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PubMed2026

cgDist: Nucleotide-level distance calculation from cgMLST allelic profiles.

Bacterial genomic surveillance requires balancing computational efficiency with genetic resolution for effective cluster investigation. cgMLST distance calculations treat all allelic differences as equivalent units, obscuring nucleotide-level variation. Furthermore, single nucleotide polymorphism-based pipelines provide finer resolution at substantially higher computational cost, which limits their routine deployment in surveillance laboratories. We present cgDist, an algorithm that calculates nucleotide-level distances directly from cgMLST allelic profiles, providing finer resolution than allele-count distances by leveraging within-allele nucleotide variation. The cache architecture stores alignment statistics, enabling distance calculation modes without computation and supporting both dataset-specific and schema-complete cache generation. This design enables incremental surveillance analysis, with performance benefits as laboratories accumulate alignment data. cgDist functions as a precision 'zoom lens' for the investigation of clusters identified through initial cgMLST screening. Rather than restructuring population relationships, this targeted approach concentrates enhanced resolution where it is most informative. The algorithm ensures that cgDist distances are greater than or equal to corresponding cgMLST distances, preserving epidemiological interpretability while adding genetic discrimination. By increasing resolution within identified clusters, cgDist may also support outbreak investigation, a potential application that remains to be evaluated on outbreak-derived data.

Algorithms↗

PubMed2026

Core genome and whole genome multi-locus sequence typing of Cronobacter isolates.

UNLABELLED: Cronobacter species, especially C. sakazakii and C. malonaticus, are opportunistic pathogens that are linked to severe infections in infants with high case fatality rates. In this study, we investigated whole genome sequencing (WGS) analysis approaches, specifically 7-gene multi-locus sequence typing (7-gene MLST), core genome MLST (cgMLST), and whole genome MLST (wgMLST) to subtype Cronobacter isolates. We analyzed a comprehensive set of 743 Cronobacter isolates derived from clinical, food, and environmental sources. We also evaluated high-quality single nucleotide polymorphism (hqSNP), cgMLST, and wgMLST to cluster epidemiologically related and differentiate sporadic C. sakazakii isolates. Our results indicate that both cgMLST and wgMLST accurately identify closely related isolates and are consistent with epidemiological findings. The allele-based analyses were also comparable with hqSNP analyses, the current gold standard. Our workflow also outputs 7-gene MLST allele calls, Cronobacter sequence types, and clonal complexes, which may be useful for historic comparisons during outbreak investigations. Following the recent classification of Cronobacter infections as nationally notifiable in the United States, our findings demonstrate the efficacy of WGS-based approaches within the PulseNet framework to improve outbreak detection and response strategies for Cronobacter. IMPORTANCE: Cronobacter species, specifically C. sakazakii and C. malonaticus, are opportunistic pathogens linked to severe infections in infants with high case fatality rates. This study highlights the critical importance of advanced molecular techniques in public health surveillance, using whole genome sequencing (WGS) methodologies such as multi-locus sequence typing (7-gene MLST), core genome MLST (cgMLST), and whole genome MLST (wgMLST). The validation of these WGS-based approaches within the PulseNet framework is timely, especially following the recent classification of Cronobacter infections as nationally notifiable in the United States. WGS methods not only enhance outbreak detection but can also inform public health guidance aimed at preventing infections and reducing mortality in vulnerable populations, especially infants. Our research supports implementation of cgMLST as a standardized approach for routine PulseNet surveillance of Cronobacter, with wgMLST and hqSNP analyses providing additional discriminatory power for outbreak investigations and high resolution phylogenetic analysis.

Multilocus Sequence Typing↗

PubMed2026

Potential dissemination and persistence of Clostridium perfringens along the slaughtering process in French cattle, pig or poultry slaughterhouses.

Clostridium perfringens is a major foodborne pathogen associated with meat products, yet its dissemination routes and persistence within slaughterhouses remain poorly understood. In this study, whole-genome sequencing combined with multilocus sequence typing (MLST), core genome MLST (cgMLST), and core single nucleotide polymorphism (SNP) analysis was applied to 286 C. perfringens isolates collected from cattle, pig, and poultry slaughterhouses in France. MLST analysis revealed extensive genetic diversity, with most isolates assigned to novel allelic profiles rather than previously described sequence types. Phylogenetic analyses based on cgMLST and SNP data revealed frequent recovery of closely related isolates from feces, meat, surfaces, and air, highlighting widespread dissemination of strains within slaughterhouses during processing. Notably, close genetic related isolates recovered from air and other sample types are consistent with air-associated dissemination within slaughterhouse environments. In addition, the detection of closely related strains across different sampling campaigns suggests the potential persistence of C. perfringens within slaughterhouse environments over time. Most isolates were classified as toxinotype A (97.9%), with a few belonging to toxinotypes D (1.0%) and G (1.0%), and in silico analyses revealed a broad distribution of virulence-associated genes. Antimicrobial resistance genes (ARGs) were commonly detected, particularly those conferring resistance to tetracyclines, although isolates carrying multiple ARGs remained infrequent. Overall, this study provides new insights into the genomic diversity, dissemination pathways, and persistence of C. perfringens in multi-species slaughterhouses. These findings highlight the potential role of air-associated dissemination in contamination dynamics and underscore the importance of improved hygiene control strategies to mitigate food safety risks along the meat production chain.

Antimicrobial resistance gene (ARG)↗

PubMed2025

Characterization of carbapenem-resistant Pseudomonas aeruginosa in Canadian hospitals: 6 years of the CANWARD study (2018-23).

OBJECTIVES: Antimicrobial resistance in Pseudomonas aeruginosa is of increasing concern in Canada, leading to limited treatment options and poor clinical outcomes. Herein we characterized carbapenem-resistant P. aeruginosa identified through the Canadian national surveillance program CANWARD. METHODS: Antimicrobial susceptibility for 1725 P. aeruginosa isolates was assessed using broth microdilution and 2024 CLSI breakpoints. WGS of carbapenem-resistant isolates was used to identify STs, resistance and virulence markers. Genetic relatedness was further assessed using cgMLST for select STs. RESULTS: From 2018 to 2023, CANWARD collected 1725 P. aeruginosa isolates, of which 371 (21.5%) were carbapenem-resistant. The majority of carbapenem-resistant P. aeruginosa were isolated from respiratory specimens of male patients aged 18-65 years living in central Canada. Only 0.8% (n = 3) of the carbapenem-resistant isolates harboured a carbapenemase gene. WGS identified mutations associated with OprD dysfunction, MexAB-OprM efflux and AmpC overexpression in 73.6%, 1.1% and 4.9% of isolates, respectively. Most isolates (98.1%) harboured at least one of the following class D β-lactamase genes: OXA-2, OXA-5, OXA-10 or OXA-50-like subfamily. Wide genetic diversity was observed with 151 different STs identified. The most common STs were ST17 (4.6%), ST27 (4.6%) and high-risk clones ST235 (4.3%), ST244 (3.5%), ST253 (6.4%) and ST357 (2.7%). cgMLST clusters were identified amongst 34.9% of the high-risk clones, suggesting clonal dissemination. CONCLUSIONS: Currently, >20% of clinical isolates of P. aeruginosa in Canada are carbapenem-resistant. Genetic evidence indicates that clonal dissemination of high-risk clones is occurring in Canada. High-risk clones are virulent and often MDR. Continued surveillance of P. aeruginosa is important.

Pseudomonas aeruginosa↗

PubMed2026

Applicability of Nanopore-only whole-genome sequencing for Pseudomonas aeruginosa outbreak investigation in the ICU setting: a multicentric study.

UNLABELLED: Pseudomonas aeruginosa outbreaks frequently occur in intensive care units (ICUs). In particular, ICU patients requiring mechanical ventilation are vulnerable to P. aeruginosa ventilator-associated pneumonia, which is associated with high morbidity and mortality. Fast and accurate genotyping during the early stage is crucial to document and manage P. aeruginosa outbreaks at the ICU. In this study, we have evaluated the applicability of Oxford Nanopore whole-genome sequencing (WGS) for outbreak investigation and antimicrobial resistance (AMR) prediction. To evaluate whether a Nanopore-only WGS workflow was able to reproduce Illumina-confirmed transmission clusters, 19 P. aeruginosa isolates from ICUs at UZ Brussels (Belgium) that were previously sequenced with Illumina were sequenced using a Nanopore-only workflow based on the latest V14 chemistry, followed by bioinformatic analysis via BugSeq and MBioSEQ Ridom Typer. Although both bioinformatic platforms showed high concordance between Illumina and Nanopore data, MBioSEQ Ridom Typer yielded the lowest allelic distance (maximum one cgMLST allele), confirming all outbreak clusters. When applying the Nanopore-only workflow to longitudinally collected isolates, low genetic heterogeneity (maximum three cgMLST alleles) was observed between isolates from the same patient. WGS and subsequent outbreak analysis of 65 respiratory P. aeruginosa isolates collected from 38 different ICU patients across six Belgian hospitals during a 9-month period showed no intra- or inter-hospital transmission. When the Nanopore-only WGS data were used to predict AMR, there was high categorical agreement (95%) between AMR genotype and phenotype. These findings highlight the potential of Nanopore WGS as a rapid and accurate tool for outbreak investigation of P. aeruginosa. IMPORTANCE: In recent years, Nanopore sequencing has found its way to clinical laboratories because of its affordability, scalability, and, most importantly, its ability to obtain sequencing results in near-real time. However, despite improved raw read accuracies with the latest generation R10.4.1 flow cells, the question remains whether the achieved accuracy is sufficient for accurate bacterial outbreak investigation, particularly in high-risk settings such as intensive care units (ICUs). In this study, we show that Nanopore-only whole-genome sequencing (WGS) is able to match Illumina-only WGS in terms of accuracy for Pseudomonas aeruginosa outbreak investigation in the ICU setting, although important sequence type-dependent and even strain-specific methylation issues need to be resolved in order to guarantee this accuracy. By providing a fast and accurate workflow for reliable P. aeruginosa outbreak investigation, this study could pave the way for large-scale implementation of Nanopore-only WGS, leading to faster outbreak response times.

Humans↗

PubMed2026

A standardized, genome-guided MLST scheme for Avibacterium paragallinarum: enhanced epidemiological typing and validation against existing methods.

Avibacterium paragallinarum, the causative agent of infectious coryza (IC), is an important respiratory pathogen of chickens with growing prevalence in commercial and backyard flocks. Current strain-typing methods, including classical serotyping and molecular approaches, such as ERIC-PCR or single-locus HPG2 typing, lack sufficient discriminatory power to investigate the epidemiology or population structure. To address this limitation, we developed a genome-guided multilocus sequence typing (MLST) scheme as a robust and portable tool for A. paragallinarum strain differentiation. Housekeeping genes were identified from 42 whole-genome sequences (WGS); 18 candidates were evaluated; and six were selected for the final MLST scheme. We used the scheme to differentiate 75 A. paragallinarum samples and compared its performance against classical HPG2-based typing, ad hoc core genome MLST (cgMLST), and the MLST scheme published by M. Guo, Y. Jin, H. Wang, X. Zhang, and Y. Wu (Vet Sci 11:208, 2024, https://doi.org/10.3390/vetsci11050208). The new MLST showed higher discriminatory power than HPG2 and outperformed Guo's scheme with higher discriminatory power, particularly for characterizing the samples originating from North and South America. It also showed strong concordance with cgMLST clustering while being more practical for routine use. Overall, the six-locus MLST identified 31 sequence types across 75 samples, revealing epidemiologically meaningful clustering at regional and national scales and capturing temporal persistence of lineages. All allele definitions and sequence types have been deposited in PubMLST, ensuring standardized nomenclature and global accessibility. This scheme represents a reproducible, cost-effective, and globally applicable tool that enhances outbreak investigation, surveillance, and population studies of A. paragallinarum, bridging the gap between low-resolution traditional methods and resource-intensive whole-genome sequencing.IMPORTANCEInfectious coryza (IC) caused by Avibacterium paragallinarum is a major respiratory disease of poultry that causes acute infection, reducing egg production and growth and resulting in significant economic losses in poultry production worldwide. Controlling IC depends on understanding how different strains spread and persist, yet current methods to differentiate strains are either unreliable or too costly for routine use. In this study, we developed a standardized multilocus sequence typing system that provides a simple, accurate, and globally accessible way to identify and compare strains of A. paragallinarum. This scheme identified important links between outbreaks at local and regional levels and showed that certain strains persisted over time. By making the scheme available through PubMLST, laboratories worldwide can use a common tool to track and investigate the pathogen. This accessible tool improves disease surveillance, supports outbreak investigations, and helps poultry producers and veterinarians respond more effectively to IC.

Multilocus Sequence Typing↗

PubMed2026

Large-scale genomic analysis places Chinese CC398 as a persistent human-associated MSSA lineage apart from the dominant global LA-MRSA clade.

Staphylococcus aureus clonal complex (CC)398 has emerged as a dominant livestock-associated methicillin-resistant S. aureus (LA-MRSA) lineage worldwide; however, its evolutionary trajectory and regional diversification remain incompletely understood. We developed a core-genome multilocus sequence typing (cgMLST) scheme with hierarchical clustering and applied it to over 30,000 S. aureus genomes, revealing frequent cross-border transmission of CC398. Subsequent time-calibrated phylogenetic analysis placed the most recent common ancestor at 1942 (95% CI: 1939-1945), with the human-to-livestock host jump around 1969 (95% CI: 1968-1972). Chinese CC398 exhibits a distinct trajectory: unlike the LA-MRSA lineages dominating Europe and North America, Chinese isolates are predominantly human-associated methicillin-susceptible S. aureus (HA-MSSA), forming unique East Asia-specific phylogroups (SAP1, SAP2, and AP1-AP3), with distinct resistance and virulence profiles. The LA lineage remains limited in China, with multinational mixed clusters emerging only after 2019. Analysis of global transmission networks revealed a significant correlation between LA-CC398 spread and international trade in fresh swine products, while no such correlation was observed for the human-associated lineage. Beyond the established lineage markers tet(M) and scn, our analysis identified additional differentially distributed genes, including cadC-a chromosomal cadmium resistance regulator-as a novel HA-lineage-enriched gene whose functional role in host adaptation remains to be determined. This study reveals that CC398 followed fundamentally different evolutionary paths in China versus Western countries, challenging a one-size-fits-all model of its dissemination.IMPORTANCEThis study illustrates how large-scale microbial genomics can resolve the evolutionary origins and regional diversification of bacterial pathogens. By applying a novel cgMLST scheme to over 30,000 S. aureus genomes, we show that CC398 followed fundamentally different evolutionary paths in China versus Western countries-challenging the prevailing model of uniform global dissemination-and that livestock-associated MRSA expansion is closely linked to international trade in fresh pork products. These findings highlight the need for integrated surveillance across human, animal, and trade interfaces to anticipate the emergence and spread of zoonotic pathogens.

Staphylococcus aureus↗

PubMed2026

Phenotypic and phylogenomic characterization of Lactococcus garvieae isolates from rainbow trout (Oncorhynchus mykiss) in Türkiye.

Lactococcosis is an important bacterial disease of farmed fish and causes substantial economic losses in rainbow trout (Oncorhynchus mykiss) aquaculture. In this study, Lactococcus garvieae isolates recovered from rainbow trout farms in Türkiye were characterized using phenotypic, molecular, and phylogenomic methods. Among 32 presumptive Lactococcus isolates recovered from 127 dead rainbow trout, four were confirmed as L. garvieae and exhibited identical biochemical characteristics, Pulsed Field Gel Electrophoresis (PFGE) profiles, and broad growth tolerance across different pH, salinity, and temperature conditions. All isolates were presumptively classified as resistant to ciprofloxacin and florfenicol, while remaining susceptible to tetracycline and penicillin. Based on the AMR profiles, strain LG2, which exhibited the most susceptible antimicrobial profile among the isolates, was selected for whole-genome sequencing (WGS). WGS of the representative isolate LG2 generated a single 2,214,687-bp chromosomal contig with 38.5% GC content and 99.0% BUSCO completeness. In silico PCR assigned LG2 to serotype I, and the genome contained an intact capsule-associated cps/kps locus. The chromosomal lsa(D) determinant and an mdt(A)-like efflux-associated gene were detected, whereas no plasmid replicons or acquired quinolone or florfenicol resistance genes were identified, indicating discordance between the phenotypic and genomic AMR results. Taxonomic verification of 236 publicly available Lactococcus assemblies yielded 41 verified public L. garvieae genomes, which, together with LG2, formed a 42-genome within-species dataset. LG2 was most closely related to the Turkish isolate OS-37, sharing 99.96% ANI and differing by three core SNPs; both belonged to ST109, whereas the other Turkish isolates belonged to ST139. cgMLST identified a conserved genomic backbone, while pan-genome analysis identified 5,655 gene clusters and an open pan-genome characterized by a large cloud-gene fraction. These findings demonstrate the importance of species verification in Lactococcus population genomics and reveal substantial accessory-genome diversity within L. garvieae. The genomic features of LG2 provide a basis for future pathogenicity and immunogenicity studies, although experimental validation is required. Overall, these findings highlight the importance of local genomic surveillance for understanding L. garvieae population structure and provide a genomic framework for future region-specific vaccine research.

Animals↗

PubMed2026

Dissemination of blaKPC-3-harbouring Klebsiella pneumoniae across ST48 and ST628 in multiple healthcare facilities in the Republic of Korea.

Klebsiella pneumoniae carbapenemase-3 (KPC-3) remains rare in South Korea, where KPC-2 is the dominant carbapenemase, making the repeated detection of a concentrated blaKPC-3 signal over five years notable. We performed genomic analyses of blaKPC-3-harbouring K. pneumoniae from a regional healthcare network. Two chromosomally distinct lineages with concordant capsule loci (ST628/KL15 and ST48/KL62) presented multidrug-resistant phenotypes, and the virulence-associated loci were confined to ST48. Single-nucleotide polymorphism (SNP) analyses revealed near-clonal relatedness within lineages, with 0-38 pairwise SNPs among ST628 isolates and 8 SNPs between the two ST48 isolates. Core-genome multilocus sequence typing (cgMLST) supported this structure, as ST628 isolates were assigned to complex type 19149 with 0-7 allelic differences, and ST48 isolates were assigned to complex type 19150 with 5 allelic differences. These patterns support vertical spread via clonal expansion across multiple facilities. Despite substantial chromosomal separation, most isolates carried the same IncFII(K) plasmid backbone and blaKPC-3, and they were nearly indistinguishable from a plasmid previously reported in South Korea. One isolate carried blaKPC-3 on a distinct multireplicon IncFIB(K)/IncFII(K) plasmid, indicating that the signal was not confined to a single plasmid backbone. In both plasmids, blaKPC-3 was embedded within Tn4401b. These findings indicate that a rare blaKPC-3 genotype can persist regionally through sustained clonal dissemination and that cross-lineage linkage is compatible with past horizontal transfer involving a conserved plasmid. These findings underscore the need for subtype-resolved, regionally coordinated genomic surveillance in connected healthcare networks to detect uncommon carbapenemase variants early.

Klebsiella pneumoniae↗

PubMed2026

In-host adaptation of Staphylococcus aureus during recurrent prosthetic joint infections: a retrospective longitudinal study.

UNLABELLED: The aim of this study was to characterize the in vivo evolution of Staphylococcus aureus strains involved in recurrent prosthetic joint infections (PJIs) both phenotypically and genomically. We conducted a monocentric retrospective study in a 1,437-bed French teaching hospital between 2013 and 2021. All patients presenting a recurrent S. aureus-related PJI-defined as at least two strains isolated from distinct clinical samples more than 90 days apart-of the knee, hip, or shoulder were included. Clinical data were reviewed, and all isolates underwent phenotypic characterization, including antimicrobial susceptibility testing, growth rate determination, biofilm production assays, metabolic profiling (API 50 CH), and virulence evaluation using the Galleria mellonella infection model. Whole-genome sequencing (WGS) was performed for all strains, followed by analyses of core-genome multilocus sequence typing (cgMLST), resistome, virulome, and mobilome composition, and single-nucleotide polymorphisms (SNPs). Thirteen patients met inclusion criteria, yielding 55 S. aureus isolates. Eight patients experienced recurrent infections caused by genetically closely related strains throughout the clinical course (median: three strains per patient; range: 2-6), whereas five patients were infected by genetically distinct strains. At baseline, isolates were genetically diverse and susceptible to methicillin and rifampicin; two showed fluoroquinolone resistance due to grlA and/or gyrA mutations. In one patient (patient C), a recurrent isolate acquired an rpoB S486L mutation, conferring rifampicin resistance after rifampicin exposure. Due to the limited sample size, it is difficult to draw definitive conclusions from the phenotypic analyses. This study highlights the adaptive evolution of S. aureus during chronic PJIs and underscores the need for further research to better understand intra-host dynamics in long-standing infections. IMPORTANCE: This study conducted in a 1,437-bed French teaching hospital analyzed the genomic and phenotypic evolution of 55 Staphylococcus aureus strains recovered in recurrent PJIs from 13 patients. The first strains showed high genotypic diversity across 12 different sequence types. Among the 13 patients, only eight experienced a true recurrence with the same strain, while five were contaminated with a different strain of S. aureus, indicating a new infection. Moreover, this study underscores the complex within-host evolution of S. aureus and highlights the phenotypical and genotypical adaptation during chronic infection.

Staphylococcus aureus↗

PubMed2025

Comparative genomics of Mycobacterium avium subsp. hominissuis strains within a group of captive lowland tapirs.

Within a group of three captive lowland tapirs (Tapirus terrestris) suffering from clinically apparent mycobacteriosis, non-tuberculous Mycobacterium avium subsp. hominissuis (MAH) strains were isolated from the animals and the tapir's enclosure. Based on MIRU-VNTR findings, which identified two closely related INMV profiles (124 and 246), a micro-evolutionary event was assumed, and four available MAH strains were submitted to whole genome sequencing (short- and long-read technologies). Surprisingly, the differences based on single nucleotide polymorphisms (SNPs) were exceptionally high between the four strains, i.e., between 841 and 11,166 bases, due to a strong impact of homologous recombination. Thus, an ad hoc core genome multilocus sequence typing (cgMLST) scheme was created and pangenome analysis was conducted for determining the genomic similarity between the strains. The INMV246 isolate obtained from sputum on the enclosure floor and one INMV124 isolate of tapir #2 showed the highest congruence, suggesting that both originated from a shared source. The other two INMV124 isolates were genomically distinct from these strains. Nevertheless, in all four strains two plasmids were detected, which were highly conserved between the strains. The study showed that the genomic variability between MAH strains isolated from the same site within a short period of time can be exceptionally high and the influence of homologous recombination needs to be considered when determining MAH strain relationships, particularly via SNP analyses.

Animals↗

PubMed2026

Whole-Genome Analysis Reveals Antimicrobial Resistance and Population Structure of Environmental and Veterinary Acinetobacter baumannii.

Acinetobacter (A.) baumannii is an important multidrug-resistant pathogen increasingly recognized across animal and environmental settings, and carbapenem-resistant A. baumannii (CRAB) is classified as a critical-priority pathogen by the World Health Organization. This study investigated the antimicrobial resistance (AMR) and genomic characteristics of 122 A. baumannii isolates comprising 72 veterinary and 50 environmental isolates collected in Andhra Pradesh, India. Antimicrobial susceptibility testing, whole-genome sequencing (WGS), resistance and virulence gene profiling, multilocus sequence typing (MLST), core-genome analysis, single nucleotide polymorphism (SNP) phylogeny, and pan-genome analysis were performed. Overall, 58.2% of isolates were multidrug-resistant (MDR), and 41.8% were extensively drug-resistant (XDR). Sequence type (ST) 52 predominated among veterinary isolates, whereas ST2 was more frequent among environmental isolates. The presence of carbapenem-resistant isolates along with the ST2 lineage enhances the similarity to clinical A. baumannii. Several intrinsic resistance genes, including blaOXA-23, armA, aph(3″)-Ib, aph(6)-Id, tet(B), mph(E), and msr(E), were more prevalent in the ST2-associated population. Virulence-associated determinants were widely conserved. Core-genome MLST (cgMLST) and core-genome SNP (cgSNP) analyses identified highly related isolates within both lineages, while pairwise SNP differences were 0-7. Pan-genome analysis identified 4204 gene clusters and distinct accessory gene patterns between ST2 and ST52. These findings indicate that resistance gene distribution was closely associated with lineage structure and support integrated genomic surveillance of A. baumannii across animal and environmental reservoirs.

Acinetobacter baumannii↗