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The increasing prevalence of multidrug-resistant organism (MDRO) carriage poses major challenges to medicine as healthcare costs increase. Recently, faecal microbiota transplantation (FMT) has been discussed as a novel and effective method for decolonising MDRO.To compare the efficacy of different FMT methods to optimise the success rate of decolonisation in patients with MDRO carriage.This prospective cohort study enrolled patients with MDRO carriages from 2018 to 2021. Patients underwent FMT via one of the following methods: oral capsule, oesophagogastroduodenoscopy (EGD), colonoscopy, or gastric tube.A total of 57 patients underwent FMT for MDRO decolonisation. The colonoscopy group required the shortest time for decolonisation, whereas the EGD group required the longest (24.9 vs. 190.4 days, p = 0.022). The decolonisation rate in the oral capsule group was comparable to that in the EGD group (84.6% vs. 85.7%, p = 0.730). An important clinical factor associated with decolonisation failure was antibiotic use after FMT (odds ratio = 6.810, p = 0.008). All four groups showed reduced proportions of MDRO species in microbiome analysis after FMT.Compared to other conventional methods, the oral capsule is an effective FMT method for patients who can tolerate an oral diet. The discontinuation of antibiotics after FMT is a key factor in the success of decolonisation.

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.

Background 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. Methods The effects of an atypical commensal Escherichia coli strain harboring an additional catalase gene (compared to typical E. coli strain) on dextran sulfate sodium (DSS)-induced colitis were explored in mice. Results The atypical E. coli (atEc) significantly restored body weight, reduced disease activity score, and improved histological scores in mice with colitis. Hydrogen peroxide levels in colitis mice were noticeably decreased when the mice were administered atEc. The proinflammatory cytokine levels were decreased and regulatory T cell numbers were increased after the administration of atEc. The abundance of Firmicutes was significantly recovered, while that of Proteobacteria decreased in atEc -treated mice compared with that in vehicle-treated wild-type mice. To investigate the role of interleukin (IL)-17A in mediating the anti-inflammatory effects of the atEc, IL-17A‒knockout mice were orally administered atEc. Clinical and immune responses and microbial composition were significantly reduced in IL-17A‒knockout mice compared with those in wild-type mice. Conclusions atEc ameliorates colonic inflammation by controlling hydrogen peroxide levels, immune responses (including regulatory T cells and IL-17A), and microbial composition. atEc could be a novel candidate of probiotic for IBD treatment.

Background: 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 as how the airway microbiome affected airway immune development. Here, we uncover a previously unidentified mode of interaction between host airway immunity and a unique strain of Staphylococcus epidermidis, a predominant species of the nasal microbiome. Results: Intranasal inoculation of S. epidermidis AIT01 accompanied the active recruitment of neutrophils and monocytes into mouse lungs on the murine model. The recruitment of the immune cells resulted in the complete protection of the murine host against Pseudomonas aeruginosa respiratory infection. Interestingly, an AIT01-secreted protein identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a well-known bacterial moonlighting protein, mediated this protective effect. Intranasal delivery of the purified protein conferred significant resistance against other additional airway infections of Gram-negative pathogens or influenza A virus. Moreover, single cell RNA-seq analysis using mouse lung cells revealed that transcription of genes involved in cytokine production, phagocytosis, chemotaxis, and immune cell migration was considerably upregulated in neutrophils and monocytes in response to the treatment with the protein. Conclusions: Our results illustrate how an indigenous nasal microbe and its secretory protein can extensively instigate an innate immune defense. We also suggest a promising prophylactic option for airway infections in the era of global pandemics. Keywords: Staphylococcus epidermidis, human nasal microbiome, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), innate immune defense

The stringent response (SR) is a highly conserved stress response in bacteria. It is composed of two factors, (i) a nucleotide alarmone, guanosine tetra- and pentaphosphate ((p)ppGpp), and (ii) an RNA polymerase-binding protein, DksA, that regulates various phenotypes, including bacterial virulence. The clinically significant opportunistic bacterial pathogen Pseudomonas aeruginosa possesses two genes, dksA1 and dksA2, that encode DksA proteins. It remains elusive, however, which of these two genes plays a more important role in SR regulation. In this work, we compared genome-wide, RNA-Seq–based transcriptome profiles of ΔdksA1, ΔdksA2, and ΔdksA1ΔdksA2 mutants to globally assess the effects of these gene deletions on transcript levels coupled with phenotypic analyses. The ΔdksA1 mutant exhibited substantial defects in a wide range of phenotypes, including quorum sensing (QS), anaerobiosis, and motility, whereas the ΔdksA2 mutant exhibited no significant phenotypic changes, suggesting that the dksA2 gene may not have an essential function in P. aeruginosa under the conditions used here. Of note, the ΔdksA1 mutants displayed substantially increased transcription of genes involved in polyamine biosynthesis, and we also detected increased polyamine levels in these mutants. Because SAM is a shared precursor for the production of both QS autoinducers and polyamines, these findings suggest that DksA1 deficiency skews the flow of SAM toward polyamine production rather than to QS signaling. Together, our results indicate that DksA1, but not DksA2, controls many important phenotypes in P. aeruginosa. We conclude that DksA1 may represent a potential target whose inhibition may help manage recalcitrant P. aeruginosa infections.

Previous studies have shown disrupted synaptic plasticity and neural activity in depression. Such alteration is strongly associated with disrupted synaptic structures. Chronic stress has been known to induce changes in dendritic structure in the basolateral amygdala (BLA) and medial prefrontal cortex (mPFC), but antidepressant effect on structure of these brain areas has been unclear. Here, the effects of imipramine on dendritic spine density and morphology in BLA and mPFC subregions of stressed mice were examined. Chronic restraint stress caused depressive-like behaviors such as enhanced social avoidance and despair level coincident with differential changes in dendritic spine structure. Chronic stress enhanced dendritic spine density in the lateral nucleus of BLA with no significant change in the basal nucleus of BLA, and altered the proportion of stubby or mushroom spines in both subregions. Conversely, in the apical and basal mPFC, chronic stress caused a significant reduction in spine density. The proportion of stubby or mushroom spines in these subregions overall reduced while the proportion of thin spines increased after repeated stress. Interestingly, most of these structural alterations by chronic stress were reversed by imipramine. In addition, structural changes caused by stress and blocking the changes by imipramine were corelated well with altered activation and expression of synaptic plasticity-promoting molecules such as phospho-CREB, phospho-CAMKII, and PSD-95. Collectively, our data suggest that imipramine modulates stress-induced changes in synaptic structure and synaptic plasticity-promoting molecules in a coordinated manner although structural and molecular alterations induced by stress are distinct in the BLA and mPFC.

IL-17 family cytokines are directly involved in host immune responses and the critical mediators for host defense against infection or inflammation. IL-17C is highly expressed in respiratory epithelium and is induced after acute bacterial lung infection. However, the definite function of IL-17C induced by Pseudomonas aeruginosa (PAO1 strain) is not fully understood, and our study was designed to demonstrate IL-17C-induced immune response against PAO1 infection in nasal epithelium. Passage-2 normal human nasal epithelial (NHNE) cells were infected with PAO1 and the relationship between IL-17C-related immune responses and the iron absorption of PAO1, depending on inoculation of recombinant human IL-17C (rhIL-17C), was assessed by measuring the siderophore activity of PAO1. Microarray data showed that IL-17C expression increased 34.7 times at 8 hours postinfection (hpi) in NHNE cells, and IL-17C mRNA levels increased until 48 hpi. The PAO1 colonies significantly increased from 8 hpi in NHNE cells, and siderophore activity of PAO1 was enhanced in the supernatants of PAO1-infected NHNE cells. Interestingly, PAO1 colonies were reduced in PAO1-infected NHNE cells treated with rhIL-17C, and supernatants from NHNE cells treated with rhIL-17C also exhibited decreased PAO1 colonies. We found that the siderophore activity of PAO1 was significantly reduced in the supernatants of NHNE cells treated with rhIL-17C where LCN2 expression was highly elevated. Our findings indicate that IL-17C mediates an antibacterial effect against PAO1 by inhibiting siderophore activity in nasal epithelium. We propose that IL-17C might be an efficient mediator to suppress PAO1 infection through disturbing iron absorption of PAO1 in nasal epithelium.

The 2 biotypes of Vibrio cholerae O1 serogroup strains—classical and El Tor—use glucose in distinct ways. Classical biotype strains perform organic acid-producing fermentation and eventually lose viability due to the self-induced creation of an acidic environment, whereas El Tor biotype strains use an alternative neutral fermentation pathway, which confers them with survival advantages. However, we report that the neutral fermentation pathway has only been recruited in prototype Wave 1 El Tor biotype strains, which have not been isolated since the mid-1990s. Current Wave 2 and Wave 3 atypical El Tor strains contain a single-base deletion in a gene that directs bacteria toward neutral fermentation, resulting in the loss of neutral fermentation and an appearance that is similar to classical biotype strains. Moreover, when sufficient glucose was supplied, Wave 1 El Tor strains maintained their use of acid-producing fermentation, in parallel with neutral fermentation, and thus lost viability in the late stationary phase. The global replacement of Wave 1 El Tor strains by Wave 2 and 3 atypical El Tor strains implies that the acidic fermentation pathway may not be disadvantageous to V. cholerae. The characteristics that we have reported might improve oral rehydration in the treatment of cholera.

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. IMPORTANCE Lactobacillus 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.

Our recent study presented that human nasal commensal Staphylococcus epidermidis could potentiate antiviral immunity in the nasal mucosa through interferon-related innate responses. Here, we found that human nasal commensal S. epidermidis promoted protease–protease inhibitor balance in favor of the host and prevented influenza A virus (IAV) replication in the nasal mucosa and lungs. A relatively higher induction of Serpine1 exhibited in S. epidermidis-inoculated nasal epithelium and S. epidermidis-induced Serpine1 significantly decreased the expression of serine proteases. Furthermore, the transcription of urokinase plasminogen activator (uPA) and Serpine1 was biologically relevant in S. epidermidis-inoculated nasal epithelium, and the induction of uPA might be related to the sequential increase of Serpine1 in human nasal epithelium. Our findings reveal that human nasal commensal S. epidermidis manipulates the cellular environment lacking serine proteases in the nasal epithelium through Serpine1 induction and disturbs IAV spread to the lungs at the level of the nasal mucosa.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes diverse human infections including chronic airway infection in patients with cystic fibrosis (CF). Comparing the genomes of CF and non-CF PA isolates has great potential to identify the genetic basis of pathogenicity. To gain a deeper understanding of PA adaptation in CF airways, we performed a genome-wide association study (GWAS) on 1,001 PA genomes. Genetic variations identified among CF isolates were categorized into (i) alterations in protein-coding regions, either large- or small-scale, and (ii) polymorphic variation in intergenic regions. We introduced each CF-associated genetic alteration into the genome of PAO1, a prototype PA strain, and validated the outcomes experimentally. Loci readily mutated among CF isolates included genes encoding a probable sulfatase, a probable TonB-dependent receptor (PA2332~PA2336), L-cystine transporter (YecS, PA0313), and a probable transcriptional regulator (PA5438). A promoter region of a heme/hemoglobin uptake outer membrane receptor (PhuR, PA4710) was also different between the CF and non-CF isolate groups. Our analysis highlights ways in which the PA genome evolves to survive and persist within the context of chronic CF infection., Author summary Understanding how human pathogens, such as Pseudomonas aeruginosa (PA), adapt to the host environment during prolonged infections can provide important information for the treatment of disease. In this study, we compared the genome sequences of PA isolates from cystic fibrosis (CF) and non-CF patients and identified genetic variation that was significantly associated with CF disease. CF-associated genetic variations included: (i) the deletion of three amino acids in a transcriptional regulator (PA5438), which leads to decreased antibiotic susceptibility, enhanced virulence expression and sustained biofilm production in the presence of transferrin, and (ii) the insertion of three amino acids in a cystine transporter (PA0313), which results in decreased pyochelin production. Furthermore, single nucleotide replacement in a promoter was identified and shown to upregulate the cellular process for iron acquisition. Our bioinformatics analyses, linked with experimental validation via genetic manipulation and phenotypic investigation of mutants, provides novel insights into the genomic plasticity of PA during chronic CF infection.

Background Major allergen sources Dermatophagoides farinae, Dermatophagoides pteronyssinus, and Tyrophagus putrescentiae have been reported to have distinct microbiomes. The purpose of this study was to investigate the effect of each mite on airway epithelial cells as a model of airway allergic disease. Methods Transcriptomic analysis (RNA-seq) of an airway epithelial cell line (BEAS-2B) was performed to compare gene expression patterns after treatment with extracts of three mite species (D. farinae, D. pteronyssinus, and T. putrescentiae). In addition, mycobiome deep sequencing of mites was employed to identify fungal species that interact with the microbiomes of the mites. Results Immune responses to bacteria were enriched only in the D. farinae-treated group as this species harbored larger numbers of bacteria than the other mites, and the high level of LPS in D. farinae caused proinflammatory cytokine production in airway epithelial cells. In addition, antibiotic metabolism pathways were enriched in D. pteronyssinus-treated cells but not in D. farinae-treated cells. Subsequent experiments revealed that D. pteronyssinus had a high fungal load that inhibited bacterial survival in this mite species. Conclusion The large amount of bacteria in D. farinae causes airway epithelial cells to produce more allergy-related cytokines than D. pteronyssinus, since fungi present in D. pteronyssinus suppress the abundance of mite-associated bacteria.

Background: Our recent study presented that human nasal commensal Staphylococcus Epidermidis could potentiate antiviral immunity in the nasal mucosa through interferon-related innate responses. Here, we found that human nasal commensal S. epidermidis promoted protease–protease inhibitor balance in favor of the host and prevented influenza A virus (IAV) replication in the nasal mucosa and lungs. Methods: We examined the contribution of intranasal administration of human nasal S. epidermidis in controlling IAV infection using murine model and culture nasal epithelial cells. IAV mRNA, viral titer, nucleoprotein levels, and histopathologic findings were evaluated. The induction of S. epidermidis-regulated serine protease inhibitors and correlation of serine proteases were also determined. Findings: A relatively higher induction of Serpine1 exhibited in S. epidermidis-inoculated nasal epithelium and S. epidermidis-induced Serpine1 significantly decreased the expression of serine proteases. Furthermore, the transcription of urokinase plasminogen activator (uPA) and Serpine1 was biologically relevant in S. epidermidis-inoculated nasal epithelium, and the induction of uPA might be related with the sequential increase of Serpine1 in human nasal epithelium. Interpretation: Our findings reveal that human nasal commensal S. epidermidismanipulates the cellular environment lacking serine proteases in the nasal epithelium through Serpine1 induction and disturbs IAV spread to the lungs at the level of the nasal mucosa. Funding: This work was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education 2016R1D1A1B01014116, 2019M3C9A6091945, 2017M3A9F3041233) and was also supported by a grant from the Korean Health Technology R&D Project through the Korean Health Industry Development Institute, funded by the Ministry of Health & Welfare of the Republic of Korea (HI20C0546). Declaration of Interest: None to declare. Ethical Approval: The institutional review board (IRB) of the Seoul National University College of Medicine approved the protocol for this study (IRB #C2012248 [943]). In vivo experiments with C57BL/6J mice were carried out according to guidelines approved by the IRB of the Seoul National University College of Medicine (IACUC #2016-0093).

Background 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. Methods Eight-week-old C57BL/6 mice were administered DSS for 4 days to induce colitis, and either normal chow (NC) or EEN was administered for the following 4 days. The mice were grouped according to the feeding pattern after DSS administration: DSS/NC and DSS/EEN groups. The clinical course was confirmed via daily observation of the weight and stool. Fecal samples were collected and 16sRNA sequencing was used. The mice were sacrificed to confirm colonic histopathology. Results Weight reduction and increase in disease activity were observed as the day progressed for 4 days after DSS administration. There was significant weight recovery and improvement in disease activity in the EEN group compared to that in the NC group. Verrucomicrobia and Proteobacteria abundances tended to increase and Bacteroidetes abundance decreased in the EEN group. In the EEN group, significant changes in the β-diversity of the microbiota were observed. In the analysis of microbiome species, abundances of Akkermansia muciniphila, Clostridium cocleatum, mucin-degrading bacteria, Flintibacter butyricus, and Parabacteroides goldsteinii, which are beneficial microbiota, were significantly increased in the EEN group compared to those in the NC group. More abundant mucins were confirmed in the colonic histopathology of the EEN group. These microbial and histopathological differences suggested that EEN might improve colitis symptoms in a murine colitis model by promoting mucin recycling and subsequently inducing the healing effect of the gut barrier. Conclusion EEN showed clinical efficacy in a murine model of colitis. Based on the increase in mucin-degrading bacteria and the pathological increase in mucin production after EEN administration, it can be observed that mucin plays an important role in the therapeutic effect of EEN., Graphical Abstract

Pseudomonas aeruginosa is a Gram-negative bacterium of clinical importance. As an opportunistic pathogen, its virulence is dependent on quorum sensing (QS). Stringent response (SR), a conserved mechanism in the bacterial kingdom, is activated under diverse stress conditions including nutrient starvation. DksA1, an RNA polymerase-binding transcriptional regulator, plays a role in P. aeruginosa SR. Our recent study clearly demonstrated that, apart from SR, DksA1 regulates a wide range of phenotypes including QS-mediated virulence. This suggests that DksA1 is a potential target to be inhibited to control P. aeruginosa infections. Here, we screened a library of 6,970 chemical compounds and identified two compounds (termed Dkstatin-1 and Dkstatin-2) that specifically suppress DksA1 activity. Two Dkstatins substantially suppressed production of elastase and pyocyanin and protected a murine host against lethal infection with PAO1, a prototype strain of P. aeruginosa. Dkstatins also suppressed the production of homoserine lactone (HSL)-based autoinducers that activate P. aeruginosa QS. The level of 3-oxo-C12-HSL produced in Dkstatin-treated cells was almost identical to that in the ΔdksAI mutant of PAO1. Our RNASeq analysis showed that transcript levels of genes involved in QS and virulence were markedly reduced in Dkstatin-treated PAO1 cells, further demonstrating that Dkstatin-mediated suppression occurs at the transcriptional level. Importantly, Dkstatins increased antibiotic susceptibilities of PAO1, particularly to the antibiotics that inhibit protein synthesis. Our co-immunoprecipitation assay demonstrated that Dkstatins interfere with the binding of DksA1 to the β subunit of RNA polymerase, which potentially explains the mode of Dkstatin action. Collectively, our results suggest that the inhibition of P. aeruginosa QS, which has often been attempted, can be achieved by DksA1 inhibitors. Dkstatins will prove to be useful when establishing infection control strategies.Author summaryBacterial cells developed numerous systems to handle environmental stresses. Stringent response (SR) is one of those systems, and it is activated under the stress of nutrient starvation. Active SR requires DksA1, a protein conserved across bacterial species. Besides this well-recognized function, DksA1 in P. aeruginosa positively regulates its virulence by activating quorum sensing (QS). This finding led us to hypothesize that inhibiting DksA1 would result in virulence attenuation of the pathogenic P. aeruginosa. Herein, we identified two molecules that effectively suppressed DksA1 function through a large-scale compound library screen. These two compounds suppressed P. aeruginosa virulence and therefore protected a murine host from a deadly infection of P. aeruginosa. Moreover, the compounds also rendered P. aeruginosa more susceptible to antibiotics. Our results demonstrate that dangerous P. aeruginosa infections can be controlled by inhibiting DksA1.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes diverse human infections such as chronic airway infection in cystic fibrosis (CF) patients. Although many sequenced genomes are available, a comprehensive comparison between genomes of CF versus non-CF PA isolates remains yet to be conducted. In order to gain a deeper understanding into the PA adaptation in the CF airway, we performed a Genome-Wide Association Study (GWAS) using a total of 1,001 PA genomes. Genetic variations uniquely identified among CF isolates were categorized into (i) alterations in protein-coding regions either large- or small-scale and (ii) polymorphic variations in intergenic regions. We introduced each CF-specific genetic alteration into the genome of PAO1, a prototype PA strain and experimentally validated their outcomes. Loci readily mutated among CF isolates include genes encoding a probable sulphatase and a probable TonB-dependent receptor (PA2332~PA2336), L-cysteine transporter (YecS, PA0313) and a probable transcriptional regulator (PA5438). A promoter region of heme/hemoglobin uptake outer membrane receptor (PhuR, PA4710) was similarly identified as meaningfully different between the CF and non-CF isolate groups. Our analysis, the first of its kind, highlights how PA evolves its genome to persist and survive within the context of chronic CF infection.

Our recent study presented evidence thatStaphylococcus epidermidis(S. epidermidis) was the most frequently encountered microbiome component in healthy human nasal mucus and thatS. epidermidiscould induce interferon (IFN)-dependent innate immunity to control acute viral lung infection. The serine protease inhibitor Serpine1 was identified to inhibit influenza A virus (IAV) spread by inhibiting glycoprotein cleavage, and the current study supports an additional mechanism of Serpine1 induction in the nasal mucosa, which can be regulated throughS. epidermidisand IFN signaling. The exposure ofin vivomice to humanS. epidermidisincreased IFN-λ secretion in nasal mucosa and prevented an increase in the burden of IAV in the lung.S. epidermidis-inoculated mice exhibited the significant induction of Serpine1in vivoin the nasal mucosa, and by targeting airway protease,S. epidermidis-inducedSerpine1 inhibited the intracellular invasion of IAV to the nasal epithelium and led to restriction of IAV spreading to the lung. Furthermore, IFN-λ secretion was involved in the regulation of Serpine1 inS. epidermidis-inoculated nasal epithelial cells andin vivonasal mucosa, and this was biologically relevant for the role of Serpine1 as an interferon-stimulated gene in the upper airway. Together, our findings reveal that human nasal commensalS. epidermidismanipulates the suppression of serine protease inin vivonasal mucosa through Serpine1 induction and protects the nasal mucosa from IAV invasion through IFN-λ signaling.IMPORTANCEPreviously, we proved that nasal microbiome could enhance IFN-related innate immune responses to protect the respiratory tract against influenza virus infection. The present study shows a great understanding of the intimate association of S. epidermidis-regulated IFN-lambda induction and serine protease inhibitor in nasal mucosa. Our data demonstrate that S. epidermidis-regulated Serpine1 suppresses the invasion of influenza virus through suppression of airway serine protease at the level of nasal mucosa and impedes IAV spread to the respiratory tract. Thus, human nasal commensalS. epidermidisrepresents a therapeutic potential for treating respiratory viral infections via the change of cellular environment in respiratory tract.

Background: Obesity is one of the major public health problems related to various chronic health conditions, with steadily increasing prevalence worldwide. Lactobacillus provides various benefits to the host body; however, its role in obesity is unknown. Results: In this study, we found higher colonisation of Lactobacillus sakei species in obesity group, which in turn was related to increased reactive oxygen species (ROS) levels induced by higher fat intake. We isolated L. sakei ob4.1 strain from the faeces of one subject with obesity and compared its genetic and molecular features with those of L. sakei DSM 20017 strain. L. sakei ob4.1 showed higher catalase activity, which was regulated by oxidative stress at gene transcriptional levels. L. sakei ob4.1 maintained colon epithelial cell-adhesion ability under ROS stimulation, and live bacteria could decrease colon epithelial inflammation in a dose-dependent manner. Establishing a mouse model revealed high-fat diet-induced colon ROS to be associated with increased colonisation of L. sakei ob4.1 through catalase activity. Four-week supplementation with this strain could reduce colon inflammation effectively, though not so for body weight or ROS levels in high fat-fed mice. Conclusion: We, therefore, concluded that changes in host gut-oxidative stress levels could link high fat-induced obesity to increased colonisation of L. sakei ob4.1 , and this strain could be potent as anti-inflammatory probiotic in obese population with gut inflammation. Keywords Lactobacillus sakei; Obesity; Gut microbiome; Reactive oxygen stress; Inflammation; High fat diet

The stringent response (SR), which is activated by accumulation of (p)ppGpp under conditions of growth-inhibiting stresses, plays an important role on growth and virulence in Vibrio cholerae. Herein, we carried out a genome-wide screen using transposon random mutagenesis to identify genes controlled by SR in a (p)ppGpp-overproducing mutant strain. One of the identified SR target genes was flaC encoding flagellin. Genetic studies using flaC and SR mutants demonstrated that FlaC was involved in bacterial growth, toxin production, and normal flagellum function under conditions of high (p)ppGpp levels, suggesting FlaC plays an important role in SR-induced pathogenicity in V. cholerae.

The pathogen Vibrio cholerae is the causative agent of cholera. Emergence of antibiotic-resistant V. cholerae strains is increasing, but the underlying mechanisms remain unclear. Herein, we report that the stringent response regulator and stress alarmone guanosine tetra- and pentaphosphate ((p)ppGpp) significantly contributes to antibiotic tolerance in V. cholerae. We found that N16961, a pandemic V. cholerae strain, and its isogenic (p)ppGpp-overexpressing mutant ΔrelAΔspoT are both more antibiotic-resistant than (p)ppGpp0 (ΔrelAΔrelVΔspoT) and ΔdksA mutants, which cannot produce or utilize (p)ppGpp, respectively. We also found that additional disruption of the aconitase B–encoding and tricarboxylic acid (TCA) cycle gene acnB in the (p)ppGpp0 mutant increases its antibiotic tolerance. Moreover, expression of TCA cycle genes, including acnB, was increased in (p)ppGpp0, but not in the antibiotic-resistant ΔrelAΔspoT mutant, suggesting that (p)ppGpp suppresses TCA cycle activity, thereby entailing antibiotic resistance. Importantly, when grown anaerobically or incubated with an iron chelator, the (p)ppGpp0 mutant became antibiotic-tolerant, suggesting that reactive oxygen species (ROS) are involved in antibiotic-mediated bacterial killing. Consistent with that hypothesis, tetracycline treatment markedly increased ROS production in the antibiotic-susceptible mutants. Interestingly, expression of the Fe(III) ABC transporter substrate–binding protein FbpA was increased 10-fold in (p)ppGpp0, and fbpA gene deletion restored viability of tetracycline-exposed (p)ppGpp0 cells. Of note, FbpA expression was repressed in the (p)ppGpp-accumulating mutant, resulting in a reduction of intracellular free iron, required for the ROS-generating Fenton reaction. Our results indicate that (p)ppGpp-mediated suppression of central metabolism and iron uptake reduces antibiotic-induced oxidative stress in V. cholerae.

The intestinal microbiota is a complex ecosystem consisting of various microorganisms that expands human genetic repertoire and therefore affects human health and disease. The metabolic processes and signal transduction pathways of the host and intestinal microorganisms are intimately linked, and abnormal progression of each process leads to changes in the intestinal environment. Alterations in microbial communities lead to changes in functional structures based on the metabolites produced in the gut, and these environmental changes result in various bacterial infections and chronic enteric inflammatory diseases. Here, we illustrate how antibiotics are associated with an increased risk of antibiotic-associated diseases by driving intestinal environment changes that favor the proliferation and virulence of pathogens. Understanding the pathogenesis caused by antibiotics would be a crucial key to the treatment of antibiotic-associated diseases by mitigating changes in the intestinal environment and restoring it to its original state.

A biofilm is a community of microbes that typically inhabit on surfaces and are encased in an extracellular matrix. Biofilms display very dissimilar characteristics to their planktonic counterparts. Biofilms are ubiquitous in the environment and influence our lives tremendously in both positive and negative ways. Pseudomonas aeruginosa is a bacterium known to produce robust biofilms. P. aeruginosa biofilms cause severe problems in immunocompromised patients, including those with cystic fibrosis or wound infection. Moreover, the unique biofilm properties further complicate the eradication of the biofilm infection, leading to the development of chronic infections. In this review, we discuss the history of biofilm research and general characteristics of bacterial biofilms. Then, distinct features pertaining to each stage of P. aeruginosa biofilm development are highlighted. Furthermore, infections caused by biofilms on their own or in association with other bacterial species (i.e., multispecies biofilms) are discussed in detail.

Nod-like receptor family, CARD domain-containing 4 (NLRC4) inflammasome activation is required for efficient clearance of intracellular pathogens through caspsase-1-dependent pyroptosis in macrophages. Although neutrophils have a critical role in protection from Pseudomonas aeruginosa infection, the mechanisms regulating inflammasome-mediated pyroptosis in neutrophils and its physiological role are largely unknown. We sought to determine the specific mechanisms regulating neutrophil pyroptosis in P. aeruginosa strain PAO1 (PAO1) lung infection and to identify the pathological role of this process. Nox2

Pseudomonas aeruginosa has been identified as an important causative agent of airway infection, mainly in cystic fibrosis. This disease is characterized by defective mucociliary clearance induced in part by mucus hyper-production. Mucin is a major component of airway mucus and is heavily O-glycosylated, with a protein backbone. Airway infection is known to be established with bacterial adhesion to mucin. However, the genes involved in mucin degradation or utilization remain elusive. In this study, we sought to provide a genetic basis of P. aeruginosa airway growth by identifying those genes. First, using RNASeq analyses, we compared genome-wide expression profiles of PAO1, a prototype P. aeruginosa laboratory strain, grown in M9-mucin (M9M) and M9-glucose (M9G) media. Additionally, a PAO1 transposon (Tn) insertion mutants library was screened for mutants defective in growth in M9M medium. One mutant with a Tn insertion in the xcpU gene (PA3100) was determined to exhibit faulty growth in M9M medium. This gene contributes to the type II secretion system, suggesting that P. aeruginosa uses this secretion system to produce a number of proteins to break down and assimilate the mucin molecule. Furthermore, we screened the PAO1 genome for genes with protease activity. Of 13 mutants, one with mutation in PA3247 gene exhibited defective growth in M9M, suggesting that the PA3247-encoded protease plays a role in mucin utilization. Further mechanistic dissection of this particular process will reveal new drug targets, the inhibition of which could control recalcitrant P. aeruginosa infections.

The stringent response (SR) is a highly conserved stress response in bacteria. It is composed of two factors, (i) a nucleotide alarmone, guanosine tetra- and pentaphosphate ((p)ppGpp), and (ii) an RNA polymerase-binding protein, DksA, that regulates various phenotypes, including bacterial virulence. The clinically significant opportunistic bacterial pathogen Pseudomonas aeruginosa possesses two genes, dksA1 and dksA2, that encode DksA proteins. It remains elusive, however, which of these two genes plays a more important role in SR regulation. In this work, we compared genome-wide, RNA-Seq–based transcriptome profiles of ΔdksA1, ΔdksA2, and ΔdksA1ΔdksA2 mutants to globally assess the effects of these gene deletions on transcript levels coupled with phenotypic analyses. The ΔdksA1 mutant exhibited substantial defects in a wide range of phenotypes, including quorum sensing (QS), anaerobiosis, and motility, whereas the ΔdksA2 mutant exhibited no significant phenotypic changes, suggesting that the dksA2 gene may not have an essential function in P. aeruginosa under the conditions used here. Of note, the ΔdksA1 mutants displayed substantially increased transcription of genes involved in polyamine biosynthesis, and we also detected increased polyamine levels in these mutants. Because SAM is a shared precursor for the production of both QS autoinducers and polyamines, these findings suggest that DksA1 deficiency skews the flow of SAM toward polyamine production rather than to QS signaling. Together, our results indicate that DksA1, but not DksA2, controls many important phenotypes in P. aeruginosa. We conclude that DksA1 may represent a potential target whose inhibition may help manage recalcitrant P. aeruginosa infections.

Background 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. Results Here, we illustrate how Bacteroides vulgatus, a dominant species of the Bacteroidetes phylum in mouse intestine, suppresses infection by Vibrio cholerae, an important human pathogen. Clindamycin (CL) is an antibiotic that selectively kills anaerobic bacteria, and accordingly Bacteroidetes are completely eradicated from CL-treated mouse intestines. The Bacteroidetes-depleted adult mice developed severe cholera-like symptoms, when infected with V. cholerae. Germ-free mice mono-associated with B. vulgatus became resistant to V. cholerae infection. Levels of V. cholerae growth-inhibitory metabolites including short-chain fatty acids plummeted upon CL treatment, while levels of compounds that enhance V. cholerae proliferation were elevated. Furthermore, the intestinal colonization process of V. cholerae was well-simulated in CL-treated adult mice. Conclusions Overall, we provide insights into how a symbiotic microbe and a pathogenic intruder interact inside host intestine. We identified B. vulgatus as an indigenous microbial species that can suppress intestinal infection. Our results also demonstrate that commensal-derived metabolites are a critical determinant for host resistance against V. cholerae infection, and that CL pretreatment of adult mice generates a simple yet useful model of cholera infection.

IL-17 family cytokines are directly involved in host immune responses and the critical mediators for host defense against infection or inflammation. IL-17C is highly expressed in respiratory epithelium and is induced after acute bacterial lung infection. However, the definite function of IL-17C induced by

Background 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. Results Exposure of NHNE cells to nasal mucosa-derived S. epidermidis increased IFN-λ mRNA and secreted protein levels in the absence of viral stimulation. In the context of IAV infection, NHNE exposure to S. epidermidis prevented an increase in the viral burden, as revealed by IAV PA mRNA abundance, IAV nucleoprotein levels, and viral titers. S. epidermidis also enhanced transcription of IFN-stimulated genes independently of Toll-like receptor 2 and further induced IFN-λ production in IAV-infected cells by promoting phosphorylation of interferon regulatory factor 7. In a murine infection model, S. epidermidis prevented the spread of IAV to the lungs by stimulating IFN-λ innate immunity and suppressing IAV replication in the nasal mucosa. Conclusion The human nasal commensal S. epidermidis mediates front-line antiviral protection against IAV infection through modulation of IFN-λ-dependent innate immune mechanisms in the nasal mucosa, thereby demonstrating the role of host-bacterial commensalism in shaping human antiviral responses. Electronic supplementary material The online version of this article (10.1186/s40168-019-0691-9) contains supplementary material, which is available to authorized users.

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.

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.

PURPOSE Tigecycline is one of the drugs used to treat multi-drug resistant Klebsiella pneumoniae (K. pneumoniae) infections, including complicated skin and soft tissue infections, complicated intra-abdominal infection, and community-acquired pneumonia in the Republic of Korea. However, since its commercial release, K. pneumoniae resistance against tigecycline has been reported, and there is a serious concern about the spread of tigecycline resistant bacteria. MATERIALS AND METHODS In this study, we collected and analyzed 342 isolates from 23 hospitals in the Republic of Korea to determine the mechanisms of tigecycline susceptibility and their clonal types. The hospitals include several from each province in the Republic of Korea, except Jeju, an island province, and nonsusceptibility among the isolates was tested by the disk diffusion method. In our lab, susceptibility was checked again using the broth dilution method, and clonal types were determined using the multilocus sequence typing protocol. Real-time PCR was performed to measure the ramR mutation in the isolates nonsusceptible to tigecycline, which would suggest an increased expression of the AcrAB multidrug pump. RESULTS Fifty-six K. pneumoniae isolates were found to be nonsusceptible, 16% of the 342 collected. Twenty-seven and nine isolates of the tigecycline nonsusceptible isolates had mutations in the ramR and rpsJ genes, respectively; while 18 nonsusceptible isolates harbored the tetA gene. Comparison of isolates with and without ramR mutation showed a significant statistical difference (p

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.

The erythrocyte membrane is composed of a phospholipid bilayer, which is known to undergo physicochemical changes during storage at low temperatures. This study was conducted to identify marker phospholipids that indicate alteration during deep-frozen storage and to determine the amount of marker phospholipids. Our research suggested a method to detect phospholipids by profiling analysis of thermally injured red blood cells (RBCs) without protecting agents. Human blood was stored at -80°C for 72 days. The RBC membrane phospholipids were extracted through a modified Bligh and Dyer method. Six selected phospholipids were analyzed and quantified using liquid chromatography-tandem mass spectrometry, and an in vitro model system was developed. The intracellular level of N-nervonoyl-D-erythro-sphingosylphosphorylcholine significantly increased in the thermally injured RBCs, and multiple biomarker candidates were evaluated by profiling analysis and mass spectrometry technology for targeted metabolomics.

Pseudomonas aeruginosa, a Gram-negative bacterium, is characterized by its versatility that enables persistent survival under adverse conditions. It can grow on diverse energy sources and readily acquire resistance to antimicrobial agents. As an opportunistic human pathogen, it also causes chronic infections inside the anaerobic mucus airways of cystic fibrosis patients. As a strict respirer, P. aeruginosa can grow by anaerobic nitrate ( [Formula: see text] ) respiration. Nitric oxide (NO) produced as an intermediate during anaerobic respiration exerts many important effects on the biological characteristics of P. aeruginosa. This review provides information regarding (i) how P. aeruginosa grows by anaerobic respiration, (ii) mechanisms by which NO is produced under such growth, and (iii) bacterial adaptation to NO. We also review the clinical relevance of NO in the fitness of P. aeruginosa and the use of NO as a potential therapeutic for treating P. aeruginosa infection.