Applied and Environmental Microbiology最新文献

Candida auris, an emerging fungal pathogen characterized by multidrug resistance and high-mortality nosocomial infections, poses a serious global health threat. However, the precise and rapid identification and characterization of C. auris remain a challenge. Here, we employed Raman spectroscopy combined with machine learning to identify C. auris isolates and its closely related species as well as to predict antifungal resistance and key virulence factors at the single-cell level. The average accuracy of identification among all Candida species was 93.33%, with an accuracy of 98% for the clinically simulated samples. The drug susceptibility of C. auris to fluconazole and amphotericin B was 99% and 94%, respectively. Furthermore, the phenotypic prediction of C. auris yielded an accuracy of 100% for aggregating cells and 97% for filamentous cells. This proof-of-concept methodology not only precisely identifies C. auris at the clade-specific level but also rapidly predicts the antifungal resistance and biological characteristics, promising a valuable medical diagnostic tool to combat this multidrug-resistant pathogen in the future.

Importance: Currently, combating Candida auris infections and transmission is challenging due to the lack of efficient identification and characterization methods for this species. To address these challenges, our study presents a novel approach that utilizes Raman spectroscopy and artificial intelligence to achieve precise identification and characterization of C. auris at the singe-cell level. It can accurately identify a single cell from the four C. auris clades. Additionally, we developed machine learning models designed to detect antifungal resistance in C. auris cells and differentiate between two distinct phenotypes based on the single-cell Raman spectrum. We also constructed prediction models for detecting aggregating and filamentous cells in C. auris, both of which are closely linked to its virulence. These results underscore the merits of Raman spectroscopy in the identification and characterization of C. auris, promising improved outcomes in the battle against C. auris infections and transmission.

Amphenicol antibiotics, such as chloramphenicol (CHL), thiamphenicol (TAP), and florfenicol (Ff), are high-risk emerging pollutants. Their extensive usage in aquaculture, livestock, and poultry farming has led to an increase in bacterial antibiotic resistance and facilitated the spread of resistance genes. Yet, limited research has been conducted on the co-resistance of CHL, TAP, and Ff. Herein, a novel amidase AphA was identified from a pure cultured strain that can concurrently mediate the hydrolytic inactivation of CHL, TAP, and Ff, yielding products p-nitrophenylserinol, thiamphenicol amine (TAP-amine), and florfenicol amine (Ff-amine), respectively. The antibacterial activity of these antibiotic hydrolysates exhibited a significant reduction or complete loss in comparison to the parent compounds. Notably, AphA shared less than 26% amino acid sequence identity with previously reported enzymes and exhibited high conservation within the sphingomonad species. Through enzymatic kinetic analysis, the AphA exhibited markedly superior affinity and catalytic activity toward Ff in comparison to CHL and TAP. Site-directed mutagenesis analysis revealed the indispensability of catalytic triad residues, particularly serine 153 and histidine 277, in forming crucial hydrogen bonds essential for AphA's hydrolytic activity. Comparative genomic analysis showed that aphA genes in some species are closely adjacent to various transposable elements, indicating that there is a high potential risk of horizontal gene transfer (HGT). This study established a hydrolysis resistance mechanism of amphenicol antibiotics in sphingomonads, which offers theoretical guidance and a novel marker gene for assessing the prevalent risk of amphenicol antibiotics in the environment.IMPORTANCEAmphenicol antibiotics are pervasive emerging contaminants that present a substantial threat to ecological systems. Few studies have elucidated resistance genes or mechanisms that can act on CHL, TAP, and Ff simultaneously. The results of this study fill this knowledge gap and identify a novel amidase AphA from the bacterium Sphingobium yanoikuyae B1, which mediates three typical amphenicol antibiotic inactivation, and the molecular mechanism is elucidated. The diverse types of transposable elements were identified in the flanking regions of the aphA gene, indicating the risk of horizontal transfer of this antibiotic resistance genes (ARG). These findings offer new insights into the bacterial resistance to amphenicol antibiotics. The gene reported herein can be utilized as a novel genetic diagnostic marker for monitoring the environmental fate of amphenicol antibiotics, thereby enriching risk assessment efforts within the context of antibiotic resistance.

Distantly related organisms may evolve similar traits when exposed to similar environments or engaging in certain lifestyles. Several members of the Lactobacillaceae [lactic acid bacteria (LAB)] family are frequently isolated from the floral niche, mostly from bees and flowers. In some floral LAB species (henceforth referred to as bee-associated LAB), distinctive genomic (e.g., genome reduction) and phenotypic (e.g., preference for fructose over glucose or fructophily) features were recently documented. These features are found across distantly related species, raising the hypothesis that specific genomic and phenotypic traits evolved convergently during adaptation to the floral environment. To test this hypothesis, we examined representative genomes of 369 species of bee-associated and non-bee-associated LAB. Phylogenomic analysis unveiled seven independent ecological shifts toward the bee environment in LAB. In these species, we observed significant reductions of genome size, gene repertoire, and GC content. Using machine leaning, we could distinguish bee-associated from non-bee-associated species with 94% accuracy, based on the absence of genes involved in metabolism, osmotic stress, or DNA repair. Moreover, we found that the most important genes for the machine learning classifier were seemingly lost, independently, in multiple bee-associated lineages. One of these genes, acetaldehyde-alcohol dehydrogenase (adhE), encodes a bifunctional aldehyde-alcohol dehydrogenase which has been associated with the evolution of fructophily, a rare phenotypic trait that is pervasive across bee-associated LAB species. These results suggest that the independent evolution of distinctive phenotypes in bee-associated LAB has been largely driven by independent losses of the same sets of genes.IMPORTANCESeveral LAB species are intimately associated with bees and exhibit unique biochemical properties with potential for food applications and honeybee health. Using a machine learning-based approach, our study shows that adaptation of LAB to the bee environment was accompanied by a distinctive genomic trajectory deeply shaped by gene loss. Several of these gene losses occurred independently in distantly related species and are linked to some of their unique biotechnologically relevant traits, such as the preference for fructose over glucose (fructophily). This study underscores the potential of machine learning in identifying fingerprints of adaptation and detecting instances of convergent evolution. Furthermore, it sheds light onto the genomic and phenotypic particularities of bee-associated bacteria, thereby deepening the understanding of their positive impact on honeybee health.

Polymicrobial diseases such as periodontal disease and caries pose significant treatment challenges due to their resistance to common approaches like antibiotic therapy. These infections exhibit increased resilience, due to microbial interactions that also disrupt host immune responses. Current research focuses on virulence and disease-promoting interactions, but less is known about interactions that could inhibit or prevent disease development. Normally human-associated microbiomes maintain homeostasis, preventing pathobionts from becoming dominant. In conditions like chronic disseminated candidiasis or severe early childhood caries (s-ECC), an overgrowth of microbes such as Candida albicans disrupts this balance. Typically, C. albicans coexists benignly within the microbial community but can become pathogenic, forming biofilms and interacting with other microbes such as cariogenic Streptococcus mutans. This interaction is particularly significant in s-ECC, where it exacerbates the disease's progression and severity. Here, we present that Corynebacterium durum, itself and through its extracellular membrane vesicles disrupts interkingdom assemblages between C. albicans and S. mutans. Mechanistically the interaction interference occurs at the genetic level with downregulated HWP1 expression, a surface protein specifically induced in the presence of S. mutans promoting the interkingdom interaction. Additionally, we show that C. durum can impede C. albicans systemic virulence in the Galleria mellonella infection model. This suggests that oral corynebacteria may act as a beneficial commensal species, exerting antifungal effects within polymicrobial communities and opening new avenues for managing polymicrobial diseases.IMPORTANCEPolymicrobial diseases such as severe early childhood caries (s-ECC) lack effective treatment options. Prevention, requiring a deeper understanding of ecological processes before the onset of disease symptoms, could be a potential strategy. In this context, we investigated how relatively abundant oral biofilm Corynebacterium species, which are associated with oral health, can interfere with the interkingdom partnership of Streptococcus mutans and Candida albicans. This partnership is a significant driver of tooth decay in s-ECC due to synergistic activities that increase cariogenicity. Our study reveals that oral corynebacteria, through the production of extracellular membrane vesicles, can disrupt the S. mutans and C. albicans partnership by inhibiting fungal hyphae formation. Additionally, the fatty acid cargo within these vesicles exhibits antifungal properties, suggesting that corynebacteria play a role in shaping microbial dynamics within the oral biofilm.

Development of genome-editing tools in diverse microbial species is an important step both in understanding the roles of those microbes in different environments, and in engineering microbes for a variety of applications. Freshwater-specific clades of Actinobacteria are ubiquitous and abundant in surface freshwaters worldwide. Here, we show that Rhodoluna lacicola and Aurantimicrobium photophilum, which represent widespread clades of freshwater Actinobacteria, are naturally transformable. We also show that gene inactivation via double homologous recombination and replacement of the target gene with antibiotic selection markers can be used in both strains, making them convenient and broadly accessible model organisms for freshwater systems. We further show that in both strains, the predicted phytoene synthase is the only phytoene synthase, and its inactivation prevents the synthesis of all pigments. The tools developed here enable targeted modification of the genomes of some of the most abundant microbes in freshwater communities. These genome-editing tools will enable hypothesis testing about the genetics and (eco)physiology of freshwater Actinobacteria and broaden the available model systems for engineering freshwater microbial communities.

Importance: To advance bioproduction or bioremediation in large, unsupervised environmental systems such as ponds, wastewater lagoons, or groundwater systems, it will be necessary to develop diverse genetically amenable microbial model organisms. Although we already genetically modify a few key species, tools for engineering more microbial taxa, with different natural phenotypes, will enable us to genetically engineer multispecies consortia or even complex communities. Developing genetic tools for modifying freshwater bacteria is particularly important, as wastewater, production ponds or raceways, and contaminated surface water are all freshwater systems where microbial communities are already deployed to do work, and the outputs could potentially be enhanced by genetic modifications. Here, we demonstrate that common tools for genome editing can be used to inactivate specific genes in two representatives of a very widespread, environmentally relevant group of Actinobacteria. These Actinobacteria are found in almost all tested surface freshwater environments, where they co-occur with primary producers, and genome-editing tools in these species are thus a step on the way to engineering microbial consortia in freshwater environments.

Paenibacillus polymyxa, a Gram-positive bacterium commonly found in soil and plant roots, plays an important role in the environment due to its nitrogen-fixing ability and is renowned for producing antibiotics like polymyxin. In this study, we present a robust framework for investigating the evolutionary and taxonomic connections of strains belonging to P. polymyxa available at the National Center for Biotechnology Information, as well as five new additional strains isolated at the University of Camerino (Italy), through pangenome analysis. These strains can produce secondary metabolites active against Staphylococcus aureus and Klebsiella pneumoniae. Employing techniques such as digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) estimation, OrthoFinder, and ribosomal multilocus sequence typing, we consistently divided these P. polymyxa strains into four clusters, which differ significantly in terms of ANI and dDDH percentages, both considered as reference indices for separating bacterial species. Moreover, the strains of Cluster 2 were re-classified as belonging to the Paenibacillus ottowii species. By comparing the pangenomes, we identified the core genes of each cluster and analyzed them to recognize distinctive features in terms of biosynthetic/metabolic potential. The comparison of pangenomes also allowed us to pinpoint differences between clusters in terms of genetic variability and the percentage of the genome dedicated to core and accessory genes. In conclusion, the data obtained from our analyses of strains belonging to the P. polymyxa species converge toward a necessary reclassification, which will require a fundamental contribution from microbiologists in the near future.

Importance: The development of sequencing technologies has led to an exponential increase in microbial sequencing data. Accurately identifying bacterial species remains a challenge because of extensive intra-species variability, the need for multiple identification methods, and the rapid rate of taxonomic changes. A substantial contribution to elucidating the relationships among related bacterial strains comes from comparing their genomic sequences. This comparison also allows for the identification of the "pangenome," which is the set of genes shared by all individuals of a species, as well as the set of genes that are unique to subpopulations. Here, we applied this approach to Paenibacillus polymyxa, a species studied for its potential as a biofertilizer and biocontrol agent and known as an antibiotic producer. Our work highlights the need for a more efficient classification of this bacterial species and provides a better delineation of strains with different properties.

Cyanobacteria are photosynthetic organisms that play important roles in carbon cycling and are promising bioproduction chassis. Here, we isolate two novel cyanobacteria with 4.6Mbp genomes, UTEX 3221 and UTEX 3222, from a unique marine environment with naturally elevated CO₂. We describe complete genome sequences for both isolates and, focusing on UTEX 3222 due to its planktonic growth in liquid, characterize biotechnologically relevant growth and biomass characteristics. UTEX 3222 outpaces other fast-growing model strains on a solid medium. It can double every 2.35 hours in a liquid medium and grows to high density (>31 g/L biomass dry weight) in batch culture, nearly double that of Synechococcus sp. PCC 11901, whose high-density growth was recently reported. In addition, UTEX 3222 sinks readily, settling more quickly than other fast-growing strains, suggesting favorable economics of harvesting UTEX 3222 biomass. These traits may make UTEX 3222 a compelling choice for marine carbon dioxide removal (CDR) and photosynthetic bioproduction from CO₂. Overall, we find that bio-prospecting in environments with naturally elevated CO₂ may uncover novel CO₂-metabolizing organisms with unique characteristics.

Importance: Cyanobacteria provide a potential avenue for both biomanufacturing and combatting climate change via high-efficiency photosynthetic carbon sequestration. This study identifies novel photosynthetic organisms isolated from a unique geochemical environment and describes their genomes, growth behavior in culture, and biochemical composition. These cyanobacteria appear to make a tractable research model, and cultures are made publicly available alongside information about their culture and maintenance. Application of these organisms to carbon sequestration and/or biomanufacturing is discussed, including unusual, rapid settling characteristics of the strains relevant to scaled culture.

The glyoxalase pathway is the primary detoxification mechanism for methylglyoxal (MG), a ubiquitous toxic metabolite that disrupts redox homeostasis. In the glyoxalase pathway, glyoxalase II (GlyII) can completely detoxify MG. Increasing the activity of the glyoxalase system can enhance the resistance of plants or organisms to abiotic stress, but the relevant mechanism remains largely unknown. In this study, we investigated the physiological functions of GlyII genes (sll1019 and slr1259) in Synechocystis sp. PCC 6803 under MG or ethanol stress based on transcriptome and metabolome data. High-performance liquid chromatography (HPLC) results showed that proteins Sll1019 and Slr1259 had GlyII activity. Under stress conditions, sll1019 and slr1259 protected the strain against oxidative stress by enhancing the activity of the glyoxalase pathway and raising the contents of antioxidants such as glutathione and superoxide dismutase. In the photosynthetic system, sll1019 and slr1259 indirectly affected the light energy absorption by strains, synthesis of photosynthetic pigments, and activities of photosystem I and photosystem II, which was crucial for the growth of the strain under stress conditions. In addition, sll1019 and slr1259 enhanced the tolerance of strain to oxidative stress by indirectly regulating metabolic networks, including ensuring energy acquisition, NADH and NADPH production, and phosphate and nitrate transport. This study reveals the mechanism by which sll1019 and slr1259 improve oxidative stress tolerance of strains by glyoxalase pathway. Our findings provide theoretical basis for breeding, seedling, and field production of abiotic stress tolerance-enhanced variety.IMPORTANCEThe glyoxalase system is present in most organisms, and it is the primary pathway for eliminating the toxic metabolite methylglyoxal. Increasing the activity of the glyoxalase system can enhance plant resistance to environmental stress, but the relevant mechanism is poorly understood. This study revealed the physiological functions of glyoxalase II genes sll1019 and slr1259 in Synechocystis sp. PCC 6803 under abiotic stress conditions and their regulatory effects on oxidative stress tolerance of strains. Under stress conditions, sll1019 and slr1259 enhanced the activity of the glyoxalase pathway and the antioxidant system, maintained photosynthesis, ensured energy acquisition, NADH and NADPH production, and phosphate and nitrate transport, thereby protecting the strain against oxidative stress. This study lays a foundation for further deciphering the mechanism by which the glyoxalase system enhances the tolerance of cells to abiotic stress, providing important information for breeding, seedling, and selection of plants with strong stress resistance.

The aim of this study was to evaluate whether community-level monitoring of respiratory and enteric viruses in wastewater can provide a comprehensive picture of local virus circulation. Wastewater samples were collected weekly at the wastewater treatment plant (WWTP) inlet and at the outlet of a nearby nursing home (NH) in Burgundy, France, during the winter period of 2022/2023. We searched for the pepper mild mottle virus as an indicator of fecal content as well as for the main respiratory viruses [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza, and respiratory syncytial virus] and enteric viruses (rotavirus, sapovirus, norovirus, astrovirus, and adenovirus). Samples were analyzed using real-time reverse transcription PCR-based methods. SARS-CoV-2 was the most frequently detected respiratory virus, with 66.7% of positive samples from the WWTP and 28.6% from the NH. Peaks of SARS-CoV-2 were consistent with the chronological incidence of infections recorded in the sentinel surveillance and the nearby hospital databases. The number of positive samples was lower in the NH than in WWTP for the three respiratory viruses. Enteric viruses were frequently detected, most often sapovirus and norovirus genogroup II, accounting both for 77.8% of positive samples in the WWTP and 57.1% and 37%, respectively, in the NH. The large circulation of sapovirus was unexpected in particular in the NH. Combined wastewater surveillance using simple optimized methods can be a valuable tool for monitoring viral circulation and may serve as a suitable early warning system for identifying both local outbreaks and the onset of epidemics. These results encourage the application of wastewater-based surveillance (WBS) to SARS-CoV2, norovirus, and sapovirus.IMPORTANCEWBS provides valuable information on the spread of epidemic viruses in the environment using appropriate and sensitive detection methods. By monitoring the circulation of viruses using reverse transcription PCR methods in wastewater from the inlet of a wastewater treatment plant and the outlet of a nearby retirement home (connected to the same collective sewer network), we aimed to demonstrate that implementing combined WBS at key community sites allows effective detection of the occurrence of respiratory (influenza, respiratory syncytial virus, and SARS-CoV-2) and enteric (norovirus, rotavirus, and sapovirus) virus infections within a given population. This analysis on a localized scale provided new information on the viral circulation in the two different sites. Implementing WBS to monitor the circulation or the emergence of infectious diseases is an important means of alerting the authorities and improving public health management. WBS could participate actively to the health of humans, animals, and the environment.

Cra (catabolite repressor/activator) is a global transcription factor (TF) that plays a pleiotropic role in controlling the transcription of several genes involved in carbon utilization and energy metabolism. Multiple studies have investigated the regulatory mechanism of Cra and its rational use for metabolic regulation, but due to the complexity of its regulation, there remain challenges in the efficient use of Cra. Here, the structure, mechanism of action, and regulatory function of Cra in carbon and nitrogen flow are reviewed. In addition, this paper highlights the application of Cra in metabolic engineering, including the promotion of metabolite biosynthesis, the regulation of stress tolerance and virulence, the use of a Cra-based biosensor, and its coupling with other transcription factors. Finally, the prospects of Cra-related regulatory strategies are discussed. This review provides guidance for the rational design and construction of Cra-based metabolic regulation systems.