Legionella anisa has been frequently isolated from engineered water systems; however, its population structure remains understudied compared to Legionella pneumophila. Here, we generated complete genome sequences for four L. anisa isolates recovered from a healthcare facility in Rimouski, Canada. Further the population structure of this species was investigated by performing comparative genomic analyses of the genomes generated in this study together with publicly available L. anisa genomes. Genome-wide phylogenetic analysis revealed the presence of three distinct clades separated by substantial genetic divergence (∼500 SNP), with the Rimouski isolates forming a tightly clustered group, suggesting a clonal lineage. Comparative pangenome analysis indicated moderate core genome conservation accompanied by a highly variable accessory genome (∼50%). The isolates characterized in this study harbored multiple plasmids encoding genes associated with conjugation, heavy metal resistance, and other stress-related functions, suggesting potential roles in environmental persistence. Previous studies have shown that L. anisa can proliferate within protozoan host cells, although outcomes vary depending on the host species. Our isolates showed efficient proliferation within Acanthamoeba castellanii, but not within Vermamoeba vermiformis, under the conditions tested. Together, these findings underscore the genomic diversity of this understudied Legionella species and provide a framework for future investigations regarding environmental persistence and potential pathogenicity.
Metagenomics offers a powerful framework for authentic, interdisciplinary learning, yet it remains underrepresented in undergraduate education due to technical and infrastructural barriers. We hypothesized that a research-based, learning-by-doing metagenomics workshop supported by accessible bioinformatics tools could enhance students' perceived skills, self-efficacy, and conceptual understanding of metagenomic analysis. To test this hypothesis, we designed and evaluated a hybrid hands-on workshop in which undergraduate and postgraduate students analyzed real environmental shotgun metagenomic datasets generated from soil samples collected during a citizen science initiative. Using the graphical workflow platform KBase, participants completed an end-to-end metagenomic analysis, from quality control and assembly to genome reconstruction, taxonomic classification, functional annotation, and scientific presentation of results. Educational outcomes were assessed through validated retrospective pre-post questionnaires, self-efficacy scales, and an open-ended conceptual understanding task. Participants showed significant increases in perceived metagenomic skills and confidence in performing metagenomic analyses, while gains in perceived learning showed a positive trend. Conceptual understanding improved across educational levels, particularly among participants with limited prior experience. Together, these findings demonstrate that authentic, data-driven metagenomics activities can effectively lower barriers to computational biology and foster meaningful learning through hands-on research experiences.
Dry and wet heat treatments are widely used physical methods for microbial inactivation, and their effectiveness in destroying bacterial spores has been extensively demonstrated. This review systematically discusses the bactericidal effects and underlying mechanisms of dry heat, wet heat, and steam-based treatments on bacterial spores. Wet heat inactivates spores through heat-induced protein denaturation and dipicolinic acid release, resulting in multi-level structural damage, including spore coat collapse, cortical peptidoglycan hydrolysis, and core hydration. On the other hand, dry heat treatment requires the creation of a high-energy thermal environment (typically > 160°C), and its inactivation mechanism involves a dual pathway: spore dehydration and mechanical stress, along with damage to the DNA repair system, ultimately causing irreversible injury. Steam treatment overcomes limitations in traditional heat transfer. While saturated steam follows the mechanism of wet heat, pulsed superheated steam integrates both dry heat and wet heat inactivation mechanisms, significantly enhancing spore inactivation efficiency. Importantly, combining thermal treatments with the bactericidal agents can create synergistic effects, further improving sterilization efficacy. These technologies show great potential for application in the food industry. However, further research is needed to optimize processing parameters, reduce operational costs, and promote their commercial application.
Hesperetin is a naturally occurring flavonoid with reported antimicrobial activity, but its antibacterial modes of action remain poorly understood. In this study, the antibacterial effects of hesperetin in Escherichia coli were investigated. Hesperetin treatment increased intracellular reactive oxygen species (ROS) levels, and scavenger-based experiments supported the contribution to downstream effects. Elevated ROS was associated with membrane depolarization and oxidative membrane damage, including lipid peroxidation, as indicated by increased malondialdehyde levels. Hesperetin treatment also increased DNA damage, including chromatin condensation and DNA fragmentation. Because recA is a key regulator of the SOS DNA repair response, a recA-deficient strain was used to assess the role of SOS-associated repair signaling; DNA damage was significantly greater in the recA-deficient strain than in the wild-type strain. In addition, programmed cell death-like features, including phosphatidylserine externalization detected by Annexin V/PI staining, were observed. Collectively, these findings indicate that the antibacterial activity of hesperetin involves ROS-associated membrane injury, oxidative DNA damage, and recA-dependent SOS-associated responses, together with programmed cell death-like features in E. coli.
Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide, and resistance to major antibiotic classes is increasing. Exploiting collateral sensitivity (CS) may be an effective approach to optimize antibiotic use and mitigate the impact of resistance. In this study, we investigated CS and collateral resistance (CR) profiles in experimentally evolved C. jejuni mutants derived from ATCC 29428 resistant to ciprofloxacin, erythromycin, gentamicin, and tetracycline. Minimum inhibitory concentrations of thirteen antibiotics were determined in the wild-type and mutant strains to characterize collateral effects. Whole-genome sequencing was performed to identify mutations potentially underlying the observed collateral effects, and bacterial growth curves were used to evaluate the fitness of the mutants. Ciprofloxacin-resistant mutants exhibited CS most consistently to aminoglycosides, potentially explained by mutations in gyrA, parC, and parE. Gentamicin-resistant mutants showed CS to macrolides, fosfomycin, and amoxicillin. Erythromycin-resistant mutants displayed CS to aminoglycosides, albeit inconsistently across biological replicates. No CS was observed in tetracycline-resistant mutants, which showed CR to other ribosome-targeting antibiotics, potentially due to mutations in rpsJ. Consistent CR was observed among antibiotics of the same class. These findings demonstrate CS and CR patterns in C. jejuni, highlighting evolutionary trade-offs that could inform more effective treatment strategies against resistant C. jejuni infections.
Triclosan is an antibiotic frequently used to selectively isolate Pseudomonas aeruginosa from environmental and animal samples. Here, we report that while nine tested P. aeruginosa strains were indeed triclosan-resistant when grown on Luria Bertani agar supplemented with triclosan, all nine exhibited decreased CFU by plating following pre-exposure to phosphate-buffer saline (PBS). Compared to growth on Luria Bertani agar in the absence of PBS incubation, PBS pre-exposure was associated with mean CFU reductions ranging from ∼25% (clinical isolate PABL048) to ∼80% (laboratory strain PA14). The potential for triclosan agar to cause significant errors in experiments designed to quantify P. aeruginosa numbers was demonstrated using a mouse model of P. aeruginosa gastrointestinal carriage. In this experiment, P. aeruginosa carriage was underestimated by up to 20-fold when fecal samples were processed using PBS. These findings suggest that triclosan should be used with caution to quantify P. aeruginosa numbers in protocols that utilize PBS.
Dysbiotic diseases of the oral microbiota, such as dental caries, endodontic infections, and periodontitis, represent a clinical challenge due to bacterial resistance and biofilm formation. In this context, plant-derived secondary metabolites such as Geraniol have emerged as promising alternatives owing to their low toxicity and multiple mechanisms of action. This study aimed to evaluate the antibacterial and antibiofilm activity of Geraniol against resistant and prevalent bacterial species from the oral microbiota of patients treated at the Dental Clinic of the Federal University of Pernambuco (UFPE). Samples were collected from patients with oral alterations, and microorganisms were identified using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Antimicrobial susceptibility was determined using the VITEK® automated system. Multidrug-resistant (MDR) isolates and reference strains were selected to assess the minimum inhibitory concentration (MIC), minimum bactericidal concentration, and antibiofilm activity of Geraniol. A total of 74 isolates were identified, including Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, and Pseudomonas stutzeri, with 60% classified as MDR. Geraniol exhibited significant inhibitory activity against Streptococcus mutans (ATCC 700610) and Enterococcus faecalis (ATCC 29212), with an MIC of 0.032 mg/ml. Furthermore, Geraniol inhibited biofilm formation in six isolates of S. mutans and E. faecalis. These findings highlight the potential of Geraniol as a therapeutic alternative for resistant oral infections, particularly in dysbiosis-associated biofilms. Future studies should explore its clinical applications and potential combinations with other agents to overcome bacterial resistance and improve outcomes in the treatment of oral diseases.
The demand for d-amino acids is increasing as their unique physiological role continues to be elucidated. meso-Diaminopimelate dehydrogenase catalyzes the reversible NADP+-dependent oxidative deamination of meso-diaminopimelate to produce l-2-amino-6-oxopimelate. Moreover, a few enzymes show a broad range of reductive amination activity toward 2-oxo acids, synthesizing d-amino acids. Here, we report the identification and characterization of a novel NADP+-dependent meso-diaminopimelate dehydrogenase from Candidatus Syntrophocurvum alkaliphilum (CSaDAPDH). The purified enzyme exhibited oxidative deamination activity toward meso-diaminopimelate, as well as reductive amination activity toward several 2-oxo acids to produce the corresponding d-amino acids. The optimum pH and temperature for oxidative deamination of meso-diaminopimelate were 9.0 and 45°C, respectively. The optimum pH for reductive amination of pyruvate was 6.5, which was the lowest optimum pH among known enzymes, and thus, may lead to the development of new enzymatic methods for d-amino acid production. CSaDAPDH retained more than 60% of its activity after incubation for 30 min at 50 °C (pH 7.0) or at pHs ranging from 5.5 to 7.0 (50°C). Moreover, using known enzymes as comparisons, the coenzyme and substrate recognition mechanisms of CSaDAPDH were elucidated based on a multiple sequence alignment and the homology model.
Whole-cell biosensors provide a cost-effective and sensitive approach for real-time monitoring of toxic metals in environmental samples. In this study, a bacterial whole-cell biosensor was engineered using Escherichia coli BL21(DE3) by integrating the cadC regulatory gene from Bacillus megaterium TWSL_4 with a green fluorescent protein (GFP) reporter to enable fluorescence-based detection of heavy metals. The biosensor gene cassette (Pcad + cadC + gfp) was first cloned into the pUC19 vector and then subcloned into the pET28a(+) expression vector to generate the recombinant plasmid pETCG28. Functional characterization showed that the engineered strain E. coli BL21/pETCG28 exhibited enhanced tolerance to heavy metals, sustaining growth at Pb²⁺ concentrations up to 1600 ppm, Cd²⁺ up to 200 ppm, and Zn²⁺ up to 60 ppm, significantly higher than the wild-type strain. Fluorescence analyses demonstrated strong concentration-dependent responses to heavy metal exposure. Corrected total cell fluorescence increased nearly fourfold between 1 ppb and 10 ppb of Pb²⁺ (R² = 0.95, p < 0.0001). Cd²⁺ exposure produced an approximately threefold increase (R² = 0.96, p < 0.0001), while Zn²⁺ generated a moderate twofold response (R² = 0.94, p < 0.0001). Optimal biosensor performance occurred at pH 7.0 and 37°C, demonstrating potential for portable environmental monitoring applications.
In Vibrio parahaemolyticus RIMD2210633, chitin metabolism is enhanced by β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamin (GlcNAc-GlcN), which is produced from N,N'-diacetylchitobiose using chitin oligosaccharide deacetylase. However, GlcNAc-GlcN metabolism is not well understood. Vibrio bacteria have two types of disaccharide transport systems: the ABC transporter and the phosphotransferase system (PTS). The ABC transporter system is involved in the transcription of proteins related to metabolism. In this study, we clarified the mechanism by which GlcNAc-GlcN is transported into the cell and activates chitin metabolism. Using two types of Vibrio spp., in the presence or absence of chitin oligosaccharide deacetylase and PTS, we investigated the transcription of chitinases and transporters using real-time quantitative PCR. Furthermore, the utilization of disaccharides was investigated using bacterial proliferation. Our results suggested that GlcNAc-GlcN may act as a transcriptional signal via ABC transporter proteins but is translocated via PTS. These findings are expected to the study of the ecology of GlcNAc-GlcN-utilizing bacteria, about which little information is available.
Photobacterium damselae subsp. piscicida (Pdp) is a host-adapted primary pathogen impacting finfish aquaculture worldwide, whose virulence evolution is driven by the mobilome. The pPHDPT3 plasmid encoding a type III secretion system is critical for Pdp virulence but unstable in vitro in European and Japanese isolates. Here we show that a stable ancestral variant is conserved in Australian isolates. The elusive pPHDPT3 variant has undergone gene loss and accumulated a ∼5.5 kb quadruple direct repeat, which could explain plasmid loss via the classic dimer catastrophe scenario. In addition, we hypothesize that a 189-aa serine recombinase encoded within this repeat, and also on pPHDP10 plasmid, may act as a plasmid resolvase, with its frequent loss further exacerbating the dimer catastrophe.
TolC is the outer membrane component of the tripartite RND efflux pump AcrAB-TolC. The underlying mechanism of TolC-dependent acid survival at physiological pH remains unclear. The present study aimed at understanding mechanism of TolC mediated acid survival in logarithmic phase culture of Enterobacter cloacae subsp. cloacae ATCC 13047 not preconditioned to acidic pH. Of the three distinct loci encoding tolC and tolC like proteins, only ECL_04 363 (hereafter referred to as EctolC2) exhibited 86% identity with TolC protein from E. coli K12; deletion of which compromised survival at pH 4.0. Tracking the periplasmic and cytoplasmic pH changes with fluorescent protein sensors indicated deletion of EctolC2 resulted in sustained cytoplasmic acidification and loss of pH homeostasis causing cell death. Exogenous supplementation of lysine significantly rescued acid induced lethality in EcΔtolC2 mutants. ΔmarRAB mutants exhibited slower death rate compared to Δrob mutants; underpinning role of the latter in upregulating EctolC2 at early time point of growth at pH 4.0. Results of the study construe that EctolC2 provides reinforcement to the outer membrane preventing extreme acidification of the periplasm and thereby restoring cytoplasmic pH homeostasis. This study provides significant insight into mechanism of TolC dependent acid survival in E. cloacae.
Microbial biomass is considered an alternative protein source that can contribute to ensure food security. Detrimentally, microbial growth processes can lead to the formation of CO2 and liquid effluent, which requires waste management. With the overall goal to contribute microbial solutions to address current societal challenges, the aim of this study was to establish a holistic bioprocess to deliver multipurpose bioingredients using food-grade Propionibacteriaceae. Strains of Propionibacterium freudenreichii, Acidipropionibacterium microaerophilum, Acidipropionibacterium acidipropionici, and Acidipropionibacterium olivae grew with glucose, glycerol, lactate, lactose, or lactose + glycerol in the presence of bicarbonate/CO2. The major fermentation metabolite was propionate with lower levels of acetate and succinate depending on strain and substrate. Based on pathway prediction, strains assimilated up to 10% mol CO2/mol glycerol while releasing up to 60% mol/mol with other substrates. Biomass contained 14%-44% protein and all essential amino acids depending on strain and growth condition. Fermentates conferred antimicrobial activity against bacteria and molds in broth dilution assays. In summary, different Propionibacteriaceae species showed potential for single cell protein and metabolite production for food-related application. We present an approach to design a bioprocess through a choice of culture, substrate, and suggest application possibilities of biomass and fermentates that avoid the generation of additional waste streams.
Nutritional exchanges fuel the evolutionary and ecological dominance of multi-partner symbioses among reef-building corals and microbial associates in oligotrophic tropical marine ecosystems. Mutualistic relationships with endosymbiotic dinoflagellates (Family Symbiodiniaceae) are central to coral holobiont metabolism, yet their metabolic contributions are sensitive to nutrient availability. Symbiodiniaceae may compensate for oligotrophic environments via metabolic exchanges with prokaryotic partners. Bacterial production of ligands with affinities for otherwise insoluble elements promotes uptake and exchanges. Bacterial secretion of small molecules with high ferric (Fe3+) iron affinities, herein referred to as siderophore production, presents one example of microbial metabolic cooperation. We isolated 78 pure bacterial culture lines from 14 Symbiodiniaceae cultures to screen for siderophore production using a Chrome Azurol S (CAS) overlay assay. Colorimetric changes observed on CAS overlays indicated ubiquitous siderophore production across 22 bacterial genera. Many of the isolated bacterial cultures corresponded to known 'core' Symbiodiniaceae microbiome. These results suggest an avenue of bacterial metabolism may facilitate biotic iron exchange among coral holobiont partners. Future characterization of the identity siderophores secreted will inform predictions on their impacts on iron exchange within the coral holobiont. Ultimately, a greater ability to acquire iron via siderophore production may improve the coral holobiont's tolerance to environmental stressors.
Pseudomonas plecoglossicida is the etiological agent of visceral white spot disease, which induces significant mortality in economically important fish such as the large yellow croaker. In this study, we integrated recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection to establish a fluorescence-based assay for rapid identification of P. plecoglossicida. The complete single-tube, two-stage RPA-CRISPR/Cas12a workflow can be performed within ~45 min. Using purified genomic DNA, the assay achieved an analytical detection limit of 1.65 copies μl-1 and showed no cross-reactivity with several other common fish pathogens. Its applicability was further evaluated using crude DNA extracts from spleen, liver, and kidney tissues of experimentally infected large yellow croakers. Overall, with its rapid turnaround, minimal equipment requirement, and high sensitivity, the RPA-CRISPR/Cas12a assay represents a promising diagnostic tool for rapid detection of P. plecoglossicida, thereby helping to control the spread of infection.
In vitro multispecies biofilm models are widely used to study oral diseases and to evaluate treatment strategies for conditions such as peri-implantitis, often relying on accurate species quantification to assess treatment efficiency. However, the influence of DNA extraction methodology on downstream quantitative analysis has not yet been addressed for such model systems. Here, we evaluated three mechanistically distinct protocols, a custom phenol-chloroform approach, and two commercial kits employing different lysis strategies. These were applied to planktonic cultures of six peri-implantitis-associated species individually, as well as to defined multispecies biofilms grown on implant surfaces. Pure culture DNA yields differed substantially between methods, revealing pronounced species-dependent variation. Species-specific quantification of biofilm replicates by quantitative PCR resulted in community profiles that appeared dominated by either commensal early- or pathogenic late-colonizers, depending solely on the extraction approach employed. These findings demonstrate that DNA extraction is a critical yet often overlooked variable, capable of fundamentally altering the apparent community composition of in vitro biofilm models. Our work is intended to serve as a warning, emphasizing the need for method validation and standardization when applying DNA-based community profiling to biofilm models before drawing conclusions on relative species abundances.
Antiracist pedagogy is a teaching philosophy aimed at critically examining the interconnection between race, power, education, and one's field of study. After participating in the Seminar for Transformation Around Antiracist Teaching (START) program at Brown University, an intergenerational biology team composed of a professor, doctoral student, and an undergraduate student developed new assignments covering broad topics in bioethics for an Introductory Microbiology course. Here, we share our process and perspectives on engaging students in the exploration of how the field of microbiology is deeply connected to history, society, and culture.
The design of specific inhibitors of metallo-β-lactamases (MBL) is one of the priority research lines due to the spread of carbapenem resistant bacterial strains which limits therapeutic options. In parallel, the investigation of new uses of known medicines is prompted by regulatory agencies. In this work, the potential MBL inhibitory potency of L-captopril (an antihypertensive drug) and dimercaprol (used in poisoning by metals) was investigated in a clinical collection of Enterobacterales and Pseudomonas spp. In addition, a comprehensive review of the available literature was conducted, to discuss jointly new and previously available data. The results indicate that activity of imipenem and meropenem combined with 400 mg/l is restored for > 80% and 60%, respectively, for Enterobacterales strains, but not in the Pseudomonas group. Dimercaprol had a limited activity in both bacterial groups. Based on these promising results of L-captopril, pharmacological calculations were performed. It is concluded that the repurposing objective could not be achieved as the overall data indicated that the concentrations of captopril required to achieve carbapenemase inhibitory activity in vivo are higher than maximum used in treatment. Communication of negative results would help to discontinue active current research on L-captopril and should focus on other potential MBL inhibitors.
Acinetobacter baumannii, a prominent nosocomial pathogen, commonly causes life-threatening disease in immunocompromised individuals with mortality rates approaching 35%. Known for its multidrug resistance, studies investigating antimicrobial peptides (AMPs) have proven promising. We have previously shown that the AMP WAM-1 exhibits potent bactericidal activity against A. baumannii, but its full therapeutic value and mechanism of action have not yet been elucidated. To evaluate the potential clinical relevance of WAM-1, we characterized its antibacterial effects, inhibition by lipopolysaccharide (LPS), and whether treatment with WAM-1 induced resistance in A. baumannii. Additionally, we explored WAM-1's mechanism of action by visualizing structural changes induced by treatment with WAM-1 and evaluating permeabilization of the bacterial outer membrane. The activity of WAM-1 was comparable to that of efficacious antibiotics and was not inhibited by the presence of exogenous LPS. Scanning electron microscopy showed evidence of membrane disruption, cellular content leakage, and cell lysis, indicating damage to the bacterial outer membrane, and depolarization of the membrane was observed. WAM-1 was found to induce resistance more slowly than tested antibiotics. Overall, our data indicates that WAM-1 may be a promising therapeutic option for A. baumannii infections and may act, at least in part, by damaging the outer membrane.
Methanosarcina acetivorans is a model methanogen because of its metabolic versatility and genetic tractability. This microbe is not known to natively utilize hydrogen as a catabolic electron donor, despite its genome encoding hydrogenases, and the fact that this microbe can be used to heterologously express functional hydrogenases. Its native hydrogenases are expressed at a very low level and are suggested to have a role in recycling hydrogen that is produced as a byproduct of nitrogen fixation. To explore whether hydrogen can act as a catabolic electron donor, we utilized a previously constructed strain (str. JB-MF) that has the operon encoding for the methyl-H4MPT:CoM methyltransferase (Mtr) disrupted which makes growth dependent on oxidation of electron donors other than methanol. We showed that this M. acetivorans strain can grow by methanol reduction to methane with hydrogen as the sole electron donor. The strain was then used to test the putative electron donors for methyl-reducing methanogenesis: hydrogen, serine, and ethanol. Hydrogen and serine were identified to act as catabolic electron donors for methyl-reducing methanogenesis, which demonstrates the expanded metabolic versatility of M. acetivorans JB-MF. The methanophenazine-reducing hydrogenase (Vht) and the F420-reducing hydrogenase (Frh) were determined to be involved in hydrogen metabolism, because deletion of either made M. acetivorans JB-MF incapable of growing using H2-dependent methyl-reduction. We demonstrate that strain JB-MF is a suitable chassis to screen alternative electron donors for methanogenesis and that this strain may be used for targeted directed evolution of oxidoreductases.
