Applied and Environmental Microbiology最新文献

Antimicrobial resistance (AMR) is a growing global public health concern affecting animals, humans, and the environment. Given its ubiquity, Escherichia coli may play a key role in the dissemination of AMR across these domains. Peri-urban regions where urban and rural systems intersect present unique challenges for controlling AMR. Despite this, limited data are available on AMR dissemination across the One Health continuum in peri-urban settings such as the Fraser Valley region of Canada. This study adopted a One Health approach to assess associations in AMR traits between E. coli from dairy production systems, nearby natural environments, and peri-urban communities within the same geographic location and timeframe. Over 1,000 isolates were recovered, and 421 were whole-genome sequenced to determine the presence of antimicrobial resistance genes (ARGs), plasmids, and virulence genes and to assess genomic relatedness. Findings revealed that AMR was not widespread: 17.8% of isolates carried at least one ARG, 10.2% were classified as multidrug-resistant, and 9.5% carried beta-lactamase genes. Phylogenomic analysis revealed high genomic diversity, with isolates belonging to 174 different sequence types (STs), including clinically important ST131 and ST10. Pairwise single-nucleotide polymorphism (SNP) comparisons identified 207 isolate pairs differing by ≤100 SNPs, indicating early-stage cross-domain movement of AMR. Overall, the findings from this study show that the prevalence of AMR E. coli is low, but there is evidence of transmission between animals and the environment, highlighting the importance of proactive, integrated surveillance and mitigation strategies to reduce the risk of future AMR dissemination across the One Health continuum.

Importance: Antimicrobial resistance (AMR) is a global public health concern that spans all three One Health domains (humans, animals, and the environment). Escherichia coli is present in humans, animals, and environmental sources-its ubiquity makes it an ideal organism to study AMR hotspots and transmission pathways across the One Health continuum. While surveillance of AMR in agricultural settings is increasing globally, little is known about transmission pathways in peri-urban agriculture areas where there is a high density of livestock farming in close proximity to residential communities. To identify potential AMR hotspots and transmission routes, this study investigated the occurrence and genomic relatedness of generic E. coli in the Fraser Valley region of British Columbia, a highly diverse agricultural region in western Canada. Our findings expand current knowledge by suggesting that early-stage transmission of AMR is occurring between the human, animal, and environmental sectors of the One Health triad, highlighting areas for improved resistance mitigation to prevent widespread dissemination.

Engineering of highly thermostable keratinase is of great theoretical interest in understanding protein stability mechanisms and practical significance for processing keratinous wastes such as feathers and wool. The thermostable subtilisin-like protease C2 is the major keratinase secreted by Thermoactinomyces vulgaris CDF but is rapidly inactivated at temperatures above 90°C. Here, we employed various methods to further stabilize protease C2. Four of the 35 selected single-point mutations designed by automated computational tools (PROSS, FireProt, ProteinMPNN, HyperMPNN, and ThermoMPNN) retained higher residual activity (~72%-84%) than protease C2 (~54%) after 1-h incubation at 85°C. The rational design of surface ion pairs and proline substitutions in β-turns generated two single-point variants with increased thermostability. Although three single-point aspartate substitutions appeared to be neutral, they could synergistically or cumulatively improve enzyme stability. The combination of these nine stabilizing mutations yielded the variant SM9 with a half-life of ~4 h at 100°C. The molecular dynamics simulations of protease C2 revealed several relatively flexible regions, including two Ca²⁺-binding sites (Ca1 and Ca2). Empirically modifying the Ca1 site and incorporating an additional two Ca²⁺-binding sites (Ca3 and Ca4) into the flexible regions yielded the variant CM1 with enhanced thermostability. By combining the mutations in SM9 and CM1, the variant CM16 was generated with a half-life of more than 9 h at 100°C. SM9 and CM16 are also highly resistant to high alkalinity, high salinity, urea, sodium dodecyl sulfate (SDS), organic solvents, and reductants, enabling them to efficiently degrade chicken feathers at temperatures near the boiling point.IMPORTANCEThe boiling-resistant enzymes are especially valuable not only for probing the molecular basis that allows proteins to function at the maximum temperature capable of supporting life but also offer the opportunity to greatly expand the enzymatic reaction conditions. Besides exploring naturally occurring boiling-resistant enzymes from hyperthermophiles, artificial engineering of enzymes with boiling resistance remains an important challenge. Our results demonstrate that the thermostability of the subtilisin-like protease C2 with keratinolytic activity can be largely improved by the combined use of automated computational design, structure-based rational design, and empirical engineering. The resulting variants are not only stable and functional at temperatures near or above 100°C but also show improved resistance to polyextreme conditions, providing new clues about the stabilization mechanism of subtilases. Moreover, by virtue of their hyperthermostability, the boiling-resistant variants could be repeatedly used for processing keratin substrates at high temperatures and find practical applications in feed, food, and leather industries.

The widespread persistence of antimicrobial resistance (AMR) plasmids presents a fundamental challenge to microbial evolution, known as the "plasmid paradox": if these plasmids cause fitness cost, why are they not eliminated by selection? The classical view, which imposed a fixed generic fitness cost, is insufficient to explain their epidemiological success. Here, we propose a new paradigm-the plasmid-host fitness landscape-a multi-dimensional model that takes into account the complex interplay between ecology and genetics. This landscape unfolds into three main axes. First, the host axis reveals that fitness costs often arise from host-dependent genetic conflicts, not a generic burden. Second, the time axis demonstrates that the fitness cost of any plasmid can be negated over time through plasmid or chromosome compensations, which leads to ameliorating initial costs and locking in resistance. Third, the environmental axis shows that the fitness cost of any plasmid can be affected by external factors like temperature and sub-inhibitory concentrations of antibiotics. These factors dynamically modulate the benefits and costs of plasmid carriage. By integrating the complex interplay between these dimensions, we argue that the plasmid fitness costs are not a fixed generic measurement, but rather a contingent trajectory across this landscape. This paradigm shifts the focus from static measurements to a dynamic, predictive science, providing a new foundation for assessing and managing the threat of mobile resistance.

Genomic and metagenomic explorations of the oceans have identified well-structured microbial assemblages showing endemic genomic adaptations with increasing depth. However, deep water column surveys have been limited, especially of the Gulf of Mexico (GoM) basin, despite its importance for human activities. To fill this gap, we report on 19 deeply sequenced (~5 Gbp/sample) shotgun metagenomes collected along a vertical gradient, from the surface to about 2,000 m deep, at three GoM stations. Beta diversity analysis revealed strong clustering by depth, and not by station. However, a community-level pangenome style gene content analysis revealed ~54% of predicted gene sequences to be station-specific within our GoM samples. Of the 154 medium-to-high-quality MAGs recovered, 145 represent novel species compared with the NCBI genomes and Tara Oceans MAGs databases. Two of these MAGs were relatively abundant at both surface and deep samples, revealing remarkable versatility across the water column. A few MAGs of freshwater origin (~6% of total detected) were relatively abundant at 600 m deep and 270 miles from the coast at one station, revealing that the effects of freshwater input in the GoM can sometimes be far-reaching and long-lasting. Notably, 1,447/16,068 of the total COGs detected were positively (Pearson's r ≥ 0.5) or negatively (Pearson's r ≤ -0.5) correlated with depth, including beta-lactamases, dehydrogenases, and CoA-associated oxidoreductases. Taken together, our results reveal substantial novel genome and gene diversity across the GoM's water column, and testable hypotheses for some of the diversity patterns observed.IMPORTANCETo what extent microbial communities are similar between different ocean basins at similar depths, and what the impact of freshwater input by major rivers may be on these communities, remain poorly understood issues with potentially important implications for modeling and managing marine biodiversity. In this study, we performed metagenomic sequencing and recovered 154 medium-to-high-quality metagenome-assembled genomes (MAGs) from three stations in the Gulf of Mexico (GoM) and from various depths up to about 2,000 m. Comparison to MAGs recovered from other ocean basins highlighted the unique diversity harbored by the GoM, which could be driven by more substantial input from the Mississippi River and by human activities, including offshore oil drilling. The data and results provided by this study should be useful for future comparative analysis of marine biodiversity and contribute to its more complete characterization.

Transport systems play a vital role in metabolism in mycoplasmas by facilitating the uptake of essential nutrients from host cells, on which mycoplasmas are highly dependent. Co-culture of Mycoplasma bovis with Madin-Darby bovine kidney cells has shown that the gene MBOVPG45_0748 is essential for the survival of M. bovis in association with host cells. To characterize the function of this protein, intracellular and extracellular metabolomic profiles of a mutant strain with a transposon inserted into the MBOVPG45_0748 gene (∆MBOVPG45_0748) were compared to those of the parent strain, PG45. The ∆MBOVPG45_0748 mutant contained lower levels of metabolites involved in purine, pyrimidine, and glycerol metabolism and elevated levels of intermediates in the pentose phosphate and glycolysis pathways. Depletion of nucleosides from the culture supernatant occurred at a significantly reduced rate in the mutant, suggesting a role of the product of MBOVPG45_0748 in nucleoside uptake. This was further supported by isotope labeling studies with ¹³C₅-thymidine, which confirmed that nucleoside uptake and interconversion were disrupted in ∆MBOVPG45_0748 and suggested a compensatory strategy for the maintenance of nucleic acid biosynthesis in the mutant. The poorer survival of ∆MBOVPG45_0748 in medium with DNA supplementation showed that the MBOVPG45_0748 protein was involved in the efficient import of nucleosides derived from degradation of extracellular DNA. Overall, these findings suggest that the MBOVPG45_0748 protein is a nucleoside transporter involved in the uptake of purines and pyrimidines and maintenance of their intracellular balance, which is essential for the survival of M. bovis in association with host cells.

Importance: Mycoplasma bovis causes pneumonia, mastitis, arthritis, keratoconjunctivitis, and reproductive tract disease in cattle, compromising animal welfare and imposing economic losses on farmers. Development of effective control strategies against M. bovis requires a better understanding of the host-microbe interactions involved in the pathogenesis of the diseases it causes. Mycoplasmas are dependent on transport systems to acquire nutrients from host cells. Thus, these systems play an important role in their survival and virulence. We used a combination of metabolomic techniques to establish that a transporter gene that is essential for the survival of M. bovis in association with host cells plays a role in nucleoside uptake. These results highlight the importance of purine and pyrimidine metabolism in the interactions between M. bovis and host cells.

Cellulose deconstruction and utilization are foundational to renewable biofuel and biochemical production. Anaerocellum bescii (formerly Caldicellulosiruptor bescii) is an extremely thermophilic cellulolytic bacterium, notable for its multi-domain cellulases and hemicellulases that efficiently degrade lignocellulosic biomass. However, the mechanisms by which A. bescii transports cello-oligosaccharides released during cellulose degradation into the cell for catabolism remain unclear. Among its 23 ATP-binding cassette (ABC) sugar transporters, we identified a conserved ABC transporter locus (athe_0595-0598) encoding two extracellular binding proteins: Athe_0597 and Athe_0598. Biophysical analyses using differential scanning calorimetry and isothermal titration calorimetry revealed that Athe_0597 binds cello-oligosaccharides of varying lengths (G2-G5), while Athe_0598 is specific to cellobiose (G2). Ligand docking simulations supported these findings and shed light on the subsite configuration of these substrate-binding proteins (SBPs). To assess its physiological importance, we genetically deleted this transporter locus in A. bescii strain HTAB187, which grew poorly on cellobiose and did not grow on cellulose. Comparison of growth with a msmK deletion strain that cannot consume oligosaccharides showed that HTAB187 retains growth on non-cello-oligosaccharides and monosaccharides. Taken together, these results integrate biophysical characterization, structural modeling, and genetic perturbation to elucidate how A. bescii transports cello-oligosaccharides released from cellulose, providing mechanistic insight relevant to consolidated bioprocessing applications.IMPORTANCEAnaerocellum bescii is the most thermophilic lignocellulolytic bacterium known and holds potential for bioprocessing lignocellulosic biomass into renewable fuels. Its diverse ATP-binding cassette (ABC) sugar transporters make it a valuable model for studying thermophilic sugar uptake. Here, we identify a single ABC transporter with two substrate-binding proteins (Athe_0597 and Athe_0598) responsible for cello-oligosaccharide uptake. Genetic deletion of this transporter locus impaired growth on cellobiose and eliminated growth on cellulose. This is the first genetic manipulation in A. bescii to modulate transport of a specific sugar. We also characterize the substrate specificity of the extracytoplasmic binding proteins associated with the locus. One binds various cellodextrins (G2-G5), while the other specifically binds cellobiose (G2). Molecular modeling depicts how each oligosaccharide is docked within the binding pocket of these proteins. Understanding the mechanism of cello-oligosaccharide uptake by A. bescii expands opportunities for its metabolic engineering and furthers our understanding of its carbohydrate utilization systems.

Agrobacterium tumefaciens, a soil bacterium, was used as a model organism to study the mechanisms of triclosan (TCS) resistance in environmental bacteria. Adaptive laboratory evolution tests were performed to select TCS-resistant strains by challenging a wild-type (WT) strain with increasing concentrations of TCS (8, 12, 16, and 20 µg/mL). Two high-dose-resistant strains, HDR-12a and HDR-20a, were isolated and used for detailed examination. In comparison to the minimum inhibitory concentration of the WT strain (10 µg/mL), HDR-12a (20 µg/mL), and HDR-20a (32 µg/mL) showed increased resistance to TCS. Whole-genome sequencing and transcriptomic analysis performed to identify mechanisms underlying the different degrees of TCS resistance among the two evolved A. tumefaciens strains revealed a nucleotide base change (missense mutation, Asn157Thr) in the transcriptional repressor triR gene as the key mechanism of TCS resistance in HDR-20a. This change reduced the DNA-binding ability of TriR, causing overexpression of the triABC operon that encodes the TCS-specific efflux pump. In contrast, HDR-12a had no mutation in the triR gene. HDR-12a exhibited transcriptomic changes in several genes involved in ATP-binding cassette (ABC) transporters and in the metabolism of sulfur, fatty acids, and carbohydrates. However, it remains unclear whether these transcriptomic changes are directly responsible for TCS resistance in HDR-12a. Both the TCS-adapted strains also showed increased resistance to chloramphenicol and erythromycin. Overall, these results demonstrate that TCS pollution in environmental hotspots can select for adaptive and cross-resistant bacteria.IMPORTANCETCS is widely used as a preservative and disinfectant in many personal healthcare products. TCS is subsequently released into aquatic and terrestrial environments. The emergence and spread of multidrug-resistant pathogens from the use of antimicrobials like TCS and the misuse of antibiotic drugs now pose a serious global public health threat. Understanding how resistance develops has implications for preventing the emergence of antimicrobial resistance. The adapted TCS-resistant strains showed cross-resistance to chloramphenicol and erythromycin. This study provides insight into how environmental exposure to triclosan can drive adaptive and cross-resistance mechanisms in a soil bacterium, highlighting its relevance to environmental antimicrobial resistance and public health risk.

Quorum sensing (QS) is a cell-cell communication mechanism widely employed by bacteria to control group behaviors in a cell density-dependent manner. QS plays a critical role in the regulation of physiological processes in the Burkholderia cepacia complex (Bcc), which consists of at least 28 closely related species. To date, several different QS systems have been identified in the Bcc, including the well-characterized N-acyl-L-homoserine lactone-type QS systems and diffusible signaling factor-type QS systems. Here, we review the research progress on QS in the Bcc, including biosynthesis, biological functions, and regulatory mechanisms. We compare the biosynthetic pathways and regulatory mechanisms of these QS signals, which reveal their specificity and universality. We also review recent antibacterial research, which focuses on targeting these QS signaling systems, and the application prospects of this strategy.