Biogeographical variation in antimicrobial resistance in rivers is influenced by agriculture and is spread through bacteriophages.

Antibiotic resistance is currently an extensive medical challenge worldwide, with global numbers increasing steadily. Recent data have highlighted wastewater treatment plants as a reservoir of resistance genes. The impact of these findings for human health can best be summarized using a One Health concept. However, the molecular mechanisms impacting resistance spread have not been carefully evaluated. Bacterial viruses, that is bacteriophages, have recently been shown to be important mediators of bacterial resistance genes in environmental milieus and are transferrable to human pathogens. Herein, we investigated the biogeographical impact on resistance spread through river-borne bacteriophages using amplicon deep sequencing of the microbiota, absolute quantification of resistance genes using ddPCR, and phage induction capacity within wastewater. Microbial biodiversity of the rivers is significantly affected by river site, surrounding milieu and time of sampling. Furthermore, areas of land associated with agriculture had a significantly higher ability to induce bacteriophages carrying antibiotic resistance genes, indicating their impact on resistance spread. It is imperative that we continue to analyse global antibiotic resistance problem from a One Health perspective to gain novel insights into mechanisms of resistance spread.

Studies on Bd0934 and Bd3507, Two Secreted Nucleases from Bdellovibrio bacteriovorus, Reveal Sequential Release of Nucleases during the Predatory Cycle.

Bdellovibrio bacteriovorus is an obligate predatory bacterium that invades and kills a broad range of Gram-negative prey cells, including human pathogens. Its potential therapeutic application has been the subject of increased research interest in recent years. However, an improved understanding of the fundamental molecular aspects of the predatory life cycle is crucial for developing this bacterium as a "living antibiotic." During intracellular growth, B. bacteriovorus secretes an arsenal of hydrolases, which digest the content of the host cell to provide growth nutrients for the predator, e.g., prey DNA is completely degraded by the nucleases. Here, we have, on a genetic and molecular level, characterized two secreted DNases from B. bacteriovorus, Bd0934 and Bd3507, and determined the temporal expression profile of other putative secreted nucleases. We conclude that Bd0934 and Bd3507 are likely a part of the predatosome but are not essential for the predation, host-independent growth, prey biofilm degradation, and self-biofilm formation. The detailed temporal expression analysis of genes encoding secreted nucleases revealed that these enzymes are produced in a sequential orchestrated manner. This work contributes to our understanding of the sequential breakdown of the prey nucleic acid by the nucleases secreted during the predatory life cycle of B. bacteriovorusIMPORTANCE Antibiotic resistance is a major global concern with few available new means to combat it. From a therapeutic perspective, predatory bacteria constitute an interesting tool. They not only eliminate the pathogen but also reduce the overall pool of antibiotic resistance genes through secretion of nucleases and complete degradation of exogenous DNA. Molecular knowledge of how these secreted DNases act will give us further insight into how antibiotic resistance, and the spread thereof, can be limited through the action of predatory bacteria.

Tuft cell IL-17RB restrains IL-25 bioavailability and reveals context-dependent ILC2 hypoproliferation.

The tuft cell-ILC2 circuit orchestrates rapid type 2 responses upon detecting microbe-derived succinate and luminal helminths. Our findings delineate key mechanistic steps, involving IP3R2 engagement and Ca 2+ flux, governing IL-25 production by tuft cells triggered by succinate detection. While IL-17RB plays a pivotal intrinsic role in ILC2 activation, it exerts a regulatory function in tuft cells. Tuft cells exhibit constitutive Il25 expression, placing them in an anticipatory state that facilitates rapid production of IL-25 protein for ILC2 activation. Tuft cell IL-17RB is crucial for restraining IL-25 bioavailability, preventing excessive tonic ILC2 stimulation due to basal Il25 expression. Suboptimal ILC2 stimulation by IL-25 resulting from tuft cell Il17rb -deficiency or prolonged succinate exposure induces a state of hypoproliferation in ILC2s, also observed in chronic helminth infection. Our study offers critical insights into the regulatory dynamics of IL-25 in this circuit, highlighting the delicate tuning required for responses to diverse luminal states.

Oxygen Exposure and Tolerance Shapes the Cell Wall-Associated Lipids of the Skin Commensal Cutibacterium acnes.

Cutibacterium acnes is one of the most abundant bacteria on the skin. Being exposed to oxygen and oxic stress, the secretion of the bacterial antioxidant protein RoxP ensures an endogenous antioxidant system for the preservation of skin health. To investigate the impact of the antioxidant RoxP on oxidation of the bacteria, wildtype and an isogenic roxp mutant were cultured in anaerobic and oxic conditions. The carbonylated status of proteins were recorded, as were the most significant modifications in a relative intensity of free fatty acids (FFA) and lipids containing fatty acids (FA), such as di- (DG) and triglycerides (TG), di- (DGDG) and sulfoquinozyldiacylglycerol (SQDG) and ceramides. Concerning the fatty acid types, it was observed that the free fatty acids contained mainly C12:0-C26:0 in hydroxy and acylated forms, the DG contained mainly C29:0-C37:0, the TG contained mainly C19:0-C33:0, and the DGDG/SQDGs contained very long fatty acids (C29:0-C37:0) demonstrating the interdependence of de novo synthesis of lipids and RoxP. The area of DGDG peaks (924.52, 929.56 and 930.58) were affected by bacterial growth conditions, with the exception of m/z 910.61. Moreover, the FFA unsaturation is wider in the SQDG species (C30:0 to C36:6) than in DG, TG or free FFA species. It could be concluded that both environmental oxidative statuses, as well as the prevalence of bacterial antioxidant systems, significantly shape the lipidome of C. acnes.

Unveiling Rare Pathogens and Antibiotic Resistance in Tanzanian Cholera Outbreak Waters.

The emergence of antibiotic resistance is a global health concern. Therefore, understanding the mechanisms of its spread is crucial for implementing evidence-based strategies to tackle resistance in the context of the One Health approach. In developing countries where sanitation systems and access to clean and safe water are still major challenges, contamination may introduce bacteria and bacteriophages harboring antibiotic resistance genes (ARGs) into the environment. This contamination can increase the risk of exposure and community transmission of ARGs and infectious pathogens. However, there is a paucity of information on the mechanisms of bacteriophage-mediated spread of ARGs and patterns through the environment. Here, we deploy Droplet Digital PCR (ddPCR) and metagenomics approaches to analyze the abundance of ARGs and bacterial pathogens disseminated through clean and wastewater systems. We detected a relatively less-studied and rare human zoonotic pathogen, Vibrio metschnikovii, known to spread through fecal--oral contamination, similarly to V. cholerae. Several antibiotic resistance genes were identified in both bacterial and bacteriophage fractions from water sources. Using metagenomics, we detected several resistance genes related to tetracyclines and beta-lactams in all the samples. Environmental samples from outlet wastewater had a high diversity of ARGs and contained high levels of blaOXA-48. Other identified resistance profiles included tetA, tetM, and blaCTX-M9. Specifically, we demonstrated that blaCTX-M1 is enriched in the bacteriophage fraction from wastewater. In general, however, the bacterial community has a significantly higher abundance of resistance genes compared to the bacteriophage population. In conclusion, the study highlights the need to implement environmental monitoring of clean and wastewater to inform the risk of infectious disease outbreaks and the spread of antibiotic resistance in the context of One Health.

Enrichment of antibiotic resistance genes within bacteriophage populations in saliva samples from individuals undergoing oral antibiotic treatments.

Spread of antibiotic resistance is a significant challenge for our modern health care system, and even more so in developing countries with higher prevalence of both infections and resistant bacteria. Faulty usage of antibiotics has been pinpointed as a driving factor in spread of resistant bacteria through selective pressure. However, horizontal gene transfer mediated through bacteriophages may also play an important role in this spread. In a cohort of Tanzanian patients suffering from bacterial infections, we demonstrate significant differences in the oral microbial diversity between infected and non-infected individuals, as well as before and after oral antibiotics treatment. Further, the resistome carried both by bacteria and bacteriophages vary significantly, with bla CTX-M1 resistance genes being mobilized and enriched within phage populations. This may impact how we consider spread of resistance in a biological context, as well in terms of treatment regimes.

Development of a Molecular Imprinting-Based Surface Plasmon Resonance Biosensor for Rapid and Sensitive Detection of Staphylococcus aureus Alpha Hemolysin From Human Serum.

Stapylococcus aureus is a common infectious agent in e.g. sepsis, associated with both high mortality rates and severe long-term effects. The cytolytic protein α-hemolysin has repeatedly been shown to enhance the virulence of S. aureus. Combined with an unhindered spread of multi drug-resistant strains, this has triggered research into novel anti virulence (i.e. anti α-hemolysin) drugs. Their functionality will depend on our ability to identify infections that might be alleviated by such. We therefore saw a need for detection methods that could identify individuals suffering from S. aureus infections where α-hemolysin was a major determinant. Molecular imprinted polymers were subsequently prepared on gold coated sensor chips. Used in combination with a surface plasmon resonance biosensor, α-hemolysin could therethrough be quantified from septic blood samples (n = 9), without pre-culturing of the infectious agent. The biosensor recognized α-hemolysin with high affinity (KD = 2.75 x 10-7 M) and demonstrated a statistically significant difference (p < 0.0001) between the α-hemolysin response and potential sample contaminants. The detection scheme proved equally good, or better, when compared to antibody-based detection methods. This novel detection scheme constitutes a more rapid, economical, and user-friendly alternative to many methods currently in use. Heightening both reproducibility and sensitivity, molecular imprinting in combination with surface plasmon resonance (SPR)-technology could be a versatile new tool in clinical- and research-settings alike.

Biotechnological Potential of Bdellovibrio and Like Organisms and Their Secreted Enzymes.

Bdellovibrio and like organisms (BALOs) are obligate predatory bacteria that selectively prey on a broad range of Gram-negative bacteria, including multidrug-resistant human pathogens. Due to their unique lifestyle, they have been long recognized as a potential therapeutic and biocontrol agent. Research on BALOs has rapidly grown over the recent decade, resulting in many publications concerning molecular details of bacterial predation as well as applications thereof in medicine and biotechnology. This review summarizes the current knowledge on biotechnological potential of obligate predatory bacteria and their secreted enzymes.

In Vivo Detection and Absolute Quantification of a Secreted Bacterial Factor from Skin Using Molecularly Imprinted Polymers in a Surface Plasmon Resonance Biosensor for Improved Diagnostic Abilities.

In this study, a surface plasmon resonance (SPR) biosensor was developed for the detection and quantification of a secreted bacterial factor (RoxP) from skin. A molecular imprinting method was used for the preparation of sensor chips and five different monomer-cross-linker compositions were evaluated for sensitivity, selectivity, affinity, and kinetic measurements. The most promising molecularly imprinted polymer (MIP) was characterized by using scanning electron microscopy, atomic force microscopy, and cyclic voltammetry. Limit of detection (LOD) value was calculated as 0.23 nM with an affinity constant of 3.3 × 10-9 M for the promising MIP. Besides being highly sensitive, the developed system was also very selective for the template protein RoxP, proven by the calculated selectivity coefficients. Finally, absolute concentrations of RoxP in several skin swabs were analyzed by using the developed MIP-SPR biosensor and compared to a competitive ELISA. Consequently, the developed system offers a very efficient tool for the detection and quantification of RoxP as an early indicator for some oxidative skin diseases especially when they are present in low-abundance levels (e.g., skin samples).

Common skin bacteria protect their host from oxidative stress through secreted antioxidant RoxP.

Cutibacterium acnes is an abundant skin commensal with several proposed mutualistic functions. A protein with strong antioxidant activity was recently identified from the C. acnes secretome. This protein, termed RoxP, facilitated aerobic bacterial growth in vitro and ex vivo. As reducing events naturally occurred outside of the bacterial cell, it was further hypothesized that RoxP could also serve to modulate redox status of human skin. The biological function of RoxP was here assessed in vitro and in vivo, through oxidatively stressed cell cultures and through protein quantification from skin affected by oxidative disease (actinic keratosis and basal cell carcinoma), respectively. 16S rDNA amplicon deep sequencing and single locus sequence typing was used to correlate bacterial prevalence to cutaneous RoxP abundances. We show that RoxP positively influence the viability of monocytes and keratinocytes exposed to oxidative stress, and that a congruent concentration decline of RoxP can be observed in skin affected by oxidative disease. Basal cell carcinoma was moreover associated with microbial dysbiosis, characterized by reduced C. acnes prevalence. C. acnes's secretion of RoxP, an exogenous but naturally occurring antioxidant on human skin, is likely to positively influence the human host. Results furthermore attest to its prospective usability as a biopharmaceutical.