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Mapping genetic and phenotypic diversity of Pseudomonas aeruginosa across clinical and environmental isolation sites.

Pseudomonas aeruginosa is a clinically significant opportunistic pathogen adept at thriving in both host-associated and environmental settings. To define the extent to which P. aeruginosa isolates specialize across niches and identify genotype-phenotype correlates, we performed whole genome sequencing and comprehensive phenotypic characterization of 125 P. aeruginosa isolates from diverse clinical and environmental sites, evaluating virulence-associated traits, including motility, cytotoxicity, biofilm formation, pyocyanin production, and antimicrobial susceptibility. We identify that genomic diversity does not correlate with isolation source or most virulence phenotypes. Instead, we find that the two major P. aeruginosa clades (Groups A and B) delineate phylogeny and cytotoxicity, with Group B strains showing significantly higher cytotoxicity than Group A. Sequence analysis revealed previously uncharacterized alleles of genes encoding type III secretion effector proteins. We observed high variability amongst strains and isolation sources in all four assayed virulence phenotypes. Antimicrobial resistance (AMR) is exclusively observed in clinical isolates, not environmental, reflecting antibiotic exposure-driven selection. Bacterial GWAS revealed a statistically significant association between cytotoxicity and exoU presence, and we identified a novel exoU allelic variant with decreased cytotoxicity, demonstrating that functional diversity within well-characterized virulence factors may still influence pathogenic outcomes. In summary, our analyses of 125 diverse isolates suggest that the ability of P. aeruginosa to thrive across diverse niches is driven by broadly conserved genetic repertoire rather than niche-specific accessory genes.

Journal Article

Effect of socio-environmental isolation on brain biochemistry, behaviour and psychoactive drug activity.

Isolation has been widely described to induce a strong aggressive behaviour in many animal species and especially in rodents. A deeper analysis of this altered behaviour induced by isolation, allows for the identification of several other changes involving numerous peripheral, behavioural and neurochemical functions. As a consequence of the manifold aspects involved in this experimental situation, the definition "isolation syndrome" seems to be much more adequate than the simplest definition of "aggressiveness by isolation". On this framework, some similarities with psychoneurosis in men are also suggested.

Aggression

The effect of prolonged isolation from environmental allergens on the clinical and laboratory manifestations of the allergic state. Observation on members of the South African Antarctic expedition.

The 1975 South African Antarctic expedition included 8 men with a history of allergy, 7 who showed cutaneous hypersensitivity to common allergens, but denied having experienced symptoms, and 6 who had neither symptoms nor positive skin tests. During a year of residence in the essentially allergen-free, barren environment of Antarctica, allergic subjects were entirely sypmtom-free. Prolonged isolation from environmental allergens did not consistently diminish the serum concentration of total or specific IgE or the intensity of positive skin test reactions, which indicates that the cells responsible for the synthesis and secretion of reaginic antibodies have a prolonged existence and do not require repeated allergenic stimulation for maintenance of function.

Allergens

Antibiotic resistance patterns of gram-negative bacteria isolated from environmental sources.

A total of 2,445 gram-negative bacteria belonging to fecal coliform, Pseudomonas, Moraxella, Acinetobacter, and Flavobacterium-Cytophaga groups were isolated from the rivers and bay of Tillamook, Oregon, and their resistances to chloramphenicol (25 microgram/ml), streptomycin (10 microgram/ml), ampicillin (10 microgram/ml), tetracycline (25 microgram/ml), chlortetracycline (25 microgram/ml), oxytetracycline (25 microgram/ml), neomycin (50 microgram/ml), nitrofurazone (12.5 microgram/ml), nalidixic acid (25 microgram/ml), kanamycin (25 microgram/ml), and penicillin G (10 IU/ml) were determined. Among fecal coliforms the bay isolates showed greater resistance to antibiotics than those from tributaries or surface runoff. No such well-defined difference was found among other bacterial groups. The antibiotic resistance patterns of gram-negative bacteria from different sources correlated well, perhaps indicating their common origin. The antibiotic resistance patterns of gram-negative bacteria of different general also correlated well, perhaps indicating that bacteria which share a common environment also share a common mode for developing antibiotic resistance.

Anti-Bacterial Agents

Insights into the origin, hybridisation and adaptation of Candida metapsilosis hybrid pathogens.

Hybridisation is a source of genetic diversity, can drive adaptation to new niches and has been found to be a frequent event in lineages harbouring pathogenic fungi. However, little is known about the genomic implications of hybridisation nor its impact on pathogenicity-related traits. A common limitation for addressing these questions is the narrow representativity of sequenced genomes, mostly corresponding to strains isolated from infected patients. The opportunistic human pathogen Candida metapsilosis is a hybrid that descends from the crossing between unknown parental lineages. Here, we sequenced the genomes of five new C. metapsilosis isolates, one representing the first African isolate for this species, and four environmental isolates from marine niches. Our comparative genomic analyses, including a total of 29 sequenced strains, shed light on the phylogenetic relationships between C. metapsilosis hybrid isolates and show that environmental strains are closely related to clinical ones and belong to different clades, suggesting multiple independent colonisations. Furthermore, we identify a new diverging clade likely emerging from the same hybridisation event that originated two other previously described hybrid clades. Lastly, we evaluate phenotypes relevant during infection such as drug susceptibility, thermotolerance or virulence. We identify low drug susceptibility phenotypes which we suggest might be driven by loss of heterozygosity events in key genes. We discover that thermotolerance is mainly clade-dependent and find a correlation with the faecal origin of some strains which highlights the adaptive potential of the fungus as commensal.

Humans

Extensive hidden prophage diversity in Enterobacter species reveals host specificity and local distribution.

Bacteriophages are key drivers of bacterial evolution, particularly through their integration as prophages within host genomes. However, the diversity and host specificity of prophages in relevant pathogens such as Enterobacter species remain poorly characterized. In this study, we revealed the diversity of prophages, mapped their distribution and explored their relationships with their bacterial hosts. We analysed 3,661 prophage sequences identified from the genomes of 20 different Enterobacter species. This analysis uncovered an extensive hidden diversity, comprising 1,617 phage genera and 2,423 phage species - nearly 80% of which were singletons - highlighting an exceptionally rich prophage landscape. We found substantial variation in prophage species richness across host species and isolation sources, with Enterobacter kobei and environmental isolates exhibiting the highest richness. Prophage populations showed strong host specificity and limited cross-species transmission. Moreover, prophages exhibited geographic structuring and significant congruence between host and prophage phylogenies, as well as with the ecological lifestyles of their bacterial hosts. Although we found phages of the same species infecting different host species, these events were infrequent. Finally, bacterial genomes encoded diverse defence systems, mainly PDC-S07, RM type I-II and gabija, whereas only 8.9% of prophages encoded anti-defence systems, mostly anti-CBASS and anti-RM. Overall, this study provides new insights into the diversity of Enterobacter prophages and underscores their ecological and clinical relevance in shaping host adaptation and phage-host dynamics.

Prophages

Phylogenetic Constraints and Environmental Filtering Jointly Drive Adaptive Evolution in Phragmites australis: From Genetic Structure to Trait Decoupling on the Mongolian Plateau.

The Mongolian Plateau, a typical arid and semi-arid zone in Eurasia, is characterized by highly heterogeneous and fragmented wetland habitats. Phragmites australis, a common wetland species in this region, exhibits remarkable adaptability. Unraveling the coordination between phylogenetic history and local environmental filtering is crucial for elucidating its adaptive mechanisms. Integrating landscape genomics and trait-based phylogenetic analyses, we analyzed transcriptome-wide SNPs, multidimensional functional traits, and environmental variables across 90 individuals from 30 natural P. australis populations. This study aims to reveal the genetic and phenotypic variation patterns underlying population genetic structure and trait variation, specifically distinguishing the roles of geographic isolation, environmental filtering, and phylogenetic history. Results reveal a significant drainage-dependent pattern in genetic structure. Populations in hydrologically connected basins show extensive admixture, whereas those in isolated endorheic basins form distinct lineages. While geographic isolation underpins genetic differentiation, environmental filtering independently explains ~33.84% of the genetic variation, driven primarily by moisture heterogeneity (precipitation seasonality and soil moisture). Crucially, we observed differentiated evolutionary trajectories across functional traits. Structural traits (e.g., plant height, leaf thickness) are phylogenetically conserved; in contrast, physiological traits (e.g., water use efficiency) are decoupled from phylogeny, showing patterns consistent with high plasticity regulated by local environments. This evolutionary decoupling strategy enables P. australis to flexibly adapt to heterogeneous habitats while maintaining structural stability. This study uncovers the synergistic mechanisms by which geographic isolation and environmental filtering jointly shape the genetic patterns of this cosmopolitan species at a regional scale, clarifies that its evolutionary responses may depend heavily on the differentiated plasticity of trait types, and provides valuable regional insights into how widespread wetland species adapt to heterogeneous environments under global change.

Mongolia Plateau

Migration, extinction, and alary morphism in water-striders (Gerris Fabr.)

A migration-extinction model has been developed for the analysis of alary polymorphism and its adaptive significance in univoltine populations of water-striders (Gerris). It is assumed that all actual and potential population sites are identical with respect to all relevant variables, i.e. that populations do not live in a 'harlequin' environment. The model can be applied to other groups with alary polymorphism. The effects of isolation, environmental stability, and environmental productivity are discussed, with numerical examples. Isolation and stability enhance the optimality of short-wingedness, while temporariness of population sites selects for long-wingedness. Optima cannot always be realized. The models reveal several critical variables which are of interest in the ecological genetics of Gerris and which should be measured in natural populations: (a) extinction probabilities (a distinction being drawn between extinction due to drying up of the population site and extinction due to other causes), (b) extent of passive dispersal, and (c) the proportion of colonized population sites in a large geographical region.

Adaptation, Biological

Investigation of Mycobacterium abscessus Cluster in Hospital, Maryland, USA, 2024.

Mycobacterium abscessus infections are frequently drug resistant and can cause severe disease, particularly among vulnerable populations in healthcare settings. In June 2024, a cluster of M. abscessus infection cases was reported at a hospital in Maryland, USA. An investigation involving hospital infection prevention staff, the Maryland Department of Health, the Centers for Disease Control and Prevention, and the Wadsworth Laboratory of the New York State Department of Health included initiation of mitigation efforts such as tap water use restrictions, point-of-use water filters, hospitalwide water flushing, and staff education regarding sink safety. Whole-genome sequencing of patient and environmental isolates, coupled with epidemiologic data, revealed a strong association (0-24 single-nucleotide polymorphism differences) between environmental and patient samples, indicating that infections were likely a result of patient exposure to M. abscessus. Ensuring initiation of mitigation initiatives likely prevented additional patient infections. Although effective in this investigation, not all mitigation strategies are equal or sustainable.

Humans

Differences in virulence of Naegleria fowleri.

All pathogenic Naegleria fowleri isolated from the environment were highly virulent to mice when instilled intranasally. Axenic cultivation gradually decreased virulence of highly virulent strains. This decrease was most pronounced in environmental isolates and of minor importance in N. fowleri isolated from human cerebrospinal fluid. The low virulent strains obtained by continuous axenic cultivation appeared after clonation to consist of individuals with different virulence. Virulence could be enhanced in low virulent strains by brain passage and passages in Vero cell cultures, but could not be induced by these methods in nonvirulent strains isolated from the environment. Different mice strains showed different sensitivities to infection with pathogenic Naegleria. In addition, older mice were less sensitive than younger animals to low virulent strains.

Aging

Bioprospecting Chromobacterium violaceum for bioremediation: an alternative to environmental lead pollution.

Lead pollution is a major environmental concern, but current decontamination technologies remain limited due to high costs. Therefore, alternative biotechnological processes have been successfully developed and applied due to their reduced cost and lower aggressiveness in the environment. The remarkable adaptive versatility of Chromobacterium violaceum in metal-contaminated environments makes this bacterium a promising candidate for Pb bioremediation. Therefore, the reference strain C. violaceum ATCC 12,472 and the environmental isolate C. violaceum SCV1, the first strain of this species isolated from a Brazilian area with natural Pb occurrence, were evaluated for Pb resistance under different Pb concentrations and exposure times. Pb biosorption was assessed by scanning electron microscopy, while strain-specific protein profiles were characterized using tandem mass spectrometry-based proteomic analysis. The results obtained revealed the potential of C. violaceum to perform lead bioremediation. Scanning electron microscopy analysis confirmed the biosorption of lead by C. violaceum strains. C. violaceum SCV1 was able to remove up to 40% more lead concentration when compared to ATCC 12,472 which suggested adaptation through natural selection process of C. violaceum SCV1. Proteome analysis revealed 1531 proteins, of which several are candidates for lead bioremediation. This is the first study on the resistance proteomics of C. violaceum against lead. The acclimatization of the bacteria linked to the identification of several proteins related to: biosorption; efflux and ionic uptake (bioaccumulation); biomolecule transport; and biomethylation, point out to this organism as a potential lead bioremediation agent.

Chromobacterium

Limitations of the Moeller lysine and ornithine decarboxylase tests.

A total of 40 fecal and environmental isolates, including 26 Escherichia coli strains, 9 members of the genus Klebsiella, and 5 members of the genus Enterobacter, were tested by enzyme assay for their endogenous and induced levels of lysine decarboxylase and ornithine decarboxylase when grown in Moeller decarboxylase medium. All of the coliforms examined had measurable lysine decarboxylase and ornithine decarboxylase activities whether or not they were positive in the Moeller test. In general, the Moeller lysine decarboxylase test reflected the inducibility of lysine decarboxylase whereas the Moeller ornithine decarboxylase test did not relect the inducibility of ornithine decarboxylase. Neither test measured the amount of intracellular enzyme; rather, they indicated whether the amount of polyamine liberated was sufficient to raise the pH of the culture medium above 7. Changing the growth conditions (i.e., the concentrations of glucose, lysine, and amino acids other than lysine) greatly influenced the lysine decarboxylase activity in coliforms. The limitations on the interpretation of the Moeller test results are discussed.

Bacteriological Techniques

Evaluation of three test procedures for identification of Staphylococcus aureus from clinical sources.

A total of 520 clinical and environmental isolates of the family Micrococcaceae that fermented glucose anaerobically were tested for their ability to produce coagulase, thermostable nuclease, and deoxyribonuclease. Of these, 450 isolates coagulated rabbit plasma, produced thermostable nuclease, and were identified as Staphylococcus aureus, 447 of which produced a 3+ to 4+ clot. The remaining three isolates produced a 2+ clot, deoxyribonuclease, and thermostable nuclease. It was found that three of the S. aureus isolates failed to produce deoxyribonuclease. A total of 70 isolates which did not coagulate rabbit plasma and which were thermostable nuclease negative were identified as S. epidermidis. Three of them produced deoxyribonuclease. It is suggested that the thermostable nuclease test be performed on all isolates producing a 2+ (or 1+) clot in the coagulase test before identifying them as S. aureus.

Anaerobiosis

Virulence-associated variants in Cryptococcus neoformans sequence type 93 are less likely to be associated with population structure compared to independent rare mutations.

Cryptococcus neoformans is a pathogenic yeast that is the causative agent of cryptococcal meningitis. While it is well known that the genotype of C. neoformans impacts patient outcomes, the reason for this association has not been well elucidated. In this study, we examined the relationship between two subpopulations in the sequence type 93 clade of C. neoformans: ST93A and ST93B. We found extensive linkage disequilibrium (LD) among the single nucleotide polymorphisms (SNPs) that differentiate ST93A from ST93B. We also found differences in the extent of linkage among SNPs within each subpopulation; LD was more extensive within ST93B than ST93A. SNPs associated with virulence were in long-range linkage disequilibrium with less frequency than recurrent SNPs not associated with virulence. We investigated the karyotype of ST93A and ST93B using contour-clamped gel electrophoresis and long-read sequencing and found that the extensive long-range linkage was not due to chromosomal rearrangements. Overall, we found that the two subpopulations in ST93 are driven by SNPs in LD. We additionally found that recurrent SNPs associated with virulence were less frequently evolutionarily linked and were two times more likely to be independent, congruent mutations rather than tied to phylogeny.IMPORTANCECryptococcus neoformans is an important pathogen that is widely distributed and ubiquitous in the environment. The majority of the human population has a latent, controlled infection suggesting that C. neoformans is uniquely adapted to cause infection. In spite of this, the reason C. neoformans is a pathogen remains unknown; interestingly, most environmental isolates are avirulent but are genetically very similar to disease-causing virulent isolates. Recent evidence from genome-wide association studies shows that small mutations in key virulence-associated genes are associated with the virulence of specific isolates. The data presented here provide an evolutionary framework for those small mutations. The mutations that impact disease are not being collected over long-term evolution. The mutations may instead occur independently during infection. Identifying these genes that are more likely to be mutated during infection will be fundamental for understanding C. neoformans virulence.

Cryptococcus neoformans

Comparative evaluation of three high-molecular-weight DNA extraction kits for Oxford Nanopore sequencing of Clostridioides difficile and Clostridium perfringens.

UNLABELLED: Clostridioides difficile and Clostridium perfringens are Gram-positive, spore-forming anaerobic pathogens affecting humans and animals, for which genomic data have been mainly generated using short-read or hybrid sequencing approaches. In this study, we evaluated three commercial non-bead-beating DNA extraction kits designed for high-molecular-weight DNA recovery for Oxford Nanopore long-read whole-genome sequencing of two C. difficile and two C. perfringens strains, including one reference strain and one clinical or environmental isolate per species. Based on sequencing performance and kit ease of use, one kit was selected for additional sequencing of plasmid-carrying strains of both species. All three kits allowed correct identification of sequence types, toxin-encoding genes, and antimicrobial resistance determinants, confirming their suitability for clinical and epidemiological applications. However, the BT MasterPure Kit provided the highest DNA concentrations, longest fragment sizes, and superior read lengths and N50 values, particularly for C. difficile, achieving >100× coverage and enabling reliable circularization of chromosomes and plasmids, including a C. difficile metronidazole resistance plasmid and C. perfringens plasmids carrying toxin and antibiotic resistance genes. The other kits produced slightly lower DNA yields, resulting in shorter reads and reduced genome coverage for C. difficile, highlighting the challenge of extracting high-quality DNA from Gram-positive, spore-forming bacteria. Overall, this study provides practical guidance for selecting DNA extraction protocols optimized for Oxford Nanopore sequencing of C. difficile and C. perfringens, supporting high-quality genome assemblies and plasmid characterization and facilitating the routine genomic surveillance of clinically relevant spore-forming pathogens. IMPORTANCE: High-quality genomic data are essential for accurate characterization of Clostridioides difficile and Clostridium perfringens, two clinically and epidemiologically important Gram-positive, spore-forming pathogens. However, long-read sequencing performance can be strongly influenced by the choice of DNA extraction method, particularly for organisms with robust cell walls, where commonly used methods can lead to fragmented DNA. In this work, DNA of four strains was extracted using three commercial high-molecular-weight DNA extraction kits and sequenced using Oxford Nanopore Technologies. The best-performing kit was also evaluated using three additional strains known to harbor plasmids in order to assess its plasmid recovery efficiency. The results demonstrated successful plasmid recovery, circularization, and characterization. DNA extraction protocols optimized for Oxford Nanopore sequencing enable the rapid and cost-effective characterization of C. difficile and C. perfringens for genomic surveillance or outbreak investigations.

Clostridioides difficile