammonia and nitrite accumulation. However, the microbial composition and in situ efficacy of these products remain poorly characterized. We evaluated the ability of enrichment cultures containing comammox Nitrospira (CX) and the ammonia-oxidizing archaeon Ca. Nitrosotenuis aquarius (AQ) to serve as aquarium nitrifying supplements, tested alongside a conventional supplement (CS) containing ammonia-oxidizing bacteria. Inoculation of new aquaria containing zebrafish (Danio rerio) with nitrifying supplements resulted in distinct nitrifier profiles within biofilters of each treatment group corresponding with significant reductions in peak ammonia and nitrite concentrations, which were most evident when applied at the maximum tested supplement dose. Treating aquaria with CX, AQ, or CS maintained ammonia and nitrite at low concentrations, and CX effectively prevented ammonia and nitrite accumulation through all stages of aquarium operation and biofilter establishment. After removing all fish from the aquaria, ammonia "spike-in" experiments demonstrated biofilters treated with CX and AQ conferred long-term protection against nitrite accumulation following acute ammonia loading events. Whereas 16S rRNA gene sequences associated with CS were rarely detected within biofilter profiles, those associated with CX and AQ established early and persisted temporally. Overall, results demonstrate how nitrifiers might be differentially adapted to home aquarium conditions and the potential of AOA and comammox Nitrospira to improve biofilter establishment and maintaining safe water quality in newly established freshwater aquaria.

Shigella flexneri is an enteric pathogen responsible for causing shigellosis, a type of bacillary dysentery of significant global health concern. The increasing prevalence of antibiotic resistance has driven the search for alternative treatments, including bacteriophage (phage) therapy. However, the effectiveness of orally administered phages for enteric pathogens depends on their ability to survive passage through the gastrointestinal tract. In this study, the ability of bacteriophage Sf14 to maintain infectivity was evaluated under simulated gastrointestinal conditions using a modified INFOGEST 2.0 in vitro digestion model. This model includes salivary, gastric, and intestinal fluids with physiologically relevant compositions of electrolytes, bile salts, and enzymes. Phages were incubated in each simulated phase at standard physiological temperature, then tested for infectivity using quantitative plaque assays. Results show that Sf14 remained stable during the short duration of the oral phase and for the duration of the intestinal phase. By contrast, survival in simulated gastric fluid required a pH of 2.75 or higher, with rapid and substantial loss of infectivity at pH 2.5 and below. These findings demonstrate that while Sf14 is stable in oral and intestinal environments, the gastric phase poses a critical barrier to phage survival. With in vivo fasting gastric pH ranging from 1.5 to 3, encapsulation would enable the most flexible oral phage therapy against enteric pathogens, but ingestion after eating or alongside antacids may be sufficient.