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Non-academic partners can be vital in successful public engagement activities on antimicrobial resistance. With collaboration between academic and non-academic partners, we developed and launched an open-access web-based application, the ‘antibiotic footprint calculator’, in both Thai and English. The application focused on a good user experience, addressing antibiotic overuse and its impact, and encouraging immediate action. The application was unveiled in joint public engagement activities. From 1 Nov 2021 to 31 July 2022 (9 month period), 2554 players estimated their personal antibiotic footprint by using the application.

Aim Pseudomonas aeruginosa, a leading opportunistic pathogen causing hospital-acquired infections, is also commonly found in agricultural settings. However, there are minimal attempts to examine the molecular and functional attributes shared by agricultural and clinical strains of P. aeruginosa. This study investigates the presence of P. aeruginosa in edible vegetable plants (including salad vegetables) and analyses the evolutionary and metabolic relatedness of the agricultural and clinical strains. Methods and Results Eighteen rhizospheric and endophytic P. aeruginosa strains were isolated from cucumber, tomato, eggplant, and chili directly from the farms. The identity of these strains was confirmed using biochemical and molecular assays. The genetic and metabolic traits of these plant-associated P. aeruginosa isolates were compared with clinical strains. DNA fingerprinting and 16S rDNA-based phylogenetic analyses revealed that the plant- and human-associated strains are evolutionarily related. Both agricultural and clinical isolates possessed plant-beneficial properties, including mineral solubilization to release essential nutrients (phosphorous, potassium, and zinc), ammonification, and the ability to release extracellular pyocyanin, siderophore, and indole-3 acetic acid. Conclusion These findings suggest that rhizospheric and endophytic P. aeruginosa strains are genetically and functionally analogous to the clinical isolates. In addition, the genotypic and phenotypic traits do not correlate with plant sources or ecosystems. Significance and Impact of the Study This study reconfirms that edible plants are the potential source for human and animal transmission of P. aeruginosa.

Microbial biofilms are composed of surface-adhered microorganisms enclosed in extracellular polymeric substances. The biofilm lifestyle is the intrinsic drug resistance imparted to bacterial cells protected by the matrix. So far, conventional drug susceptibility tests for biofilm are reagent and time-consuming, and most of them are in static conditions. Rapid and easy-to-use methods for biofilm formation and antibiotic activity testing need to be developed to accelerate the discovery of new antibiofilm strategies. Herein, a Lab-On-Chip (LOC) device is presented that provides optimal microenvironmental conditions closely mimicking real-life clinical biofilm status. This new device allows homogeneous attachment and immobilization of Pseudomonas aeruginosa PA01-EGFP cells, and the biofilms grown can be monitored by fluorescence microscopy. P. aeruginosa is an opportunistic pathogen known as a model for drug screening biofilm studies. The influence of flow rates on biofilms growth was analyzed by flow simulations using COMSOL® 5.2. Significant cell adhesion to the substrate and biofilm formation inside the microchannels were observed at higher flow rates 100 µL/h. After biofilm formation, the effectiveness of silver nanoparticles (SNP), chitosan nanoparticles (CNP), and a complex of chitosan-coated silver nanoparticles (CSNP) to eradicate the biofilm under a continuous flow was explored. The most significant loss of biofilm was seen with CSNP with a 65.5% decrease in average live/dead cell signal in biofilm compared to the negative controls. Our results demonstrate that this system is a user-friendly tool for antibiofilm drug screening that could be simply applied in clinical laboratories.Key Points• A continuous-flow microreactor that mimics real-life clinical biofilm infections was developed.• The antibiofilm activity of three nano drugs was evaluated in dynamic conditions.• The highest biofilm reduction was observed with chitosan-silver nanoparticles.

The lung microbiome impacts on lung function, making any smoking-induced changes in the lung microbiome potentially significant. The complex co-occurrence and co-avoidance patterns between the bacterial taxa in the lower respiratory tract (LRT) microbiome were explored for a cohort of active (AS), former (FS) and never (NS) smokers. Bronchoalveolar lavages (BALs) were collected from 55 volunteer subjects (9 NS, 24 FS and 22 AS). The LRT microbiome composition was assessed using 16S rRNA amplicon sequencing. Identification of differentially abundant taxa and co-occurrence patterns, discriminant analysis and biomarker inferences were performed. The data show that smoking results in a loss in the diversity of the LRT microbiome, change in the co-occurrence patterns and a weakening of the tight community structure present in healthy microbiomes. The increased abundance of the genus Ralstonia in the lung microbiomes of both former and active smokers is significant. Partial least square discriminant and DESeq2 analyses suggested a compositional difference between the cohorts in the LRT microbiome. The groups were sufficiently distinct from each other to suggest that cessation of smoking may not be sufficient for the lung microbiota to return to a similar composition to that of NS. The linear discriminant analysis effect size (LEfSe) analyses identified several bacterial taxa as potential biomarkers of smoking status. Network-based clustering analysis highlighted different co-occurring and co-avoiding microbial taxa in the three groups. The analysis found a cluster of bacterial taxa that co-occur in smokers and non-smokers alike. The clusters exhibited tighter and more significant associations in NS compared to FS and AS. Higher degree of rivalry between clusters was observed in the AS. The groups were sufficiently distinct from each other to suggest that cessation of smoking may not be sufficient for the lung microbiota to return to a similar composition to that of NS.

The use of whole metagenomic data to infer the relative abundance of all its microbes is well established. The same data can be used to determine the replication rate of all eubacterial taxa with circular chromosomes. Despite their availability, the replication rate profiles (metareplicome) have not been fully exploited in microbiome analyses. Another relatively new approach is the application of causal inferencing to analyse microbiome data that goes beyond correlational studies. A novel scalable pipeline called MeRRCI (Metagenome, metaResistome, and metaReplicome for Causal Inferencing) was developed. MeRRCI combines efficient computation of the metagenome (bacterial relative abundance), metaresistome (antimicrobial gene abundance) and metareplicome (replication rates), and integrates environmental variables (metadata) for causality analysis using Bayesian networks. MeRRCI was applied to an infant gut microbiome data set to investigate the microbial community’s response to antibiotics. Our analysis suggests that the current treatment stratagem contributes to preterm infant gut dysbiosis, allowing a proliferation of pathobionts. The study highlights the specific antibacterial resistance genes that may contribute to exponential cell division in the presence of antibiotics for various pathogens, namely Klebsiella pneumoniae, Citrobacter freundii, Staphylococcus epidermidis, Veilonella parvula and Clostridium perfringens . These organisms often contribute to the harmful long-term sequelae seen in these young infants.

Background Chronic infection with high-risk human papillomavirus is a necessary cause for cervical carcinogenesis. This study examined prevalence of nonavalent vaccine preventable HPV types over four months among sexually active women in the United States. Methods This sub-study obtained meta-data for 80 of the 1,365 women (18–25 years), enrolled in the BRAVO study, a randomized, open-label trial of home screening and treatment of asymptomatic bacterial vaginosis at high-risk for sexually transmitted infections conducted between 2008 and 2013. Participants were randomized to treatment or standard-of-care, and followed every 2-months for 12 months. Stored vaginal swabs from the first three visits were tested for the nine vaccine preventable HPV types using quantitative PCR. Prevalence and associated 95% confidence intervals for the HPV types were assessed using R (version 3.6.1). Results The average age of the participants was 21.5 (SD ± 2.11) years, with 60% having ever been pregnant and all were African-American. Majority (71%) reported ≥ two sex partners in the prior year with 89% having unprotected vaginal sex and 45% having a new sex partner in the prior year. About 30% had ≥ one of the nine nonavalent vaccine HPV types at all three time points over a period of four months, 15% at two of any three visits, 19% at one of the three visits and 36% were negative for all nine vaccine HPV types at all time points. The most frequently detected HPV vaccine types were 52, 58, 16, and 18. The prevalence of any vaccine HPV types, and high-risk HPV types was 63.8% and 58.8%, respectively. Conclusions Our findings suggest that HPV vaccination which is currently recommended for all unvaccinated persons through age 26 years, is likely to be more beneficial than previously thought as nonavalent HPV vaccine was not available during the time these data were collected.

Attention Deficit Hyperactivity Disorder (ADHD) is an increasingly prevalent neuropsychiatric disorder characterized by hyperactivity, inattention, and impulsivity. Symptoms emerge from underlying deficiencies in neurocircuitry, and recent research has suggested a role played by the gut microbiome. The gut microbiome is a complex ecosystem of interdependent taxa with an exponentially complex web of interactions involving these taxa, plus host gene and reaction pathways, some of which involve neurotransmitters with roles in ADHD neurocircuitry. Studies have analyzed the ADHD gut microbiome using macroscale metrics such as diversity and composition, and have proposed several biomarkers. Few studies have delved into the complex underlying dynamics ultimately responsible for the emergence of such metrics, leaving a largely incomplete, sometimes contradictory, and ultimately inconclusive picture.We aim to help complete this picture by venturing beyond taxa abundances and into taxa relationships (i.e. cooperation and competition), using a publicly available gut microbiome dataset from 30 Control (15 female, 15 male) and 28 ADHD (15 female, 13 male) undergraduate students. We conduct our study in two parts. We first perform the same macroscale analyses prevalent in ADHD gut microbiome literature (diversity, differential, biomarker, and composition) to observe the degree of correspondence, or any new trends. We then estimate two-way ecological relationships by producing Control and ADHD Microbial Co-occurrence Networks (MCNs), using SparCC correlations (p < 0.01). We perform community detection to find clusters of taxa estimated to mutually cooperate along with their centroids, and centrality calculations to estimate taxa most vital to overall gut ecology. We conclude by summarizing our results, and provide conjectures on how they can guide future experiments, some methods for improving our experiments, and general implications for the field.

Introduction. Pseudomonas aeruginosa causes fatal infections in immunocompromised individuals and patients with pulmonary disorders. Gap Statement. Agricultural ecosystems are the vast reservoirs of this dreaded pathogen. However, there are limited attempts to analyse the pathogenicity of P. aeruginosa strains associated with edible plants. Aim. This study aims to (i) elucidate the virulence attributes of P. aeruginosa strains isolated from the rhizosphere and endophytic niches of cucumber, tomato, eggplant and chili;and (ii) compare these phenotypes with that of previously characterized clinical isolates. Methodology. Crystal-violet microtitre assay, swarm plate experiment, gravimetric quantification and sheep blood lysis were performed to estimate the biofilm formation, swarming motility, rhamnolipid production and haemolytic activity, respectively, of P. aeruginosa strains. In addition, their pathogenicity was also assessed based on their ability to antagonize plant pathogens (Xanthomonas oryzae, Pythium aphanidermatum, Rhizoctonia solani and Fusarium oxysporum) and kill a select nematode (Caenorhabditis elegans). Results. Nearly 80 % of the plant-associated strains produced rhamnolipid and exhibited at least one type of lytic activity (haemolysis, proteolysis and lipolysis). Almost 50 % of these strains formed significant levels of biofilm and exhibited swarming motility. The agricultural strains showed significantly higher and lower virulence against the bacterial and fungal pathogens, respectively, compared to the clinical strains. In C. elegans, a maximum of 40 and 100% mortality were induced by the agricultural and clinical strains, respectively. Conclusion. This investigation shows that P. aeruginosa in edible plants isolated directly from the farm express virulence and pathogenicity. Furthermore, clinical and agricultural P. aeruginosa strains antagonized the tested fungal phytopathogens, Pythium aphanidermatum, Rhizoctonia solani and Fusarium oxysporum. Thus, we recommend using these fungi as simple eukaryotic model systems to test P. aeruginosa pathogenicity.

The increased propensity of harmful algal blooms (HABs) and exposure from HABs-cyanotoxin causes human toxicity. It has been associated with the progression of several diseases that encompass the liver, kidneys, and immune system. Recently, a strong association of cyano-HAB toxicity with the altered host gut microbiome has been shown. We tested the hypothesis that prior exposure to cyanotoxin microcystin may alter the microbiome and induce microbiome-host-resistome crosstalk. Using both wild-type and humanized mice, we show that the mice exposed to microcystin had an altered microbiome signature that harbored antimicrobial resistance genes. Host resistome phenotypes such as mefA, msrD, mel, ant6, and tet40 increased in diversity and relative abundance following microcystin exposure. Interestingly, the increased abundance of these genes was traced to resistance to common antibiotics such as tetracycline, macrolides, glycopeptide, and aminoglycosides, crucial for modern-day treatment for several diseases. Increased abundance of these genes was positively associated with increased expression of PD1, a T-cell homeostasis marker, and pleiotropic inflammatory cytokine IL-6 with a concomitant negative association with immunosurveillance markers IL7 and TLR2. Microcystin exposure also caused decreased TLR2, TLR4, and REG3G expressions, increased immunosenescence, and higher systemic levels of IL-6 in both wild-type and humanized mice. In conclusion, the results show a first-ever characterization of the host resistome of microcystin exposure and its connection to host immune status and antibiotic resistance. The results may be crucial for understanding the ability of exposed subjects to fight future bacterial infections and the progression of the debilitating disease in hospital settings.

Pseudomonas aeruginosais an urgent threat pathogen due to its evolving resistance to multiple antibiotics. Agricultural soil and plants are the vast reservoirs of this much-dreaded opportunistic bacterium. A few human isolates of P. aeruginosa are known to infect plants and insects. However, there is no report on the occurrence of multi-drug resistant P. aeruginosa in edible vegetable crops. This study compared 18 P. aeruginosa isolates from the rhizosphere and endophytic niches of four different vegetable crops (cucumber, tomato, eggplant, and chili) with three known clinical strains. All the isolates were tested for virulence traits such as resistance to various antibiotics classes, motility, biofilm, and production of virulence factors (rhamnolipid, pyocyanin, hemolysin, proteases, and lipases). Hierarchical clustering based on Ward minimum variance with Manhattan distance matrix grouped the strains into three clusters based on their phenotypic traits. Strains were exhibiting the highest virulence co-clustered with the human pathogenic isolates. These strains were resistant to cephalosporins, aminoglycosides, macrolides, nitrofurans, tetracyclines, and sulfonamides. These extensively drug-resistant (XDR) strains were only susceptible to polymyxin (colistin) and quinolone (cephalosporin). This study shows that the virulence traits are shared between plant- and human-isolates of P. aeruginosa. More importantly, the occurrence of XDR strains in vegetable crops is a serious global threat.

The pathophysiology of Gulf War Illness (GWI) remains elusive even after three decades. The persistence of multiple complex symptoms along with metabolic disorders such as obesity worsens the health of present Gulf War (GW) Veterans often by the interactions of the host gut microbiome and inflammatory mediators. In this study, we hypothesized that the administration of a Western diet might alter the host metabolomic profile, which is likely associated with the altered bacterial species. Using a five-month symptom persistence GWI model in mice and whole-genome sequencing, we characterized the species-level dysbiosis and global metabolomics, along with heterogenous co-occurrence network analysis, to study the bacteriome–metabolomic association. Microbial analysis at the species level showed a significant alteration of beneficial bacterial species. The beta diversity of the global metabolomic profile showed distinct clustering due to the Western diet, along with the alteration of metabolites associated with lipid, amino acid, nucleotide, vitamin, and xenobiotic metabolism pathways. Network analysis showed novel associations of gut bacterial species with metabolites and biochemical pathways that could be used as biomarkers or therapeutic targets to ameliorate symptom persistence in GW Veterans.

Objective: Recently emerged beta-coronavirus SARS-CoV-2, has resulted in the current pandemic designated COVID-19. COVID-19 manifests as severe illness exhibiting systemic inflammatory response syndrome, acute respiratory distress syndrome (ARDS), thrombotic events, and shock, exacerbated further by co-morbidities and age. Recent clinical evidence suggests that the development of ARDS and subsequent pulmonary failure result from a complex interplay between cell types (endothelial, epithelial and immune) within the lung promoting inflammatory infiltration and a pro-coagulative state. How the complex molecular events mediated by SARS-CoV-2 in infected lung epithelial cells lead to thrombosis and pulmonary failure, is yet to be fully understood. Methods: We address these questions here, using publicly available transcriptomic data in the context of lung epithelia affected by SARS-CoV-2 and other respiratory infections, in vitro. We then extend our results to the understanding of in vivo lung, using a publicly available COVID-19 lung transcriptomic study. Results and Conclusions: Our analysis indicates that there exists a complex interplay between the fibrinolytic system particularly plasmin, and the complement and platelet-activating systems upon SARS-CoV-2 infection, with a potential for therapeutic intervention.

Upon infection, foreign antigenic proteins stimulate the host’s immune system to produce antibodies targeting the pathogen. These antibodies bind to regions on the antibody called epitopes. Structural similarity (molecular mimicry) of epitopes between an infecting pathogen and host proteins or other pathogenic proteins the host has previously encountered can impact the host immune response to the pathogen and may lead to cross-reactive antibodies. The ability to identify potential regions of molecular mimicry in a pathogen can illuminate immune effects which are especially important to pathogen treatment and vaccine design. Here we present Epitopedia, a software pipeline that facilitates the identification of regions that may exhibit potential three-dimensional molecular mimicry between an antigenic pathogen protein and known immune epitopes as catalogued by the immune epitope database (IEDB). Epitopedia is open-source software released under the MIT license and is freely available on GitHub, including a Docker container with all other software dependencies preinstalled. We performed an analysis describing how various secondary structure states, identity between pentapeptide pairs, and identity between the parent sequences of pentapeptide pairs affects RMSD. We found that pentapeptides pairs in a helical conformation had considerably lower RMSD values than those in Extended or Coil conformations. We also found that RMSD is significantly increased when pentapeptide pairs are from non-homologous sequences.

Molecular mimicry between viral antigens and host proteins can produce cross-reacting antibodies leading to autoimmunity. Previous studies have shown that antibodies that cross-react with thrombopoietin, a regulator of platelet production, can induce thrombocytopenia which is characterized by low platelet count. Thrombocytopenia is associated with increased mortality in COVID-19 patients and is a rare side effect following vaccination against SARS-CoV-2. Here we show molecular mimicry between the Spike protein from SARS-CoV-2, the causative agent of COVID-19, and human thrombopoietin. Our computational analyses identify a shared five-amino acid long sequence motif (TQLPP) with similar structure and antibody-binding properties for these proteins, strongly indicative of molecular mimicry with potential for cross-reactivity. Our results are supported by reports of the presence of antibodies against epitopes containing TQLPP in both COVID-19 and pre-pandemic patient samples, indicating antibody cross-reactivity between Spike and a human protein. Since the Spike protein is the antigenic component of the SARS-CoV-2 vaccines, altering the motif may reduce the risk for thrombocytopenia and improve long-term protection against evolving variants. Our findings highlight the importance of considering peptide-level molecular mimicry when developing therapeutic interventions and designing vaccines to avoid potential autoimmune reactions.

Long non-coding RNA plays a vital role in changing the expression profiles of various target genes that leads to cancer development. Thus, identifying prognostic lncRNAs related to different cancers might help in developing cancer therapy. To discover the critical lncRNAs that can identify the origin of different cancers, we proposed to use the state-of-the-art deep learning algorithm Concrete Autoencoder (CAE) in an unsupervised setting, which efficiently identifies a subset of the most informative features. However, CAE does not identify reproducible features in different runs due to its stochastic nature. We proposed a multi-run CAE (mrCAE) to identify a stable set of features to address this issue. The assumption is that a feature appearing in multiple runs carries more meaningful information about the data under consideration. The genome-wide lncRNA expression profiles of 12 different types of cancers, a total of 4,768 samples available in The Cancer Genome Atlas (TCGA), were analyzed to discover the key lncRNAs. The lncRNAs identified by multiple runs of CAE were added to the final list of key lncRNAs, which are capable of identifying 12 different cancers. Our results showed that mrCAE performs better in feature selection than single-run CAE, standard autoencoder (AE), and other state-of-the-art feature selection techniques. This study discovered a set of top-ranking 128 lncRNAs that could identify the origin of 12 different cancers with an accuracy of 95%. Survival analysis showed that 76 of 128 lncRNAs have the prognostic capability in differentiating high- and low-risk groups of patients in different cancers. The proposed mrCAE outperformed AE, which select latent features and were thought to be the best tools for dimensionality reduction. By selecting actual features instead of pseudo-features, mrCAE can be valuable for precision medicine. The identified prognostic lncRNAs (this work), mRNAs, miRNAs, and DNA methylated genes (future work) can lead to biomarkers and therapies for different cancers.

SummaryPseudomonas aeruginosa is a leading opportunistic pathogen capable of causing fatal infections in immunocompromised individuals and patients with degenerative lung diseases. Agricultural soil and plants are the vast reservoirs of this dreaded pathogen. However, there have been limited attempts to analyze the pathogenicity of P. aeruginosa strains associated with edible vegetable plants. This study aims to elucidate the virulence attributes of P. aeruginosa strains isolated from the rhizosphere and endophytic niches of cucumber, tomato, eggplant, and chili collected from agricultural fields. Virulence of the agricultural strains was compared to three previously characterized clinical isolates. Our results showed that 50% of the plant-associated strains formed significant levels of biofilm and exhibited swarming motility. Nearly 80% of these strains produced considerable levels of rhamnolipid and exhibited at least one type of lytic activity (hemolysis, proteolysis, and lipolysis). Their virulence was also assessed based on their ability to suppress the growth of plant pathogens (Xanthomonas oryzae, Pythium aphanidermatum, Rhizoctonia solani, and Fusarium oxysporum) and kill a select nematode (Caenorhabditis elegans). The plant-associated strains showed significantly higher virulence against the bacterial phytopathogen whereas the clinical strains had significantly higher antagonism against the fungal pathogens. In C. elegans slow-killing assay, the clinical strains caused 50-100% death while a maximum of 40% mortality was induced by the agricultural strains. This study demonstrates that some of the P. aeruginosa strains associated with edible plants harbor multiple virulence traits. Upon infection of humans or animals, these strains may evolve to be more pathogenic and pose a significant health hazard.

Molecular mimicry between viral antigens and host proteins can produce cross-reacting antibodies leading to autoimmunity. The coronavirus SARS-CoV-2 causes COVID-19, a disease curiously resulting in varied symptoms and outcomes, ranging from asymptomatic to fatal. Autoimmunity due to cross-reacting antibodies resulting from molecular mimicry between viral antigens and host proteins may provide an explanation. Thus, we computationally investigated molecular mimicry between SARS-CoV-2 Spike and known epitopes. We discovered molecular mimicry hotspots in Spike and highlight two examples with tentative high autoimmune potential and implications for understanding COVID-19 complications. We show that a TQLPP motif in Spike and thrombopoietin shares similar antibody binding properties. Antibodies cross-reacting with thrombopoietin may induce thrombocytopenia, a condition observed in COVID-19 patients. Another motif, ELDKY, is shared in multiple human proteins, such as PRKG1 involved in platelet activation and calcium regulation, and tropomyosin, which is linked to cardiac disease. Antibodies cross-reacting with PRKG1 and tropomyosin may cause known COVID-19 complications such as blood-clotting disorders and cardiac disease, respectively. Our findings illuminate COVID-19 pathogenesis and highlight the importance of considering autoimmune potential when developing therapeutic interventions to reduce adverse reactions.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the cause of an infection known as coronavirus infectious disease 2019 (COVID-19). COVID-19 has become a global source of morbidity, mortality and social disruption since its emergence in East Asia in late 2019 and subsequent pandemic spread. Typical symptoms include cough, sore throat, fever, and sudden loss of taste and smell. Persistent, post-infection sequelae have been noted in a minority of cases. Severe complications and deaths occur mostly in older adults. Laboratory confirmation can be performed by viral RNA and antigen detection in nasal swabs or by detecting specific neutralizing antibodies. There is no effective and approved antiviral treatment, but several vaccines with favourable safety and efficacy profiles are being used in mass vaccination programmes. Vaccine-based COVID control should be seen as an addition to existing hygiene measures such as physical distancing, increased hand hygiene, cough etiquette, and barrier protection with personal protective equipment for frontline healthcare workers and other high-risk professions.

Causal inference in biomedical research allows us to shift the paradigm from investigating associational relationships to causal ones. Inferring causal relationships can help in understanding the inner workings of biological processes. Association patterns can be coincidental and may lead to wrong conclusions about causality in complex systems. Microbiomes are highly complex, diverse, and dynamic environments. Microbes are key players in human health and disease. Hence knowledge of critical causal relationships among the entities in a microbiome, and the impact of internal and external factors on microbial abundance and their interactions are essential for understanding disease mechanisms and making appropriate treatment recommendations. In this paper, we employ causal inference techniques to understand causal relationships between various entities in a microbiome, and to use the resulting causal network to make useful computations. We introduce a novel pipeline for microbiome analysis, which includes adding an outcome or “disease” variable, and then computing the causal network, referred to as a “disease network”, with the goal of identifying disease-relevant causal factors from the microbiome. Internventional techniques are then applied to the resulting network, allowing us to compute a measure called the causal effect of one or more microbial taxa on the outcome variable or the condition of interest. Finally, we propose a measure called causal influence that quantifies the total influence exerted by a microbial taxon on the rest of the microiome. Our pipeline is robust, sensitive, different from traditional approaches, and able to predict interventional effects without any controlled experiments. The pipeline can be used to identify potential eubiotic and dysbiotic microbial taxa in a microbiome. We validate our results using synthetic data sets and using results on real data sets that were previously published.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the cause of an infection known as coronavirus infectious disease 2019 (COVID-19). COVID-19 has become a global source of morbidity, mortality and social disruption since its emergence in East Asia in late 2019 and subsequent pandemic spread. Typical symptoms include cough, sore throat, fever, and sudden loss of taste and smell. Persistent, post-infection sequelae have been noted in a minority of cases. Severe complications and deaths occur mostly in older adults. Laboratory confirmation can be performed by viral RNA and antigen detection in nasal swabs or by detecting specific neutralizing antibodies. There is no effective and approved antiviral treatment, but several vaccines with favourable safety and efficacy profiles are being used in mass vaccination programmes. Vaccine-based COVID control should be seen as an addition to existing hygiene measures such as physical distancing, increased hand hygiene, cough etiquette, and barrier protection with personal protective equipment for frontline healthcare workers and other high-risk professions.

BackgroundMicrobe-microbe and host-microbe interactions in a microbiome play a vital role in both health and disease. However, the structure of the microbial community and the colonization patterns are highly complex to infer even under controlled wet laboratory conditions. In this study, we investigate what information, if any, can be provided by aBayesian Network(BN) about a microbial community. Unlike the previously proposedCo-occurrence Networks(CoNs), BNs are based on conditional dependencies and can help in revealing complex associations.ResultsIn this paper, we propose a way of combining a BN and a CoN to construct asigned Bayesian Network(sBN). We report a surprising association between directed edges in signed BNs and knowncolonization orders.ConclusionsBNs are powerful tools for community analysis and extracting influences and colonization patterns, even though the analysis only uses an abundance matrix with no temporal information. We conclude that directed edges in sBNs when combined with negative correlations are consistent with and strongly suggestive of colonization order.

Vaginal dysbiosis-induced by an overgrowth of anaerobic bacteria is referred to as bacterial vaginosis (BV). The dysbiosis is associated with an increased risk for acquisition of sexually transmitted infections. Women with symptomatic BV are treated with oral metronidazole (MET), but its effectiveness remains to be elucidated. This study used whole-genome sequencing (WGS) to determine the changes in the microbiota among women treated with MET. WGS was conducted on DNA obtained from 20 vaginal swabs collected at four time points over 12 months from five randomly selected African American (AA) women. The baseline visit included all women who were diagnosed with asymptomatic BV and were untreated. All subjects were tested subsequently once every 2 months and received a course of MET for each BV episode during the 12 months. The BV status was classified according to Nugent scores (NSs) of vaginal smears. The microbial and resistome profiles were analysed along with the sociodemographic metadata. Despite treatment, none of the five participants reverted to normal vaginal flora — two were consistently positive for BV, and the rest experienced episodic cases of BV. WGS analyses showed Gardnerella spp. as the most abundant organism. After treatment with MET, there was an observed decline of Lactobacillus and Prevotella species. One participant had a healthy vaginal microbiota based on NS at one follow-up time point. Resistance genes including tetM and lscA were detected. Though limited in subjects, this study shows specific microbiota changes with treatment, presence of many resistant genes in their microbiota, and recurrence and persistence of BV despite MET treatment. Thus, MET may not be an effective treatment option for asymptomatic BV, and whole metagenome sequence would better inform the choice of antibiotics.

SUMMARYRecently emerged beta-coronavirus, SARS-CoV-2 has resulted in the current pandemic designated COVID-19. COVID-19 manifests as severe illness exhibiting systemic inflammatory response syndrome, acute respiratory distress syndrome (ARDS), thrombotic events, and shock, exacerbated further by co-morbidities and age1–3. Recent clinical reports suggested that the pulmonary failure seen in COVID-19 may not be solely driven by acute ARDS, but also microvascular thrombotic events, likely driven by complement activation4,5. However, it is not fully understood how the SARS-CoV-2 infection mechanisms mediate thrombotic events, and whether such mechanisms and responses are unique to SARS-CoV-2 infection, compared to other respiratory infections. We address these questions here, in the context of normal lung epithelia, in vitro and in vivo, using publicly available data. Our results indicate that plasmin is a crucial mediator which primes interactions between complement and platelet-activating systems in lung epithelia upon SARS-CoV-2 infection, with a potential for therapeutic intervention.

Motivation: Metagenomics sequencing data can be used to compute not just the relative abundance profile, but also the replication rates of every taxon in the microbiome sample. We investigate how the dynamics implied by the replication rates can be used to understand the antibiotic response in microbiomes, given the significant variation in the types of antibiotics and the types of response by different taxa. The analysis is further expanded by factoring in the resistome of the microbiomes, which can be readily profiled from the metagenomic sequence data. The fact that some antibiotics such as β -lactams target replicating cells makes it even more critical to use replication rates to analyze the antibiotic response. Results: We introduce a novel approach for metagenomic analysis that integrates microbial community profiling, replication rate calculation, and causal structural learning to analyze the antibiotic response. First, we developed PeTRi, which involves efficient cluster computation of bacterial replication rates from metagenomic sequence data. Second, we integrate the abundance profile, replication profile, resistome profile, and environmental variables to perform causality analysis. Finally, we applied the integrated analysis to the data from an infant gut microbiome study. Conclusions from our analysis are as follows: (i) Microbes tend to lower their replication rates in response to β -lactams; (ii) The presence of antibiotic resistance genes combined with the causality analysis strongly suggest that genes fosA5, oqxA, kpnF, arnA, and acrA provides resistance for the taxon K. pneumoniae, allowing it to replicate and dominate the microbiome after the drug ticarcillin-clavulanate was administered; and (iii) Human and donor milk strongly influence the resistome of the infant gut microbiome.

Inferring causal effects is critically important in biomedical research as it allows us to move from the typical paradigm of associational studies to causal inference, and can impact treatments and therapeutics. Association patterns can be coincidental and may lead to wrong inferences in complex systems. Microbiomes are highly complex, diverse, and dynamic environments. Microbes are key players in health and diseases. Hence knowledge of genuine causal relationships among the entities in a microbiome, and the impact of internal and external factors on microbial abundance and interactions are essential for understanding disease mechanisms and making treatment recommendations.In this paper, we investigate fundamental causal inference techniques to measure the causal effects of various entities in a microbiome. In particular, we show how to use these techniques on microbiome datasets to study the rise and impact of antibiotic-resistance in microbiomes. Our main contributions include the following. We introduce a novel pipeline for microbiome studies, new ideas for experimental design under weaker assumptions, and data augmentation by context embedding. Our pipeline is robust, different from traditional approaches, and able to predict interventional effects without any controlled experiments. Our work shows the advantages of causal inference in identifying potential pathogenic, beneficial, and antibiotic-resistant bacteria. We validate our results using results that were previously published.

Additional file 3 sBN of Saliva

Additional file 5 sBN of Supragingival plaque

Additional file 7 sBN of Keratinized Gingiva with 99.99% taxa

Additional file 2 sBN of Palatine tonsils

Additional file 4 sBN of Subgingival plaque

Additional file 6 sBN of Throat

Additional file 1 sBN of Buccal mucosa

Patients with Alpha-1 Antitrypsin Deficiency (A1AD) have abnormally low levels of the protein Alpha-1 Antitrypsin (AAT) in their blood, because of a double mutation that makes the protein misfold and instead collect in the liver (sometimes even causing cirrhosis). The currently accepted single dosage (SD) of AAT supplements does not produce AAT blood concentrations anywhere near normal levels; they typically only reach the effect of having a single mutation. Some have therefore advocated for a double dosage (DD) of these treatments, which generally would be enough to approach these normal concentrations. Levels of cytokines, produced by the immune system in response to an attack, have already been observed to drop dramatically when A1AD patients consuming single dosage started taking double dosage, and then either remain the same or increase again upon return to a single dosage regimen. In this study we administer the same dosage sequence to A1AD patients (SD, DD, SD) for one month each and view the effects on their lung microbiome and metabolome. We analyze both at the end of each stage, comparing and contrasting and discovering potential biomarkers for each stage, and concluding with a discussion of potential implications.

PURPOSE: The presence of alginate-overproducing (Alg(+)) strains of Pseudomonas aeruginosa in cystic fibrosis patients is indicative of chronic infection. The Alg(+) phenotype is generally due to a mutation in the mucA gene, encoding an innermembrane protein that sequesters AlgT/U, the alginate-specific sigma factor. AlgT/U release from the anti-sigma factor MucA is orchestrated via a complex cascade called regulated intramembrane proteolysis. The goal of this study is to identify new players involved in the regulation of alginate production. METHODOLOGY: Previously, a mutant with a second-site suppressor of alginate production (sap), sap27, was isolated from the constitutively Alg(+) PDO300 that harbours the mucA22 allele. A cosmid from a P. aeruginosa minimum tiling path library was identified via en masse complementation of sap27. The cosmid was transposon mutagenized to map the contributing gene involved in the alginate production. The identified gene was sequenced in sap27 along with algT/U, mucA, algO and mucP. The role of the novel gene was explored using precise in-frame algO and algW deletion mutants of PAO1 and PDO300. RESULTS/KEY FINDINGS: The gene responsible for restoring the mucoid phenotype was mapped to lptD encoding an outer-membrane protein. However, the sequencing of sap27 revealed a mutation in algO, but not in lptD. In addition, we demonstrate that lipopolysaccharide transport protein D (LptD)-dependent alginate production requires AlgW in PAO1 and AlgO in PDO300. CONCLUSION: LptD plays a specific role in alginate production. Our findings suggest that there are two pathways for the production of alginate in P. aeruginosa, one involving AlgW in the wild-type, and one involving AlgO in the mucA22 mutant.

Background: Communicating about antimicrobial resistance (AMR) to the public is challenging. Methods: We developed a dictionary of terms commonly used to communicate about AMR. For each term, we developed learning points to explain AMR and related concepts in plain language. We conducted a pilot evaluation in 374 high school students in Ubon Ratchathani, Thailand. In three 50-minute sessions, students were asked to answer five true/false questions using a paper-based questionnaire. The first session assessed their understanding of AMR at baseline, the second after searching the internet, and the third after the provision of the printed AMR dictionary and its web address. Results: We developed the AMR dictionary as a web-based application (www.amrdictionary.net). The Thai version of the AMR dictionary included 35 terms and associated learning points, seven figures displaying posters promoting AMR awareness in Thailand, and 66 recommended online videos. In the pretest, the proportion of correct responses to each question ranged from 10% to 57%; 10% of the students correctly answered that antibiotics cannot kill viruses and 57% correctly answered that unnecessary use of antibiotics makes them ineffective. After the internet searches, the proportions of correct answers increased, ranging from 62% to 89% (all p Conclusions: Understanding of AMR among Thai high school students is limited. The AMR dictionary can be a useful supportive tool to increase awareness and improve understanding of AMR. Our findings support the need to evaluate the effectiveness of communication tools in the real-world setting.

Background This study aims to investigate the effect of metronidazole for the treatment of recurrent and persistent bacterial vaginosis (BV). Methods Stored vaginal swabs of 80 African American (AA) women were randomly selected from a previously conducted clinical trial for this pilot study. Women with BV were treated with metronidazole. Vaginal smears were categorized by the Nugent score (NS) [0–3, normal; 4–6, intermediate state; 7–10, BV]. Women were classified as recurrent BV (RBV), persistent BV (PBV) or no BV based on three consecutive NS. RBV occurs when an episode of BV occurs after successful treatment of a prior episode. PBV occurs in instances when BV treatment fails to restore healthy Lactobacillus levels. All women were asymptomatic for BV at baseline and followed every two months for four months. Results After four months, 22.5% (CI: 13%, 32%) of women did not have BV, 7.5% (CI: 2%, 13%) had RBV and 70% had PBV (CI: 60%, 80%). 30% of treated women did not have BV compared to 15% of untreated women (p=0.18). BV recurred among 12.5% of treated women and 2.5% of untreated women (p=0.2). BV persisted among 57.5% of treated women and 82.5% of untreated women (p=0.03). Women that were treated had 0.33 decreased odds (95%CI: 0.12, 0.92, p≤0.05) of having PBV as compared to untreated women. The mean age was 21.4 years (SD: 2.11 years). Prior antibiotic use among the sample was low (3.8%), and 75% of women were not treated for BV during their lifetime. Among those who were previously treated for BV, 60% were treated more than five times. Douching was reported by 49% of the sample. Conclusion These preliminary findings suggest, standard BV treatment may not be effective among women with RBV or adherence to treatment may be low among women with asymptomatic BV. Disclosure No significant relationships.

Background Bacterial vaginosis (BV) increases the risk of many sexually transmitted infections. The co-occurrence of persistent BV and high-risk HPV (HrHPV) increases the risk of developing cervical cancer. This study aims to investigate the co-occurrence of HrHPV and BV among young women in the US. Methods Stored vaginal swabs were acquired from a previously completed clinical trial. The kinds of bacteria present in the samples were identified by classifying 16S rRNA gene sequences in each sample using high-throughput pyrosequencing. HPV genotyping was performed using quantitative polymerase chain reaction using TaqMan probes in a customized plate (ThermoFisher Scientific; Waltham, Massachusetts). BV was classified using Nugent Scores of Gram Stain. Results Eighty reproductive age African American (AA) women were included in the analysis. The point prevalence of HrHPV was 48.1% (95%CI: 37–59%). The mean age of the participants was 21.4 years (SD: 2.11), 81.2% graduated high school. Prior antibiotic use was low (3.8%), and 75% were not treated for BV during their lifetime. Among those who had been treated previously for BV, most women were treated ≥ five times (60%). According to Nugent Scores, 70% had BV, 13.7% had intermediate flora and 16.3% were healthy. Among HrHPV positive women, 66.7% were infected with single HrHPV genotype, 33.3% with multiple HrHPV genotypes. Concurrent HrHPV and BV infection was found among 33.3% of the sample. However, there was no significant difference between the prevalence of HrHPV among women with and without BV. Conclusion Co-occurence of hrHPV and BV among this group of young African American women was relatively high. Considering that these conditions are very common among women worldwide, further research in this field is imperative. More studies are needed to accurately evaluate temporal sequence of acquisition of both conditions in any attempt to establish a causal relationship. Disclosure No significant relationships.

Muropeptides are a group of bacterial natural products generated from the cell wall in the course of its turnover. These compounds are cell-wall recycling intermediates and are also involved in signaling within the bacterium. However, the identity of these signaling molecules remains elusive. The identification and characterization of 20 muropeptides from Pseudomonas aeruginosa is described. The least abundant of these metabolites is present at 100 and the most abundant at 55,000 molecules per bacterium. Analysis of these muropeptides under conditions of induction of resistance to a β-lactam antibiotic identified two signaling muropeptides (N-acetylglucosamine-1,6-anhydro-N-acetylmuramyl pentapeptide and 1,6-anhydro-N-acetylmuramyl pentapeptide). Authentic synthetic samples of these metabolites were shown to activate expression of β-lactamase in the absence of any β-lactam antibiotic, thus indicating that they serve as chemical signals in this complex biochemical pathway.

In the last 70 years, we have seen a radical change in our perception and understanding of the microbial world. During this period, we learned from Woese and Fox there exists a third kingdom called 'Archea' based on the phylogenetic studies of the 16S rRNA that revolutionized microbiology (Woese & Fox, 1977; Woese et al., 1978). Furthermore, we were forced to reckon with the fact that Koch and Pasteur's way of growing cells in test-tubes or flasks planktonically does not necessarily translate to the real-life scenario of bacterial lifestyle, where they prefer to live and function as a closely knit microbial community called biofilm. Thanks are due to Costerton, who led the crusade on the concept of biofilms and expanded its scope of inquiry, which forced scientists and clinicians worldwide to rethink how we evaluate and apply the data. Then progressively, disbelief turned into belief, and now it is universally accepted that the micro-organisms hobnob with the members of their community to communicate and coordinate their behaviour, especially in regard to growth patterns and virulence traits via signalling molecules. Just when we thought that we were losing the battle against bacteria, antimicrobials were discovered. We then witnessed the rise and fall of antibiotics and the development of antibiotic resistance. Due to space and choice limitation, we will focus on the three areas that caused this major paradigm shift (i) antimicrobial resistance (AMR), (ii) biofilm and (iii) quorum sensing (QS), and how the Journal of Medical Microbiology played a major role in advancing the shift.

Otitis media (OM) is an inflammation of the middle ear associated with infection. Despite appropriate therapy, acute OM (AOM) can progress to chronic suppurative OM (CSOM) associated with ear drum perforation and purulent discharge. The effusion prevents the middle ear ossicles from properly relaying sound vibrations from the ear drum to the oval window of the inner ear, causing conductive hearing loss. In addition, the inflammatory mediators generated during CSOM can penetrate into the inner ear through the round window. This can cause the loss of hair cells in the cochlea, leading to sensorineural hearing loss. Pseudomonas aeruginosa and Staphylococcus aureus are the most predominant pathogens that cause CSOM. Although the pathogenesis of AOM is well studied, very limited research is available in relation to CSOM. With the emergence of antibiotic resistance as well as the ototoxicity of antibiotics and the potential risks of surgery, there is an urgent need to develop effective therapeutic strategies against CSOM. This warrants understanding the role of host immunity in CSOM and how the bacteria evade these potent immune responses. Understanding the molecular mechanisms leading to CSOM will help in designing novel treatment modalities against the disease and hence preventing the hearing loss.

Persistent high-risk Human papillomavirus (HPV) is a necessary but not sufficient cause for development of cervical cancer. Changes in the vaginal microbiota including reduction of Lactobacillus and increased microbial diversity may facilitate HPV infection and persistence as well as the pathogenesis of cervical cancer. Our objective was to characterize the vaginal microbiota among women with and without persistent HPV infection with HPV 6, 11, 16, 18, 31, 33, 45, 52, 58 in a cohort of young U.S. women. Methods: This analysis used data from a longitudinal study of 1365 women followed for 12 months every two months apart from six locations across the US. HPV genotyping was performed using quantitative PCR using TaqMan probes in a customized plate (ThermoFisher Scientific). Bacterial communities were profiled by 16S rRNA gene sequences from the V3-V4 region using high-throughput pyrosequencing. Persistence was defined by examining for HPV presence in two consecutive visits. Results: Participants included 80 African-American women tested at 3 consecutive time-points. The mean age of participants was 21.4 years. About 43.7% (95% CI: 32.7%–55.3%) had persistent HPV infection, 20% (11.9%–30.4%) were able to clear the infection, and 36.2% (25.8%–47.8%) were consistently negative for HPV infection at all three time points. Atopobium and Peptoniphilus were significantly more abundant in women who were HPV negative suggesting possible protective effects. Prevotella bivia was enriched among women with persistent HPV. Conclusion: Based on these data, we can hypothesize that Prevotella richness is significantly associated with HPV persistence, suggesting a possible role in chronic HPV infection and development of cervical cancer.

The progression of cystic fibrosis (CF) from an acute to a chronic disease is often associated with the conversion of the opportunistic pathogen Pseudomonas aeruginosa from a nonmucoid form to a mucoid form in the lung. This conversion involves the constitutive synthesis of the exopolysaccharide alginate, whose production is under the control of the AlgT/U sigma factor. This factor is regulated posttranslationally by an extremely unstable process and has been commonly attributed to mutations in the algT (algU) gene. By exploiting this unstable phenotype, we isolated 34 spontaneous nonmucoid variants arising from the mucoid strain PDO300, a PAO1 derivative containing the mucA22 allele commonly found in mucoid CF isolates. Complementation analysis using a minimal tiling path cosmid library revealed that most of these mutants mapped to two protease-encoding genes, algO, also known as prc or PA3257, and mucP Interestingly, our algO mutations were complemented by both mucP and algO, leading us to delete, clone, and overexpress mucP, algO, mucE, and mucD in both wild-type PAO1 and PDO300 backgrounds to better understand the regulation of this complex regulatory mechanism. Our findings suggest that the regulatory proteases follow two pathways for regulated intramembrane proteolysis (RIP), where both the AlgO/MucP pathway and MucE/AlgW pathway are required in the wild-type strain but where the AlgO/MucP pathway can bypass the MucE/AlgW pathway in mucoid strains with membrane-associated forms of MucA with shortened C termini, such as the MucA22 variant. This work gives us a better understanding of how alginate production is regulated in the clinically important mucoid variants of Pseudomonas aeruginosaIMPORTANCE Infection by the opportunistic pathogen Pseudomonas aeruginosa is the leading cause of morbidity and mortality seen in CF patients. Poor patient prognosis correlates with the genotypic and phenotypic change of the bacteria from a typical nonmucoid to a mucoid form in the CF lung, characterized by the overproduction of alginate. The expression of this exopolysaccharide is under the control an alternate sigma factor, AlgT/U, that is regulated posttranslationally by a series of proteases. A better understanding of this regulatory phenomenon will help in the development of therapies targeting alginate production, ultimately leading to an increase in the length and quality of life for those suffering from CF.

Otitis media (OM) is a common disease affecting humans, especially paediatric populations. OM refers to inflammation of the middle ear and can be broadly classified into two types, acute and chronic. Bacterial infection is one of the most common causes of OM. Despite the introduction of vaccines, the incidence of OM remains significantly high worldwide. In this mini-review article, we discuss the recent treatment modalities for OM, such as suspension gel, transcutaneous immunization, and intranasal and transtympanic drug delivery, including therapies that are currently undergoing clinical trials. We provide an overview of how these recent advancements in therapeutic strategies can facilitate the circumvention of current treatment challenges involving preadolescence soft palate dysfunction, biofilm formation, tympanic membrane (ear drum) barrier and the attainment of efficacious drug concentrations in the middle ear. While traditional first-line immunization strategies are generally not very efficacious against biofilms, new technologies that use transdermal or intranasal drug delivery via chitosan–PsaA nanoparticles have shown promising results in experimental animal models of OM. Sustained drug delivery systems such as penta-block copolymer poloxamer 407–polybutylphosphoester (P407-PBP) or poloxamer 407 (e.g. OTO-201, with the brand name ‘OTIPRIO’) have demonstrated that treatments can be reduced to a single topical application. The emergence of effective new treatment modalities opens up promising new avenues for the treatment of OM that could lead to improved quality of life for many children and their families.

The bacterial cell-wall that forms a protective layer over the inner membrane is called the murein sacculus – a tightly cross-linked peptidoglycan mesh unique to bacteria. Cell-wall synthesis and recycling are critical cellular processes essential for cell growth, elongation and division. Both de novo synthesis and recycling involve an array of enzymes across all cellular compartments, namely the outer membrane, periplasm, inner membrane and cytoplasm. Due to the exclusivity of peptidoglycan in the bacterial cell-wall, these players are the target of choice for many antibacterial agents. Our current understanding of cell-wall biochemistry and biogenesis in Gram-negative organisms stems mostly from studies of Escherichia coli. An incomplete knowledge on these processes exists for the opportunistic Gram-negative pathogen, Pseudomonas aeruginosa. In this review, cell-wall synthesis and recycling in the various cellular compartments are compared and contrasted between E. coli and P. aeruginosa. Despite the fact that there is a remarkable similarity of these processes between the two bacterial species, crucial differences alter their resistance to β-lactams, fluoroquinolones and aminoglycosides. One of the common mediators underlying resistance is the amp system whose mechanism of action is closely associated with the cell-wall recycling pathway. The activation of amp genes results in expression of AmpC β-lactamase through its cognate regulator AmpR which further regulates multi-drug resistance. In addition, other cell-wall recycling enzymes also contribute to antibiotic resistance. This comprehensive summary of the information should spawn new ideas on how to effectively target cell-wall processes to combat the growing resistance to existing antibiotics.

SummaryOtitis media (OM) is a public health problem in both developed and developing countries. It is the leading cause of hearing loss and represents a significant healthcare burden. In some cases, acute OM progresses to chronic suppurative OM (CSOM), characterized by effusion and discharge, despite antimicrobial therapy. The emergence of antibiotic resistance and potential ototoxicity of antibiotics has created an urgent need to design non-conventional therapeutic strategies against OM based on modern insights into its pathophysiology. In this article, we review the role of innate immunity as it pertains to OM and discuss recent advances in understanding the role of innate immune cells in protecting the middle ear. We also discuss the mechanisms utilized by pathogens to subvert innate immunity and thereby overcome defensive responses. A better knowledge about bacterial virulence and host resistance promises to reveal novel targets to design effective treatment strategies against OM. The identification and characterization of small natural compounds that can boost innate immunity may provide new avenues for the treatment of OM. There is also a need to design novel methods for targeted delivery of these compounds into the middle ear, allowing higher therapeutic doses and minimizing systemic side effects.

Pseudomonas aeruginosa is a dreaded pathogen in many clinical settings. Its inherent and acquired antibiotic resistance thwarts therapy. In particular, derepression of the AmpC β-lactamase is a common mechanism of β-lactam resistance among clinical isolates. The inducible expression of ampC is controlled by the global LysR-type transcriptional regulator (LTTR) AmpR. In the present study, we investigated the genetic and structural elements that are important for ampC induction. Specifically, the ampC (P ampC ) and ampR (P ampR ) promoters and the AmpR protein were characterized. The transcription start sites (TSSs) of the divergent transcripts were mapped using 5′ rapid amplification of cDNA ends-PCR (RACE-PCR), and strong σ 54 and σ 70 consensus sequences were identified at P ampR and P ampC , respectively. Sigma factor RpoN was found to negatively regulate ampR expression, possibly through promoter blocking. Deletion mapping revealed that the minimal P ampC extends 98 bp upstream of the TSS. Gel shifts using membrane fractions showed that AmpR binds to P ampC in vitro whereas in vivo binding was demonstrated using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). Additionally, site-directed mutagenesis of the AmpR helix-turn-helix (HTH) motif identified residues critical for binding and function (Ser38 and Lys42) and critical for function but not binding (His39). Amino acids Gly102 and Asp135, previously implicated in the repression state of AmpR in the enterobacteria, were also shown to play a structural role in P. aeruginosa AmpR. Alkaline phosphatase fusion and shaving experiments suggest that AmpR is likely to be membrane associated. Lastly, an in vivo cross-linking study shows that AmpR dimerizes. In conclusion, a potential membrane-associated AmpR dimer regulates ampC expression by direct binding.