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PubMed2025

Genomic analysis of the liverpool epidemic strain of pseudomonas aeruginosa infecting persons with cystic fibrosis reveals likely Canadian origins.

INTRODUCTION: The Liverpool Epidemic Strain (LES) of Pseudomonas aeruginosa is one of several known strains to be transmissible between persons with cystic fibrosis (CF) (pwCF) and the only known strain to have infected large proportions of CF populations on two continents. Despite its prevalence, efforts to understand its spread have proven elusive. METHODS: We leveraged a prospective collection of P. aeruginosa isolates from pwCF attending the Southern Alberta Adult CF clinic from 1986 to 2020 to identify all individuals with LES infection. LES isolates collected every 1-2 years from each pwCF were sequenced and compared with 171 published LES genomes by phylogenetic analysis. RESULTS: Of 395 pwCF screened, ten pwCF infected with the LES were identified, from whom 46 LES isolates were sequenced. The earliest LES isolate was recovered in 1986, ∼2 years earlier than the previously oldest published LES isolate recovered in the UK. Phylogenetic analysis identified a diverse set of isolates at the root of the LES phylogeny that formed four clades, one of which gave rise to a "classic LES" clade. Canadian isolates formed a paraphyletic group that included the root of this clade and out of which the UK LES clade emerged. We estimated the date of the most recent common ancestor (MRCA) of the UK LES clade as 1977. CONCLUSIONS: Our study provides genomic evidence in support of a silent epidemic of LES infection occurring in the late 1970s among pwCF first originating in Canada and being spread to the UK, where transmission markedly accelerated.

Humans↗

PubMed2026

Emergence of cefiderocol resistance in carbapenem-resistant Escherichia coli ST167 prior to clinical use: A multifactored resistance landscape.

OBJECTIVES: Cefiderocol is a novel siderophore cephalosporin with potent activity against multidrug-resistant Gram-negative bacteria. Here, we reported the prevalence and mechanisms of cefiderocol resistance in carbapenem-resistant Escherichia coli (CREC) in China before its clinical use. METHODS: A total of 443 non-duplicate CREC isolates collected from 67 hospitals in China (2013-2021) underwent antimicrobial susceptibility testing according to CLSI guidelines. Whole-genome sequencing, transcriptomic analysis, siderophore quantification, and targeted genetic manipulation were performed to investigate the underlying resistance mechanisms. RESULTS: Among the 443 CREC isolates, 102 (23.0%) were resistant to cefiderocol, and 34 (7.6%) showed intermediate susceptibility. Multivariable logistic regression identified ST167 lineage (OR, 3.05; 95% CI, 1.12-8.29; P = 0.028), blaNDM-5 carriage (OR, 9.04; 95% CI, 2.96-27.57; P < 0.001), and cirA truncation (OR, 49.56; 95% CI, 20.33-120.79; P < 0.001) as independent factors associated with cefiderocol resistance. Among ST167 isolates, cefiderocol-resistant isolates showed increased yersiniabactin carriage and siderophore production but comparable TonB-dependent transporter expression profiles. Phylogenetic analysis revealed that cefiderocol-resistant ST167 isolates clustered into a distinct subclade enriched with resistance-associated determinants, including a recurrent FhuA P50S substitution detected in 59/64 (92.2%) resistant isolates. Functional assays showed that the P50S substitution increased cefiderocol minimum inhibitory concentration (0.032-0.125 µg/mL), particularly in an NDM-5-producing background (0.032-0.5 µg/mL). CONCLUSIONS: Cefiderocol resistance is highly prevalent among high-risk ST167 CREC isolates before the clinical introduction of cefiderocol in China, highlighting the need for continued surveillance of this epidemic lineage. Cefiderocol resistance is mediated by multiple resistance determinants, and we identify the recurrent FhuA P50S substitution as a novel contributor to reduced cefiderocol susceptibility.

Antimicrobial resistance↗

PubMed2024

Expansion of Oropouche virus in non-endemic Brazilian regions: analysis of genomic characterisation and ecological drivers.

BACKGROUND: Oropouche virus (OROV) is an arbovirus endemic in the Amazon region that closely resembles other arboviruses in terms of human disease, leading to potential misdiagnoses. The virus ecology has mostly restricted its occurrence to the Amazon biome; however, after a large 2023-24 OROV epidemic in the Brazilian Amazon region, outbreaks are being reported across Brazil and in other countries in Latin America. Here, we investigate the OROV spread outside Amazonia. METHODS: In this genomic and epidemiological study, OROV cases from January, 2023, to July, 2024, provided by the General Coordination of Public Health Laboratories of Brazil on Aug 1, 2024, were compared by geographical location (Amazon vs non-Amazon) and municipal population size, and a linear mixed model was employed to assess the relationship between agricultural area size and cases. OROV-positive samples from central laboratories of five non-Amazonian Brazilian states were sequenced using an amplicon-based approach. Bayesian phylogeographical analysis was performed with near full-length viral genomes, incorporating individual travel histories when relevant. The estimated dates of viral introductions in each sampled location were then contextualised with public epidemiological data. FINDINGS: Epidemic data show that outside the Amazon region, OROV cases frequency was 3·9-times higher in small municipalities than in large municipalities. The planted areas of some agricultural products, such as banana plantations, were positively correlated (r=0·39, p<0·0001) with OROV cases. The linear mixed model revealed that, besides banana, cassava also has larger (p<0·05) planted areas in municipalities with OROV cases when compared with those with no cases. The phylogenetic analysis of 32 new OROV genomes reconstructed multiple exportation events of the newly identified reassortant lineage from the Amazon to other Brazilian regions between January and March, 2024. At least three of the previously described OROV phylogenetic clades circulating in the Amazon were the source of viral introductions. Molecular clock analysis estimated that viral introductions happened from 50 days to 100 days before detecting the outbreaks in each state. INTERPRETATION: Our results confirm that the novel OROV reassortant lineage spread from the Amazon to other regions in early 2024, successfully establishing local transmission. The fact that outbreaks were observed in small municipalities, instead of large urban centres, suggests that local ecological conditions that are ideal for OROV vector occurrence, such as the banana plantation environment, might be important factors driving its spread in Brazil. FUNDING: DECIT, CNPq, FAPEAM, and Inova-Fiocruz. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Brazil↗

PubMed1979

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. I) Isolation and characterization of lactate dehydrogenases from thermophilic and mesophilic bacilli.

Lactate dehydrogenases from thermophilic bacilli (Bacillus stearothermophilus, Bacillus caldotenax) and from mesophilic bacilli (Bacillus X1, Bacillus subtilis) have been isolated by a two-step purification procedure. Only one type (LDH-P4) composed of four identical subunits (Mr 34 000 or 36 000) was found in each bacillus. The tetrameric enzymes were characterized with respect to thermostability, pH and temperature dependence of the pyruvate reduction and the L-lactate oxidation, substrate specificity, saturation kinetics (Km values of pyruvate, lactate, NAD, NADH), pyruvate and oxamate inhibition, and activation by fructose bisphosphate. The thermophilic and mesophilic enzymes differ characteristically in these parameters. Preliminary structural data (amino acid composition, comparative N-terminal sequence analysis) show the expected close phylogenetic relationship (high degree of sequence homology), but also typical differences between thermophilic and mesophilic dehydrogenases, a suitable basis for further comparative studies.

Bacillus↗

PubMed1992

Molecular epidemiology of HIV transmission in a dental practice.

Human immunodeficiency virus type 1 (HIV-1) transmission from infected patients to health-care workers has been well documented, but transmission from an infected health-care worker to a patient has not been reported. After identification of an acquired immunodeficiency syndrome (AIDS) patient who had no known risk factors for HIV infection but who had undergone an invasive procedure performed by a dentist with AIDS, six other patients of this dentist were found to be HIV-infected. Molecular biologic studies were conducted to complement the epidemiologic investigation. Portions of the HIV proviral envelope gene from each of the seven patients, the dentist, and 35 HIV-infected persons from the local geographic area were amplified by polymerase chain reaction and sequenced. Three separate comparative genetic analyses--genetic distance measurements, phylogenetic tree analysis, and amino acid signature pattern analysis--showed that the viruses from the dentist and five dental patients were closely related. These data, together with the epidemiologic investigation, indicated that these patients became infected with HIV while receiving care from a dentist with AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Phylogeny of the Bovidae (Artiodactyla, Mammalia), based on mitochondrial ribosomal DNA sequences.

Portions of the 12S and 16S mitochondrial ribosomal genes for 16 species representing nine tribes in the mammal family Bovidae were compared with six previously published orthologous sequences. Phylogenetic analysis of variable nucleotide positions under different constraints and weighting schemes revealed no robust groupings among tribes. Consensus trees support previous hypotheses of monophyly for four clades, including the traditional subfamily Bovinae. However, the basal diversification of bovid tribes, which was largely unresolved by morphological, immunodiffusion, allozyme, and protein sequence data, remains unresolved with the addition of DNA sequence data. The intractability of this systematic problem is consistent with a rapid radiation of the major bovid groups. Several analyses of our data show that monophyly of the Bovidae, which was weakly supported by previous morphological and molecular work, is questionable.

Animals↗

PubMed1992

Small subunit ribosomal RNA of Blastomyces dermatitidis: sequence and phylogenetic analysis.

We determined the small subunit (18S) ribosomal RNA sequence of the dimorphic fungus Blastomyces dermatitidis. The sequence was compared to that of fourteen other eukaryotic organisms, ten of which were higher fungi, and an evolutionary tree was constructed based on these sequences. B. dermatitidis aligned most closely with the Ascomycetes Neurospora crassa and Podospora anserina, in agreement with previous phylogenetic analysis based on morphological criteria. Phase-specific cDNA clones derived by reverse transcription of RNA isolated from the yeast and mycelial phases of B. dermatitidis were also sequenced. The 18S ribosome sequence was found to be the same in both phases. Heterogeneity was found at both the genomic and RNA level at position 1352.

Base Sequence↗

PubMed2025

Phylogenetic Methods Meet Deep Learning.

Deep learning (DL) has been widely used in various scientific fields, but its integration into phylogenetics has been slower, primarily due to the complex nature of phylogenetic data. The studies that apply DL to sequencing data often limit analyses to four-taxon trees. Many of these studies serve as "proof of principle" and perform similarly to traditional phylogeny reconstruction methods. New ways of using training data, such as encoding with compact bijective ladderized vectors or transformers, enable the handling of much larger trees and genomic data sets. This short perspective focuses on the application of DL in phylogenetics, introducing prevalent DL architectures. We highlight potential problems in the field by discussing the risks of using simulation-based training data and emphasize the importance of reproducibility and robustness in computational estimates. Finally, we explore promising research areas, including the combination of phylogenetics and population genetics in DL, the analysis of neighbor dependencies, and the potential to significantly reduce computational cost compared to traditional methods. This perspective illustrates the potential of DL in complementing traditional phylogeny reconstruction methods and aiding the advancement of phylogenetic analysis, especially in performing computationally demanding tasks such as model selection or estimating branch support values.

Humans↗

PubMed2025

Detection and characterization of neonatal cytomegalovirus through nanopore sequencing using flongle flow cells: Pilot study in Philadelphia, Pennsylvania.

BACKGROUND: Cytomegalovirus (CMV) remains a significant infection in neonates and its early detection can aid with further treatment (antiviral, audiology). However, current diagnostics do not provide genetic information. OBJECTIVE: We explored the use of the portable and comprehensive sequencing method from Oxford Nanopore Technologies, utilizing low-cost Flongle flow cells to detect and perform sequence-level characterization of neonatal urine samples that tested positive for CMV by PCR. STUDY DESIGN: We performed a pilot study based on a retrospective cohort study of neonates who were positive for CMV by PCR, who were admitted at two birth hospitals in Philadelphia, PA. We leveraged deep and long-read sequencing results to analyze the reads in two forms: by comparing them against a reference-based strain and by reconstructing the genome through de novo assembly with phylogenetic tree analysis. RESULTS: We assayed seven clinical samples, including a positive and negative control sample, from newborns ranging from 23 weeks' gestation to term, with testing performed for microcephaly, hearing test results, small gestational age, and thrombocytopenia. Each sample showed multiple differences compared to the reference strain, and the phylogenetic tree analysis of the de novo assembly depicted the genetic diversity of the samples. CONCLUSION: This pilot study shows that nanopore sequencing with low-cost Flongle flow cells can detect and characterize CMV strains from clinical neonatal urine samples. This, coupled with current screening and diagnostic criteria, could further our genomic understanding of neonatal CMV, such as viral genome diversity, genotype-phenotype associations, and spread of strains.

Humans↗

PubMed2026

Genomic characterization and phylogenetic placement of Matryoshka RNA virus 1 associated with Plasmodium vivax malaria in Africa.

Plasmodium vivax is a major cause of human malaria. It harbours Matryoshka RNA virus 1 (MaRNAV-1), a bi-segmented positive-sense RNA virus. MaRNAV-1 was first described in P. vivax and is now recognized as part of a wider group of Matryoshka viruses. These viruses also infect other haemosporidian parasites such as Leucocytozoon and Haemoproteus. The presence of MaRNAV-1 in African-origin human P. vivax, however, has not been clearly established. This study investigated whether MaRNAV-1 is present in public African-origin P. vivax transcriptomic datasets. Any viral sequences recovered were characterized using comparative genomic and phylogenetic analyses. A secondary in silico analysis targeted African-origin P. vivax RNA-seq runs from public repositories. Although the search covered Africa, only Ethiopian datasets could be confidently identified, retrieved and compiled at the time. After quality control and screening for MaRNAV-1 RNA-dependent RNA polymerase (RdRp) signals, three high-confidence runs were selected for further analysis. Reference-guided reconstruction, ORF prediction, blast-based validation and RdRp phylogenetic analysis were performed. MaRNAV-1 was identified in three Ethiopian P. vivax malaria transcriptomes. This was supported by strong segment-level mapping, near-complete coverage, high mean depth and minimal low-depth masking. The recovered genomes showed the expected bisegmented organization of MaRNAV-1. Segment I was highly conserved and encoded the canonical RdRp in all three consensus sequences. Segment II showed the conserved organization of two overlapping hypothetical ORFs in all three consensus sequences. Blast analyses confirmed close similarity to MaRNAV-1 reference sequences. Phylogenetic inference grouped the Ethiopian sequences within the broader P. vivax-associated MaRNAV-1 lineage, alongside other recognized MaRNAV lineages distinct from more divergent narna-like viruses. These findings provide genomic evidence for MaRNAV-1 in publicly available African-origin P. vivax transcriptomic datasets and add to the emerging evidence for the virus in the African malaria context.

MaRNAV↗

PubMed1992

Phylogenetic and chemotaxonomic characterization of Acidaminococcus fermentans.

The phylogenetic position of Acidaminococcus fermentans was determined by comparative sequence analysis of the 16S rRNA. This Gram-negative bacterium is a member of the Sporomusa cluster that is defined by other Gram-negative bacteria, i.e. Sporomusa, Megasphaera, Selenomonas, Butyrivibrio, Pectinatus, and Zymophilus. The branching point of this group within the radiation of Gram-positive bacteria of the Clostridium/Bacillus subphylum and adjacent to Peptococcus niger could be confirmed. Chemotaxonomic data were provided for a more detailed characterization of A. fermentans.

Amino Acids, Diamino↗

PubMed2026

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↗

PubMed2026

Characterisation of Trichuris incognita n sp in Côte d'Ivoire: a morphological, genomic, and genome-wide association with drug sensitivity study.

BACKGROUND: Trichuriasis is a neglected tropical disease that affects up to 500 million individuals and can cause considerable morbidity. For decades, trichuriasis was thought to be caused by one species of whipworm, Trichuris trichiura. The aim of this study was to investigate the origin of differences in response rates to the best available anthelmintic treatment for trichuriasis-a combination of albendazole and ivermectin-in Côte d'Ivoire by analysing the parasite population. METHODS: In this morphological, genomic, and genome-wide association study (GWAS) with drug sensitivity we used long-read and short-read sequencing approaches and assembled a high-quality reference genome of Trichuris incognita n sp isolated in a primary interventional study conducted in the Lagunes district of Côte d'Ivoire. Children aged 6-12 years were screened between July 14, 2022, and July 31, 2022; children positive for T trichiura on duplicate Kato-Katz smears and with infection intensity of 200 eggs per gram or more were eligible and treated first with albendazole (400 mg) and ivermectin (200 μg/kg) then with oxantel pamoate (20 mg/kg). We constructed a species tree of the Trichuris genus using 12 434 orthologous groups. We sequenced individual worms, which were used to confirm the phylogenetic placement and investigate patterns of adaptation through comparative genomic analyses. Finally, we conducted a GWAS to compare albendazole-ivermectin sensitive worms to drug non-sensitive worms. FINDINGS: 670 children were screened, of whom 243 were enrolled and from whom 271 worms were isolated after the first treatment and 827 worms after the second treatment. Sufficient DNA was recovered from 747 worms of which 721 were suitable for further bioinformatic analysis; of these, 179 were albendazole-ivermectin sensitive worms and 542 were drug non-sensitive worms. We present and characterise a new, human-infecting Trichuris species named T incognita n sp, which is morphologically indistinguishable from T trichiura, but forms a distinct phylogenetic clade, closer to Trichuris suis than to the canonical human-infective T trichiura. Comparative genomic analysis of genes suspected to confer resistance to either albendazole or ivermectin in helminths revealed a high number of β-tubulin orthologs, present in the whole population of T incognita n sp, compared with the canonical T trichiura species, but these genes were not associated with a resistant phenotype. The GWAS did not provide conclusive evidence of adaptation to drug pressure within the same species. INTERPRETATION: Our results demonstrate that trichuriasis can be caused by multiple whipworm species, and that differences in response rates might result from species responding differently to drug treatment, rather than from the intraspecies establishment of resistance. This discovery, coupled with the high tolerability of T incognita n sp to albendazole-ivermectin, marks a substantial shift in how we understand and approach whipworm infections. FUNDING: European Research Council.

Trichuris↗

PubMed2026

Emergence of carbapenemase-producing Escherichia coli in acute care hospitals in 32 European countries (the CCRE survey): a prospective, multicentre, cross-sectional, epidemiological, microbiological, and genomic surveillance study.

BACKGROUND: The emergence of carbapenem resistance in Escherichia coli is of major concern due to the high propensity of spread of this species and scarce treatment options. Herein, we examined the occurrence and spread of carbapenem-resistant E coli based on the carbapenem-resistant and/or colistin-resistant Enterobacterales (CCRE) survey performed across European countries in 2019. METHODS: We analysed epidemiological, microbiological, and whole-genome sequencing data of 548 E coli isolates from individual patients from 156 hospitals in 32 European countries over 6 months in 2019. These hospitals collected the first ten successive isolates of carbapenem-resistant or carbapenem-susceptible increased exposure (carbapenem-R/I) Klebsiella pneumoniae species complex or E coli, and carbapenem-susceptible (carbapenem-S) comparator isolates of the same species. Antimicrobial susceptibility testing was performed for 19 antimicrobial agents. Whole-genome sequencing was performed centrally using Illumina technology. Isolates from the CCRE survey were compared with those from the European Survey of Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) study. FINDINGS: Of the 548 E coli isolates, 211 (38·5%) were carbapenem-resistant or susceptible, increased exposure (carbapenem-R/I), and 337 (61·5%) were carbapenem-susceptible (carbapenem-S). Five sequence types (STs) accounted for 96 (45·5%) of 211 carbapenem-R/I isolates: ST131 (27), ST410 (20), ST38 (19), ST167 (16), and ST648 (14). Carbapenemase genes were identified in 182 (86·3%) carbapenem-R/I isolates, a pronounced increase from the 2013-14 EuSCAPE study (36 of 99, 36·4%). The most common genes were blaNDM-5 (62 of 182, 34·1%) and blaOXA-48 (40 of 182, 22·0%). blaNDM-5 carriage increased substantially compared with that in EuSCAPE (two of 99, 2·02%). Phylogenetic analysis showed substantial clonal spread of globally disseminated blaNDM-5-harbouring lineages, with numerous introductions into Europe but minimal onward transmission. INTERPRETATION: High-risk STs of E coli carrying carbapenemase genes are rapidly spreading globally, although our results indicate that, in 2019, most cases in Europe were sporadic. We urge vigilant monitoring, including genomic surveillance, and strengthening of control efforts, to reduce mortality and morbidity associated with the impending rise in carbapenem-R/I E coli cases. FUNDING: European Centre for Disease Prevention and Control and the Centre for Genomic Pathogen Surveillance.

Humans↗

PubMed1992

Correlation analysis of amino acid usage in protein classes.

We present a comparative study of residue usage correlations of various organism protein sets of diverse phylogenetic species and of open reading frames of several large human viral genomes. Our correlation analysis reveals three major tendencies: (i) charge compensation reflected by the high correlation of basic with acidic residues; (ii) the positive correlations of functionally and structurally similar amino acids including many pairs of hydrophobic amino acids, all pairs of aromatic amino acids, the anionic pair (glutamate and aspartate), but not the cationic pair (lysine and arginine), moderately the hydroxyl pair (serine and threonine), the small amino acids (glycine and alanine), and many (but not all) of those having high values in the Dayhoff substitutability matrix (characteristics such as amino acid polarity or codon usage agreement, except for the wobble position, do not necessarily imply significant positive correlations); (iii) a widespread negative correlation of the aggregate strong codon group amino acids (Ala, Gly, Pro) versus the weak codon group amino acids (Lys, Ile, Tyr, Asn, Phe). Discussion and speculations relate amino acid usage correlations to protein function/structure, cellular localization, proximity in amino acid biosynthetic pathways, amino acid relative abundances, tRNA and aminoacyl synthetase availabilities, and evolutionary processes.

Amino Acids↗

PubMed2025

Bacterial skin colonization with a specific Cutibacterium avidum clade as a risk factor for periprosthetic joint infections-a multicenter study.

Cutibacterium avidum is increasingly recognized as a causative agent of periprosthetic joint infections (PJIs), yet data on its pathogenic potential and distinguishing features from commensal strains remain limited. In this multicenter study, we compared 11 C. avidum isolates from PJIs with 32 isolates from healthy skin collected across four European hospitals. We investigated phylogenetic relationships, antibiotic susceptibility, biofilm formation, and bacterial fitness. Phylogenomic analysis revealed two main clades within the C. avidum population. All PJI isolates belonged exclusively to Clade 1, which also included skin isolates. Within Clade 1, gene content analysis showed no consistent genetic differences between PJI and skin isolates. All isolates exhibited moderate to strong biofilm formation, with no significant differences in either data set. Minimal inhibitory concentration (MIC) and minimal biofilm inhibitory concentration (MBIC) values were low and largely concordant, while minimal biofilm eradication concentration (MBEC) values were elevated for all antibiotics except rifampin. One isolate was resistant to clindamycin due to the erm(X) gene. Rifampin consistently showed the lowest MIC, minimal bactericidal concentration, MBIC, and MBEC values. Bacterial fitness, assessed via bacterial quantitative fitness analysis, was significantly lower in PJI isolates compared to skin isolates when all strains were analyzed (P = 0.039), but this difference was not statistically significant when restricted to Clade 1. In conclusion, C. avidum isolates are strong biofilm producers irrespective of clinical origin. PJI isolates are restricted to a single phylogenetic clade, yet lack distinct biofilm or fitness traits within that clade, suggesting that multiple Clade 1 strains may have the potential to cause PJIs. IMPORTANCE Cutibacterium avidum has long been considered a skin commensal, but it is increasingly associated with prosthetic joint infections (PJIs). Despite its clinical emergence, little is known about its virulence potential or how invasive strains differ from commensal ones. This multicenter study provides the most comprehensive comparative analysis to date, integrating phenotypic and genomic data from both PJI-associated and skin-derived isolates. We show that all isolates are strong biofilm formers and that invasive isolates exhibit reduced growth fitness-a phenotype linked to persistence and treatment failure in other pathogens. Notably, all PJI isolates belonged to a single phylogenetic clade, suggesting that specific lineages of C. avidum may be more likely to cause infection. These findings help clarify the biology of this emerging pathogen and provide a foundation for improved diagnostics, susceptibility testing, and future infection prevention and treatment strategies.

Biofilms↗

PubMed2026

Evolutionary adaptation of CCD4 enzymes in Buddleja alternifolia for crocetin biosynthesis.

INTRODUCTION: Carotenoid cleavage dioxygenase 4 (CCD4) enzymes play central roles in carotenoid turnover and apocarotenoid biosynthesis in plants. Despite their importance, the evolutionary mechanisms underlying diversification of CCD4 catalytic functions remain poorly understood. This study investigated the CCD gene family in Buddleja alternifolia, with particular emphasis on the expansion and functional evolution of the CCD4 subfamily. METHODS: A genome-wide identification and comparative analysis of CCD genes were performed in B. alternifolia. Genomic organization, phylogenetic relationships, and syntenic patterns were analyzed to investigate gene family expansion. Functional characterization of 11 BaCCD4 paralogs was conducted through biochemical assays, while structural analyses were used to identify sequence features associated with differences in substrate cleavage specificity. Gene expression profiling was performed to assess patterns of tissue-specific regulation. RESULTS: Twenty-three CCD genes were identified, including 12 CCD4 paralogs, representing one of the largest CCD4 expansions reported within Lamiales. Syntenic and genomic analyses revealed that recent tandem duplication events, particularly within a CCD4-rich region on chromosome 10, were the primary drivers of this expansion. The presence of pseudogenes in the same region supported an ongoing birth-and-death evolutionary process. Functional analyses demonstrated extensive biochemical diversification among BaCCD4 enzymes despite their high sequence similarity. Several paralogs catalyzed asymmetric carotenoid cleavage leading to citraurin production, whereas two paralogs, KAG8367281 and KAG8375220, exhibited symmetric zeaxanthin cleavage activity, producing crocetin dialdehyde, the direct precursor of crocins. Notably, these crocetin-producing enzymes belonged to closely related paralogous pairs whose counterparts displayed distinct cleavage specificities, indicating rapid neofunctionalization after duplication. Structural analyses suggested that subtle sequence variations, including indels affecting loop regions adjacent to the substrate access channel, may underlie changes in regioselectivity. Expression profiling further revealed tissue-specific expression patterns consistent with functional divergence among paralogs. DISCUSSION: These findings indicate that crocetin-forming activity in B. alternifolia likely evolved through progressive modifications of ancestral CCD4 functions rather than through a single evolutionary event. The remarkable expansion and diversification of the CCD4 subfamily provide evidence for the role of gene duplication and neofunctionalization in shaping carotenoid cleavage specificity. Collectively, this work establishes B. alternifolia as a valuable model for investigating the molecular evolution of CCD4 enzymes and the emergence of specialized apocarotenoid metabolism in plants.

CCD4 evolution↗

PubMedDate not supplied

Signal, noise, and reliability in molecular phylogenetic analyses.

DNA sequences and other molecular data compared among organisms may contain phylogenetic signal, or they may be randomized with respect to phylogenetic history. Some method is needed to distinguish phylogenetic signal from random noise to avoid analysis of data that have been randomized with respect to the historical relationships of the taxa being compared. We analyzed 8,000 random data matrices consisting of 10-500 binary or four-state characters and 5-25 taxa to study several options for detecting signal in systematic data bases. Analysis of random data often yields a single most-parsimonious tree, especially if the number of characters examined is large and the number of taxa examined is small (both often true in molecular studies). The most-parsimonious tree inferred from random data may also be considerably shorter than the second-best alternative. The distribution of tree lengths of all tree topologies (or a random sample thereof) provides a sensitive measure of phylogenetic signal: data matrices with phylogenetic signal produce tree-length distributions that are strongly skewed to the left, whereas those composed of random noise are closer to symmetrical. In simulations of phylogeny with varying rates of mutation (up to levels that produce random variation among taxa), the skewness of tree-length distributions is closely related to the success of parsimony in finding the true phylogeny. Tables of critical values of a skewness test statistic, g1, are provided for binary and four-state characters for 10-500 characters and 5-25 taxa. These tables can be used in a rapid and efficient test for significant structure in data matrices for phylogenetic analysis.

Animals↗