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Portable metagenomics for preventive surveillance and outbreak control in livestock and poultry: Pathogen detection, resistome profiling, and antimicrobial stewardship.

Conventional diagnostics for livestock and poultry outbreaks commonly rely on culture or targeted PCR panels, which may be too slow or too narrow to guide early control decisions. Portable metagenomics, particularly real-time nanopore sequencing, offers a route to broad pathogen detection, antimicrobial-resistance gene profiling, and outbreak investigation within an integrated workflow. This implementation-focused review evaluates how near-point-of-care metagenomics may support preventive veterinary medicine through earlier detection, surveillance, cohorting, biosecurity decisions, and antimicrobial stewardship. We synthesize sample-to-answer workflows for enteric and respiratory disease in food-producing animals, including sampling, nucleic-acid extraction, host depletion or target enrichment, library preparation, sequencing, bioinformatics, quality control, and interpretation. Applications in calf diarrhea, bovine respiratory disease, poultry outbreaks, mastitis, and resistome monitoring are considered alongside the central limitation that detection alone does not establish causation. Pathogen and resistance-gene signals must therefore be interpreted with clinical signs, lesions, epidemiology, controls, and confirmatory testing. We also propose a minimum reporting checklist, intended as a practical framework rather than a validated consensus standard. Portable metagenomics is not a replacement for conventional diagnostics, but appropriately validated workflows can reduce uncertainty during time-sensitive outbreaks and support more judicious antimicrobial use.

Animals

Whole genome sequence data set of methicillin-resistant Staphylococcus aureus isolated from a milkman associated with cows with subclinical mastitis in Kiruhura district, Uganda.

The whole-genome sequence data set for methicillin-resistant Staphylococcus aureus, which was isolated from a milkman associated with cows with subclinical mastitis in the Kiruhura district of Uganda, is presented here. The assembled genome size was 2822,509 bp, with a 33% GC, 2 Contigs, a Contig N50 of 2818,424, and 1 Contig L50. You can access the genome sequence and related metadata at https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_056782255.1/. This dataset can be used again for resistance gene mapping, comparing genomic analysis, and comprehending genetic diversity among MRSA isolates from Ugandan milkmen.

Antimicrobial-resistant genes Staphylococcus aureu

Mobile genetic elements-driven partitions of mega-plasmids resistome in Salmonella Infantis.

Salmonella enterica serovar Infantis (S. Infantis) becomes the primary pathogen among the top Salmonella serotypes, contributing to numerous cases of foodborne illness annually in the United States. S. Infantis infection has spread rapidly worldwide, especially the clones with pESI-like plasmids. However, the underlying mechanisms regarding the transmission of S. Infantis, particularly mobile genetic elements (MGEs), mediated horizontal gene transfer, are limited. The objective of this study was to evaluate the relationship, if any, among MGEs, antibiotic-resistant genes (ARGs), and virulence factors (VFs) within S. Infantis via genomic analysis. A total of 91 S. Infantis complete genomes with high sequencing quality were selected for downstream bioinformatic analysis. The results showed that the majority of VFs were located in the bacterial chromosomes, while most ARGs were carried by S. Infantis mega-plasmids in an MGE-favored manner. Integrons and transposons were closely associated with certain ARGs, but prophages within mega-plasmids displayed a diverse ARG profile. Collectively, MGE-mediated horizontal gene transfer might lead to ARG acquisition by mega-plasmids, subsequently contributing to the resistome of S. Infantis. Our findings provide insights into the development of MGE-associated resistome in S. Infantis that could inform more effective prevention and intervention strategies to control this pathogen, further ensuring public health and safety.IMPORTANCEThe rapid emergence and transmission of antibiotic-resistant foodborne pathogens pose a significant risk to public health, necessitating the discovery of underlying mechanisms to control multidrug-resistant pathogens. Salmonella enterica serovar Infantis (S. Infantis) has become a pathogen of clinical and epidemiological relevance in recent years, ranking as the top prevalent serovar associated with foodborne illnesses and exhibiting resistance to several antibiotics. The current investigation of multidrug resistance (MDR) S. Infantis strains primarily emphasized the presence of mega-plasmids. However, the question of how mega-plasmids contribute to the transmission of antibiotic-resistant genes (ARG) is unaddressed. Utilizing the genomic characterization of S. Infantis complete genomes with high quality, our study revealed that the resistome of S. Infantis mega-plasmids-the primary ARG reservoirs of S. Infantis-followed a specific pattern of mobile genetic elements (MGEs). Monitoring the spread of MGE-carried ARGs within mega-plasmids should be considered in future surveillance.

Interspersed Repetitive Sequences

The spatial and temporal distribution of Staphylococcus aureus along a tropical Hawaiian watershed.

Staphylococcus aureus is a leading cause of community-acquired skin and soft-tissue infections worldwide. One major route of exposure is recreating in marine waters, but knowledge is limited regarding the drivers of S. aureus in surface waters that discharge into marine environments. This study explores spatial and temporal distributions of S. aureus, including antimicrobial-resistant and virulence genes, using both culture-dependent and molecular techniques across a tropical Hawaiian watershed with a gradient of human influence. Negative binomial generalized linear mixed models revealed that the interaction between spatial and temporal factors was the strongest predictor of S. aureus and associated genes. Cultured S. aureus was highest at mid-watershed sites in summer, which included a popular swimming hole, suggesting human shedding as a significant source. Molecular detection of S. aureus (femA gene) yielded concentrations two orders of magnitude higher than cultured concentrations and peaked at estuarine sites with the greatest nutrients and water residence times. In the winter at upstream sites with no public access, staphylococci antibiotic-resistant (mecA) and S. aureus virulence gene (etb) were elevated, indicating highly pathogenic S. aureus strains in surface waters may originate from zoonotic sources. Our findings indicate that human and zoonotic sources contribute antibiotic-resistant and virulent S. aureus to watersheds, with streams facilitating environmental transmission to marine waters. This watershed-scale assessment enables the prediction of spatial and temporal conditions associated with elevated S. aureus concentrations, thereby reducing exposure and infections.

Staphylococcus aureus

Detection of opportunistic bacterial pathogens with intrinsic amoxicillin- and cephalosporin-resistance in wild koala faecal microbiomes.

Opportunistic bacterial pathogens frequently associated with human clinical infections, including antimicrobial-resistant strains, are infiltrating the microbiomes of wild animals, where they have the potential to negatively impact wildlife health. Bacterial genes conferring resistance to amoxicillin have previously been reported in koala (Phascolarctos cinereus) faecal DNA. Koalas are facing several key threats, including wildfires, and affected individuals may receive amoxicillin therapy to treat burn wounds. This study aimed to identify the species of amoxicillin-resistant bacteria in koala gut microbiomes and determine if they are opportunistic pathogens. Faecal samples collected from 98 wild-caught koalas were cultured using amoxicillin-supplemented media to isolate amoxicillin-resistant Gram-negative enteric bacteria. Isolates were screened using 16S rRNA PCR and Sanger sequencing to identify opportunistic pathogenic species, which then underwent whole-genome sequencing and antimicrobial susceptibility testing. Intrinsically amoxicillin-resistant opportunistic pathogens were obtained from 9.2% (9/98) of koala faecal samples and comprised Klebsiella oxytoca (6/98, 6.1%), Klebsiella pneumoniae (1/98, 1.0%) and Citrobacter spp. (2/98, 2.0%). Seven of nine amoxicillin-resistant opportunistic pathogens also exhibited cephalosporin resistance. Four K. oxytoca isolates belonged to lineages associated with human clinical infections, which also have the potential to cause disease in koalas, including fatal systemic infections in pouch young. The presence of amoxicillin- and cephalosporin-resistant strains may also increase the risk of gut dysbiosis and opportunistic infections when penicillins or cephalosporins are required to treat bacterial infections in koalas, highlighting the importance of good antimicrobial stewardship. The study findings demonstrate the One Health perspective of microbial pathogens and the intertwined microbial ecology between humans and wildlife.

Animals

Transferable IncHI2-Associated blaLAP-2 and blaCTX-M-55 Resistance Platforms in Foodborne Salmonella.

Extended-spectrum β-lactamase genes in foodborne Salmonella enterica can disseminate through mobile multidrug-resistance platforms. IncHI2 plasmids are important resistance vehicles capable of carrying complex resistance regions and facilitating their horizontal transfer across diverse bacterial backgrounds, but the transfer and genomic organization of IncHI2 elements co-carrying blaLAP-2 and blaCTX-M-55 remain insufficiently characterized. This study investigated two multidrug-resistant foodborne isolates recovered in Shanghai in 2022: Salmonella Agona ST13 isolate Sal22C150 and Salmonella Havana ST1527 isolate Sal22P208. Antimicrobial susceptibility testing, whole-genome sequencing, conjugation, plasmid-retention analysis, comparative genomics, as well as strain- and plasmid-level phylogenetic analyses were performed. Both isolates exhibited broad antimicrobial resistance, including resistance to extended-spectrum cephalosporins. In both isolates, blaLAP-2 and blaCTX-M-55 co-transferred with the IncHI2 replicon to Escherichia coli J53 at frequencies of (4.95 ± 0.41) × 10-5 and (4.46 ± 0.42) × 10-6 transconjugants per donor cell, respectively. All tested plasmid markers remained detectable through 20 passages without antimicrobial selection. Complete assembly of Sal22P208 confirmed the location of the three β-lactamase genes on the 275,096 bp IncHI2 plasmid pSal22P208. The plasmid contained a conserved conjugative backbone and mosaic accessory regions carrying 15 antimicrobial-resistance determinants together with mercury- and tellurium-resistance loci. SNP-based analysis placed pSal22P208 within a closely related cluster containing six reference IncHI2 plasmids differing by fewer than 30 SNPs and recovered from Salmonella and E. coli of animal, food, and human origin, suggesting a broad distribution of this plasmid lineage across diverse bacterial and ecological backgrounds. Sal22P208 additionally contained a Tn3-associated chromosomal multidrug-resistance region between rpmJ and rpmE that shared extensive structural similarity with a region in Citrobacter braakii LBA3. These findings highlight the role of transferable IncHI2 resistance platforms in the horizontal dissemination and short-term post-transfer maintenance of linked resistance determinants, while chromosomally integrated resistance regions may provide an additional route for the accumulation and inheritance of multidrug resistance in foodborne Salmonella.

IncHI2 plasmid

Evaluation of Oxford nanopore sequencing for antimicrobial resistance surveillance in Salmonella: comparison with phenotypic antimicrobial susceptibility in a large-scale study.

UNLABELLED: Salmonella is a major zoonotic foodborne pathogen, and antimicrobial resistance (AMR) in Salmonella presents a significant public health challenge. Compared with conventional antimicrobial susceptibility testing (AST), whole-genome sequencing (WGS) provides a more rapid and comprehensive approach to AMR characterization, thereby informing antimicrobial selection and supporting public health surveillance. In this study, Oxford Nanopore Technology (ONT)-based WGS was performed on 1,490 Salmonella isolates collected through nationwide surveillance in Taiwan in 2025. Genotypic resistance inferred from WGS data was compared with phenotypic AST results to assess the performance of ONT-WGS. Overall, WGS-inferred resistance showed high concordance with phenotypic resistance for most antimicrobials. However, major genotype-phenotype discordance was observed, attributed to four categories: (i) breakpoint-dependent classification, (ii) reduced or absent phenotypic expression of resistance genes, (iii) minimum inhibitory concentration (MIC) modulation by ramAp, and (iv) absence of known AMR determinants. Notable discrepancies included tigecycline resistance without known genetic determinants, nalidixic acid resistance linked to ramAp-mediated MIC elevation, and a high prevalence of colistin resistance (35.7%) in S. Enteritidis, with most resistant isolates lacking identifiable AMR determinants. Additionally, a significant proportion of ESBL- and AmpC-producing isolates were classified as susceptible or intermediate to cefotaxime and ceftazidime under CLSI criteria, highlighting the potential for misclassification and treatment failure. These findings demonstrate that ONT-WGS enables accurate and comprehensive AMR characterization by directly identifying resistance determinants and avoiding potential misclassification associated with breakpoint-based AST interpretations. When interpreted appropriately, WGS can support better antimicrobial selection and serve as a valuable alternative to conventional susceptibility testing. IMPORTANCE: Accurate prediction of antimicrobial resistance is essential for appropriate therapy and effective surveillance of Salmonella. However, discordance between genotype-based predictions and phenotypic antimicrobial susceptibility testing (AST) can complicate clinical interpretation. In this nationwide study of 1,490 Salmonella isolates, we show that Oxford Nanopore Technology-based whole-genome sequencing (ONT-WGS) provides rapid and comprehensive detection of antimicrobial resistance determinants with high concordance to phenotypic AST. We further identify four major mechanisms underlying genotype-phenotype discordance, including breakpoint-dependent classification, reduced or absent phenotypic expression of resistance genes, minimum inhibitory concentration (MIC) modulation by ramAp, and the absence of known AMR determinants. These findings demonstrate how WGS can complement conventional AST, improve interpretation of challenging susceptibility results, and strengthen genomic surveillance of emerging antimicrobial-resistant Salmonella.

Microbial Sensitivity Tests

Assessment of antibiotic resistance genes in soils polluted by chemical and technogenic ways with poly-aromatic hydrocarbons and heavy metals.

Anthropogenic activities are leaving lots of chemical footprints on the soil. It alters the physiochemical characteristics of the soil thereby modifying the natural soil microbiome. The prevalence of antimicrobial-resistance microbes in polluted soil has gained attention due to its obvious public health risks. This study focused on assessing the prevalence and distribution of antibiotic-resistance genes in polluted soil ecosystems impacted by industrial enterprises in southern Russia. Metagenomic analysis was conducted on soil samples collected from polluted sites using various approaches, and the prevalence of antibiotic-resistance genes was investigated. The results revealed that efflux-encoding pump sequences were the most widely represented group of genes, while genes whose products replaced antibiotic targets were less represented. The level of soil contamination increased, and there was an increase in the total number of antibiotic-resistance genes in proteobacteria, but a decrease in actinobacteria. The study proposed an optimal mechanism for processing metagenomic data in polluted soil ecosystems, which involves mapping raw reads by the KMA method, followed by a detailed study of specific genes. The study's conclusions provide valuable insights into the prevalence and distribution of antibiotic-resistance genes in polluted soils and have been illustrated in heat maps.

Soil Pollutants

Virus-mediated fate of antimicrobial resistance genes in livestock manure anaerobic digestion.

Antimicrobial resistance (AMR) poses a critical global health challenge, with livestock manure acting as a significant environmental reservoir for antimicrobial resistance genes (ARGs). Anaerobic digestion (AD) is a pivotal process for mitigating ARG dissemination at the livestock-environment-human interface. This study aims to elucidate the global dynamics of ARGs in AD systems, focusing on virus-host interactions and arms race, to identify actionable strategies for AMR control. We analyzed 205 metagenomic (4.5 Tb) and 36 meta-transcriptomic (640 Gb) datasets, including 15 newly generated datasets, revealing that pig manure AD harbors the highest ARG abundance (0.668 ARGs/16S rRNA), while AD systems generally exhibit limited transcriptional activation of ARGs. We constructed a viral dataset for livestock manure AD (GVD_LMAD), comprising 59,316 DNA and 727 RNA viral operational taxonomic units (vOTUs). Virus-host interactions established by CRISPR-Cas spacer, tRNA and homology matches revealed 889 lytic infections of antimicrobial-resistant bacteria (ARB) compared to only 18 ARG transduction events. Further analysis showed that the relative abundance of vOTUs assigned to the reduction role (4.11% ± 3.19%) was substantially higher than that of reproduction (0.72% ± 0.64%) and transduction (0.19% ± 0.30%), demonstrating that, among viral processes, lysis outweighs transduction in contributing to ARG abundance reduction in AD. Furthermore, an antiviral defense system (ADS) catalogue (GADSC_LMAD), derived from 2760 high-quality metagenome-assembled genomes (MAGs) containing 39,307 ADS, with ADS prevalence in ARB (7.8 ± 6.0 per MAG), indicating an intensified virus-host arms race in AD that may shield ARB from phage lysis. The resulting CRISPR-Cas immune network with expressed spacers targets foreign ARG-carrying sequences (primarily plasmids and ICEs), suggesting a mechanism that restricts horizontal gene transfer (HGT) via conjugation and transformation, despite shielding ARB from phage lysis. Collectively, these findings highlight that viral communities significantly contribute to ARG reduction through phage lysis relative to transduction, while the ADS-mediated arms race, despite protecting ARB, constructs a biological firewall that potentially limits HGT of ARGs. This study provides novel insights into virus-host dynamics as a key mechanism for controlling ARG dissemination in AD systems.

Animals

Antimicrobial-resistant Staphylococcus aureus isolated from Australian wildlife admitted to a veterinary hospital.

Although antimicrobial resistance (AMR) is a growing One Health concern, little is known about AMR in Staphylococcus aureus from Australian wildlife. This study investigated the occurrence, phenotypic AMR profiles, and genetic characteristics of S. aureus from six representative Australian wildlife species admitted to a wildlife hospital in Western Australia, including the western grey kangaroo (Macropus fuliginosus), quenda (Isoodon fusciventer), pelican (Pelecanus conspicillatus), galah (Eolophus roseicapilla), shingleback skink (Tiliqua rugosa) and long-necked turtle (Chelodina colliei). Staphylococcus aureus was isolated from 11.7% (21/180, 95% CI: 7.4%-17.3%) of the animals on admission. Whole genome sequencing identified 13 multi-locus sequence types (STs) and various virulence factors, including the human-specific immune evasion cluster (IEC). Resistance to at least one antimicrobial class was observed in 63.6% of the isolates. The blaZ, erm(T), aac(6')-aph(2″), and tet(L) AMR genes were detected in 63.6%, 13.6%, 4.5%, and 4.5% of S. aureus, respectively. After 7 days of hospitalisation, S. aureus was isolated from 16.5% (16/97, 95% CI: 9.7%-25.4%) of the animals, including two methicillin-resistant S. aureus (MRSA) isolated from two pelicans. The two MRSA were identified as community-associated MRSA clones (mecA-positive ST1-IV and ST93-IV), suggesting direct or indirect transmission between humans and wildlife during hospitalisation may have occurred. This study highlighted Australian wildlife may be a potential reservoir for genetically diverse antimicrobial-resistant S. aureus. AMR surveillance including wildlife using a One Health approach may be required.

Animals

Genomic and functional characterization of novel therapeutic lytic bacteriophages targeting multidrug-resistant Enterobacter cloacae.

The alarming rates at which extensively drug-resistant (XDR) and pandrug-resistant (PDR) Enterobacter cloacae in hospitals are increasing has begun to severely limit treatment options, and thus the urgency for alternative interventions, including bacteriophage therapy. The purpose of the study was to isolate and molecularly characterize phages that can infect E. cloacae, and, furthermore, to assess the antimicrobial efficacy of the four novel lytic bacteriophages (MMRP1, MMRP2, MMRP3, and MMRP4) against antimicrobial-resistant E. cloacae isolates and to evaluate their potential as alternative therapeutic strategies. These novel phages were characterized by plaque morphology, transmission electron microscopy (TEM), host range testing, thermal and chloroform stability assays, bacterial reduction assays, and whole-genome sequencing (WGS). Among 27 clinical isolates, MDR, XDR, and PDR phenotypes were observed in 20 (74.1%), six (22.2%), and one (3.7%) isolates, respectively. All four phages produced clear lytic plaques (0.5-3.0 mm) with titers reaching up to 6 × 1010 PFU/mL, and the phage cocktail lysed 81.4% (22 of 27 isolates) of clinical isolates with high host specificity. TEM revealed that all four E. cloacae-infecting phages (MMRP1, MMRP2, MMRP3, and MMRP4) belong to the class Caudoviricetes, exhibiting icosahedral capsids, tailed morphology, and double-stranded DNA genomes, consistent with current ICTV classification criteria. Whole genome sequencing and comparative phylogenetic analysis further resolved the taxonomic placement of these phages at the family level, positioning MMRP1 within the family Demerecviridae and MMRP4 within the family Straboviridae. All phages were stable from -20 to 40 °C and were unaffected by exposure to chloroform. Phage cocktail reduced bacterial OD₆₀₀ to ≤ 0.3 within 4 h in the bacterial reduction test. WGS revealed large circular dsDNA genomes of ~132 kbp (MMRP1) and ~149 kbp (MMRP4), GC content of 38%, and modular architectures encoding structural, lytic, and replication gene modules. The most striking and highlighted suggestion that in vitro evaluation of MMRP1 and MMRP4 are highly recommended to more deeper future experimental studies to combat MDR E. cloacae nosocomial infections supported by genomic foundation and eventually, the possibility to be suitable for phage-engineering applications in clinical settings.

Enterobacter cloacae

Genomic characterization of carbapenemase-producing Enterobacterales from wastewater reveals the convergence of KPC-2 and GES-16 in Brazil.

Carbapenemase-producing Enterobacterales (CPE) pose a significant public health concern due to the limited therapeutic options and increasing dissemination outside clinical settings. Wastewater treatment plants (WWTPs) have been proposed as relevant environmental reservoirs for antimicrobial-resistant bacteria and mobile genetic elements. In this study, we performed genomic characterization of CPE strains recovered from raw wastewater samples from the influents of different WWTPs. Antimicrobial susceptibility testing revealed multidrug resistance, including coresistance to carbapenems and polymyxins, among Klebsiella pneumoniae, Enterobacter asburiae, and Enterobacter kobei strains. Whole-genome sequencing identified the convergence of the blaKPC-2 and blaGES-16 genes, as well as the presence of blaGES-5 in E. kobei sequence type (ST) 540 strains. Moreover, the blaKPC-2 gene was detected in K. pneumoniae strains belonging to high-risk clones ST11 (capsular types KL64 and KL15) and ST307 (capsular type KL102), and E. asburiae ST384. The blaGES-5 and blaGES-16 genes were associated with class 1 integrons, while the blaKPC-2 gene was embedded within transposons (Tn4401a, Tn4401i, and Tn3-like) and insertion sequences (ISKpn27 and ISKpn6). Notably, genomic analyses and literature review demonstrated that the blaGES-16 gene remains unique to Brazil. The putative pathogenic potential of carbapenem-resistant K. pneumoniae ST11 was also assessed. These findings support the environmental circulation of clinically relevant Enterobacterales genotypes and emphasize the potential role of WWTPs as conduits for CPE dissemination, if not adequately operated. Therefore, genomic surveillance in extra-hospital settings may contribute to a better understanding of antimicrobial resistance ecology and inform One Health mitigation strategies.

Brazil

Phylogeographic analysis of Staphylococcus nepalensis reveals global occurrence of antimicrobial-resistant lineages carrying the sal(E) resistance gene.

BACKGROUND: Staphylococcus nepalensis is an emerging species first described in 2003 from the respiratory tract of goats in Nepal. We report the identification of S. nepalensis of a hypersaline lagoon in Brazil, along with in-depth phylogeographical and resistome analysis of publicly available genomes. METHODS AND RESULTS: During a local survey from hypersaline aquatic environments in Rio de Janeiro, Brazil, two staphylococcal strains were recovered, designated as COLB and AM1. These isolates were subjected to antimicrobial susceptibility testing, genomic sequencing, and comprehensive phylogenomic analyses. Genomic analysis confirmed the taxonomic identity of COLB and AM1 as S. nepalensis. Both isolates harbored the sal(E) conferring resistance to pleuromutilins and streptogramin A, whereas tet(K) conferring to tetracyclines. Additionally, AM1 carried lnu(A), consistent with the reduced susceptibility to clindamycin (MIC = 2 µg/mL) relative to COLB. Genes associated with arsenic and copper tolerance, and the replicons rep7a and rep19c, were confirmed. Phylogenomic analysis indicated that COLB and AM1 were clonally related (1 cgSNP-difference) but distinct from global isolates. Phylogeographic analysis revealed wide geographic occurrence, with some lineages carrying blaZ and mecA associated with beta-lactamase production and methicillin resistance, respectively. Strikingly, sal(E) is conserved across all S. nepalensis genomes. CONCLUSIONS: The findings confirm the presence of S. nepalensis in South America as early as 2016 and documented among available genomes from environmental, human, and animal-associated sources. Furthermore, reveal the circulation of some lineages carrying clinically relevant antimicrobial genes, underscoring the importance of accurate species identification and continuous genomic surveillance and potential One Health relevance.

Phylogeography

Genomic insights into the first blaKPC-2-carrying Klebsiella pneumoniae isolate reported in Chile: limited local dissemination of the globally distributed ST101 lineage.

OBJECTIVE: To genomically characterize Kpn-KPC-1, the first blaKPC-2-carrying Klebsiella pneumoniae isolate reported in Chile, and contextualize it within the global ST101 lineage. METHODS: Kpn-KPC-1 was analyzed by whole-genome sequencing. Its resistome, virulome, plasmid content, and blaKPC-2 genetic context were characterized, and 537 publicly available ST101 genomes were used for comparative phylogenomics. RESULTS: Kpn-KPC-1 belonged to ST101 and carried blaKPC-2 within the conventional Tn4401a transposon on a mosaic plasmid encoding multiple replication initiators and conjugation-associated genes. The isolate also harbored an OmpK36 porin alteration and accessory resistance- and virulence-associated determinants, including ICEKp3/ybt-9, K17, and O1αβ,2α. Phylogenomic analysis placed Kpn-KPC-1 within a predominantly European clade, closest to Italian isolates recovered between 2012 and 2018. The absence of additional Chilean ST101 isolates related to Kpn-KPC-1 supports limited local dissemination of this lineage. Globally, ST101 was enriched in carbapenemase genes, particularly blaOXA-48-like and blaKPC variants. CONCLUSIONS: Kpn-KPC-1 represents a transient introduction of a carbapenemase-prone, high-risk ST101 lineage rather than the founder of a locally disseminated clone in Chile. Genome-level analysis resolved the blaKPC-2 context in this historical isolate, linking the carbapenemase gene to Tn4401a on a mosaic multidrug-resistance plasmid within an imported ST101 background. These findings underscore the value of retrospective genomics for reconstructing early antimicrobial-resistance introduction events.

Carbapenem-resistant enterobacterales

Capsular Polysaccharide Is Essential for the Virulence of the Antimicrobial-Resistant Pathogen Enterobacter hormaechei.

Nosocomial infections caused by multidrug-resistant (MDR) Enterobacter cloacae complex (ECC) pathogens are on the rise. However, the virulence strategies employed by these pathogens remain elusive. Here, we study the interaction of ECC clinical isolates with human serum to define how this pathogen evades the antimicrobial action of complement, one of the first lines of host-mediated immune defense. We identified a small number of serum-sensitive strains, including Enterobacter hormaechei strain NR3055, which we exploited for the in vitro selection of serum-resistant clones. Comparative genomics between the serum-sensitive NR3055 strain and the isolated serum-resistant clones revealed a premature stop codon in the wzy gene of the capsular polysaccharide biosynthesis locus of NR3055. The complementation of wzy conferred serum resistance to NR3055, prevented the deposition of complement proteins on the bacterial surface, inhibited phagocytosis by human neutrophils, and rendered the bacteria virulent in a mouse model of peritonitis. Mice exposed to a nonlethal dose of encapsulated NR3055 were protected from subsequent lethal infections by encapsulated NR3055, whereas mice that were previously exposed to unencapsulated NR3055 succumbed to infection. Thus, capsule is a key immune evasion determinant for E. hormaechei, and it is a potential target for prophylactics and therapeutics to combat these increasingly MDR human pathogens. IMPORTANCE Infections caused by antimicrobial resistant bacteria are of increasing concern, especially those due to carbapenem-resistant Enterobacteriaceae pathogens. Included in this group are species of the Enterobacter cloacae complex, regarding which there is a paucity of knowledge on the infection biology of the pathogens, despite their clinical relevance. In this study, we combine techniques in comparative genomics, bacterial genetics, and diverse models of infection to establish capsule as an important mechanism of Enterobacter pathogens to resist the antibacterial activity of serum, a first line of host defense against bacterial infections. We also show that immune memory targeting the Enterobacter capsule protects against lethal infection. The further characterization of Enterobacter infection biology and the immune response to infection are needed for the development of therapies and preventative interventions targeting these highly antibiotic resistant pathogens.

Humans

Whole-genome sequences of Pseudomonas aeruginosa strains isolated from patients and inanimate hospital reservoirs in Chattogram, Bangladesh.

Antimicrobial-resistant Pseudomonas aeruginosa (PA) is a critical concern for the global population. This study identified four isolates and reported the draft genomes annotated as ST645, ST2238, and the high-risk clone ST773. The isolates featured diverse antimicrobial resistance (AMR) gene profiles that emphasize the need for continuous molecular surveillance in Bangladesh.

Chattogram

Chloramphenicol and tetracycline synergize with bacteriophage SeKF_13 to inactivate antimicrobial-resistant Salmonella Typhimurium.

UNLABELLED: Non-typhoidal Salmonella is estimated to cause up to 1 billion cases of global foodborne illness per year. Salmonella Typhimurium is a serovar of gravest worldwide concern as it is capable of infecting animal and human hosts and can also acquire antimicrobial resistance (AMR) determinants at a rapid rate. Recent advances in phage research have positioned them as especially useful for inactivation of Salmonella where antibiotics have proven no longer effective. Even more recently, phage-antibiotic synergy (PAS) has been proposed as a solution for AMR Salmonella, where synergistic combinations of phages and antibiotics are more effective than application of phage or antibiotic alone. Utilizing an in-house phage isolate, SeKF_13, we sought to determine the existence of PAS against a strain of Salmonella enterica serovar Typhimurium 14028 2a that is clinically resistant to bacteriostatic antibiotics chloramphenicol and tetracycline. Checkerboard assays revealed the presence of synergy when sub-lethal (sub-MIC) levels of either tetracycline or chloramphenicol were combined with phage SeKF_13 (P < 0.05; two-way ANOVA). Compared to tetracycline or chloramphenicol alone, the addition of phage also decreased the MICs of both antibiotics twofold. We also monitored the development of resistance and found that PAS significantly suppressed emergence of resistance compared to the antibacterial agents alone (P < 0.05; Tukey's HSD). Whole-genome sequencing revealed that SeKF_13 is devoid of genes encoding integrase, antimicrobial resistance, and virulence, ensuring safety in future applications. Together, our results suggest that combined treatment of phage and antibiotic can improve antimicrobial efficacy against antibiotic-resistant Salmonella enterica. IMPORTANCE: Salmonella enterica is a foodborne pathogen that causes one of the highest rates of foodborne illness worldwide. They are also capable of becoming resistant to antimicrobials very rapidly (i.e., antimicrobial resistance; AMR) due to their ability to acquire AMR determinants, undermining the effectiveness of current treatments. Bacteriophages (phages), viral predators of bacteria, have been proven to be effective in some cases, but recently, phage-antibiotic synergy has been proposed as a more effective solution than phages or antibiotics alone. We found this was, indeed, the case; using phage SeKF_13 and tetracycline or chloramphenicol (to which the Salmonella strain was resistant), we found that combination treatment was significantly more effective than either treatment alone. These results demonstrate that combined treatment of phage and antibiotic can bolster treatment efficacy against AMR Salmonella.

Salmonella typhimurium

Investigating the zoonotic origins of ESBL-producing E. coli in community-acquired urinary tract infections in Ecuador.

Extended-spectrum &#x3b2;-lactamase-producing Escherichia coli (ESBL-producing E. coli) pose a growing global health threat. Although Latin America has been identified as a global hotspot of antimicrobial resistance, the zoonotic contribution to drug-resistant infections in the region remains poorly defined. We analyzed 137 clinical ESBL-producing E. coli isolates from urinary tract infections (UTIs) in Quito, Ecuador, applying a Bayesian latent class model informed by host-associated mobile genetic elements to estimate the fraction of infections attributable to food-animal sources. We estimated that 25.5% (35/137) of UTI isolates were putative zoonotic cases. This proportion rose to 42.5% after excluding ST131-H30, a human-associated pandemic lineage. Putative zoonotic isolates were enriched for animal-associated &#x3b2;-lactamase genes (e.g., blaTEM-1B, blaCTX-M-65), lacked human-associated markers such as blaOXA-1, and exhibited diverse antimicrobial resistance gene profiles resembling those observed among food-animal isolates. These isolates were also enriched for ColV-associated virulence genes typically linked to avian pathogenic E. coli. Putative zoonotic strains contributed substantially to third-generation cephalosporin-resistant UTIs in Quito, Ecuador, challenging assumptions derived from high-income settings that such infections are driven predominantly by human-to-human transmission. These findings highlight the importance of integrated One Health surveillance and mitigation, particularly in low- and middle-income countries where gaps in water, sanitation, and hygiene (WASH) may interact with antimicrobial use in food production to amplify antimicrobial resistance transmission.IMPORTANCEESBL-producing E. coli have rapidly emerged as a major global antimicrobial resistance threat. In Latin America, cephalosporins are commonly used in food-animal production, fueling the emergence of ESBL-producing E. coli. In low- and middle-income countries, excessive antimicrobial use driven by poorly regulated over-the-counter sales, combined with inadequate water, sanitation, and hygiene (WASH) infrastructure, can facilitate antimicrobial-resistant pathogen transmission from food animals to humans. Using a novel statistical-genomic approach, we found that over one in four cephalosporin-resistant UTIs in Quito, Ecuador, may be caused by E. coli strains originating from food animals. Our findings highlight the public health risks associated with antimicrobial use in food-animal production and the role of environmental and infrastructure-related vulnerabilities. As global demand for animal protein continues rising in middle-income countries, controlling zoonotic antimicrobial resistance transmission becomes increasingly urgent for protecting human health through integrated One Health strategies.

ESBL-producing E. coli