A simple solid media assay for detection of synergy between bacteriophages and antibiotics.

The emergence of antibiotic-resistant bacteria (ARB) has necessitated the development of alternative therapies to deal with this global threat. Bacteriophages (viruses that target bacteria) that kill ARB are one such alternative. Although phages have been used clinically for decades with inconsistent results, a number of recent advances in phage selection, propagation, and purification have enabled a reevaluation of their utility in contemporary clinical medicine. In most phage therapy cases, phages are administered in combination with antibiotics to ensure that patients receive the standard-of-care treatment. Some phages may work cooperatively with antibiotics to eradicate ARB, as often determined using non-standardized broth assays. We sought to develop a solid media-based assay to assess cooperativity between antibiotics and phages to offer a standardized platform for such testing. We modeled the interactions that occur between antibiotics and phages on solid medium to measure additive, antagonistic, and synergistic interactions. We then tested the method using different bacterial isolates and identified a number of isolates where synergistic interactions were identified. These interactions were not dependent on the specific organism, phage family, or antibiotic used. A priori susceptibility to the antibiotic or the specific phage were not requirements to observe synergistic interactions. Our data also confirm the potential for the restoration of vancomycin to treat vancomycin-resistant Enterococcus (VRE) when used in combination with phages. Solid media assays for the detection of cooperative interactions between antibiotics and phages can be an accessible technique adopted by clinical laboratories to evaluate antibiotic and phage choices in phage therapy.IMPORTANCEBacteriophages have become an important alternative treatment for individuals with life-threatening antibiotic-resistant bacteria (ARB) infections. Because antibiotics represent the standard-of-care for treatment of ARB, antibiotics and phages often are delivered together without evidence that they work cooperatively. Testing for cooperativity can be difficult due to the equipment necessary and a lack of standardized means for performing the testing in liquid medium. We developed an assay using solid medium to identify interactions between antibiotics and phages for gram-positive and gram-negative bacteria. We modeled the interactions between antibiotics and phages on solid medium, and then tested multiple replicates of vancomycin-resistant Enterococcus (VRE) and Stenotrophomonas in the assay. For each organism, we identified synergy between different phage and antibiotic combinations. The development of this solid media assay for assessing synergy between phages and antibiotics will better inform the use of these combinations in the treatment of ARB infections.

A simple solid media assay for detection of synergy between bacteriophages and antibiotics.

The emergence of antibiotic resistant bacteria (ARB) has necessitated the development of alternative therapies to deal with this global threat. Bacteriophages (viruses that target bacteria) that kill ARB are one such alternative. While phages have been used clinically for decades with inconsistent results, a number of recent advances in phage selection, propagation and purification have enabled a reevaluation of their utility in contemporary clinical medicine. In most phage therapy cases, phages are administered in combination with antibiotics to ensure that patients receive the standard-of-care treatment. Some phages may work cooperatively with antibiotics to eradicate ARB, as often determined using non-standardized broth assays. We sought to develop a solid media-based assay to assess cooperativity between antibiotics and phages to offer a standardized platform for such testing. We modeled the interactions that occur between antibiotics and phages on solid medium to measure additive, antagonistic, and synergistic interactions. We then tested the method using different bacterial isolates, and identified a number of isolates where synergistic interactions were identified. These interactions were not dependent on the specific organism, phage family, or antibiotic used. A priori susceptibility to the antibiotic or the specific phage were not requirements to observe synergistic interactions. Our data also confirm the potential for the restoration of vancomycin to treat Vancomycin Resistant Enterococcus (VRE) when used in combination with phages. Solid media assays for the detection of cooperative interactions between antibiotics and phages can be an accessible technique adopted by clinical laboratories to evaluate antibiotic and phage choices in phage therapy.

Common antibiotics, azithromycin and amoxicillin, affect gut metagenomics within a household.

BACKGROUND: The microbiome of the human gut serves a role in a number of physiological processes, but can be altered through effects of age, diet, and disturbances such as antibiotics. Several studies have demonstrated that commonly used antibiotics can have sustained impacts on the diversity and the composition of the gut microbiome. The impact of the two most overused antibiotics, azithromycin, and amoxicillin, in the human microbiome has not been thoroughly described. In this study, we recruited a group of individuals and unrelated controls to decipher the effects of the commonly used antibiotics amoxicillin and azithromycin on their gut microbiomes. RESULTS: We characterized the gut microbiomes by metagenomic sequencing followed by characterization of the resulting microbial communities. We found that there were clear and sustained effects of the antibiotics on the gut microbial community with significant alterations in the representations of Bifidobacterium species in response to azithromycin (macrolide antibiotic). These results were supported by significant increases identified in putative antibiotic resistance genes associated with macrolide resistance. Importantly, we did not identify these trends in the unrelated control individuals. There were no significant changes observed in other members of the microbial community. CONCLUSIONS: As we continue to focus on the role that the gut microbiome plays and how disturbances induced by antibiotics might affect our overall health, elucidating members of the community most affected by their use is of critical importance to understanding the impacts of common antibiotics on those who take them. Clinical Trial Registration Number NCT05169255. This trial was retrospectively registered on 23-12-2021.

Fecal virome transplantation is sufficient to alter fecal microbiota and drive lean and obese body phenotypes in mice.

The gastrointestinal microbiome plays a significant role in modulating numerous host processes, including metabolism. Prior studies show that when mice receive fecal transplants from obese donors on high-fat diets (HFD) (even when recipient mice are fed normal diets after transplantation), they develop obese phenotypes, demonstrating the prominent role that gut microbiota play in determining lean and obese phenotypes. While much of the credit has been given to gut bacteria, the impact of gut viruses on these phenotypes is understudied. To address this shortcoming, we gavaged mice with viromes isolated from donors fed HFD or normal chow over a 4-week study. By characterizing the gut bacterial biota via 16S rRNA amplicon sequencing and measuring mouse weights over time, we demonstrate that transplanted viruses affect the gut bacterial community, as well as weight gain/loss. Notably, mice fed chow but gavaged with HFD-derived viromes gained more weight than their counterparts receiving chow-derived viromes. The converse was also true: mice fed HFD but gavaged with chow-derived viromes gained less weight than their counterparts receiving HFD-derived viromes. Results were replicated in two independent experiments and phenotypic changes were accompanied by significant and identifiable differences in the fecal bacterial biota. Due to methodological limitations, we were unable to identify the specific bacterial strains responsible for respective phenotypic changes. This study confirms that virome-mediated perturbations can alter the fecal microbiome in vivo and indicates that such perturbations are sufficient to drive lean and obese phenotypes in mice.

Fecal virome transplantation is sufficient to alter fecal microbiota and drive lean and obese body phenotypes in mice.

Background: The gastrointestinal microbiome plays a significant role in numerous host processes and has an especially large impact on modulating the host metabolism. Prior studies have shown that when mice receive fecal transplants from obese donors that were fed high-fat diets (HFD) (even when recipient mice are fed normal diets after transplantation), they develop obese phenotypes. These studies demonstrate the prominent role that the gut microbiota play in determining lean and obese phenotypes. While much of the credit has been given to gut bacteria, studies have not measured the impact of gut viruses on these phenotypes. To address this shortcoming, we gavaged mice with viromes isolated from donors fed HFD or normal chow. By characterizing the mice’s gut bacterial biota and weight-gain phenotypes over time, we demonstrate that viruses can shape the gut bacterial community and affect weight gain or loss. Results: We gavaged mice longitudinally over 4 weeks while measuring their body weights and collecting fecal samples for 16S rRNA amplicon sequencing. We evaluated mice that were fed normal chow or high-fat diets, and gavaged each group with either chow-derived fecal viromes, HFD-derived fecal viromes, or phosphate buffered saline controls. We found a significant effect of gavage type, where mice fed chow but gavaged with HFD-derived viromes gained significantly more weight than their counterparts receiving chow-derived viromes. The converse was also true: mice fed HFD but gavaged with chow-derived viromes gained significantly less weight than their counterparts receiving HFD-derived viromes. These results were replicated in two separate experiments and the phenotypic changes were accompanied by significant and identifiable differences in the fecal bacterial biota. Notably, there were differences in Lachnospirales and Clostridia in mice fed chow but gavaged with HFD-derived fecal viromes, and in Peptostreptococcales, Oscillospirales, and Lachnospirales in mice fed HFD but gavaged with chow-derived fecal viromes. Due to methodological limitations, we were unable to identify specific bacterial species or strains that were responsible for respective phenotypic changes. Conclusions: This study confirms that virome-mediated perturbations can alter the fecal microbiome in an in vivo model and indicates that such perturbations are sufficient to drive lean and obese phenotypes in mice.

Bacteriophage-Mediated Perturbation of Defined Bacterial Communities in an In Vitro Model of the Human Gut.

The study of bacteriophage communities reproducing in the gastrointestinal tract is limited by the quality of model systems supporting experimental manipulation in vitro. Traditionally, studies aiming to experimentally address phage-bacteria dynamics have utilized gnotobiotic mice inoculated with defined bacterial communities. While mouse models simulate complex interactions between microbes and their host, they also forestall the study of phage-bacteria dynamics in isolation of host factors. Here, we established a method for manipulating phage-bacteria dynamics using an in vitro chemostat bioreactor model of the distal human gut. We create defined communities representing a subset of bacteria in the feces of two human individuals, cultivated these communities in chemostat bioreactors, developed methods to purify the autochthonous viromes associated with each cultured community, and trialed a system for transmitting live or heat-killed viruses between chemostat bioreactors to decipher outcomes of virus-mediated perturbation. We found that allochthonous viromes were detectable via metagenomic sequencing against the autochthonous virome background and that shifts in bacterial community diversity and composition were detectable in relation to time posttreatment. These microbiome composition changes spanned multiple phyla, including Bacteroidetes, Firmicutes, and Actinobacteria. We also found that compositional changes occurred when using live viruses regardless of whether intrasubject or intersubject viruses were used as the perturbation agents. Our results supported the use of chemostat bioreactors as a platform for studying complex bacteria-phage dynamics in vitro. IMPORTANCE Bacteriophages are relatively ubiquitous in the environment and are highly abundant in the human microbiome. Phages can be commonly transmitted between close contacts, but the impact that such transmissions may have on their bacteria counterparts in our microbiomes is unknown. We developed a chemostat cultivation system to simulate individual-specific features of human distal gut microbiota that can be used to transmit phages between ecosystems and measure their impacts on the microbiota. We used this system to transfer phage communities between chemostats that represented different human subjects. We found that there were significant effects on overall microbiota diversity and changes in the relative abundances of Bacteroidetes, Firmicutes, and Actinobacteria, when intersubject perturbations were performed, compared to intrasubject perturbations. These changes were observed when perturbations were performed using live phages, but not when heat-killed phages were used, and they support the use of chemostat systems for studying complex human bacteria-phage dynamics.

Molecular Mirror Technology Facilitates High-Throughput, Accurate SARS-CoV-2 Testing.

Tests to diagnose acute SARS-CoV-2 infection are at the center of controlling the COVID-19 pandemic. Rapid tests benefit from providing quick results but suffer from lower sensitivity, while PCR tests usually take longer to provide more reliable results and can be difficult to scale to meet population needs. We evaluated the diagnostic efficacy of a Molecular Mirror assay (MMA) using nucleic acid extraction and a nucleic acid extraction-free method to determine its ability to identify SARS-CoV-2 in nasal specimens from individuals suspected of having SARS-CoV-2. We compared the MMA using nucleic acid extraction to the emergency use authorization (EUA)-approved TaqPath reverse transcriptase PCR (RT-PCR) assay to determine its performance characteristics. From 412 total specimens (including 115 previous positives and 297 previous negatives), we found that the positive percent agreement (PPA) was 99.1% (confidence interval [CI], 97.4% to 100.0%) and the negative percent agreement (NPA) was 99.3% (95% CI, 98.4% to 100.0%) for SARS-CoV-2 detection. Using the extraction-free method, we analyzed 109 specimens (51 previous positives and 58 previous negatives) and found that the PPA for the more rapid version of the assay was 87.8% (95% CI, 78.5% to 96.9%) and the NPA was 100.0% (95% CI, 100.0%) for virus detection. The extraction method has performance comparable to what is observed in many PCR-based assays. The extraction-free method has lower PPA but has the advantage of being more rapid and having a higher throughput. Our data offer a proof of concept that nuclear magnetic resonance (NMR) detection can be used in SARS-CoV-2 diagnostic testing and may allow for alternative supply chains to increase testing options. IMPORTANCE Accurate diagnostics for SARS-CoV-2 infections have been critical for responding to the COVID-19 pandemic. Both high-sensitivity/specificity PCR-based tests and lower-sensitivity/specificity rapid antigen assays have been the subject of worldwide supply chain limitations as individual facilities and countries have struggled to meet their population testing needs. We evaluated the diagnostic efficacy of a Molecular Mirror assay (MMA), which uses nuclear magnetic resonance to detect the presence of SARS-CoV-2 nucleic acids both with and without full nucleic acid extractions. We found that compared to a U.S. emergency use authorization (EUA) approved assay (TaqPath) that uses reverse transcriptase PCR (RT-PCR), the MMA had high PPA and NPA with full nucleic acid extractions, and acceptable positive percent agreement (PPA) and negative percent agreement (NPA) with an extraction-free protocol. In a landscape marred by supply chain shortages across the world, altered SARS-CoV-2 detection methods such as the MMA can add to testing supplies while providing quality SARS-CoV-2 testing results.

Metabolic differentiation and intercellular nurturing underpin bacterial endospore formation.

Despite intensive research, the role of metabolism in bacterial sporulation remains poorly understood. Here, we demonstrate that Bacillus subtilis sporulation entails a marked metabolic differentiation of the two cells comprising the sporangium: the forespore, which becomes the dormant spore, and the mother cell, which dies as sporulation completes. Our data provide evidence that metabolic precursor biosynthesis becomes restricted to the mother cell and that the forespore becomes reliant on mother cell-derived metabolites for protein synthesis. We further show that arginine is trafficked between the two cells and that proposed proteinaceous channels mediate small-molecule intercellular transport. Thus, sporulation entails the profound metabolic reprogramming of the forespore, which is depleted of key metabolic enzymes and must import metabolites from the mother cell. Together, our results provide a bacterial example analogous to progeny nurturing.

Bacterial-fungal interactions revealed by genome-wide analysis of bacterial mutant fitness.

Microbial interactions are expected to be major determinants of microbiome structure and function. Although fungi are found in diverse microbiomes, their interactions with bacteria remain largely uncharacterized. In this work, we characterize interactions in 16 different bacterial-fungal pairs, examining the impacts of 8 different fungi isolated from cheese rind microbiomes on 2 bacteria (Escherichia coli and a cheese-isolated Pseudomonas psychrophila). Using random barcode transposon-site sequencing with an analysis pipeline that allows statistical comparisons between different conditions, we observed that fungal partners caused widespread changes in the fitness of bacterial mutants compared to growth alone. We found that all fungal species modulated the availability of iron and biotin to bacterial species, which suggests that these may be conserved drivers of bacterial-fungal interactions. Species-specific interactions were also uncovered, a subset of which suggested fungal antibiotic production. Changes in both conserved and species-specific interactions resulted from the deletion of a global regulator of fungal specialized metabolite production. This work highlights the potential for broad impacts of fungi on bacterial species within microbiomes.

Rapid Inhibition Profiling Identifies a Keystone Target in the Nucleotide Biosynthesis Pathway.

Understanding the mechanism of action (MOA) of new antimicrobial agents is a critical step in drug discovery but is notoriously difficult for compounds that appear to inhibit multiple cellular pathways. We recently described image-based approaches [bacterial cytological profiling and rapid inducible profiling (RIP)] for identifying the cellular pathways targeted by antibiotics. Here we have applied these methods to examine the effects of proteolytically degrading enzymes involved in pyrimidine nucleotide biosynthesis, a pathway that produces intermediates for transcription, DNA replication, and cell envelope synthesis. We show that rapid removal of enzymes directly involved in deoxyribonucleotide synthesis blocks DNA replication. However, degradation of cytidylate kinase (CMK), which catalyzes reactions involved in the synthesis of both ribonucleotides and deoxyribonucleotides, blocks both DNA replication and wall teichoic acid biosynthesis, producing cytological effects identical to those created by simultaneously inhibiting both processes with the antibiotics ciprofloxacin and tunicamycin. Our results suggest that RIP can be used to identify and characterize potential keystone enzymes like CMK whose inhibition dramatically affects multiple pathways, thereby revealing important metabolic connections. Identifying and understanding the role of keystone targets might also help to determine the MOAs of drugs that appear to inhibit multiple targets.

SCH79797 improves outcomes in experimental bacterial pneumonia by boosting neutrophil killing and direct antibiotic activity.

Objectives: The role of protease-activated receptor 1 (PAR1) in the pathogenesis of pneumonia and sepsis is ambiguous given the existing literature. As PAR1 is classically activated by the coagulation-based protease thrombin and leads to vascular leakage, our hypothesis was that PAR1 blockade with SCH79797 would be therapeutically beneficial in an experimental model of murine Gram-negative pneumonia. Methods: In this study, we administered SCH79797 via the intrapulmonary route 6 h after the establishment of Escherichia coli pneumonia and observed a significant improvement in survival, lung injury, bacterial clearance and inflammation. We focused on neutrophils as a potential target of the PAR1 antagonist, since they are the predominant inflammatory cell type in the infected lung. Results: Neutrophils appear to express PAR1 at low levels and the PAR1 antagonist SCH79797 enhanced neutrophil killing. Part of this effect may be explained by alterations in the generation of reactive oxygen species (ROS). SCH79797 also led to robust neutrophil extracellular trap (NET) generation and cathelicidin-related antimicrobial peptide (CRAMP) release by neutrophils. Surprisingly, SCH79797 also had a potent, direct antibiotic effect with disruption of the E. coli cell membrane. However, the newer-generation PAR1 antagonist, vorapaxar (SCH530348), had no appreciable effect on neutrophil activity or direct bacterial killing, which suggests the effects seen with SCH79797 may be PAR1 independent. Conclusions: In summary, we observed that intrapulmonary treatment with SCH79797 has significant therapeutic effects in a model of E. coli pneumonia that appear to be due, in part, to both neutrophil-stimulating and direct antibacterial effects of SCH79797.