ABSTRACT Expanded-spectrum cephalosporin-resistant Enterobacterales (ESC-R-E) have disseminated beyond clinical settings, but their occurrence and genomic epidemiology in wildlife remain poorly characterized, particularly in low- and middle-income countries. ESC-R may result from the production of extended-spectrum β-lactamases (ESBLs) or AmpC-type cephalosporinases. We investigated the prevalence, resistance mechanisms, and population structure of ESC-R-E in free-ranging wildlife, captive wild animals, and zoo workers in Algeria. Between October 2021 and June 2023, 1,899 fecal samples were collected across six Algerian provinces. ESC-R-E isolates were recovered by selective culture and characterized by antimicrobial susceptibility testing, and whole-genome sequencing. Overall, 73 ESC-R-E isolates (3.9%) were identified, including seven OXA-48-like carbapenemase co-producers (0.4%). Carriage was significantly higher in captive animals (13.5%) than in free-ranging wildlife (1.6%) or zoo workers (7.7%). Among the 66 ESBL-producing isolates analyzed, CTX-M enzymes predominated ( n = 61), particularly CTX-M-15 ( n = 53). Klebsiella pneumoniae was the predominant species ( n = 33), with ST17 accounting for 27/33 isolates. Closely related CTX-M-15-producing ST17 isolates were recovered from captive animals, free-ranging wildlife, and a zoo worker and carried an IncFIB(K) plasmid harboring bla CTX-M-15 within a multidrug resistance region closely related to globally disseminated plasmids. Globally disseminated high-risk Escherichia coli clones were also detected, namely ST167, ST361, ST410, and a persistent ST38 lineage in Algerian wildlife since 2016. These findings demonstrate the circulation of high-risk Enterobacterales lineages and mobile resistance plasmids in Algerian wildlife-associated ecosystems and highlight the need for integrated One Health surveillance of antimicrobial resistance at the human-animal-environment interface. IMPORTANCE Wildlife is increasingly recognized as a potential reservoir and sentinel for antimicrobial resistance, yet genomic data from low- and middle-income countries remain scarce. This study provides one of the largest genomic investigations of expanded-spectrum cephalosporin-resistant Enterobacterales in wildlife from North Africa. We demonstrate that internationally disseminated high-risk Klebsiella pneumoniae and Escherichia coli lineages, together with globally distributed CTX-M-15-carrying plasmids, are circulating among captive animals, free-ranging wildlife, and humans in zoological environments. The recovery of closely related K. pneumoniae ST17 isolates from different host species highlights the potential for antimicrobial resistance dissemination across the human-animal-environment interface. These findings extend the known ecological range of clinically important resistant Enterobacterales beyond healthcare settings and identify wildlife-associated ecosystems as part of the broader antimicrobial resistance network. Our study underscores the value of integrating wildlife into One Health surveillance programs to better understand and mitigate the environmental spread of antimicrobial resistance.
ABSTRACT Antibiotic resistance has renewed interest in bacteriophages as antimicrobials, yet the complexity of phage-host interactions often leads to inconsistent infection outcomes, underscoring the need to unravel the molecular basis of phenotypic diversity among closely related phages. Here, we compared two closely related T7-like phages, polyvalent phages C5 and N30, both belonging to Teseptimavirus, which share 89.67% genomic identity and an identical host range. While C5 exhibited a higher adsorption rate, plaque size, and efficiency of plating, N30 showed stronger bacteriostatic activity and larger burst sizes, highlighting the importance of a comprehensive evaluation of phage infection traits for therapeutic or biocontrol applications. Comparative genomics revealed high overall similarity between phages C5 and N30 but notable divergence in a hypothetical protein and a histidine-asparagine-histidine (HNH) endonuclease. Broader analysis across Teseptimavirus further uncovered two distinct genomic positions of the HNH endonuclease: either adjacent to virion assembly proteins in the genome middle or next to the terminase large subunit at the genome end, suggesting functional specialization in capsid assembly and DNA packaging. Tertiary structure prediction and comparison of the HNH endonucleases revealed moderate structural similarity despite moderate sequence divergence. Furthermore, a single nucleotide substitution in the HNH gene of N30 markedly increased its efficiency of plating in one tested host strain, demonstrating that terminase-associated HNH endonuclease plays a pivotal role in plaque formation. Together, these findings provide the first evidence of dual HNH endonuclease positioning within Teseptimavirus and identify the HNH endonuclease as a key genetic determinant of plaque formation, offering new insights into the molecular basis of phage infection variability and host adaptation.IMPORTANCEClosely related bacteriophages often display strikingly different infection phenotypes, yet the genetic basis of this variation remains poorly understood. Here, we uncover a previously unrecognized feature within the genus Teseptimavirus: the HNH endonuclease can occupy two distinct genomic positions, either associated with virion assembly or DNA packaging modules, suggesting functional specialization. We further demonstrate that this enzyme is closely linked to infection phenotypes, as even a single nucleotide substitution in the HNH gene significantly alters the efficiency of plating. These findings identify the HNH endonuclease as a key determinant of phage infectivity and reveal that its genomic context may shape functional outcomes. This work provides new insight into how subtle genetic differences drive phenotypic divergence among closely related phages and highlights the HNH endonuclease as a promising target for rational phage optimization in therapeutic and biocontrol applications.
ABSTRACT Anaerobic degradation of polycyclic aromatic hydrocarbons (PAHs) proceeds through CoA-activated intermediates and β-oxidation-like reactions; however, the enzymes responsible for key hydration and hydrolytic steps remain insufficiently defined. In the sulfate-reducing enrichment culture N47, the thn operon encodes multiple putative hydratases and hydrolases proposed to participate in anaerobic naphthalene degradation. Here, we cloned, heterologously expressed, and purified six candidate enzymes (ThnA, ThnH, ThnI, ThnL, ThnM, and ThnU) to elucidate their substrate specificities. Among them, ThnL showed robust activity toward 2-carboxycyclohexylideneacetyl-CoA, catalyzing rapid hydration of the enoyl-CoA double bond to form the tertiary alcohol 1-(1-hydroxy-2-carboxycyclohexyl)acetyl-CoA. ThnL exhibited kinetic parameters of Kₘ = 0.11 mM and Vₘₐₓ = 350 µmol min⁻¹ mg⁻¹. Two additional hydratases, ThnU and ThnI, converted the substrate only weakly (0.26% and 0.08% of ThnL activity), indicating promiscuous rather than physiological roles. ThnL also catalyzed the hydrolytic C1–C2 ring cleavage of 2-oxocyclohexane-1-carbonyl-CoA to pimelyl-CoA, revealing a bifunctional hydratase/hydrolase activity, although this reaction is unlikely to be physiologically relevant based on growth assays. This study shows that the ThnL-catalyzed hydration of 2-carboxycyclohexylideneacetyl-CoA to the tertiary alcohol 1-(1-hydroxy-2-carboxycyclohexyl)acetyl-CoA completes the final preparatory step before the second-ring cleavage in anaerobic naphthalene degradation.IMPORTANCEPolycyclic aromatic hydrocarbons (PAHs), such as naphthalene, are widespread environmental pollutants that persist in oxygen-limited environments, such as marine sediments and contaminated aquifers. Although microorganisms are known to degrade these compounds anaerobically, the enzymatic mechanisms underlying key steps of these pathways remain poorly understood. In particular, the enzymes responsible for hydration reactions preceding ring cleavage in anaerobic naphthalene degradation have not been experimentally verified. In this study, we characterized several candidate enzymes encoded by the thn operon from a sulfate-reducing enrichment culture and identified ThnL as the enzyme catalyzing the hydration of 2-carboxycyclohexylideneacetyl-CoA. This finding clarifies a crucial preparatory step before the second-ring cleavage during anaerobic naphthalene metabolism and advances our understanding of microbial PAH degradation in anoxic environments.
ABSTRACT Red yeasts serve as indispensable microbial resources for the production of carotenoids and lipids; however, environmental stress factors often limit their performance during industrial fermentation. The zinc-finger transcription factors Msn2/Msn4 act as central regulators of stress responses in yeasts. However, researchers have not yet systematically elucidated the similarities and differences in the mechanisms underlying the regulatory effects of Msn2/Msn4 under different stress conditions. In this study, we compared the shared and stress-specific regulatory functions of RkMsn4 in Rhodosporidium kratochvilovae YM25235 under conditions of glucose starvation, 15 °C low temperature, and 4% NaCl salt stress. RkMsn4 contributed to the activity of the oxidative defense systems, including antioxidant enzymes, carotenoids, and glutathione, and the remodeling of membrane lipids, including phospholipids and unsaturated fatty acids (UFAs) across all three stress conditions. Additionally, RkMsn4 regulated stress-specific metabolic regulation by modulating branched-chain amino acid (BCAA) and aromatic amino acid (AAA) metabolism under glucose starvation conditions and promoting the utilization of exogenous sucrose and fructose under salt stress. By conducting weighted gene co-expression network analysis (WGCNA), we identified candidate genes potentially associated with carotenoid and lipid metabolism. These findings provide practical insights for improving tolerance to stress and metabolic productivity of red yeasts in industrial bioprocesses.IMPORTANCERed yeasts are important microorganisms for the production of carotenoids and lipids, but their performance is often limited by environmental stress. Understanding how red yeasts respond to environmental stress is crucial for advancing their application in industrial fermentation. This study reveals the stress response mechanisms mediated by the transcription factor RkMsn4 in Rhodosporidium kratochvilovae YM25235 under glucose starvation, low temperature, and salt stress, as well as its regulatory role in carotenoid and lipid metabolism. These findings provide valuable insights for enhancing the stress resistance and productivity of red yeasts in industrial applications.
ABSTRACT Coastal wetland ecosystems (CWEs), including mangroves, saltmarshes, and seagrasses, support vital biogeochemical cycles mediated by microbes, yet their global microbial diversity, distribution, and potential functions remain elusive. Thus, we analyzed 1,381 16S rRNA gene amplicon data sets targeting the V3–V4 and V4 regions to examine microbial diversity and assembly processes across these habitats. Results revealed significant differences in microbial diversity and composition among these ecosystems, with mangroves exhibiting the highest richness and diversity. The habitat-specific indicator taxa were Rhodothermia, Anaerolineaceae, and SBR1031 in mangroves, Flavobacteriaceae, Burkholderiales, and Woeseiaceae in saltmarshes, and Desulfosarcinaceae, Pseudomonadaceae, Firmicutes, and Bacillales in seagrasses through linear discriminant analysis effect size (LEfSe) and random forest analyses. Co-occurrence network analysis revealed a robust structure comprising 1,521 nodes and 64,463 edges, dominated by Gammaproteobacteria, Desulfobacteria, Bacteroidia, and Desulfobulbia. KEGG-based functional prediction revealed distinct functional profiles across CWEs. Mangrove communities exhibited higher predicted potentials for carbon fixation and complex hydrocarbon metabolism, whereas seagrass communities showed higher potentials for sulfate metabolism. Saltmarsh communities were enriched in predicted functions related to the degradation of certain aromatic compounds. Using iCAMP, drift was most pronounced in seagrass communities (63.10%), whereas its contribution was lower but remained substantial in mangroves and saltmarshes. Deterministic processes also contributed considerably to community assemblies in mangroves (43.04%) and saltmarshes (44.42%). This underscores the dependence of dominant assembly processes on local environmental contexts. Our findings establish a basis for elucidating the structure and function of microbial communities in CWEs, offering insights for future hypothesis-driven research and enhancing predictive capacity amid growing anthropogenic and climatic pressures.IMPORTANCECoastal wetlands, including mangroves, saltmarshes, and seagrasses, protect shorelines and play a critical role in regulating the global climate. Microbial communities maintain the stability and essential functions of these ecosystems. This study provided the first comprehensive global map of microbial communities in the three ecosystems. By analyzing over 1,381 samples, the results revealed that different habitats relied on different diversity, composition, and dominant taxa. Mangroves showed the highest richness and diversity. KEGG-based functional predictions suggested that microbial communities in the CWEs shared core predicted metabolic functions but differed in their predicted functional potentials. The null model revealed that the assembly process of the three habitats is mainly shaped by stochastic ecological drift. However, deterministic selection also played a significant role in structuring saltmarsh and mangrove communities, suggesting that environmental factors commonly influenced the community assembly. Understanding these patterns is crucial for conservation because it allows scientists to better predict how coastal protection and carbon storage face increasing pressure from human activity.
