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.

Hypervirulent Klebsiella pneumoniae represents a growing clinical threat, particularly where multidrug resistance limits treatment options. Here, we report the isolation and characterization of three novel lytic phages from Benin (Kp1Bj_HH11_M23, Kp2Bj_LN294_M23, and Kp10Bj_LN54_14). These Myoviruses, belonging to the genera Marfavirus and Jiaodavirus, display broad host range activity against multidrug-resistant and hypervirulent K. pneumoniae strains. Genomic analysis confirmed the absence of virulence and antimicrobial resistance genes. The phages exhibit rapid adsorption, short latency periods, and high burst sizes (119-2208 PFU/cell). All three phages significantly inhibited biofilm formation and reduced established biofilms in vitro. Their stability across a wide range of temperatures and pH further supports their potential for therapeutic development. Together, these data highlight the value of locally sourced phages as candidates for tackling region-specific antimicrobial resistance challenges.

BackgroundEscherichia coli is a common environmental and zoonotic bacterium that serves as an important indicator of fecal contamination and antimicrobial resistance (AMR) in poultry production systems. The emergence of multidrug-resistant (MDR) E. coli in poultry farm environments poses a significant One Health threat to animal, human, and environmental health. ObjectiveThis study aimed to isolate, identify, determine the prevalence, assess the antibiotic susceptibility pattern, and molecularly confirm Escherichia coli isolated from poultry farm environments in Lalmonirhat district, Bangladesh. Materials and MethodsA cross-sectional study was conducted from January to June 2024 using 60 environmental samples comprising water (n = 20), soil (n = 20), and bird-dropping (n = 20) samples collected from poultry farms in five upazilas of Lalmonirhat district. Isolation and identification of E. coli were performed using standard cultural, morphological, and biochemical techniques. Antimicrobial susceptibility was determined by the Kirby-Bauer disc diffusion method following CLSI guidelines. Ten randomly selected isolates were confirmed by polymerase chain reaction (PCR) targeting the species-specific uidA gene. ResultsThe overall prevalence of E. coli was 50% (30/60). Source-wise prevalence was highest in bird-dropping samples (75%), followed by water (45%) and soil (30%). Area-wise prevalence ranged from 33.33% in Patgram to 66.67% in Hatibandha. The isolates exhibited the highest resistance to oxytetracycline (66.67%) and ciprofloxacin (60%), while the highest susceptibility was observed to erythromycin (63.33%), neomycin (56.67%), and amoxicillin (56.67%). All ten isolates subjected to PCR produced the expected 486 bp uidA gene amplicon, confirming their identity as E. coli. ConclusionThe findings demonstrate a considerable prevalence of antimicrobial-resistant E. coli in poultry farm environments of Lalmonirhat district. Strengthening farm biosecurity, improving hygiene and waste management, implementing antimicrobial stewardship, and maintaining continuous molecular surveillance are essential to minimize the dissemination of resistant E. coli within a One Health framework.

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.

SynopsisO_ST_ABSBackgroundC_ST_ABSThe complexity of diagnosing and treating biofilm-associated infections necessitates a comprehensive strategy to mitigate the rising rates of antimicrobial resistance (AMR). Microtiter plate methods are used globally for determination of biofilm eradication concentrations (MBEC) but few have been adapted to observe pharmacodynamic observations. Here, we describe a method which allows for both static and pharmacodynamic assays of biofilm evaluation. MethodsA total of 150 clinical isolates from Southmead Hospital were assessed, representing five bacterial species (N=30 per bacterial species): Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus and Klebsiella pneumoniae. MBECs were determined using a developed method using 96 well plates and glass beads. MBECs of seven different antibiotics were compared to those determined using the established Calgary biofilm device (CBD). Dynamic pharmacodynamic evaluations to produce Biofilm Time Kill curve (BTKC) based on published planktonic time kill curve (TKC) data and ISO recommendations were carried out using the glass bead model for K. pneumoniae and ciprofloxacin, S. aureus and levofloxacin and S. pneumoniae and vancomycin. Quantification of biofilm biomass was assessed at 0, 2, 4, 8 and 24 hours and compared to planktonic culture survival under comparable challenge conditions. ResultsComparing MBEC results for all bacterial strains and antibiotic challenges showed no statistical difference between the glass bead and CBD methods (P <0.05). Biofilm BTKC AUBKC were inferior to planktonic equivalents but demonstrated specific pharmacodynamic patterns of biofilm reduction efficacy. MBEC correlated with biofilm BTKC penetration in line with clinical observations for S. aureus vs vancomycin and S. pneumoniae vs levofloxacin. ConclusionsThe glass bead biofilm models provide robust, reproducible alternatives to the traditional methods of determining MBEC and bridge the gap with biofilm pharmacodynamic evaluations. These methods also provide a low-cost option to current methods as only standard laboratory equipment is required, allowing for the generation of comprehensive data sets. This ensures greater translatability to complex in vitro models and clinical scenarios.

Shiga toxin-producing Escherichia coli (STEC) persist in cattle and can colonize the bovine recto-anal junction, making it an important reservoir for transmission. Curli are extracellular amyloid fibers associated with biofilm formation and environmental persistence, but their contribution to STEC adherence to bovine epithelial cells is unclear. This study examined the relationship between curli production, biofilm formation, and adherence of O157, O26, and O111 STEC isolates to bovine recto-anal junction (RAJ) squamous epithelial (RSE) cells. Thirty isolates were evaluated for RSE adherence, curli-associated phenotypes, and biofilm formation under environmental and host-associated growth conditions. O157 and O111 isolates predominantly exhibited strong aggregative adherence to RSE cells, whereas O26 isolates showed more heterogeneous, primarily diffuse adherence. O111 isolates produced the strongest biofilms under environmental conditions and displayed relatively stable curli phenotypes, while O157 and O26 isolates showed greater variation. However, environmental biofilm formation did not consistently correlate with RSE adherence. Biofilm formation was also markedly reduced under host-simulated conditions in DMEM-LG at 26, 37, and 39{degrees}C, including among isolates that produced strong environmental biofilms. Differences in Shiga toxin genotype and expression likewise did not account for the major adherence patterns, as O111 isolates lacking stx2 retained strong aggregative adherence. These findings indicate that curli-associated biofilm formation is primarily influenced by environmental conditions and does not directly predict STEC adherence to bovine RSE cells. The results suggest that environmental persistence and host-cell attachment represent distinct phenotypes and that STEC adherence to bovine epithelial cells is likely mediated by additional strain- and serotype-specific factors.

Enterococcus faecalis is a leading cause of infective endocarditis, particularly in older and hospitalized patients. Treatment is challenging due to the intrinsic resistance of E. faecalis to many clinically important antibiotics and the increasing prevalence of multidrug-resistant strains. Bacteriophage (phage)-based therapies, including phage cocktails and phage-antibiotic combinations, offer a promising approach for treating resistant bacterial infections. In this study, we isolated ten novel phages from wastewater by screening on E. faecalis clinical isolates and tested phage activity both individually and in phage-phage and phage-antibiotic combinations against 18 E. faecalis clinical isolates collected from patients with infective endocarditis. We found that 15/18 (83%) isolates were susceptible to at least one phage tested, and that the same proportion of isolates were susceptible to a cocktail of three phages with complementary activities. We also observed improved bacterial growth inhibition and increased killing when phages were combined with ampicillin or daptomycin, two antibiotics commonly used to treat E. faecalis infections. Finally, we found that treatment with the three-phage cocktail improved the survival of Galleria mellonella infected with a pathogenic E. faecalis endocarditis isolate. Overall, these findings suggest that phages could be a helpful addition to the therapeutic repertoire for treating E. faecalis infective endocarditis. ImportanceNew approaches are needed for treating infective endocarditis, a life-threatening heart infection. Here we focus on Enterococcus faecalis, a resilient bacterium that is increasingly resistant to standard-of care antibiotic regimens, and explore the use of phage therapy for treating E. faecalis endocarditis. We found that combining phages together in a cocktail or pairing them with existing antibiotics can effectively kill E. faecalis clinical isolates from patients with infective endocarditis, and that treatment of infected waxworm moth larvae with a phage cocktail improved their survival. The results of this study contribute to the development of phage-based therapeutic approaches for E. faecalis endocarditis, which could ultimately lead to improved outcomes for patients facing this severe infection.

Despite increased recognition of the diverse resident microbiome of the urinary tract (i.e., the urobiome) in postmenopausal women, the roles and functions of these microbes remain largely unknown. Further empirical research is needed to understand the physiology, interactions, and antibiotic resistance evolution of urobiome members with pathogenic potential. However, experimental work relies on viable, culturable isolates. Standard urine culturing practices are designed for identifying a narrow set of known urinary microbes, and are thus poorly suited for cultivating taxa from the resident urobiome. Here we expand the urine culturing toolkit to reliably recover diverse urobiome taxa for downstream empirical research. Urine samples collected from postmenopausal women with recurrent urinary tract infections were shipped at ambient temperature to a central point for culturing. Microbial viability was maintained using boric acid preservative tubes during multi-day transport of sample aliquots. Selective media incubated under specialized conditions were used to promote recovery of diverse urobiome members, including fastidious taxa. Under 5% CO2 -enriched atmospheric conditions and with longer incubation times, we leveraged a chromogenic agar (UTIC) to further differentiate isolates based on colony color and morphology. We evaluated the workflow for its ability to isolate and characterize urobiome taxa, as determined by morphological differentiation and taxonomic identification. Across 108 urine samples, 6.3 {+/-} 3.2 distinct isolates were recovered, with no detectable relationship between sample shipment duration and isolate richness. On chromogenic agar, colony growth and color intensity was improved with CO2 -enriched atmospheric conditions and extended incubation times. We identified diverse taxa that are typically underrepresented in standard diagnostic culture and provide novel morphological characterizations for members of the genera Actinotignum, Aerococcus, Facklamia, Lactobacillus, Latilactobacillus, Limosilactobacillus, and Streptococcus species, which have not been previously described on UTIC chromogenic agar. Using this novel workflow, we recovered a diverse collection of urobiome isolates from urine samples shipped over multiple days. We also demonstrated the utility of a chromogenic agar for the visual differentiation of key urobiome taxa. While sequencing approaches have enhanced our understanding of urobiome composition, culturing is needed to investigate microbial interactions, virulence mechanisms, and antimicrobial susceptibility. This protocol adds to the growing toolkit for the cultivation of diverse urobiome isolates needed to support downstream empirical studies and advance urinary tract infection research.

Microbial-mediated plastic degradation has the potential to address the persistent global problems of plastic waste and pollution. Previous work has shown that soils can harbour microbes capable of plastic degradation, but we expect there is a broader diversity of soil microbes capable of metabolizing plastics than identified to date using more traditional cultivation-based screening methods. Here we demonstrate a novel approach to identify putative plastic degrading microbes in soil. We paired in situ, real-time measurements of microbial plastic degradation on conductive sensors with subsequent microbial community profiling of the sensor-associated biofilms exhibiting appreciable degradation. To illustrate the utility of our approach, we focus on microbial degradation of the bioplastic polymer PHBV, poly(3-hydroxybutuyrate-co-3-hydroxyvalerate). We screened a range of soils with the in situ sensors to identify a subset of five soils with high PHBV degradation rates, and confirmed that PHBV degradation was due to microbial activity. We then extracted DNA directly from sensors placed in soils with high measured rates of PHBV degradation and used marker gene sequencing to identify the bacterial and fungal taxa associated with the observed PHBV degradation. We confirmed via in vitro culturing that microbes isolated from the sensors have a demonstrated capacity for PHBV metabolism. Together, these results highlight the benefit and feasibility of using low-cost, in-soil sensors to simultaneously collect real-time data on plastic degradation rates in soil and identify previously unrecognized microbial taxa capable of degrading and metabolizing plastic polymers in situ.

ObjectiveThis study aimed to establish an efficient isolation, culture, and preservation system for lactic acid bacteria from Mongolian horse feces by systematically evaluating the effects of sampling methods, culture conditions, media types, and cryopreservation protocols. MethodsFecal samples were collected using rectal and natural defecation methods, and bacteria were isolated by the spread plate technique, with isolates subcultured and identified via 16S rRNA gene sequencing. The performance of five commercial media (A-E), aerobic versus anaerobic conditions, and different glycerol concentrations (10% and 30%), temperatures (-20 {degrees}C and -80 {degrees}C), and storage durations (7, 15, and 30 days) were compared. ResultsRectal sampling showed significantly higher lactic acid bacterial survival and greater species diversity (12 vs. 8). Anaerobic culture yielded substantially higher counts and diversity (707 strains, 12 species) compared to aerobic conditions (36 strains, 5 species). Medium C (M.R.S. AGAR) exhibited the best isolation performance, producing the highest number of strains (311) and species (11), with selective enrichment for both bacilli and cocci. Glycerol effectively improved strain survival during sample cryopreservation, with the optimal short-term storage ([&le;]15 days) achieved at -20 {degrees}C with 30% glycerol. Functional screening of randomly selected strains from optimized conditions demonstrated promising growth, acid and bile salt tolerance, and antimicrobial activity. ConclusionRectal sampling combined with anaerobic incubation using Medium C is recommended for optimal isolation. For samples that cannot be processed immediately, short-term preservation at -20 {degrees}C with 30% glycerol prior to isolation is effective. This study provides practical technical references for the acquisition and preservation of equine intestinal lactic acid bacteria resources.

Antimicrobial resistance in Pseudomonas aeruginosa has emerged as a major clinical concern. Ciprofloxacin has potent intrinsic activity against P. aeruginosa, but resistance to this antibiotic is increasingly reported in clinical settings. In this study, P. aeruginosa ATCC 27853 strain was exposed to gradually increasing ciprofloxacin concentrations for 50 passages ([~]100 days). The final selected resistant strain (CIP-R) with 256-fold of minimum inhibitory concentration was studied further. Whole-genome sequencing revealed 12 genomic alterations, including known mutations in the quinolone resistance-determining genes gyrA (Thr83Ile), parC (Thr177Asn), parE (Glu459Lys). A duplication mutation in nfxB (Tyr153_Gly154dup) was also observed. In addition to these genes, we observed mutations in pilA, tadB, psdR, NP446_RS32055 (TRAP transporter permease), multiple dppA3 variants, and a prophage-associated hypothetical gene (NP446_RS24255), which have not been reported previously. The gyrA Thr83Ile substitution was conserved in 86.38% of ciprofloxacin-resistant clinical isolates retrieved from the NCBI database. Resistance acquisition was accompanied by slower growth, impaired swimming and swarming motility, diminished surface attachment, reduced biofilm formation. The resistant strain has enhanced {beta}-lactamase activity, and resistance to levofloxacin, cefepime, and meropenem together with sensitivity to piperacillin-tazobactam and aztreonam. This study highlights the gyrA Thr83Ile mutation as a key genomic marker for molecular screening of ciprofloxacin resistance and reveals secondary adaptive trade-offs that can be targeted for clinical diagnostic and therapeutic decision-making. In conclusion, achieving high-level ciprofloxacin resistance in P. aeruginosa involves non-target-site genomic adaptations and physiological trade-offs beyond classical target mutations.

Polycyclic aromatic hydrocarbons (PAHs) are hazardous organic contaminants for which microbial bioaugmentation is a promising remediation strategy, but poor persistence of introduced microorganisms can limit efficacy. Encapsulation may improve persistence, yet the influence of capsule design, microbial species, and environmental conditions on performance remains poorly understood. We evaluated alginate encapsulation of the PAH-degrading bacteria Pseudomonas putida and Novosphingobium aromaticivorans across nutrient conditions and capsule formulations. Encapsulation effects varied by species and medium, influencing growth rate, maximum cell density, overall growth, and lag time; notably, encapsulation shortened lag time of N. aromaticivorans in sRB15 medium (36.9 h to 3.9-5.3 h). Enumeration methods also affected apparent cell recovery. After 8 weeks, encapsulation had no significant effect on P. putida but resulted in increased concentrations of N. aromaticivorans relative to planktonic cultures (1.22 x 10; vs. 2.05 x 10; CFU/mL). Capsule composition further influenced cell retention: increasing alginate approximately doubled capsule-associated cell concentrations, while chitosan coatings reduced cell concentrations within capsules without affecting external concentrations. These findings demonstrate that the benefits of encapsulation are species- and environment-dependent and that capsule formulation can be tuned to influence bacterial persistence and release, informing the design of encapsulated inoculants for bioaugmentation applications.

Antimicrobial resistance poses a global health threat, yet the fate of antimicrobial resistance genes (ARGs) and microbial communities from wastewaters in receiving rivers is rarely quantified. This study investigated the degradation kinetics of ARGs in sewage-polluted river water mesocosms and under sulfamethoxazole and copper stress, separately and in combination. Seven clinically relevant ARGs (blaCTX-M, blaNDM, qnrS, sul2, tetW, ermF, and aph(3'')-Ib), a mobile genetic element marker (intI1) and bacterial marker genes (uidA, 16S rDNA) were quantified by qPCR. Bacterial community changes were monitored using 16S rDNA amplicon sequencing. All target genes decayed with approximately first-order kinetics in all conditions, with qnrS (half-life: 8.0 h) and blaCTX-M (8.4 h) declining most rapidly and intI1 (32.5 h) and sul2 (46.5 h) declining most slowly. Ordination showed that ARG composition shifted primarily over time rather than by antibiotic/metal treatment. Bacterial communities shifted from initially Campylobacterota dominated ([&ge;] 90%) to Pseudomonadota, Bacillota, and Actinomycetota dominated communities over 168 h. Quantitative microbiome profiling showed a 95% reduction in ASV richness. PICRUSt2 analysis suggested progressive enrichment of aerobic and several metabolic pathways, as the mesocosms transitioned from highly polluted anoxic conditions to oxygenated conditions. Shifts in microbial community composition were coupled with changes in the resistome as identified by Procrustes analyses (r = 0.841, p = 0.001) and other analyses. Together, results show that river self-purification can rapidly reduce ARG loads, at least in warmer climates. This study provides approximate kinetic parameters for mathematical models to predict the fate of ARGs in rivers.

Pseudolysogeny is a latent state in which phage development is delayed after infection and has been proposed to promote phage persistence under unfavorable conditions. Virulent phage T3 has been reported to establish pseudolysogeny after infecting starved E. coli, then resume lytic replication following transfer to nutrient-rich media, a phenotype linked to the T3 SAMase gene. Here, we revisited the findings of Krueger et al. (1975) to test pseudolysogeny in T3 and examine phage propagation under nutrient-limited conditions. Both T3 and T7 showed impaired propagation under nutrient limitation, with the most stringent conditions causing substantial losses in recoverable infective centers. T3 was modestly more resilient than T7 under these conditions, but we were unable to reproduce the reported phenotype in which T3 remained latent while T7 replicated normally. Supplementation of minimal medium with small amounts of LB supported propagation of both phages, and a repeat experiment designed to more closely match the historical protocol, including post-adsorption reduction of extracellular phage carryover, likewise failed to reveal a T3-specific pseudolysogenic state. Together, our results indicate that, in this experimental system, phage propagation dynamics are more consistently explained by nutrient conditions and media switching than by starvation prior to infection. These findings suggest that the previously reported T3 pseudolysogeny phenotype may depend on additional environmental or methodological factors and underscore the importance of revisiting historically reported phage behaviors using modern controls.

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.

Klebsiella aerogenes, a member of the Enterobacteriaceae, is a causative agent of healthcare-associated infections, and outbreaks caused by drug-resistant K. aerogenes have been reported worldwide. The range of antimicrobial agents available for treating infections caused by carbapenem-resistant K. aerogenes is limited. While in vivo animal experiments using clinical K. aerogenes isolates to evaluate antimicrobial therapy could facilitate selection of the most effective treatment, conducting infection experiments involving large numbers of mammals such as mice is challenging due to ethical concerns related to animal welfare. Silkworms are invertebrates increasingly used as experimental models for infectious disease research to evaluate antimicrobial efficacy. In this study, we aimed to establish a silkworm infection model using a clinical K. aerogenes isolate to evaluate its utility for determining effective antimicrobial doses. K. aerogenes strains were isolated from a patient at a Japanese hospital, and a silkworm infection model was established using the clinical isolate. The non-metallo-beta-lactamase-producing strain K. aerogenes TUM25562, isolated from a patient with a complicated urinary tract infection, was susceptible to meropenem (MEPM) and gentamicin (GM) in vitro. During treatment, additional isolates with increased resistance to MEPM and subsequently to both MEPM and GM emerged. K. aerogenes TUM25562 caused dose-dependent mortality in silkworms. Treatment with clinically equivalent weight-based doses of MEPM or GM did not cure the infected silkworms. The median effective (ED50) doses of MEPM and GM were therefore investigated using the silkworm infection model. Administration of higher doses corresponding to four times the ED50 significantly prolonged the survival of infected silkworms. These results suggest that a silkworm infection model using clinical K. aerogenes isolates may provide a practical approach for evaluating antimicrobial efficacy and determining effective antimicrobial doses.

BackgroundLive bird markets (LBMs) may facilitate the persistence and dissemination of Escherichia coli and antimicrobial-resistant bacteria because of intensive bird handling, environmental contamination and inadequate sanitation. However, information on E. coli contamination and antimicrobial susceptibility in LBM environments in Rajshahi District, Bangladesh, remains limited. ObjectiveThis study aimed to determine the prevalence, identify the cultural and biochemical characteristics, and assess the antimicrobial susceptibility pattern of E. coli isolated from water, soil and bird-dropping samples collected from LBMs in Rajshahi District. MethodsA total of 60 environmental samples, comprising 20 water, 20 soil and 20 bird-dropping samples, were collected from LBMs across all ten upazillas of Rajshahi District between January and June 2023. E. coli was isolated and identified using cultural characteristics, Gram staining and biochemical tests. Antimicrobial susceptibility was determined by the Kirby- Bauer disc diffusion method against seven antimicrobial agents using CLSI interpretive criteria. ResultsE. coli was detected in 33 of 60 samples, giving an overall prevalence of 55.00%. Prevalence was highest in bird-dropping samples (75.00%), followed by water (55.00%) and soil (35.00%). Among the 33 isolates, resistance was highest to oxytetracycline (78.79%) and amoxicillin (63.64%), followed by ciprofloxacin (48.48%), doxycycline (33.33%), levofloxacin (9.09%), erythromycin (9.09%) and neomycin (6.06%). Sensitivity was highest to neomycin (60.61%), followed by levofloxacin and erythromycin (51.51% each). ConclusionThe high prevalence of E. coli and substantial resistance to several commonly used antimicrobials indicate considerable microbiological and antimicrobial-resistance concerns in LBM environments. Improved sanitation, biosecurity, hygienic poultry handling and prudent antimicrobial use are warranted to reduce environmental contamination and potential transmission of resistant bacteria.

Antimicrobial resistance (AMR) represents a major global health threat, with plasmid-borne mcr genes driving colistin resistance and exposing critical gaps in One-Health surveillance across human, animal, and environmental reservoirs. The most prevalent variant, mcr-1, remains difficult to monitor in resource-limited settings due to the lack of rapid, affordable, and field-deployable molecular tools. Here, we developed C12amcr, an integrated molecular toolbox that combines pre-amplification PCR with a fluorescent CRISPR-Cas12a assay targeting a conserved region of mcr-1 and a custom low-cost, hand-held 3D-printed portable fluorometer. Under optimized conditions, the assay achieved a limit of detection of 630 cells/mL. In poultry feces spiked with mcr-1-positive E. coli, C12amcr detected as few as 1,800 cells/mL. When tested on 22 community-derived E. coli isolates, the assay showed 100% concordance with both next-generation sequencing for mcr-1 detection and phenotypic colistin susceptibility testing by broth microdilution. The accompanying portable fluorometer performed equivalently to a laboratory microplate reader while enabling fully decentralized workflows compatible with portable PCR platforms. By integrating locally produced molecular reagents, straightforward protocols, and an accessible field-ready fluorescence reader, C12amcr overcomes key barriers to decentralized AMR surveillance and provides a practical, scalable solution for One-Health monitoring in resource-limited settings.