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A leakage-aware genomic prediction pipeline for meropenem resistance in Klebsiella pneumoniae using transformer-based resistome representation learning.

MOTIVATION: Antimicrobial resistance (AMR) in Klebsiella pneumoniae, particularly to carbapenems such as meropenem, is a major global health problem. Machine learning is increasingly used to predict resistance from genomic markers; however, many models fail to capture high-level gene-gene interactions and may exhibit inflated performance due to lineage-biased prediction. Existing genomic prediction models largely rely on flat feature representations that fail to capture epistatic gene interactions, and commonly suffer from inflated performance estimates due to phylogenetic data leakage. To address these limitations simultaneously, a leakage-aware hybrid TabTransformer-CatBoost pipeline was developed, combining self-attention-based resistome representation learning with gradient boosting classification under clade-aware data partitioning. A self-attention encoder converts sparse gene presence-absence profiles into contextualized latent embeddings, which are subsequently classified using gradient boosting to capture lineage-aware AMR patterns. RESULTS: The proposed architecture outperformed classical baselines including Logistic Regression, Random Forest, XGBoost, and optimized CatBoost models. Internal accuracy reached 92.59% for the Chained Hybrid configuration (area under the receiver operating characteristic curve, AUROC = 0.8670, F1 = 0.8537). Performance gains primarily originated from the embedding stage, as confirmed by ablation analysis. External validation across independent multinational cohorts (n = 305) demonstrated generalizability (AUROC = 0.8105; F1 = 0.7552). Permutation testing produced near-zero Matthews Correlation Coefficient (MCC) = 0.0091, indicating predictions reflect genuine biological signal rather than noise. These results establish attention-based genomic embedding with gradient boosting as a scalable, interpretable, and leakage-aware framework for clinical AMR prediction. AVAILABILITY AND IMPLEMENTATION: The source code for the TabTransformer-CatBoost framework, including preprocessing pipelines and pre-trained embeddings, is available at https://github.com/SibelKervanci/kp-meropenem-tabtransformer.

Journal Article

Farming reshapes the gut resistome, virulome, and mobilome of Cervidae.

The rapid expansion of cervid farming raises concerns about antimicrobial resistance (AMR) dissemination, yet its impact on the Cervidae gut microbiome remains poorly characterized. We integrated 89 newly sequenced fecal metagenomes with 599 publicly available datasets, comprising 285 metagenomes from farmed cervids and 370 from wild cervids, to construct a catalog of 15,494 non-redundant metagenome-assembled genomes (MAGs) representing 2,401 species. Our analysis demonstrates that farming profoundly reshapes the gut microbiome's functional composition. Specifically, farmed cervids exhibited significantly higher relative abundance, diversity, and heterogeneity of antimicrobial resistance genes (ARGs) compared to wild counterparts. We observed a robust synergistic relationship between ARGs, virulence factor genes, and mobile genetic element (MGE)-associated genes, identifying 70 ARG-MGE combinations as evidence of potential horizontal gene transfer. Plasmid profiling further suggested that a subset of ARGs may be associated with conjugative plasmids, with plasmid-associated ARGs being significantly more abundant in farmed than in wild cervids. Virome analyses indicated that bacteriophages, particularly Siphoviridae, may serve as mobile reservoirs for ARGs. Notably, Cervidae shared 268 ARG types with humans, including 23 high-risk genes associated with resistance to clinically important antibiotics (e.g. tetX1, vanRD, and bla-CTX-M-178), with Escherichia coli as a key cross-host carrier. These findings highlight that human-impacted cervid gut microbiomes are significant environmental reservoirs of clinically relevant AMR, underscoring the necessity for enhanced antibiotic stewardship and resistance surveillance in managed wildlife within a One Health framework.

Animals

Molecular surveillance of foodborne bacterial pathogens and resistome in food products from Hong Kong.

Foodborne infections pose an increasing public health challenge worldwide. The problem has been aggravated by the dissemination of antimicrobial resistance genes among zoonotic pathogens, which results in a sharp increase in antibiotic resistance rate recorded among the major foodborne pathogens. To obtain an overview of the extent to which food products purchased in the markets in Hong Kong were contaminated by foodborne pathogens, we collected 95 raw meat samples from wet markets and isolated 236 bacterial strains of various species, with Escherichia coli being the most dominant species (131 strains). Contamination of food products by multiple foodborne pathogens was commonly observed. These include both Gram-positive and Gram-negative bacteria that exhibit various levels of resistance, with some possessing multiple clinically important antibiotic resistance genes. Seventeen bacterial strains of various species isolated from three food samples were comprehensively analysed by the Oxford Nanopore R10.4 technology. Novel conjugative plasmids carrying antimicrobial resistance gene-bearing mobile genetic elements were commonly detectable in the test strains. Some of the plasmids were shown to have originated from other environmental sources or other bacterial species, indicating that raw foods in the local market may serve as a reservoir of resistance-encoding genetic elements from which such elements are disseminated to various microbial pathogens. These findings suggest a need to perform periodic but comprehensive surveillance of multidrug-resistant bacterial pathogens and the major antimicrobial resistance genes in common food products, so as to disrupt the transmission routes of such organisms and the resistance-encoding genetic elements that they harbour.

Hong Kong

Soil Acidification Enriches Antibiotic Resistome.

Soil acidification represents a critical global change issue. Its impacts on antibiotic resistance genes (ARGs), however, remain poorly understood. Here we first analyzed a published global dataset comprising 1012 sampling sites and found a significant negative correlation between soil pH and the total richness and relative abundance of ARGs. To validate the observed pattern, we subjected three soils (with initial pH 7.8-7.9) each to 4 acidification levels (pH 7, 6, 5, and 4) for 30 days and subsequent recovery for another 30 days in microcosms. Shotgun metagenomic sequencing revealed that acidification (pH 6, 5, and 4) significantly increased the total richness and relative abundance of ARGs, as well as the relative abundances of 175 ARG subtypes, across all three soils. These 175 acidification-enriched ARGs together accounted for more than 70% of all the ARGs under severely acidified conditions (pH 5 and 4). Moreover, 93% of the bacteria carrying acidification-enriched ARGs also carried various virulence factor genes homologs associated with pathogenicity in reference databases, resulting in increased risk score. The total relative abundance of the acidification-enriched ARGs was primarily associated with changes in bacterial community traits (community composition, acidification-enriched metabolic functions, and genome size), followed by the increase in availability of toxic metals. When soil recovered from severe acidification (pH 5 and 4), the total relative abundance of the acidification-enriched ARGs significantly declined, demonstrating that the effect of soil acidification is partially reversible. This study reveals an underrecognized risk of ARGs caused by soil acidification, highlighting that the prevention and mitigation of soil acidification are crucial for combating antibiotic resistance.

Hydrogen-Ion Concentration

Seasonal hydrological dynamics affected the diversity and assembly process of the antibiotic resistome in a canal network.

The significant threat of antibiotic resistance genes (ARGs) to aquatic environments health has been widely acknowledged. To date, several studies have focused on the distribution and diversity of ARGs in a single river while their profiles in complex river networks are largely known. Here, the spatiotemporal dynamics of ARG profiles in a canal network were examined using high-throughput quantitative PCR, and the underlying assembly processes and its main environmental influencing factors were elucidated using multiple statistical analyses. The results demonstrated significant seasonal dynamics with greater richness and relative abundance of ARGs observed during the dry season compared to the wet season. ARG profiles exhibited a pronounced distance-decay pattern in the dry season, whereas no such pattern was evident in the wet season. Null model analysis indicated that deterministic processes, in contrast to stochastic processes, had a significant impact on shaping the ARG profiles. Furthermore, it was found that Firmicutes and pH emerged as the foremost factors influencing these profiles. This study enhanced our comprehension of the variations in ARG profiles within canal networks, which may contribute to the design of efficient management approaches aimed at restraining the propagation of ARGs.

Seasons

Phenotype-genotype discordance in antimicrobial resistance profiles of Gram-negative uropathogens recovered from catheter-associated urinary tract infections in Egypt.

OBJECTIVES: Catheter-associated urinary tract infections (CAUTIs) are among the most common healthcare-associated infections in low- and middle-income countries (LMICs), but there are few resistome data available for relevant uropathogens. The goal of this study was to characterize the antimicrobial resistance (AMR) phenotypes and genotypes of a large collection of Gram-negative bacteria recovered from CAUTIs in a hospital in Mansoura, Egypt. METHODS: Phenotypic AMR profiles and whole-genome sequence data were generated for 132 isolates. Resistomes were predicted using ResFinder, CARD and AMRFinder. Similarity of uropathogen genomic data was determined using sourmash (kmer signatures). Escherichia coli genomic data were subject to a pangenome analysis using Panaroo. RESULTS: Sixty-seven E. coli (Phylogroup B2; 53.7%, 36/67), 14 Pseudomonas aeruginosa, 11 Klebsiella pneumoniae, 9 Proteus mirabilis, 8 Providencia spp., 5 Enterobacter hormaechei and 18 rare CAUTI-associated isolates were identified. Several (22/132) isolates were multidrug-resistant, while almost half (62/132) were extensively drug-resistant. Phenotype-genotype discordance was found to be an important consideration in resistome studies in Egypt, with a total concordance of 91% (1115/1225), 85.7% (1273/1485) and 80.5% (1196/1485) for ResFinder, CARD and AMRFinder, respectively. Pseudomonas, at the species level, exhibited the greatest discordance. At the antimicrobial level, meropenem was subject to greatest discordance. New AMR variants were found for Egypt for Pseudomonas (blaOXA-486, blaOXA-488, blaOXA-905, blaIMP-43, blaPDC-35, blaPDC-45, blaPDC-201) and E. coli (blaTEM-176, blaTEM-190). CONCLUSIONS: This study shows that there is phenotype-genotype discordance in AMR profiling among CAUTI isolates, highlighting the need for comprehensive approaches in resistome studies. We also show the genomic diversity of Gram-negative uropathogens contributing to disease burden in a little-studied LMIC setting.

Egypt

Comprehensive profiling of antibiotic resistance genes and functional clusters of orthologous groups annotation of gut microbiota in Indonesian Kedu chickens.

Antibiotic resistance is a growing global health concern, with poultry systems acting as important reservoirs of antibiotic resistance genes (ARGs). However, resistome and functional profiles of indigenous chickens raised under traditional systems remain underexplored. This study aimed to characterize the antibiotic resistome, virulence factor genes, and metabolic potential of gut microbiota in Indonesian Kedu chickens using a shotgun metagenomic approach. Digesta samples from five gastrointestinal segments of 21 healthy adult chickens were analyzed through high-throughput sequencing. ARGs were identified using the Comprehensive Antibiotic Resistance Database (CARD) and Antibiotic Resistance Genes Databases (ARDB), while virulence factors and functional genes were annotated using Virulence Factor Database (VFDB), Clusters of Orthologous Groups (COG), and Carbohydrate-Active EnZymes (CAZy) databases. Results revealed a diverse resistome dominated by multidrug resistance and efflux pump mechanisms, with prominent genes associated with fluoroquinolone, tetracycline, β-lactam, and glycopeptide resistance. The detection of clinically relevant ARGs suggests that genetic determinants associated with antimicrobial resistance are present in the gut microbiota of traditionally raised Kedu chickens, although metagenomic data alone cannot determine whether these genes are actively expressed or confer phenotypic resistance. Virulence factor analysis showed functions related to adherence, immune evasion, iron acquisition, quorum sensing, and efflux activity, reflecting strong microbial adaptability. Functional profiling demonstrated enrichment in translation, carbohydrate and amino acid metabolism, genome maintenance, and cell envelope biogenesis. Additionally, CAZyme analysis indicated a high capacity for complex polysaccharide degradation, supporting efficient utilization of fiber-rich traditional diets. In conclusion, this study provides a comprehensive metagenomic overview of antibiotic resistance and functional potential in Kedu chicken gut microbiota, emphasizing the importance of incorporating indigenous poultry into antimicrobial resistance surveillance within a One Health framework.

Antibiotic resistance genes

Whole-Genome Analysis of Multidrug-Resistant Escherichia coli from Bloodstream Infections in Iraqi Cancer Patients.

BACKGROUND: In Iraq, oncology patients with bloodstream infections face escalating treatment challenges due to rising antimicrobial resistance in Escherichia coli, compounded by limited diagnostic capacity and restricted therapeutic options. However, data from oncology settings in Iraq are limited. This study aimed to characterize and compare the antimicrobial resistance gene profiles of multidrug-resistant and antibiotic-sensitive E. coli isolates from cancer patients, to inform infection control and stewardship strategies. METHODS: A prospective, multicenter investigation was conducted in three oncology hospitals in Baghdad. Fifty-five Escherichia coli bloodstream isolates, 36 multidrug-resistant (MDR) (65.5%) and 19 antibiotic-sensitive (34.5%), underwent phenotypic susceptibility testing using VITEK® 2 and disk diffusion. Whole-genome sequencing (Illumina MiSeq) was performed, and antimicrobial resistance genes (ARGs) were identified using AMRFinderPlus and classified by functional category. RESULTS: MDR isolates showed a broad resistome dominated by efflux systems. Across all isolates, a total of 36 unique antimicrobial resistance genes were identified, underscoring substantial resistome diversity., efflux pump genes were detected in 100%, β-lactamase genes in 88.9%, macrolide resistance genes in 66.7%, tetracycline resistance genes in 55.6%, and aminoglycoside resistance genes in 41.7%. Representative determinants included AcrAB-TolC/EmrAB-TolC/MdtABC-TolC (efflux) and BlaEC family/CMY-42/TEM types (β-lactams). Among sensitive isolates, only 11 antibiotic-associated genes, mainly efflux or regulators (e.g., acrF, emrD, emrR, emrY, tolC; regulators marR, evgA; target parC; others baeS, cpxA, cysB), overlapped with MDR, suggesting a shared core that is insufficient alone for phenotypic resistance without additional high-level mechanisms. CONCLUSIONS: Multidrug-resistant E. coli from oncology patients harbor dense, efflux-driven resistomes supplemented by diverse β-lactamases and other resistance determinants, while sensitive isolates retain a limited core set of genes. Genomic surveillance in cancer centers is critical for anticipating resistance emergence and informing targeted antimicrobial strategies.

Humans

Metabolic reprogramming and taxonomic drivers in bacterial vaginosis: A large-scale metagenomic meta-analysis.

OBJECTIVE: Bacterial vaginosis (BV) represents a profound ecological shift from a Lactobacillus-dominated microbiota to a diverse polymicrobial biofilm associated with adverse outcomes. While taxonomic signatures are well-documented, the functional mechanisms driving this transition remain obscured. This study elucidates the genomic potential for metabolic reprogramming and the putative "functional handover" underpinning the stability of the dysbiotic state. METHODS: A computational meta-analysis of 3557 vaginal microbiomes from diverse global cohorts was performed using the standardized MGnify pipeline. A high-resolution subset of 187 whole-genome shotgun (WGS) metagenomes was stratified to compare functional potential across demographic groups. Taxon-function interaction networks were constructed, utilizing a dual-filter statistical approach (p&#x202f;<&#x202f;0.05 and effect size ranking), to map the shift from homeostatic maintenance to dysbiotic metabolic potential. RESULTS: BV was characterized by a fundamental shift from "maintenance" pathways to high-turnover "growth-oriented" genomic repertoires. While ABC transporter-like domains were present in healthy communities, dysbiosis was marked by a quantitative expansion and diversification of these systems alongside P-loop NTPases. Network analysis revealed a putative "functional handover": while Gardnerella serves as the adherent structural scaffold, the metabolic burden appears to be associated with secondary anaerobes, specifically BVAB1 and Sneathia, which exhibit strong genomic correlations with nutrient transport and stress response pathways. Crucially, microbiomes from women of African ancestry (Black cohort) exhibited a distinct functional profile with genomic signatures consistent with functions previously associated with resistome expansion (e.g., tetracycline/macrolide resistance), contrasting with Asian cohorts. CONCLUSION: BV is a state of metabolic reprogramming where genomic functional dominance is transferred from Lactobacillus to a cooperative network of anaerobic opportunists. Identifying BVAB1 and Sneathia as candidate metabolic engines, supported by a Gardnerella scaffold, challenges current therapeutic paradigms and highlights the potential for precision medicine targeting specific functional drivers and resistome profiles across diverse populations.

Humans

The fate of antibiotics and antibiotic resistance genes in Large-Scale chicken farm Environments: Preliminary view of the performance of National veterinary Antimicrobial use reduction Action in Guangdong, China.

In 2018, China implemented the Veterinary Antimicrobial Use Reduction Action to curb the rapid development of antibiotic resistance (AR). However, the AR-related pollutions in animal farms after the reduction policy has been poorly investigated. Here, we performed a comprehensive investigation combining UPLC-MS/MS, metagenomic, and bacterial genomic analyses in eight representative large-scale chicken farms in Guangdong, China. Our results showed that antibiotics and ARGs contaminations were more severe in broiler farms than in layer farms. Notably, diverse tet(X) variants were prevalent in the chicken farms. These tet(X)s was carried by diverse E. coli lineages and obviously correlated with ISCR2 and IS1B transposases. The resistomes in chicken farms was significantly correlated with microbial community, and multiple factor analyses indicated that the joint effect of antibiotics-microbial community-MGEs was the most dominant driver of ARGs. Host tracking identified a variety of ARG bacterial hosts and the co-occurrence of ARGs-MRGs-MGEs. Source tracking indicated that the inherent component represented the main feature of resistomes in different hosts, while ARG transfer between the chicken gut and farm environments were frequent. A multiperspective evaluation of AR risk revealed that the early effect of antibiotic reduction was exhibited by the mitigation of maximum level of risky ARGs, prevalence of environmental AR pathogens, and HGT potential of ARGs mediated by phage structures. Overall, our findings provide insights into the antibiotic and ARG profiles in large-scale chicken farms with different rearing strategies and demonstrate a preliminary view of the performance of antibiotic reduction actions in China.

Chickens

Contaminant-degrading bacteria are super carriers of antibiotic resistance genes in municipal landfills: A metagenomics-based study.

Municipal landfills are hotspot sources of antimicrobial resistance (AMR) and are also important habitats of contaminant-degrading bacteria. However, high diversity of antibiotic resistance genes (ARGs) in landfills hinders assessing AMR risks in the affected environment. More concerned, whether there is co-selection or enrichment of antibiotic-resistant bacteria and contaminant-degrading bacteria in these extremely polluted environments is far less understood. Here, we collected metagenomic datasets of 32 raw leachate and 45 solid waste samples in 22 municipal landfills of China. The antibiotic resistome, antibiotic-resistant bacteria and contaminant-degrading bacteria were explored, and were then compared with other environmental types. Results showed that the antibiotic resistome in landfills contained 1,403 ARG subtypes, with the total abundance over the levels in natural environments and reaching the levels in human feces and sewage. Therein, 49 subtypes were listed as top priority ARGs for future surveillance based on the criteria of enrichment in landfills, mobilizable and present in pathogens. By comparing to those in less contaminated river environments, we elucidated an enrichment of antibiotic-resistant bacteria with contaminant-degrading potentials in landfills. Bacteria in Pseudomonadaceae, Moraxellaceae, Xanthomonadaceae and Enterobacteriaceae deserved the most concerns since 72.2&#xa0;% of ARG hosts were classified to them. Klebsiella pneumoniae, Acinetobacter nosocomialis and Escherichia coli were abundant multidrug-resistant pathogenic species in raw leachate (&#x223c;10.2&#xa0;% of total microbiomes), but they rarely carried contaminant-degradation genes. Notably, several bacterial genera belonging to Pseudomonadaceae had the most antibiotic-resistant, pathogenic, and contaminant-degrading potentials than other bacteria. Overall, the findings highlight environmental selection for contaminant-degrading antibiotic-resistant pathogens, and provide significant insights into AMR risks in municipal landfills.

Metagenomics

Whole-genome sequencing links a Salmonella Newport ST164 outbreak on Fernando de Noronha to prior circulation in the Brazilian poultry supply chain.

Foodborne outbreaks at geographically isolated tourist destinations pose distinctive One Health challenges, combining limited local surveillance capacity, complex intercontinental supply chains, and high visitor turnover. In May 2021, a diarrheal outbreak linked to a gastronomic festival in Fernando de Noronha, that is a remote UNESCO World Heritage island off northeastern Brazil, was attributed to Salmonella enterica serovar Newport ST164. We applied an integrated genomic approach and epidemiological investigation to propose a transmission chain contextualizing and refining case definition of the S. Newport epidemic clone within national and international diversity. Whole-genome sequencing (WGS), SNP-based phylogenomic, pangenome analysis, Salmonella pathogenicity island (SPI) profiling, and resistome characterization was performed on 17 epidemiologically attributed outbreak isolates and 68 contextual genomes from Brazil, France, the United Kingdom, and the United States. The SNP analysis identified a 13 genome clonal core with less than 20 different SNPs demonstrating the possible connection between 9 patient isolates, 2 food isolates, and 2 food handler isolates, consistent with the involvement of colonised kitchen staff in cross-contamination of the ready-to-eat mussel dish. Three poultry isolates in 2020 from a mainland producer, &#x223c;2180&#xa0;km from Fernando de Noronha, differed only 13 to 17 Core-SNPs from the outbreak core, suggesting prior lineage circulation in the supply chain. Pangenome analysis also supports this evidence revealing near-complete genomic overlap of 4544 shared genes within the 5745 gene clusters (99.9%) between outbreak and non-outbreak backgrounds that mostly differentiate by a defense/prophage-associated accessory module. The resistome comprised intrinsic efflux determinants without acquired resistance and showed 35.3% of intermediate ciprofloxacin susceptibility. This One Health based study provides a WGS genomic reconstruction of a S. Newport ST164 outbreak at a remote tourist island, supporting the possibility of circulation from poultry-associated mainland reservoirs and findings consistent with cross-contamination at a gastronomic seafood festival.

Brazil

Optimizing a culture-enriched hybrid metagenomics pipeline to assess the AMR footprint of livestock manure in anaerobic digestate.

The role of environmental samples from livestock production systems, including manure and anaerobic digestate, as reservoirs of antimicrobial resistance genes (ARGs) is likely underestimated because conventional metagenomic approaches can overlook low-abundance ARGs and often lack the resolution to associate these genes with their microbial hosts and co-localized mobile genetic elements (MGEs). We evaluated whether culture-enriched metagenomics (CEMG), with and without antibiotic selection, enhances ARG detection in anaerobic digestate and improves the resolution of ARG-MGE-host associations using hybrid short- and long-read metagenomic assembly. CEMG increased ARG recovery; mean ARG abundance rose from 15.4 counts per million (CPM) in metagenomic fresh digestate (FD) to 124 CPM in CEMG without antibiotics and 160 CPM in antibiotic-selective CEMG. In FD, only 9 unique ARGs were detected, whereas CEMG recovered 112, including ARGs of clinical importance, such as glycopeptide resistance, beta-lactamase genes, and the cfr 23S rRNA methyltransferase conferring cross-resistance to multiple antibiotic classes. Antibiotic selection induced targeted, class-specific shifts in ARG profiles, with ARGs associated with tetracycline resistance consistently enriched across treatments. Hybrid metagenomic assembly resolved the genomic context of 784 ARGs, of which 59.3% were co-localized with at least one class of MGEs, predominantly plasmids and integrative conjugative elements/integrative mobilizable elements. Biocide and metal resistance genes frequently co-occurred with ARGs on the same contigs. Together, these findings demonstrate that antibiotic-selective culture enrichment enhances resistome surveillance by improving detection of low-abundance ARGs, while hybrid assembly provides critical genomic context for assessing their mobility and host associations.IMPORTANCELivestock manure and its byproducts, such as anaerobic digestate, are recognized as important environmental reservoirs of antimicrobial resistance genes (ARGs) and resistant bacteria, yet current metagenomic approaches may underestimate this risk by failing to detect low-abundance but clinically relevant ARGs. Here, we show that integrating culture enrichment with hybrid metagenomics improves ARG recovery and reveals ARG co-localization with mobile genetic elements and putative bacterial hosts. This approach captures a cultivable and condition-responsive fraction of the resistome that is not readily accessible through direct metagenomic sequencing alone, providing a more informative framework for environmental AMR surveillance.

anaerobic digestion

Drug resistant Klebsiella pneumoniae from patients and hospital effluent: a correlation?

BACKGROUND: The application of wastewater-based epidemiology has gained traction as a cost effective tool in antimicrobial resistance (AMR) surveillance with studies showing a correlation between the presence of resistant bacteria from hospital sewage and patients. This study compared Klebsiella pneumoniae from patients and hospital effluent in terms of antibiotic resistance patterns, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and phylogenomic relationships. RESULTS: Pooled effluent samples were collected from the final effluent point of a regional hospital and K. pneumoniae isolates were identified on selective media. Clinical isolates were also collected from the same hospital. Antimicrobial susceptibility testing (AST) was performed using the VITEK&#xae; 2 system. DNA was extracted prior to whole genome sequencing (WGS). The resistome, mobilome, and phylogenetic lineages of sequenced isolates were assessed using bioinformatics analysis. A total of 10 randomly selected presumptive and 10 clinical K. pneumoniae constituted the sample and were subjected to AST. Total resistance was observed in the clinical samples to cefuroxime, cefotaxime, piperacillin/tazobactam, gentamicin, tobramycin and trimethoprim/sulfamethoxazole. The effluent isolates exhibited total susceptibility to most antibiotics but showed resistance to amoxicillin/clavulanic acid and piperacillin/tazobactam (100%), and tigecycline (10%). The effluent isolates did not exhibit a diverse resistome, while the clinical isolates harboured genes conferring resistance to aminoglycoside (aph(6)-Id, aph(3'')-Ib, aac(6')-Ib-cr, aadA16), &#xdf;-lactam (blaSVH group, blaOXA group, blaTEM group), and fluoroquinolone (oqxA, oqxB) antibiotics. Only class 1 integrons were identified. Phylogenetic analysis revealed that effluent isolates from this study were not closely related to the clinical isolates. CONCLUSION: This study showed no correlation between the resistance profiles of the clinical and effluent isolates. The relationship between AMR in hospital effluent and clinical resistance may depend on the antimicrobial agents and bacterial species studied.

Klebsiella pneumoniae

Long-read sequencing reveals putatively mobilizable resistance genes and multi-drug resistance plasmids underestimated by short-read metagenomics.

While shotgun metagenomics is often used to profile antibiotic resistome in gut microbial communities, few studies have investigated if the choice of sequencing platform and assembly strategy affect what mobile genetic elements and antimicrobial resistance genes are recovered. In this study, we compared three platforms (Illumina, Oxford Nanopore, and PacBio HiFi) and seven assembly strategies on gut metagenomes from cattle, pig, and human as case studies. Long-read assemblies recovered 5- to 7-fold more plasmid sequence than Illumina in cattle and pig (mean 17.0 Mb vs. 3.1 Mb), while Illumina performed comparably in the less diverse human gut where high per-species coverage enabled effective short-read plasmid assembly. Long reads also detected more resistance genes on plasmid contigs. Hybrid assembly results depended on the algorithm: scaffolding-based OPERA-MS preserved long-read contiguity and recovered more plasmid-borne resistance genes, while the short-read-centric metaSPAdes hybrid mode produced fragmented assemblies. After collapsing haplotype redundancy, PacBio HiFi identified 2 and 49 unique multi-drug resistance plasmid lineages in cattle and pig, respectively. On the other hand, only 2 and 4 were identified from Illumina. Long reads also placed far more ARGs in a putative mobilization context (50-73%) compared to 14-21% for short reads. Platform and assembly strategy are thus key variables in mobilome and resistome characterization and should be accounted for in antimicrobial resistance surveillance.

Animals

Tomato bacterial wilt disease outbreaks are accompanied by an increase in soil antibiotic resistance.

The presence of soil-borne disease obstacles and antibiotic resistance genes (ARGs) in soil leads to serious economic losses and health risks to humans. One area in need of attention is the evolution of ARGs as pathogenic soil gradually develops, which introduces uncertainty to the dynamic ability of conventional farming models to predict ARGs. Here, we investigated variations in tomato bacterial wilt disease accompanied by the resistome by metagenomic analysis in soils over 13 seasons of monoculture. The results showed that the abundance and diversity of ARGs and mobile genetic elements (MGEs) exhibited a significant and positive correlation with R. solanacearum. Furthermore, the binning approach indicated that fluoroquinolone (qepA), tetracycline (tetA), multidrug resistance genes (MDR, mdtA, acrB, mexB, mexE), and &#x3b2;-lactamases (ampC, blaGOB) carried by the pathogen itself were responsible for the increase in overall soil ARGs. The relationships between pathogens and related ARGs that might underlie the breakdown of soil ARGs were further studied in R. solanacearum invasion pot experiments. This study revealed the dynamics of soil ARGs as soil-borne diseases develop, indicating that these ecological trends can be anticipated. Overall, this study enhances our understanding of the factors driving ARGs in disease-causing soils.

Soil Microbiology

Integrated metagenomic, culture-based, and whole genome sequencing analyses of antimicrobial resistance in wastewater and drinking water treatment plants in Barcelona, Spain.

The misuse and overuse of antimicrobials drive the emergence of antimicrobial resistance (AMR), a critical global health concern. While wastewater treatment plants (WWTPs) are essential for removing microorganisms and contaminants, they also serve as hotspots for antibiotic-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs), facilitating their persistence and dissemination. This study investigated AMR in two WWTPs and one drinking water treatment plant (DWTP) in the Baix Llobregat area of Barcelona, Spain. Four sampling campaigns were conducted during winter and summer 2023 across different treatment stages. Due to drought conditions, reclaimed water from the Baix Llobregat WWTP was discharged upstream of the DWTP intake to supplement water resources for indirect potable reuse. A total of 991 cultivable ARB were obtained, enabling phenotypic and genotypic characterisation. The most prevalent included Aeromonas spp. (44.3&#xa0;%), Enterobacterales (27.9&#xa0;%), Pseudomonas spp. (19.1&#xa0;%), Acinetobacter spp. (4.8&#xa0;%), Shewanella spp. (2.2&#xa0;%), Stenotrophomonas spp. (1&#xa0;%), and others (0.7&#xa0;%). Among these, 57.3&#xa0;% were multidrug-resistant and 2.7&#xa0;% were extensively drug-resistant. Furthermore, 34.6&#xa0;% produced extended-spectrum beta-lactamases, 14.1&#xa0;% harboured carbapenemase genes, and 2.9&#xa0;% exhibited colistin resistance. Shotgun metagenomic analysis revealed high taxonomic diversity, without dominant genera across treatment stages. The resistome was dominated by ARGs conferring resistance to beta-lactams, aminoglycosides, and macrolides, alongside genes linked to biocide resistance and heavy metal tolerance. Spearman correlation analysis of selected sequenced strains suggested a weak to moderate co-occurrence between ARGs and biocide or heavy metal tolerance genes. These findings underline WWTPs as AMR hotspots and reinforce the need to monitor DWTP source water within the One Health framework.

Wastewater

Comprehensive Microbiome Analyses for Regenerative Endodontic Therapy.

INTRODUCTION: Comprehensive microbiome analyses include the study of all microbial taxa, including bacteria, archaea, viruses, and fungi, as well as their functional activities and antibiotic resistance gene expression. Regenerative endodontic therapy presents a clinical situation where the most effective antimicrobial approaches are needed in order to ensure clinical success. METHODS AND RESULTS: In this paper, different contemporary technologies for the identification of endodontic microorganisms, such as with next generation sequencing, and their functional characterization, such as with whole genome sequencing, are described. The role of transcriptomics, as well as resistome analysis, are also discussed. Furthermore, the manner in which all this work and knowledge could be incorporated into clinical endodontics in general, and regenerative endodontic therapy as a special treatment, is outlined. CONCLUSIONS: Comprehensive microbiome analysis can lead to the development of more effective and personalized antimicrobial treatment.

Endodontics