Opportunistic Human Pathogen Research Articles

Giant transposons promote strain heterogeneity in a major fungal pathogen.

Fungal infections are difficult to prevent and treat in large part due to strain heterogeneity, which confounds diagnostic predictability. Yet, the genetic mechanisms driving strain-to-strain variation remain poorly understood. Here, we determined the extent to which Starships-giant transposons capable of mobilizing numerous fungal genes-generate genetic and phenotypic variability in the opportunistic human pathogen Aspergillus fumigatus. We analyzed 519 diverse strains, including 11 newly sequenced with long-read technology and multiple isolates of the same reference strain, to reveal 20 distinct Starships that are generating genomic heterogeneity over timescales relevant for experimental reproducibility. Starship-mobilized genes encode diverse functions, including known biofilm-related virulence factors and biosynthetic gene clusters, and many are differentially expressed during infection and antifungal exposure in a strain-specific manner. These findings support a new model of fungal evolution wherein Starships help generate variation in genome structure, gene content, and expression among fungal strains. Together, our results demonstrate that Starships are a previously hidden mechanism generating genotypic and, in turn, phenotypic heterogeneity in a major human fungal pathogen.IMPORTANCENo "one size fits all" option exists for treating fungal infections in large part due to genetic and phenotypic variability among strains. Accounting for strain heterogeneity is thus fundamental for developing efficacious treatments and strategies for safeguarding human health. Here, we report significant progress toward achieving this goal by uncovering a previously hidden mechanism generating heterogeneity in the human fungal pathogen Aspergillus fumigatus: giant transposons, called Starships, that span dozens of kilobases and mobilize fungal genes as cargo. By conducting a systematic investigation of these unusual transposons in a single fungal species, we demonstrate their contributions to population-level variation at the genome, pangenome, and transcriptome levels. The Starship compendium we develop will not only help predict variation introduced by these elements in laboratory experiments but will serve as a foundational resource for determining how Starships impact clinically relevant phenotypes, such as antifungal resistance and pathogenicity.

Adaptation of the tetracycline-repressible system for modulating the expression of essential genes in Cryptococcus neoformans.

The opportunistic human fungal pathogen Cryptococcus neoformans has an enormous impact on human health as the causative agent of cryptococcal meningitis, and there is a dire need to expand our current antifungal arsenal. Essential gene products often serve as ideal targets for antimicrobials, and identifying and characterizing essential genes in a pathogen of interest is critical for drug development. Unfortunately, characterization of essential genes in C. neoformans is limited due to its haploid nature and lack of genetic tools for generating effective conditional-expression mutants. To date, the copper-repressible promoter pCTR4 is the most widely used system to regulate essential gene expression; however, its expression is leaky and copper has pleiotropic effects. In diverse fungal species, including Saccharomyces cerevisiae, Candida albicans, and Candida auris, the tetracycline-repressible promoter system is a powerful tool to regulate gene expression; however, it has yet to be adapted for C. neoformans. In this study, we successfully implemented the tetracycline-repressible system in C. neoformans to regulate the expression of the essential genes HSP90 and FKS1. Supplementation of cultures with the tetracycline analog doxycycline efficiently depleted HSP90 at both transcript and protein levels and inhibited C. neoformans growth and viability. Similarly, the depletion of FKS1 with doxycycline enhanced sensitivity of the strain to the echinocandin caspofungin, an antifungal that targets the glucan synthase but is generally ineffective against C. neoformans. Thus, this work unveils a novel approach to generate conditional-expression mutants in C. neoformans, providing unprecedented potential to systematically study essential gene function in this important human fungal pathogen.IMPORTANCEInvasive fungal infections cause millions of deaths annually, while the number of antifungals available to combat these pathogens is limited to only three classes: polyenes, azoles, and echinocandins. The largest source of novel antifungal drug targets are essential gene products, which are required for cellular viability. However, tools to identify and characterize essential genes in C. neoformans are extremely limited. Here, we adapted the tetracycline-repressible promoter system, that has been widely used in other organisms, to study essential gene function in C. neoformans. By placing this regulatable promoter upstream of the essential genes HSP90 and FKS1, we confirmed that the growth of the strains in the presence of the tetracycline analog doxycycline results in the depletion of essential gene expression. This approach provides a significant advance for the systematic study of essential genes in C. neoformans.

Stenotrophomonas maltophilia of clinical origin display higher temperature tolerance comparing with environmental isolates

ABSTRACT Stenotrophomonas maltophilia is a gram-negative, multidrug-resistant, opportunistic human pathogen responsible for hard-to-treat infections in immunocompromised individuals. Besides being recognized as an important clinical pathogen, S. maltophilia is also widespread in the natural environment, with knowledge of the pathogenic potential of the environmental S. maltophilia pool still lacking. In this study, we aimed to identify the differences in virulence-related traits between clinical and environmental S. maltophilia isolates by assessing their genotypic and phenotypic features. For this purpose, 40 S. maltophilia isolates from natural environment and 34 clinical isolates obtained from patients were analysed. We observed a high degree of genotypic diversity among the isolates irrespective of their origin. Although antibiotic resistance- and virulence-related genes were more prevalent in the clinical isolates, the majority of the analysed genes were also present in the environmental isolates. Most importantly, the phenotypic features, specifically the ability to form biofilms and display twitching motility at human body temperature were predominantly characteristic to the clinical isolates. Our study indicates that adaptation to endure human body temperature is a feature strongly linked to S. maltophilia strains of clinical origin, and is significant when differentiating harmless environmental bacteria from pathogenic S. maltophilia isolates.

Core histones govern echinocandin susceptibility in Candida glabrata.

Echinocandin antifungals, which impede cell wall synthesis, are often used to treat Candida bloodstream infections. The human opportunistic fungal pathogen Candida (Nakaseomyces) glabrata is increasingly being reported to exhibit co-resistance to echinocandins and ergosterol biosynthesis-inhibitory azole drugs in hospitals worldwide. However, the role of histones, protein-building blocks of the nucleosome, in governing echinocandin resistance in C. glabrata is not understood. Herein, we show that the reduced gene dosage of core histone proteins, but not of the linker histone, leads to echinocandin susceptibility, which is partly due to increased ROS levels. Additionally, our data implicate histone H3 acetylation at lysine-56 in the caspofungin response of C. glabrata. Since the emerging echinocandin resistance is an impediment to successful antifungal therapy, our findings open up a new research avenue of pharmacological targeting of histone proteins that could potentially block echinocandin resistance and attenuate C. glabrata survival in the host.

Seven-year evaluation of Stenotrophomonas maltophilia bacteremia in a university-affiliated hospital.

Stenotrophomonas maltophilia is the only opportunistic human pathogen in the genus Stenotrophomonas. These bacteria can be isolated from healthcare settings, possibly resulting in bacterial colonization of medical devices, which cause nosocomial infections. S. maltophilia bacteremia results in high mortality and morbidity. This study aimed to determine the outcomes of S. maltophilia bacteremia and its associated mortality risk factors. This was a retrospective study conducted at the University of Medical Sciences, Bakirkoy Dr. Sadi Konuk Training and Research Hospital (Istanbul, Turkey) from December 2015 to December 2022. Medical records, demographic information, clinical conditions, laboratory data, antimicrobial susceptibility, and clinical outcomes were retrospectively analyzed for the period between December 2015 and December 2022. Data from 87 patients (median age 61 years) with bacteremia were included in this study. The most common underlying conditions were diabetes mellitus (DM; 32.2%), hypertension (29.9%), and malignancies (19.5%). The most common sources of bacteremia were central venous catheters (CVCs; 44%), primary blood infection (32%), respiratory tract infection (20.7%), and wound site/abscess infection (2.3%). Seventy-eight percent of the patients were admitted to the intensive care unit (ICU) when bacteremia was detected. The 28-day mortality rate was 55%. The mortality rate was significantly higher in the ICU (p < 0.001). Elevated values of procalcitonin and C-reactive protein, and the presence of CVC were significant risk factors for mortality in those with S. maltophilia bacteremia. S. maltophilia bacteremia should be carefully monitored in patients with CVCs. Timely removal of CVCs can result in decreased mortality.

Uncovering the connection between tunicamycin-induced respiratory deficiency and reduced fluconazole tolerance in Candida glabrata.

Candida glabrata is a prevalent opportunistic fungal pathogen in humans, and fluconazole (FLC) is one of the most commonly used antifungal agents. However, the molecular mechanisms underlying FLC tolerance in C. glabrata remain largely unexplored. This study aims to identify novel mechanisms regulating FLC tolerance, with a particular focus on tunicamycin (TUN)-induced respiratory deficiency. We employed three distinct experimental approaches to investigate the impact of TUN on FLC tolerance: (1) co-treatment with TUN and FLC, (2) exclusive exposure to TUN, and (3) induction of petite formation through alternative methods. Additionally, gene expression analyses were conducted to evaluate the regulation of key genes involved in the ergosterol biosynthesis pathway. Our findings reveal that TUN exposure significantly abolishes FLC tolerance in C. glabrata, primarily through the induction of petite formation, which is characterized by mitochondrial dysfunction. Notably, TUN treatment resulted in the downregulation of critical ergosterol biosynthesis genes, including ERG1 and ERG11, indicating a metabolic shift in response to endoplasmic reticulum (ER) stress. Furthermore, both TUN-induced and ethidium bromide-induced petites displayed cross-resistance to TUN and FLC but showed reduced tolerance to FLC. These results underscore the pivotal role of TUN-induced ER stress in modulating FLC tolerance via respiratory deficiency and alterations in ergosterol metabolism. Our study emphasizes the importance of mitochondrial integrity in maintaining drug tolerance in C. glabrata and suggests potential therapeutic strategies targeting metabolic pathways associated with antifungal tolerance. A deeper understanding of these mechanisms may enhance our capacity to effectively combat fungal infections.

Antibiotic resistance profiles of oral flora in hippopotami (Hippopotamus amphibius): implications for treatment of human bite wound infections

BackgroundThe common hippopotamus (Hippopotamus amphibius) is found in aquatic environments throughout sub-Saharan Africa and is known to cause attacks on humans living or working close to water bodies. Victims surviving an attack often suffer from the consequences of severe wound infections caused by the animal’s sharp canine teeth.ObjectiveIsolation of normal flora bacteria from the oral cavity of common hippopotami (Hippopotamus amphibious) followed by antibiotic susceptibility testing to aid in the identification of a targeted antibiotic treatment regimen for hippopotamus attack victims.MethodsOral swabs were collected from 34 free-ranging hippopotami in three reserves within the Greater Kruger National Park Complex in South Africa and cultured for aerobic and anaerobic bacteria. Antibiotic susceptibility testing was conducted using the disc diffusion method (Kirby-Bauer method) and a panel of 16 antibiotic drugs representing 10 antibiotic categories.ResultsCulturing of 50 oral swab samples from 34 hippopotami yielded 188 aerobic isolates belonging to 30 bacterial genera and 41 bacterial species (Gram-negative: 70.7%; Gram-positive: 29.3%) and 16 obligate anaerobic isolates from two genera. Three bacterial species, namely Aeromonas hydrophila, Aeromonas sobria and Shewanella putrefaciens accounted for 52% of the aerobic isolates. The anaerobic isolates were identified as Prevotella melaninogenica and Clostridium spp.Antimicrobial susceptibility testing was performed for 112 aerobic isolates (Gram-negative: 93 (83%); Gram-positive: 19 (17%)) representing all isolated bacterial species. High levels of antibiotic resistance were observed among the Gram-negative species especially to most beta-lactam antibiotics (50.5% to 80.7%). Multidrug resistance was detected in 22.6% of Gram-negative isolates and in 24.1% of all isolates.ConclusionsThis study provides the first investigation of the oral flora bacteria of the common hippopotamus. Among the 32 mostly aerobic bacterial genera the most abundant bacterial species were A. hydrophila, A. sobria and S. putrefaciens. They are typical inhabitants of the aquatic habitat of the hippopotamus and of zoonotic importance as opportunistic human pathogens. The antibiotic susceptibility profiles demonstrated that quinolones, aminoglycosides, and tetracyclines were highly efficacious against these bacterial species which otherwise showed moderate to high levels of resistance to the traditional bite wound treatment with amoxicillin/clavulanate and 1st and 2nd generation cephalosporins.

Make-or-break prime editing for genome engineering in Streptococcus pneumoniae

CRISPR-Cas9 has revolutionized genome engineering by allowing precise introductions of DNA double-strand breaks (DSBs). However, genome engineering in bacteria is still a complex, multi-step process requiring a donor DNA template for repair of DSBs. Prime editing circumvents this need as the repair template is indirectly provided within the prime editing guide RNA (pegRNA). Here, we developed make-or-break Prime Editing (mbPE) that allows for precise and effective genetic engineering in the opportunistic human pathogen Streptococcus pneumoniae. In contrast to traditional prime editing in which a nicking Cas9 is employed, mbPE harnesses wild type Cas9 in combination with a pegRNA that destroys the seed region or protospacer adjacent motif. Since most bacteria poorly perform template-independent end joining, correctly genome-edited clones are selectively enriched during mbPE. We show that mbPE is RecA-independent and can be used to introduce point mutations, deletions and targeted insertions, including protein tags such as a split luciferase, at selection efficiencies of over 93%. mbPE enables sequential genome editing, is scalable, and can be used to generate pools of mutants in a high-throughput manner. The mbPE system and pegRNA design guidelines described here will ameliorate future bacterial genome editing endeavors.

PqsE adapts the activity of the Pseudomonas aeruginosa quorum-sensing transcription factor RhlR to both autoinducer concentration and promoter sequence identity.

Pseudomonas aeruginosa is an opportunistic human pathogen that poses a significant health threat. Many pathogenic behaviors of P. aeruginosa are under control of the bacterial cell-cell communication system known as quorum sensing (QS). One of the QS master regulators, RhlR, is a receptor/transcription factor that not only relies on binding of its canonical ligand, N-butyrylhomoserine lactone (C4-HSL), but additionally requires a protein-protein interaction with the enzyme, PqsE. We constructed heterologous reporter strains in Escherichia coli that allow measurements of the reliance of RhlR on C4-HSL and/or PqsE binding for the ability to activate transcription of three RhlR-regulated genes: rhlA (PqsE independent), phzM (PqsE dependent), and azeB (PqsE inhibited). Analogous assays measuring activation of the three genes in P. aeruginosa were performed, and the patterns observed correlated tightly with the heterologous reporter assays. These results confirm that the binding of PqsE to RhlR is able to fine-tune RhlR transcription factor activity in a promoter-specific manner and prove that this ability is independent of other factors present in P. aeruginosa.IMPORTANCEPseudomonas aeruginosa is an opportunistic human pathogen that can cause fatal infections. There exists an urgent need for new, effective antimicrobial agents to combat P. aeruginosa. The PqsE-RhlR protein-protein interaction is essential for P. aeruginosa to be able to make toxins, form biofilms, and infect host organisms. In this study, we use both non-native models in Escherichia coli and measurements of gene expression/toxin production in P. aeruginosa to show that the PqsE-RhlR interaction enables fine-tuned gene expression and a heightened ability of P. aeruginosa to adapt to external conditions. These findings will be highly valuable as continued efforts are made to design inhibitors of the PqsE-RhlR interaction and test them as potential antimicrobial agents against P. aeruginosa infections.

The adjacent ATP-binding protein-encoding genes of the Enterococcus faecalis phosphate-specific transport (pst) locus have non-overlapping cellular functions.

Phosphate is critical for all microbial life. In many bacteria, inorganic phosphate (Pi) is imported by the high-affinity, low-velocity Pst-PhoU system. The pstB gene encodes the ATPase that powers Pi import. The pst-phoU operon in many Firmicutes, including the human commensal and opportunistic pathogen Enterococcus faecalis, contains adjacent pstB genes, pstB1 and pstB2. No studies on the relative biological contributions of tandem pstB paralogs in any microbe have been published. This genetic study indicates that E. faecalis pstB1 and pstB2 do not have equivalent functions. The pstB2 gene encodes an ATPase that is required for Pi import, while the ATPase encoded by pstB1 has an accessory role in Pi import that can be duplicated by the presence of excess PstB2.

Inhibitory effects of reumycin produced by Streptomyces sp. TPMA0082 on virulence factors of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes a wide range of infections. The increasing multidrug-resistance of P. aeruginosa poses a critical challenge for medical care. P. aeruginosa employs virulence factors and biofilms to establish infections in humans and protect itself from environmental stress or antibiotics. These factors are regulated by a quorum sensing mechanism involving multiple regulatory systems that act interdependently through signaling molecules. Therefore, interference with quorum sensing systems can suppress the pathogenicity of P. aeruginosa. In this study, quorum sensing inhibitors were explored from secondary metabolites derived from 111 strains of actinomycetes by targeting the las system, which is thought to be upstream of the quorum sensing cascade in P. aeruginosa. As a result, reumycin was isolated from the culture broth of Streptomyces sp. TPMA0082. Reumycin, a molecule containing a pyrimidotriazine ring, inhibited the binding of the autoinducer to the LasR receptor in the las system, thereby suppressing the production of P. aeruginosa virulence factors, including pyocyanin, rhamnolipids, elastase, motility, and biofilms, without affecting bacterial growth. Toxoflavin, a reumycin derivative with a methyl group at the N1 position, exhibited strong antibacterial activity. Fervenulin, a reumycin derivative with a methyl group at the N8 position, had a negative impact on the logarithmic growth phase of the bacteria and exhibited lower inhibitory activity against virulence factor production compared to reumycin. These findings suggest that the position and number of methyl groups attached to the pyrimidotriazine structure significantly influence its biological activity, exerting distinct effects on quorum sensing inhibition and antibacterial activity.

Transcription factors DksA and PsrA are synergistic contributors to Legionella pneumophila virulence in Acanthamoeba castellanii protozoa.

The environmental bacterium Legionella pneumophila, an intracellular parasite of free-living freshwater protozoa as well as an opportunistic human pathogen, has a biphasic lifestyle. The switch from the vegetative replicative form to the environmentally resilient transmissive phase form is governed by a complex stringent response-based regulatory network that includes RNA polymerase co-factor DksA. Here, we report that, through a dysfunctional DksA mutation (DksA1), a synergistic interplay was discovered between DksA and transcription regulator PsrA using the Acanthamoeba castellanii protozoan infection model. Surprisingly, in trans expression of PsrA partially rescued the growth defect of a dksA1 strain. Whilst in trans expression of DksA expectantly could fully rescue the growth defect of the dksA1 strain, it could also surprisingly rescue the growth defect of a ΔpsrA strain. Conversely, the severe intracellular growth defect of a ΔdksA strain could be rescued by in trans expression of DksA and DksA1, but not PsrA. In vitro phenotypic assays show that either DksA or DksA1 was required for extended culturability of bacterial cells, but normal cell morphology and pigmentation required DksA only. Comparative structural modelling predicts that the DksA1 mutation affects the coordination of Mg2+ into the active site of RNAP, compromising transcription efficiency. Taken together, we propose that PsrA transcriptionally assists DksA in the expression of select transmissive phase traits. Additionally, in vitro evidence suggests that the long-chain fatty acid metabolic response is mediated by PsrA together with DksA, inferring a novel regulatory link to the stringent response pathway.

High-Throughput Culture and DNA Isolation Methods for Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic human fungal pathogen that is widely spread in the environment. The need to screen a large number of environmental or clinical isolates for phenotypic and/or genotypic purposes can be challenging and time consuming when using the protocols that are currently available. We have developed a novel approach that allows one to inoculate cultures, grow individual A. fumigatus isolates, and subsequently isolate DNA of sufficient quality for PCR and/or whole genome sequencing, all in a 96-well format. Compared to currently available methods, our protocols reduce both cost and labor significantly and are compatible with any experimental setup that uses a 96-well format. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: 96-well-format mini slant culture and preparation of spore suspension for Aspergillus fumigatus Basic Protocol 2: PCR-quality DNA isolation Basic Protocol 3: High-purity DNA isolation after mini mycelial mat culture.

Identification of a Potential High-Risk Clone and Novel Sequence Type of Carbapenem-Resistant Pseudomonas aeruginosa in Metro Manila, Philippines.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a significant opportunistic human pathogen, posing a considerable threat to public health due to its antimicrobial resistance and limited treatment options. The incidence of CRPA is high in the Philippines; however, genomic analysis of CRPA in this setting is limited. Here, we provide the phenotypic and molecular characterization of 35 non-duplicate CRPA obtained from three tertiary hospitals in Metro Manila, Philippines, from August 2022 to January 2023. Six sequence types (STs), including international high-risk clones ST111 and ST357, were identified. This article highlights the first report in the Philippines on the identification of P. aeruginosa harboring Klebsiella pneumoniae Carbapenemase-2 (KPC-2), coproduced with Verona Integron-encoded Metallo-beta-lactamase-2 (VIM-2) and Oxacillinase-74 (OXA-74). Notably, this is also the first report of KPC in the Philippines identified in P. aeruginosa. New Delhi Metallo-beta-lactamase-7 (NDM-7), coproduced with Cefotaxime-Munich-15 (CTX-M-15) and Temoneira-2 (TEM-2), was also identified from a novel ST4b1c. The relentless identification of NDM in the Philippines' healthcare setting poses a significant global public health risk. The initial detection of the P. aeruginosa strain harboring KPC exacerbated the situation, indicating the inception of potential dissemination of these resistance determinants within P. aeruginosa in the Philippines.

Asperuloside promotes innate immunity via IRE-1/XBP-1 mediated unfolded protein response.

Asperuloside promotes innate immunity via IRE-1/XBP-1 mediated unfolded protein response.

An Introduction to the Influence of Nutritional Factors on the Pathogenesis of Opportunist Fungal Pathogens in Humans.

Fungi such as Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans are opportunistic pathogens in humans. They usually infect individuals whose immune system is compromised due to either a primary infection, e.g., HIV/AIDS, or as part of treatment for another condition, e.g., stem cell or solid organ transplant. In hosts with a weakened immune system, these fungi can cause life-threatening infections. Unlike true pathogens, opportunistic pathogens do not have specific mechanisms to overcome a healthy host, requiring a different approach to understand how they cause infection. The ability of fungi to adapt to various environmental conditions, including the human host, is critical for virulence. In humans, micronutrient metals, such as iron, are sequestered to reduce serum concentrations, which helps to inhibit microbial growth. Other human tissues may increase metal concentrations to toxic levels to prevent infection by pathogens. The ability of fungi to acquire or detoxify nutrients, such as iron or copper, from the host is essential for the establishment of infection. In this review, the role of fungal nutrition will be discussed in relation to opportunistic fungal pathogens. It will focus on the acquisition of micronutrients, e.g., iron, copper, and zinc, and how this enables these fungi to circumvent host nutritional immunity.

Detection of Klebsiella pneumoniae in Veterinary and Food Matrices Using Loop-Mediated Isothermal Amplification.

Klebsiella pneumoniae is an opportunistic human pathogen of high relevance due to its ability to acquire antibiotic resistance. This pathogen is included, along with Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter spp., in the ESKAPE group, which consists of the most important bacterial pathogens resistant to antibiotics in clinical setups. Due to the importance of the rapid identification of infection-causative agents, a novel method for the rapid identification of K. pneumoniae was developed in the present work. This novel method was based on loop-mediated isothermal amplification (LAMP) and evaluated in real-time LAMP, as well as in end-point colorimetric LAMP. Additionally, the method was evaluated in two different clinical samples, namely, blood and urine, along with a food sample, namely, milk; four DNA purification protocols were also evaluated (thermal lysis, chelex, magnetic beads, and glass milk). The results revealed differences in the performance of the LAMP assays depending on the specific combination of the matrix-DNA purification protocol. Overall, the protocol reporting the best results in all the matrices was the one based on chelex, with which it was possible to reach an LOD50 below 10 CFU/mL after a short pre-enrichment step of 6 h in TSB. The method demonstrated reliability, sensitivity, and simplicity and could be performed by non-trained personnel thanks to the colorimetric format.

Comparative genomics reveals genetic diversity and differential metabolic potentials of the species of Arachnia and suggests reclassification of Arachnia propionica E10012 (=NBRC_14587) as novel species.

The genus Arachnia, including Arachnia propionica and Arachnia rubra, are part of the normal oral and respiratory microbiota but can act as opportunistic pathogens in humans. This study investigates the functional, phylogenomic and taxonomic characteristics of 10 completely sequenced Arachnia strains, to elucidate their evolutionary relationships and divergence patterns, focusing on genomic variability and functional diversity. Phylogenetic analyses revealed distinct patterns, with Arachnia propionica strains showing significant divergence compared to the conserved Arachnia rubra strains. Notably, E10012 (=NBRC 14587) emerged as a distinct lineage with unique adaptations, while NCTC11666 exhibited a unique phylogenetic position, suggesting subspecies-level classification. Functional analyses highlighted variability among Arachnia propionica strains, with E10012 (=NBRC 14587) showing genes linked to choline metabolism and metal resistance, and NCTC11666 enriched in carbohydrate-active enzymes like GH179. In contrast, Arachnia rubra demonstrated genomic conservation, indicative of evolutionary specialization. This study reveals that strains E10012 (=NBRC 14587) and NCTC11666 displayed unique genomic features and distinct phylogenetic positioning, suggesting their reclassification as potential novel species and subspecies respectively. This underscores the balance between genomic conservation and diversification in Arachnia, reflecting their ecological adaptability and functional roles in the oral microbiome.

An mRNA Display Approach for Covalent Targeting of a Staphylococcus aureus Virulence Factor.

Staphylococcus aureus (S. aureus) is an opportunistic human pathogen that causes over one million deaths around the world each year. We recently identified a family of serine hydrolases termed fluorophosphonate binding hydrolases (Fphs) that play important roles in lipid metabolism and colonization of a host. Because many of these enzymes are only expressed in Staphylococcus bacteria, they are valuable targets for diagnostics and therapeutics. Here, we developed and screened highly diverse cyclic peptide libraries using mRNA display with a genetically encoded oxadiazolone (Ox) electrophile that was previously shown to potently and covalently inhibit multiple Fph enzymes. By performing multiple rounds of counter selections with WT and catalytic dead FphB, we were able to tune the selectivity of the resulting selected cyclic peptides containing the Ox residue toward the active site serine. From our mRNA display hits, we developed potent and selective fluorescent probes that label the active site of FphB at single digit nanomolar concentrations in live S. aureus bacteria. Taken together, this work demonstrates the potential of using direct genetically encoded electrophiles for mRNA display of covalent binding ligands and identifies potent new probes for FphB that have the potential to be used for diagnostic and therapeutic applications.

Identification Pseudomonas aeruginosa by 16s rRNA gene for Differentiation from Other Pseudomonas Species that isolated from Patients and environment

Pseudomonas aeruginosa is a common and major opportunistic human pathogen, its causes many and dangersinfectious diseases due to death in some timesex: cystic fibrosis , wounds inflammation , burns inflammation , urinary tract infection , other many infections otitis external , Endocarditis , nosocomial infection and also causes other blood infections (Bacteremia). thereforebecomes founding fast and exact identification of P. aeruginosafrom samples culture very important.However, identification of this species may be problematic due to the marked phenotypic variabilitydemonstrated by samples isolates and the presence of other closely related species. To facilitate species identification, we used 16S ribosomal DNA(rRNA) sequence data toidentify genus- and species-specific 16S rRNA signature sequences,its account a stable part of the geneticcode. Based on these sequences we designed simple, rapid, and accurate PCR assays that allow the differentiation of P. aeruginosa from Pseudomonas species and other pathogen genus ,also this test considered as the most specific than the other diagnostic tests like API (20) which give 70% while the 16SrRNA test give more than 90 %.