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PubMed2025

Comparison of whole-genome sequencing-based analysis methods for taxonomic classification of isolates unclassified by MALDI-TOF MS.

Taxonomic identification of clinical isolates is routinely achieved using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). If the species cannot be reliably identified, whole-genome sequencing can be applied. The aim of this study was to compare the results of approaches for taxonomic assignment for classification of isolates that are difficult to identify. Fifty-seven isolates were included in the study. The isolates were whole-genome sequenced and de novo assembled. Assembly-based classification was performed with the Genome Taxonomy Database Toolkit (GTDB-Tk), BLAST against 16S rRNA gene databases, the Type (Strain) Genome Server (TYGS), and ribosomal MLST (rMLST). Read-based classification was performed with MetaPhlAn4 and Kraken2. Thirty-two isolates were assigned to the same species with all four assembly-based classifiers, while the remaining 25 showed diverging assignments. When evaluating the results for the latter isolates, GTDB-Tk performed better than the other classifiers regarding which assignments were most likely correct. Of the read-based classifiers, MetaPhlAn4 performed better than Kraken2. Our evaluation identified GTDB-Tk to be the strongest tool for taxonomic assignment of isolates that are difficult to identify. Disagreements between classifiers are likely due to database limitations, wrongly assigned taxonomy, or unreliable 16S rRNA gene-based assignments.

Spectrometry, Mass, Matrix-Assisted Laser Desorpti↗

PubMed2025

Molecular epidemiology of panton valentine leukocidin-producing Staphylococcus aureus infections, Djibouti, 2018-2023.

Between 2018 and 2023, a genomic study was conducted in a military camp in Djibouti to investigate the molecular epidemiology of Panton-Valentine Leukocidin-producing Staphylococcus aureus. Among 43 isolates, Sequence Type 152 was predominant (72%), mainly associated with spa types t355 and t4235. Core-genome Multi-Locus Sequence Typing revealed two concurrent transmission dynamics: localized inter-human outbreaks and repeated introductions from external sources. Comparative genomics with other African Sequence Type 152 isolates showed similar levels of diversity, suggesting a widespread continental dissemination. These findings highlight the importance of genomic surveillance to better understand and control the spread of this virulent lineage in Africa.

Leukocidins↗

PubMed2026

Methicillin-resistant Staphylococcus aureus (MRSA) infection in hospitalized patients is dominated by community-acquired strains: genomic epidemiological evidence.

OBJECTIVE: This study aimed to systematically investigate the molecular epidemiological characteristics of methicillin-resistant Staphylococcus aureus (MRSA) in Ningxia hospitals, to elucidate their genetic evolutionary relationships, and to delineate the genomic and phenotypic profiles of the dominant lineages. METHODS: Clinical isolates of MRSA strains collected between 01/01/2024 and 30/06/2024 were analyzed, employing second-generation gene sequencing technology, combined with MLST and SCCmec typing, along with evaluation of drug resistance and virulence genes. A phylogenetic tree was constructed to analyze strain homology. RESULTS: A total of 74 non-duplicate Staphylococcus aureus strains (67 MRSA and 7 MSSA) were collected. The most common clonal strain was ST59-IVa, accounting for 46.27%. This strain exhibited a high prevalence of resistance genes mecA and blaZ, at 91.04%. All five ST22-IVa strains were found to lack mecA and erm genes but showed β-lactam resistance, while possessing both lukS/F-PV (PVL) and tsst-1 virulence genes, indicating a significant toxicity risk. Genetic evolution analysis revealed that ST3355, ST4513, and ST59 were closely related, all belonging to SCCmec types IVa; the other ST types exhibited mutations at various loci, with ST5 as the central node, resulting in a wider array of ST and SCCmec typing. CONCLUSION: The ST59-IVa clone is the predominant MRSA strain in Ningxia hospitals, exhibiting multidrug resistance and virulence gene profiles consistent with national trends. However, the emergence of hypervirulent ST22-IVa strains with atypical resistance mechanisms warrants increased vigilance. We recommend enhancing the rational use of antibiotics in hospitals and implementing molecular surveillance for these highly virulent strains.

Methicillin-Resistant Staphylococcus aureus↗

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

Differential virulence potential of different clades of multidrug-resistant Klebsiella pneumoniae ST258.

Klebsiella pneumoniae (KP) isolates belonging to multi-locus sequence type 258 (ST258) are a frequent cause of hospital-associated outbreaks and display extensive multidrug resistance. The KP ST258 lineage consists of two genetically distinct clades, called Clade 1 and Clade 2. These two clades are genetically related to one another, but are historically distinguished by having different capsular polysaccharide types. While bacteria belonging to both clades are isolated from clinical infections, Clade 2 is isolated more frequently compared to Clade 1. To investigate drivers of this difference in clade prevalence, we collected 172 clinical KP ST258 isolates from patients at a single medical center. Clinical review showed that patients infected with Clade 2 isolates were more acutely ill than Clade 1-infected patients, despite having fewer comorbidities. We also found that Clade 2 isolates were more resistant to killing by human serum, despite binding more complement protein C3 than Clade 1 isolates. Additionally, mice infected with a Clade 2 isolate had increased bacterial dissemination from the lungs to the liver and spleen than mice infected with a Clade 1 isolate, and this dissemination required an intact capsule locus. Increased dissemination in mice was not due to differential serum killing, as mouse serum was unable to kill isolates of either clade, but dissemination was associated with decreased macrophage uptake of the Clade 2 isolate. Taken together, these data suggest that KP ST258 Clade 2 is more virulent than Clade 1, although the specific mechanisms at play appear to differ between mice and humans.IMPORTANCEKP ST258 is an epidemic lineage of multidrug-resistant gram-negative bacteria that has caused numerous outbreaks in hospitals around the world. The KP ST258 population is divided into two genetically related but distinct clades, which differ primarily in their capsule type. In this study, we found that patients infected with one of the KP ST258 clades were more acutely ill than patients infected with the other clade. We also observed clade-specific differences both in killing by human serum and in bacterial dissemination in a mouse model of pneumonia. Finally, we identified important limitations in the use of mouse models to study host defenses against multidrug-resistant KP infection. Overall, this work underscores the importance of capsule composition in KP ST258 virulence, identifies differences in the host response to KP infection between mice and humans, and highlights a potential role for complement-targeting immunotherapeutics in the treatment of KP infections.

Klebsiella pneumoniae↗

PubMed2025

Comparative genomics of carbapenem-resistant Acinetobacter baumannii isolated from pediatric patients in a tertiary care hospital.

Acinetobacter baumannii is a short gram-negative bacillus, notable for its intrinsic multidrug resistance and genomic plasticity, which facilitates the acquisition of additional resistance genes via mobile genetic elements. Due to its increasing carbapenem resistance, the World Health Organization has classified it as a critical priority pathogen. This study performed a comparative genomic analysis of 20 carbapenem-resistant A. baumannii clinical strains isolated from the Hospital Infantil de México Federico Gómez (CRAB-HIMFG), alongside 11 genomes from other Mexican strains. The pangenome was determined to be open, and core genome single-nucleotide polymorphism-based analysis grouped the CRAB-HIMFG strains within CC758/IC5 and CC92/IC2. A novel sequence type (ST) in the MLST-Pasteur scheme was identified, related to STPas156, and in the MLST-Oxford scheme, associated with STOxf758 and STOxf1054. Virulence and resistance genes comprised 0.61% to 2.23% of the pangenome. Oxacillinase genes and efflux pumps primarily mediated carbapenem resistance, while virulence genes included those encoding biofilm and type IV pili. Capsule typing revealed a correlation with established international clones, IC2 and IC5. Plasmids exhibited high diversity, harboring maintenance modules and toxin-antitoxin systems, with the dissemination of resistance genes linked to insertion sequences. Biofilm formation and twitching motility were not always expressed, as they depend on additional environmental factors. Our study shows that comparative genomics is an essential tool to analyze clinically and epidemiologically significant genomes, providing critical insights into gene distribution, genomic architecture, and horizontal gene transfer mechanisms in microbial populations.IMPORTANCEIn recent years, a reported increase in the mortality rate associated with infections caused by A. baumannii, along with a rise in carbapenem resistance, poses a serious clinical challenge. The WHO considered this microorganism critical for research into alternative therapies and epidemiological surveillance. Despite advances in bioinformatics, genomic studies have yet to fully elucidate the structural rearrangements and secretion systems of A. baumannii. This knowledge gap hinders our understanding of its remarkable genomic plasticity and its ability to acquire and spread resistance and virulence genes through horizontal gene transfer.

Acinetobacter baumannii↗

PubMed2025

Genomic and phenotypic insights into ST164 blaNDM-1-positive Acinetobacter baumannii from intestinal colonization in China.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a critical global threat, especially in ICUs. Yet, reports on ST164 CRAB harboring blaNDM-1 remain scarce. This study investigates two clinical CRAB isolates, L4773hy and L4796hy, derived from intestinal colonization in Hangzhou, China, focusing on their phenotypic and genomic characteristics as well as the broader transmission of ST164 A. baumannii. METHODS: Bacterial identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility was assessed via agar and broth microdilution. Whole-genome sequencing employed Illumina NovaSeq 6000 and Oxford Nanopore platforms. Resistance genes, insertion elements, transposons, and integrons were detected using ResFinder, PlasmidFinder, VFDB, ISFinder, pdifFinder, and IntegronFinder. Strains were typed by MLST, and a phylogenetic tree was constructed with kSNP3.0. Genetic environment diagrams were generated using Easyfig 2.2.5. RESULTS: Two blaNDM-1-carrying A. baumannii isolates exhibiting extensive resistance to carbapenems, cephalosporins, and fluoroquinolones. Whole-genome sequencing and genetic environment analysis revealed the presence of a conserved structural sequence (ISAba14-ISAba14-aphA-ISAba125-blaNDM-1-bleMBL) on their chromosomes. Phylogenetic and clonal dissemination analysis showed that ST164 CRAB is primarily distributed in China and exhibits clonal spread. Pathogenicity studies indicated that blaNDM-1-positive ST164 strains have enhanced survival under immune pressure but do not display increased virulence in infection models. CONCLUSION: This study provides the genomic and phenotypic characterization of intestinally colonized ST164 blaNDM-1 positive CRAB in Hangzhou, China. The elucidation of the genetic environment of blaNDM-1 further confirms the clonal dissemination of ST164 isolates, highlighting the importance of enhanced surveillance and infection control measures to mitigate the spread of these multidrug-resistant pathogens.

Acinetobacter baumannii↗

PubMed2025

Molecular epidemiological surveillance for non-tuberculous mycobacterial pulmonary disease: a single-center prospective cohort study.

UNLABELLED: Bacterial species cultured from sputum change during treatment or observation for non-tuberculous mycobacterial pulmonary disease; however, strain-level changes remain unrecognized. Variable number tandem repeat typing is a standard technique for strain identification; nonetheless, its labor-intensive and time-consuming nature limits routine clinical use. Therefore, we aimed to elucidate species-subspecies and strain dynamics in non-tuberculous mycobacteria and develop a simple sequence-based strain-level determination method. We performed a single-center prospective cohort study of 112 patients with non-tuberculous mycobacterial pulmonary disease. Whole-genome sequencing was performed on two sputum samples collected at enrollment and at the end of follow-up, followed by variable number tandem repeat (VNTR) typing. We also developed a simple long-read sequencing-based digital VNTR (dVNTR) typing method and evaluated its efficacy. Our results demonstrate that core genome multi-locus sequencing typing revealed species/subspecies changes in 13 patients (11.6%); VNTR typing detected strain changes in 16 patients (14.3%) without species/subspecies changes. Overall, pathogen shifts occurred in 29 patients (shift [+] group, 25.9%), whereas 83 had no detectable pathogen shift (shift [-] group, 74.1%). Interestingly, macrolide and amikacin susceptibility changed in both groups, but resistance remained higher in shift (-) patients. dVNTR results aligned with those of conventional VNTR typing. In conclusion, since susceptibility factors remain unclear, routine species/subspecies identification and molecular typing, such as VNTR, are optimal for patient care. Core genome multi-locus sequencing typing with a dVNTR identified pathogen shifts, innovating non-tuberculous mycobacterial pulmonary disease management.Clinical TrialsThis study is registered with UMIN as UMIN 000056067. IMPORTANCE: Pulmonary non-tuberculous mycobacterial disease is a chronic infection in which the causative pathogens may change at the species, subspecies, or strain level over time. Accurate tracking of these changes is essential for optimizing treatment; however, conventional clinical practice lacks efficient methods for monitoring such dynamics. Our study revealed pathogen changes in approximately one-quarter of patients over 1.5 years, prompting the development of a novel surveillance system that integrates next-generation sequencing for both species-subspecies identification and strain-level molecular epidemiology. This innovation enables real-time monitoring of pathogen dynamics, allowing clinicians to promptly adjust treatment strategies and improve patient care through more informed decision-making.

Humans↗

PubMed2024

Rapid inference of antibiotic susceptibility phenotype of uropathogens using metagenomic sequencing with neighbor typing.

UNLABELLED: Timely diagnostic tools are needed to improve antibiotic treatment. Pairing metagenomic sequencing with genomic neighbor typing algorithms may support rapid clinically actionable results. We created resistance-associated sequence elements (RASE) databases for Escherichia coli and Klebsiella spp. and used them to predict antibiotic susceptibility in directly sequenced (Oxford Nanopore) urine specimens from critically ill patients. RASE analysis was performed on pathogen-specific reads from metagenomic sequencing. We evaluated the ability to predict (i) multi-locus sequence type (MLST) and (ii) susceptibility profiles. We used neighbor typing to predict MLST and susceptibility phenotype of E. coli (64/80) and Klebsiella spp. (16/80) from urine samples. When optimized by lineage score, MLST predictions were concordant for 73% of samples. Similarly, a RASE-susceptible prediction for a given isolate was associated with a specificity and a positive likelihood ratio (LR+) for susceptibility of 0.65 (95% CI, 0.54-0.76) and 2.26 (95% CI, 1.75-2.92), respectively, with an increase in the probability of susceptibility of 10%. A RASE-non-susceptible prediction was associated with a sensitivity and a negative likelihood ratio (LR-) for susceptibility of 0.79 (95% CI, 0.74-0.84) and 0.32 (95% CI, 0.24-0.43) respectively, with a decrease in the probability of susceptibility of 20%. Numerous antibiotic classes could reasonably be reconsidered empiric therapy by shifting empiric probabilities of susceptibility across relevant treatment thresholds. Moreover, these predictions can be available within 6 h. Metagenomic sequencing of urine specimens with neighbor typing provides rapid and informative predictions of lineage and antibiotic susceptibility with the potential to impact clinical decision-making. IMPORTANCE: Urinary tract infections (UTIs) are a common diagnosis in hospitals and are often treated empirically with broad-spectrum antibiotics. These broad-spectrum agents can select for resistance in these bacteria and co-colonizing organisms. The use of narrow-spectrum agents is desirable as an antibiotic stewardship measure; however, it is counterbalanced by the need for adequate therapy. Identification of causative organisms and their antibiotic susceptibility can help direct treatment; however, conventional testing requires days to produce actionable results. Methods to quickly and accurately predict susceptibility phenotypes for pathogens causing UTI could thus improve both patient outcomes and antibiotic stewardship. Here, expanding on previous work showing accurate prediction for certain Gram-positive pathogens, we demonstrate how the use of RASE from metagenomic sequencing can provide informative and rapid phenotype prediction results for common Gram-negative pathogens in UTI, highlighting the future potential of this method to be used in clinical settings to guide empiric antibiotic selection.

Humans↗

PubMed2024

Genomic diversity of Campylobacter jejuni and Campylobacter coli isolated from the Ethiopian dairy supply chain.

Campylobacteriosis outbreaks have previously been linked to dairy foods. While the genetic diversity of Campylobacter is well understood in high-income countries, it is largely unknown in low-income countries, such as Ethiopia. This study therefore aimed to conduct the first genomic characterization of Campylobacter isolates from the Ethiopian dairy supply chain to aid in future epidemiological studies. Fourteen C. jejuni and four C. coli isolates were whole genome sequenced using an Illumina platform. Sequences were analyzed using the bioinformatics tools in the GalaxyTrakr platform to identify MLST types, and single nucleotide polymorphisms, and infer phylogenetic relationships among the studied isolates. Assembled genomes were further screened to detect antimicrobial resistance and virulence gene sequences. Among 14 C. jejuni, ST 2084 and ST 51, which belong to the clonal complexes ST-353 and ST-443, respectively, were identified. Among the 4 sequenced C. coli isolates, two isolates belonged to ST 1628 and two to ST 830 from the clonal complex ST-828. The isolates of C. jejuni ST 2084 and ST 51 carried β-lactam resistance gene blaOXA-605, a fluoroquinolone resistance-associated mutation T86I in the gryA gene, and a macrolide resistance-associated mutation A103V in 50S L22. Only ST 2084 isolates carried the tetracycline resistance gene tetO. Conversely, all four C. coli ST 830 and ST 1628 isolates carried tetO, but only ST 1628 isolates also carried blaOXA-605. Lastly, C. jejuni ST 2084 isolates carried a total of 89 virulence genes, and ST 51 isolates carried up to 88 virulence genes. Among C. coli, ST 830 isolates carried 71 genes involved in virulence, whereas two ST 1628 isolates carried up to 82 genes involved in virulence. Isolates from all identified STs have previously been isolated from human clinical cases, demonstrating a potential food safety concern. This finding warrants further monitoring of Campylobacter in dairy foods in Ethiopia to better understand and manage the risks associated with Campylobacter contamination and transmission.

Campylobacter coli↗

PubMed2026

Comparison of culture and culture-free methods for comprehensive identification of mycobacteria: a single-center prospective study.

The genus Mycobacterium, including Mycobacterium tuberculosis and over 200 nontuberculous mycobacteria (NTM), shows wide variability in clinical outcomes and drug susceptibility. Although culture-based identification remains the gold standard, slow mycobacterial growth delays diagnosis and treatment. In this study, we evaluated a novel culture-free method for subspecies-level identification directly from sputum. In this single-center prospective cohort study at Osaka Toneyama Medical Center, we analyzed 125 sputum samples from 115 patients with NTM pulmonary disease and 10 with non-NTM respiratory conditions. Samples were decontaminated using N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) or succinic acid. We compared the reference culture method (mycobacterial culture plus whole-genome sequencing) and a culture-free direct target capture sequencing method. Core genome multi-locus sequence typing identified subspecies in both workflows, covering 186 mycobacterial species, including M. tuberculosis. The 115 NTM cohort specimens yielded 57 smear-positive and 93 culture-positive results. The identified subspecies included 48 Mycobacterium avium subsp. hominissuis, 22 Mycobacterium intracellulare subsp. intracellulare, 5 subsp. chimaera, 7 Mycobacterium abscessus subsp. abscessus, 5 subsp. massiliense, 1 M. tuberculosis, and 5 other NTM species. The culture-free method showed a high identification rate for smear-positive specimens (75.4%) but a low identification rate for smear-negative specimens (13.9%). NALC-NaOH pretreatment resulted in higher accuracy (90.5%) than did succinic acid pretreatment (66.7%). Thus, our culture-free subspecies-level identification method achieved high accuracy, especially in alkaline-treated smear-positive sputum samples, achieving rates above 90%. This method is recommended in clinical practice for patients who require rapid diagnosis and timely initiation of appropriate treatment, bypassing time-consuming culture steps.IMPORTANCEAccurate identification of Mycobacterium species and subspecies is crucial for effective treatment, as drug susceptibility and clinical outcomes vary significantly among them. However, conventional diagnosis relies on culture-based methods that can take several weeks, critically delaying appropriate therapy. This study validates a novel culture-free method using target capture sequencing for the comprehensive, subspecies-level identification of over 186 mycobacterial species directly from sputum specimens. Our findings revealed the high accuracy of this approach for smear-positive specimens, especially with alkaline pretreatment. This rapid method is applicable in clinical settings and enables timely and precise treatment decisions, greatly benefiting patients who require urgent intervention.

Humans↗

PubMed2015

Impermanence of bacterial clones.

Bacteria reproduce asexually and pass on a single genome copied from the parent, a reproductive mode that assures the clonal descent of progeny; however, a truly clonal bacterial species is extremely rare. The signal of clonality can be interrupted by gene uptake and exchange, initiating homologous recombination that results in the unique sequence of one clone being incorporated into another. Because recombination occurs sporadically and on local scales, these events are often difficult to recognize, even when considering large samples of completely sequenced genomes. Moreover, several processes can produce the appearance of clonality in populations that undergo frequent recombination. The rates and consequences of recombination have been studied in Escherichia coli for over 40 y, and, during this time, there have been several shifting views of its clonal status, population structure, and rates of gene exchange. We reexamine the studies and retrace the evolution of the methods that have assessed the extent of DNA flux, largely focusing on its impact on the E. coli genome.

Clone Cells↗

PubMed2025

Molecular insights into carbapenemase-producing Enterobacterales from Senegal.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) are an urgent global health threat, especially in resource-limited countries. Here we determined the prevalence and the molecular characteristics of CPE isolated from infections in Dakar, Senegal. METHOD: From January 2019 to December 2020, Enterobacterales with reduced susceptibility to ertapenem (diameter < 25 mm) from infections were collected at the Pasteur Institute of Dakar. Carbapenemases were detected using biochemical and immunochromatographical assays. WGS was used to determine resistome, MLST, plasmids, virulence genes and genetic relatedness. RESULTS: Of the 1045 Enterobacterales collected during the study period, 86 had a diameter around ertapenem of <25 mm (8%) and 19 were confirmed as CPE (2%). These included Escherichia coli (n = 6) [ST410 (n = 3), ST405 (n = 2) and ST2083], Enterobacter spp. (n = 6) [ST231 (n = 3), ST245, ST760 and ST960] and Klebsiella spp. (n = 5) (ST22, ST25, ST231, ST1535, ST4843), Citrobacter freundii ST22 (n = 1) and Citrobacter koseri with unknown ST (n = 1). blaOXA-48 (n = 7; 35%), blaOXA-181 (n = 7; 35%) and blaNDM-5 (n = 6; 30%) genes were identified. C. freundii ST22 harboured blaNDM-5, blaOXA-48 and blaCTX-M-15 genes. Some E. coli isolates belonging to the high-risk clone ST410 were closely related (<20 SNPs) to isolates recovered in France from patients returning from Senegal, suggesting transnational spread. In addition, 5/6 carbapenemase-producing E. coli isolates possessed a four amino acid insertion in PBP3, conferring reduced susceptibility to aztreonam/avibactam and cefiderocol. CONCLUSIONS: This study highlights the spread of NDM-5 and OXA-181 in Senegal, and reports the first co-occurrence of NDM-5 and OXA-48 in sub-Saharan Africa. The spread of CPE, especially in high-risk clones, underscores the urgent need for continued surveillance and targeted interventions.

Senegal↗

PubMed2026

Genomic Characterization of Aeromonas dhakensis Isolated From a Fatal Dolphin Case.

Aeromonas dhakensis has emerged as a significant pathogen affecting both aquatic animals and humans; however, genomic data for isolates from marine mammals remain scarce. In this study, we characterised the genome of A. dhakensis strain KDL-001, isolated from a fatal dolphin case, using whole-genome sequencing and comparative genomics. Taxonomic analyses, including MLST and average nucleotide identity (ANI), confirmed the isolate as A. dhakensis. Core-genome phylogeny further revealed that KDL-001 is closely related to strains derived from fish and aquatic environments. Notably, in silico screening of virulence-associated genes showed that the virulence-associated gene profile of the dolphin isolate was broadly comparable to those of other A. dhakensis strains, with no isolate-specific virulence-associated genes being identified within the limits of this analysis. These findings demonstrate that the dolphin-derived isolate is genomically comparable to previously described A. dhakensis strains and possesses conserved virulence-associated genes commonly found within the species.

Animals↗

PubMed2026

A new highly discriminatory typing scheme for Treponema pallidum reveals similar levels of genetic variability across lineages.

UNLABELLED: The global resurgence of treponematoses, particularly syphilis, poses a growing public health challenge. Despite advances in sequencing technologies, obtaining complete Treponema pallidum genome sequences for epidemiological studies remains challenging due to clinical sampling and methodological constraints. There is, therefore, a need for rapid, cost-effective, and accessible typing methods. Based on the analysis of 121 T. pallidum genomes spanning all three subspecies (TPA, TPE, and TEN) from diverse regions, we selected seven highly variable genes (tp0136, tp0326, tp0548, tp0705, tp0858, tp0865, and tp1031) to form a new typing system, combined with analysis of macrolide resistance mutations in the 23S rRNA gene. The scheme was validated on 542 global T. pallidum samples, using either Sanger reads or whole genome sequence data, obtaining 82 sequence types (STs) among the 415 fully typed samples. Macrolide resistance mutations were frequently detected, highlighting the need for ongoing epidemiological surveillance. Phylogenetic analyses based on concatenated multilocus typing (MLST) loci recovered the expected subspecies and lineage structure. Consistently, almost all sequence types formed monophyletic groups, indicating strong concordance between MLST-based classification and whole-genome phylogenies. In addition, population genetic analyses revealed comparable levels of within-lineage diversity across subspecies and lineages, despite pronounced differences in geographic distribution, and identified distinct regional genetic clusters consistent with localized transmission dynamics. Importantly, the scheme employs a single-step PCR for all seven targets, facilitating implementation in standard laboratories and is publicly accessible through PubMLST. Overall, our novel MLST scheme offers a rapid, cost-effective tool to advance molecular epidemiology of T. pallidum, facilitate transmission and resistance tracking, and support global surveillance to strengthen public health interventions for syphilis and endemic treponematoses control. IMPORTANCE: We have developed a new multilocus typing (MLST) scheme useful for all Treponema pallidum lineages after the analysis of 121 complete genome sequences of this species. The new scheme can be used directly with uncultured clinical samples, thus providing an excellent contribution to the molecular surveillance of syphilis and other treponematoses. The application of this MLST scheme to over 500 samples from all lineages and main geographical regions has revealed similar levels of genetic variation within them. Furthermore, the analyses show a complex pattern of spread, with global and local contributions to the observed distribution of genetic variation in the syphilis-producing sublineages. The new scheme represents a significant improvement over previous proposals and also reveals unsuspected levels of variability in T. pallidum lineages.

Treponema pallidum↗

PubMed2025

Understanding recurrence in Mycobacterium avium complex pulmonary disease: genotypic strategies to support clinical decision-making.

Pulmonary disease caused by Mycobacterium avium complex (MAC-PD) is a chronic, recurrent disease, and its high recurrence rate after treatment makes clinical management difficult. Distinguishing whether recurrence is due to persistence of existing strains or reinfection with new strains is essential for establishing treatment strategies, preventing overuse of antimicrobials, and establishing infection control measures. According to reports, 54%-74% of MAC-PD recurrence is due to reinfection, which may be mainly related to environmental reservoirs such as household water supply. In this review, we present various clinical scenarios in which MAC-PD recurrence may occur and examine genotyping techniques as a strategy to distinguish and respond to them. From traditional methods such as IS1245-based restriction fragment length polymorphism, pulsed-field gel electrophoresis, and hsp65 and rpoB gene sequencing to high-resolution analysis techniques such as multilocus sequence testing and whole-genome sequencing, the latest molecular typing methods are comprehensively summarized. Integrating these genotype data into clinical settings, standardizing single-nucleotide polymorphism-based interpretation thresholds, and promoting the establishment of a global MAC strain database will make a substantial contribution to more accurately distinguishing the recurrence mechanisms of MAC-PD and establishing personalized treatment strategies.IMPORTANCEThe global burden of nontuberculous mycobacterial pulmonary disease (PD) is increasing, with Mycobacterium avium (MAC)-PD being the most prevalent and clinically challenging form. Its low treatment success rates, high frequency of recurrence, and persistent environmental exposure complicate both diagnosis and management. A critical clinical issue is determining whether recurrence represents true relapse, due to persistence of the original strain, or reinfection with a new strain, as this guides treatment and prevents overtreatment. Genotypic strategies capable of resolving strain-level differences can improve diagnostic accuracy, prevent misclassification, and ultimately support more informed treatment decisions. Therefore, integrating genotyping data into clinical workflows, standardizing single-nucleotide polymorphism thresholds, and establishing a global MAC strain database will not only support personalized treatment but also enhance the broader public health response to this disease.

Humans↗

PubMed1992

Analysis of relationships among isolates of Citrobacter diversus by using DNA fingerprints generated by repetitive sequence-based primers in the polymerase chain reaction.

Oligonucleotide probes which match consensus sequences of the repetitive extragenic palindromic (REP) element hybridize to genomic DNA of diverse bacterial species. Primers based on the REP sequence generate complex band patterns with genomic DNA in the polymerase chain reaction (PCR), a technique named REP-PCR. We used REP-PCR with genomic DNA to fingerprint 47 isolates of Citrobacter diversus. Previously, 37 were assigned electrophoretic types (ETs) by multilocus enzyme electrophoresis and 35 were evaluated by using outer membrane protein profiles. Fingerprints were compared by visual inspection and by similarity coefficients (SimCs) based on the number of common bands versus total bands between two given isolates. DNA fingerprints were highly similar visually for patient pairs and outbreak-related sets. SimCs for these were > or = 0.952. Fingerprints of isolates with different ETs generally were distinctive. Among 21 unrelated isolates representing 15 ETs, only 6 of 210 comparisons had SimCs of > or = 0.952. REP-PCR rapidly generated DNA fingerprints which were highly similar for epidemiologically linked isolates of C. diversus and distinct for previously characterized strains within this species. The ability of this method to discriminate between C. diversus isolates with the same biotype was similar to that of multilocus enzyme electrophoresis and outer membrane protein profiles. REP-PCR may be useful in evaluation of apparent outbreaks of this or other bacterial species which possess these extragenic, repetitive elements.

Bacterial Typing Techniques↗

PubMed1992

Sequence diversity within the argF, fbp and recA genes of natural isolates of Neisseria meningitidis: interspecies recombination within the argF gene.

Studies of natural populations of Neisseria meningitidis using multilocus enzyme electrophoresis have shown extensive genetic variation within this species, which, it has been proposed, implies a level of sequence diversity within meningococci that is greater than that normally considered as the criterion for species limits in bacteria. To obtain a direct measure of the sequence diversity among meningococci, we obtained the nucleotide sequences of most of the argF, recA and fbp genes of eight meningococci of widely differing electrophoretic type (from the reference collection of Caugant). Sequence variation between the meningococcal strains ranged from 0-0.6% for fbp, 0-1.3% for argF, and 0-3.3% for recA. These levels of diversity are no greater than those found within Escherichia coli 'housekeeping' genes and suggest that multilocus enzyme electrophoresis may overestimate the extent of nucleotide sequence diversity within meningococci. The average sequence divergence between the Neisseria meningitidis strains and N. gonorrhoeae strain FA19 was 1.0% for fbp and 1.6% for recA. The argF gene, although very uniform among the eight meningococcal isolates, had a striking mosaic structure when compared with the gonococcal argF gene: two regions of the gene differed by greater than 13% in nucleotide sequence between meningococci and gonococci, whereas the rest of the gene differed by less than 1.7%. One of the diverged regions was shown to have been introduced from the argF gene of a commensal Neisseria species that is closely related to Neisseria cinerea. The source of the other region was unclear.

Amino Acid Sequence↗