Pseudomonas aeruginosa displays intrinsic resistance to many antibiotics and known to acquire actively genetic mutations for further resistance. In this study, we attempted to understand genomic and transcriptomic landscapes of P. aeruginosa clinical isolates that are highly resistant to multiple antibiotics. We also aimed to reveal a mode of antibiotic resistance by elucidating transcriptional response of genes conferring antibiotic resistance. To this end, we sequenced the whole genomes and profiled genome-wide RNA transcripts of three different multi-drug resistant (MDR) clinical isolates that are phylogenetically distant from one another. Multi-layered genome comparisons with genomes of antibiotic-susceptible P. aeruginosa strains and 70 other antibiotic-resistance strains revealed both well-characterized conserved gene mutations and distinct distribution of antibiotic-resistant genes (ARGs) among strains. Transcriptions of genes involved in quorum sensing and type VI secretion systems were invariably downregulated in the MDR strains. Virulence-associated phenotypes were further examined and results indicate that our MDR strains are clearly avirulent. Transcriptions of 64 genes, logically selected to be related with antibiotic resistance in MDR strains, were active under normal growth conditions and remained unchanged during antibiotic treatment. These results propose that antibiotic resistance is achieved by a "constitutive" response scheme, where ARGs are actively expressed even in the absence of antibiotic stress, rather than a "reactive" response. Bacterial responses explored at the transcriptomic level in conjunction with their genome repertoires provided novel insights into (i) the virulence-associated phenotypes and (ii) a mode of antibiotic resistance in MDR P. aeruginosa strains.

Recognition of conserved bacterial components provides immediate and efficient immune responses and plays a critical role in triggering antigen-specific adaptive immunity. To date, most microbial components that are detected by host innate immune system are non-proteinaceous structural components. In order to identify novel bacterial immunostimulatory proteins, we developed a new high-throughput approach called "EPSIA", Expressed Protein Screen for Immune Activators. Out of 3,882 Vibrio cholerae proteins, we identified phosphatidylserine decarboxylase (PSD) as a conserved bacterial protein capable of activating host innate immunity. PSD in concentrations as low as 100 ng/ml stimulated RAW264.7 murine macrophage cells and primary peritoneal macrophage cells to secrete TNFalpha and IL-6, respectively. PSD-induced proinflammatory response was dependent on the presence of MyD88, a known adaptor molecule for innate immune response. An enzymatically inactive PSD mutant and heat-inactivated PSD induced approximately 40% and approximately 15% of IL-6 production compared to that by native PSD, respectively. This suggests that PSD induces the production of IL-6, in part, via its enzymatic activity. Subsequent receptor screening determined TLR4 as a receptor mediating the PSD-induced proinflammatory response. Moreover, no detectable IL-6 was produced in TLR4-deficient mouse macrophages by PSD. PSD also exhibited a strong adjuvant activity against a co-administered antigen, BSA. Anti-BSA response was decreased in TLR4-deficient mice immunized with BSA in combination with PSD, further proving the role of TLR4 in PSD signaling in vivo. Taken together, these results provide evidence for the identification of V. cholerae PSD as a novel TLR4 agonist and further demonstrate the potential application of PSD as a vaccine adjuvant.

Recent evidence suggests that the commensal microbes act as a barrier against invading pathogens and enteric infections are the consequences of multi-layered interactions among commensals, pathogens, and the host intestinal tissue. However, it remains unclear how perturbations of the gut microbiota compromise host infection resistance, especially through changes at species and metabolite levels.

Obesity is a major public health problem related to various chronic health conditions. Lactobacillus species has been reported in obese individuals; however, its role is unknown. We compared the abundance and composition of Lactobacillus species by analyzing feces from 64 healthy control subjects and 88 obese subjects. We isolated one Lactobacillus strain from the feces of a subject with obesity and further analyzed its genetic and molecular features. We found that an increased abundance and higher prevalence of Lactobacillus sakei distinguished the fecal microbiota of the obese group from that of healthy subjects and that it was related to the increased levels of reactive oxygen species (ROS) induced by higher fat intake. The L. sakei ob4.1 strain, isolated from the feces of a subject with obesity, showed high catalase activity, which was regulated by oxidative stress at the gene transcription level. L. sakei ob4.1 maintained colon epithelial cell adhesion ability under ROS stimulation, and treatment with saturated fatty acid increased colon epithelial ROS levels in a dose-dependent manner; however, L. sakei ob4.1 did not change the level of fat-induced colon epithelial ROS. Exposing mice to a high-fat diet revealed that high-fat-diet-induced colon ROS was associated with the increased colonization of L. sakei ob4.1 through catalase activity. Four-week supplementation with this strain in mice fed a high-fat diet did not change their body weights or ROS levels. A high-fat diet induces changes in the colon environment by increasing ROS levels, which provides a colonization benefit to an L. sakei strain with high catalase activity. IMPORTANCELactobacillus provides many health benefits; its various species are widely used as probiotics. However, an increased abundance of Lactobacillus has been reported in obesity, and the role of Lactobacillus strains in obesity remains unknown. We found a high abundance of the Lactobacillus sakei species in a group of obese subjects and examined its relationship with a high-fat diet and reactive oxygen species (ROS) in the feces. To find the underlying mechanism, we analyzed and characterized an L. sakei strain isolated from a severely obese individual. We found that higher gut oxidative stress could link high-fat-diet-induced obesity and L. sakei. This translational research identifies the roles of the host gut environment in the colonization and survival of L. sakei.

Short-chain fatty acids, especially butyrate, play beneficial roles in sustaining gastrointestinal health. However, due to limitations associated with direct consumption of butyrate, there has been interest in using prodrugs of butyrate. Tributyrin (TB), a triglyceride composed of three butyrate molecules and a glycerol, is a well-studied precursor of butyrate. We screened a metagenome library consisting of 5760 bacterial artificial chromosome clones, with DNA inserts originating from mouse microbiomes, and identified two clones that efficiently hydrolyse TB into butyrate. Nucleotide sequence analysis indicated that inserts in these two clones are derived from unknown microbes. BLASTp analysis, however, revealed that each insert contains a gene homologous to acetylesterase or esterase genes, from Clostridium spp. and Bacteroides spp., respectively. Predicted structures of these two proteins both contain serine-histidine-aspartate catalytic triad, highly conserved in the family of esterases. Escherichia coli host expressing each of the two candidate genes invariably produced greater amounts of butyrate in the presence of TB. Importantly, administration of TB together with cloned E. coli cells alleviated inflammatory symptoms in a mouse model of acute colitis. Based on these results, we established an efficient on-site and real-time butyrate production system that releases butyrate in a controlled manner inside the intestine.

Indigenous microbes inside the host intestine maintain a complex self-regulating community. The mechanisms by which gut microbes interact with intestinal pathogens remain largely unknown. Here we identify a commensal Escherichia coli strain whose expansion predisposes mice to infection by Vibrio cholerae, a human pathogen. We refer to this strain as 'atypical' E. coli (atEc) because of its inability to ferment lactose. The atEc strain is resistant to reactive oxygen species (ROS) and proliferates extensively in antibiotic-treated adult mice. V. cholerae infection is more severe in neonatal mice transplanted with atEc compared with those transplanted with a typical E. coli strain. Intestinal ROS levels are decreased in atEc-transplanted mice, favouring proliferation of ROS-sensitive V. cholerae. An atEc mutant defective in ROS degradation fails to facilitate V. cholerae infection when transplanted, suggesting that host infection susceptibility can be regulated by a single gene product of one particular commensal species.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of bla(VIM-2), bla(IMP-1), and bla(OXA-23), which are critical components for carbapenem resistance.

Vibrio cholerae, a Gram-negative bacterium, is the causative agent of pandemic cholera. Previous studies have shown that the survival of the seventh pandemic El Tor biotype V. cholerae strain N16961 requires production of acetoin in a glucose-rich environment. The production of acetoin, a neutral fermentation end-product, allows V. cholerae to metabolize glucose without a pH drop, which is mediated by the production of organic acid. This finding suggests that inhibition of acetoin fermentation can result in V. cholerae elimination by causing a pH imbalance under glucose-rich conditions. Here, we developed a simple high-throughput screening method and identified an inducer of medium acidification (iMAC). Of 8364 compounds screened, we identified one chemical, 5-(4-chloro-2-nitrobenzoyl)-6-hydroxy-1,3-dimethylpyrimidine-2,4(1H,3H)-dione, that successfully killed glucose-metabolizing N16961 by inducing acidic stress. When N16961 was grown with abundant glucose in the presence of iMAC, acetoin production was completely suppressed and concomitant accumulation of lactate and acetate was observed. Using a beta-galactosidase activity assay with a single-copy palsD::lacZ reporter fusion, we show that that iMAC likely inhibits acetoin production at the transcriptional level. Thin-layer chromatography revealed that iMAC causes a significantly reduced accumulation of intracellular (p)ppGpp, a bacterial stringent response alarmone known to positively regulate acetoin production. In vivo bacterial colonization and fluid accumulation were also markedly decreased after iMAC treatment. Finally, we demonstrate iMAC-induced bacterial killing for 22 different V. cholerae strains belonging to diverse serotypes. Together, our results suggest that iMAC, acting as a metabolic modulator, has strong potential as a novel antibacterial agent for treatment against cholera.

To address the drug-resistance of bacterial pathogens without imposing a selective survival pressure, virulence and biofilms are highly attractive targets. Here, we show that terrein, which was isolated from Aspergillus terreus, reduced virulence factors (elastase, pyocyanin, and rhamnolipid) and biofilm formation via antagonizing quorum sensing (QS) receptors without affecting Pseudomonas aeruginosa cell growth. Additionally, the effects of terrein on the production of QS signaling molecules and expression of QS-related genes were verified. Interestingly, terrein also reduced intracellular 3,5-cyclic diguanylic acid (c-di-GMP) levels by decreasing the activity of a diguanylate cyclase (DGC). Importantly, the inhibition of c-di-GMP levels by terrein was reversed by exogenous QS ligands, suggesting a regulation of c-di-GMP levels by QS; this regulation was confirmed using P. aeruginosa QS mutants. This is the first report to demonstrate a connection between QS signaling and c-di-GMP metabolism in P. aeruginosa, and terrein was identified as the first dual inhibitor of QS and c-di-GMP signaling.

Exclusive enteral nutrition (EEN) induces remission in pediatric Crohn's disease (CD). The exact mechanism of EEN therapy in CD is unknown, but alteration of the intestinal microflora after EEN is thought to affect mucosal healing. To determine the link between EEN therapy and therapeutic efficacy in CD, we established a murine model of dextran sulfate sodium (DSS)-induced colitis and applied EEN therapy.

Recent evidence suggests that Fusobacterium nucleatum (Fn) is associated with the development and progression of colorectal cancer. We aimed to delineate the clinical implications of Fn in metastatic colon cancer. We performed quantitative polymerase chain reaction (qPCR) using DNA samples from synchronous metastatic colon cancer patients with either formalin-fixed paraffin-embedded (FFPE) archival primary site tumor samples or fresh colon tissues. Progression-free survival (PFS)1 and PFS2 were defined as PFS of first- and second-line palliative settings. qPCR for Fn was successfully performed using 112 samples (FFPE, n = 61; fresh tissue, n = 51). Forty-one and 68 patients had right-sided and left-sided colon cancer, respectively. Patients with Fn enriched right-sided colon cancers had shorter PFS1 (9.7 vs. 11.2 months) than the other subgroups (HR 3.54, 95% confidence interval [CI] 1.05-11.99; P = 0.04). Fn positive right-sided colon was also associated with shorter PFS2 (3.7 vs. 6.7 months; HR 2.34, 95% CI 0.69-7.91; P = 0.04). In the univariate analysis, PFS1 was affected by differentiation and Fn positive right-sided colon cancer. The multivariate analysis showed that differentiation (HR 2.68, 95% CI 1.40-5.14, P = 0.01) and Fn positive right-sided colon (HR 0.40, 95% CI 0.18-0.88, P = 0.02) were associated with PFS1. Fn enrichment in right sided colon was not associated with overall survival (OS). Fn enrichment has significantly worse prognosis in terms of PFS1 and PFS2 in patients with right-sided metastatic colon cancers.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract whose occurrence is attributed to various factors, including genetic factors, immune response, microbial changes, and oxidative stress. Microbial-targeted therapy has emerged as an alternative to immunosuppressive therapy for IBD.

In this study, we aimed to design a novel and effective bacteriophage cocktail that can target both wild-type bacteria and phage-resistant mutants. To achieve this goal, we isolated four phages (U2874, phi_KPN_H2, phi_KPN_S3, and phi_KPN_HS3) that recognized different bacterial surface molecules using phage-resistant bacteria. We constructed three phage cocktails and tested their phage resistance-suppressing ability against multidrug-resistant Klebsiella pneumoniae. We argue that the phage cocktail that induces resensitization of phage susceptibility exhibited superior phage resistance-suppressing ability. Moreover, we observed trade-off effects that manifested progressively in phage-resistant bacteria. We hypothesize that such trade-off effects can augment therapeutic efficacy. We also recommend collating phage host range data against phage-resistant mutants in addition to wild-type bacteria when establishing phage banks to improve the efficiency of phage therapy. Our study underscores the importance of phage host range data in constructing effective phage cocktails for clinical use.

Pseudomonas aeruginosa is capable of establishing airway infections. Human airway mucus contains a large amount of lysozyme, which hydrolyzes bacterial cell walls. P. aeruginosa, however, is known to be resistant to lysozyme. Here, we performed a genetic screen using a mutant library of PAO1, a prototype P. aeruginosa strain, and identified two mutants (ΔbamB and ΔfabY) that exhibited decrease in survival after lysozyme treatment. The bamB and fabY genes encode an outer membrane assembly protein and a fatty acid synthesis enzyme, respectively. These two mutants displayed retarded growth in the airway mucus secretion (AMS). In addition, these mutants exhibited reduced virulence and compromised survival fitness in two different in vivo infection models. The mutants also showed susceptibility to several antibiotics. Especially, ΔbamB mutant was very sensitive to vancomycin, ampicillin, and ceftazidime that target cell wall synthesis. The ΔfabY displayed compromised membrane integrity. In conclusion, this study uncovered a common aspect of two different P. aeruginosa mutants with pleiotropic phenotypes, and suggests that BamB and FabY could be novel potential drug targets for the treatment of P. aeruginosa infection.

Staphylococcus epidermidis is one of the most abundant colonizers of healthy human mucosa including that in the respiratory tract. As the respiratory microbiome has been linked to host immune responses, this study sought to determine the role of nasal mucosa-associated S. epidermidis in innate immune responses against the influenza A virus (IAV). S. epidermidis strains were isolated from nasal mucus samples of healthy individuals. The effects of these mucosa-derived commensal strains on interferon (IFN)-dependent innate immunity and IAV infection dynamics were tested in vitro using normal human nasal epithelial (NHNE) cells and human turbinate mucosa. The effects of S. epidermidis on antiviral immunity were also tested in vivo using an acute IAV infection mouse model.

In the inner ear, endolymph fluid surrounds the organ of Corti, which is important for auditory function; notably, even slight environmental changes mediated by trauma or infection can have significant consequences. However, it is unclear how the immune response is modulated in these tissues. Here, we report the local immune surveillance role of cleaved cochlin LCCL (Limulus factor C, Cochlin, and Lgl1) during Pseudomonas aeruginosa infection in the cochlea. Upon infection, the LCCL domain is cleaved from cochlin and secreted into the perilymph. This cleaved fragment sequesters infiltrating bacteria in the scala tympani and subsequently recruits resident immune cells to eliminate the bacteria. Importantly, hearing loss in a cochlin knockout mouse model is remedied by treatment with a cochlin LCCL peptide. These findings suggest cleaved cochlin LCCL constitutes a critical factor in innate immunity and auditory function and may be a potential therapeutic target to treat chronic otitis media-induced hearing loss.

Evidence suggests that the human respiratory tract, as with the gastrointestinal tract, has evolved to its current state in association with commensal microbes. However, little is known about how the airway microbiome affects the development of airway immune system. Here, we uncover a previously unidentified mode of interaction between host airway immunity and a unique strain (AIT01) of Staphylococcus epidermidis, a predominant species of the nasal microbiome. Intranasal administration of AIT01 increased the population of neutrophils and monocytes in mouse lungs. The recruitment of these immune cells resulted in the protection of the murine host against infection by Pseudomonas aeruginosa, a pathogenic bacterium. Interestingly, an AIT01-secreted protein identified as GAPDH, a well-known bacterial moonlighting protein, mediated this protective effect. Intranasal delivery of the purified GAPDH conferred significant resistance against other Gram-negative pathogens (Klebsiella pneumoniae and Acinetobacter baumannii) and influenza A virus. Our findings demonstrate the potential of a native nasal microbe and its secretory protein to enhance innate immune defense against airway infections. These results offer a promising preventive measure, particularly relevant in the context of global pandemics.

Staphylococcus aureus is a gram-positive bacterium that causes a wide range of infections. Recently, the spread of methicillin-resistant S. aureus (MRSA) strains has seriously reduced antibiotic treatment options. Anti-virulence strategies, the objective of which is to target the virulence instead of the viability of the pathogen, have become widely accepted as a means of avoiding the emergence of new antibiotic-resistant strains. To increase the number of anti-virulence therapeutic options, it is necessary to identify as many novel virulence-associated genes as possible in MRSA. Co-functional networks have proved useful for mapping gene-to-phenotype associations in various organisms. Herein, we present StaphNet (www.inetbio.org/staphnet), a genome-scale co-functional network for an MRSA strain, S. aureus subsp. USA300_FPR3757. StaphNet, which was constructed by the integration of seven distinct types of genomics data within a Bayesian statistics framework, covers approximately 94% of the coding genome with a high degree of accuracy. We implemented a companion web server for network-based gene prioritization of the phenotypes of 31 different S. aureus strains. We demonstrated that StaphNet can effectively identify genes for virulence-associated phenotypes in MRSA. These results suggest that StaphNet can facilitate target discovery for the development of anti-virulence drugs to treat MRSA infection.

Protection from NO gas, a toxic byproduct of anaerobic respiration in Pseudomonas aeruginosa, is mediated by nitric oxide (NO) reductase (NOR), the norCB gene product. Nevertheless, a norCB mutant that accumulated approximately 13.6 microM NO paradoxically survived anaerobic growth. Transcription of genes encoding nitrate and nitrite reductases, the enzymes responsible for NO production, was reduced >50- and 2.5-fold in the norCB mutant. This was due, in part, to a predicted compromise of the [4Fe-4S](2+) cluster in the anaerobic regulator ANR by physiological NO levels, resulting in an inability to bind to its cognate promoter DNA sequences. Remarkably, two O(2)-dependent dioxygenases, homogentisate-1,2-dioxygenase (HmgA) and 4-hydroxyphenylpyruvate dioxygenase (Hpd), were derepressed in the norCB mutant. Electron paramagnetic resonance studies showed that HmgA and Hpd bound NO avidly, and helped protect the norCB mutant in anaerobic biofilms. These data suggest that protection of a P. aeruginosa norCB mutant against anaerobic NO toxicity occurs by both control of NO supply and reassignment of metabolic enzymes to the task of NO sequestration.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen whose virulence is dependent on quorum sensing (QS). DksA1, an RNA polymerase-binding transcriptional regulator, plays a role in determining a number of phenotypes, including QS-mediated virulence. We therefore envisioned that DksA1 inhibitors may help to control P. aeruginosa infection. Here, we screened a library of 6970 chemical compounds and identified two compounds (henceforth termed Dkstatins) that specifically suppressed DksA1 activity. Treatment with these two compounds also substantially decreased the production of elastase and pyocyanin, dominant virulence determinants of P. aeruginosa, and protected murine hosts from lethal infection from a prototype strain of P. aeruginosa, PAO1. The Dkstatins also suppressed production of homoserine lactone (HSL)-based autoinducers that activate P. aeruginosa QS. The level of 3-oxo-C12-HSL produced by Dkstatin-treated wildtype PAO1 closely resembled that of the ΔdksA1 mutant. RNA-Seq analysis showed that transcription levels of QS- and virulence-associated genes were markedly reduced in Dkstatin-treated PAO1 cells, indicating that Dkstatin-mediated suppression occurs at the transcriptional level. Importantly, Dkstatins increased the antibiotic susceptibilities of PAO1, particularly to protein synthesis inhibitors, such as tobramycin and tetracycline. Co-immunoprecipitation assays demonstrated that these Dkstatins interfered with DksA1 binding to the β subunit of RNA polymerase, pointing to a potential mechanism of action. Collectively, our results illustrate that inhibition of P. aeruginosa QS may be achieved via DksA1 inhibitors and that Dkstatins may serve as potential lead compounds to control infection.