scholarly journals Toxin Synthesis by Clostridium difficile Is Regulated through Quorum Signaling

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Charles Darkoh ◽  
Herbert L. DuPont ◽  
Steven J. Norris ◽  
Heidi B. Kaplan
Keyword(s):  
Clostridium Difficile ◽  
Gut Microbiota ◽  
Antibiotic Therapy ◽  
Molecular Mechanisms ◽  
Public Health Problem ◽  
The United States ◽  
Multidrug Resistant ◽  
Accessory Gene ◽  
Content Type ◽  
Accessory Gene Regulator

ABSTRACTClostridium difficileinfection (CDI) is dramatically increasing as a cause of antibiotic- and hospital-associated diarrhea worldwide. C. difficile, a multidrug-resistant pathogen, flourishes in the colon after the gut microbiota has been altered by antibiotic therapy. Consequently, it produces toxins A and B that directly cause disease. Despite the enormous public health problem posed by this pathogen, the molecular mechanisms that regulate production of the toxins, which are directly responsible for disease, remained largely unknown until now. Here, we show that C. difficile toxin synthesis is regulated by an accessory gene regulator quorum-signaling system, which is mediated through a small (<1,000-Da) thiolactone that can be detected directly in stools of CDI patients. These findings provide direct evidence of the mechanism of regulation of C. difficile toxin synthesis and offer exciting new avenues both for rapid detection of C. difficile infection and development of quorum-signaling-based non-antibiotic therapies to combat this life-threatening emerging pathogen.IMPORTANCEClostridium difficileinfection (CDI) is the most common definable cause of hospital-acquired and antibiotic-associated diarrhea in the United States, with the total cost of treatment estimated between 1 and 4.8 billion U.S. dollars annually. C. difficile, a Gram-positive, spore-forming anaerobe, flourishes in the colon after the gut microbiota has been altered by antibiotic therapy. As a result, there is an urgent need for non-antibiotic CDI treatments that preserve the colonic microbiota. C. difficile produces toxins A and B, which are directly responsible for disease. Here, we report that C. difficile regulates its toxin synthesis by quorum signaling, in which a novel signaling peptide activates transcription of the disease-causing toxin genes. This finding provides new therapeutic targets to be harnessed for novel nonantibiotic therapy for C. difficile infections.

scholarly journals A Prolonged Outbreak of KPC-3-Producing Enterobacter cloacae and Klebsiella pneumoniae Driven by Multiple Mechanisms of Resistance Transmission at a Large Academic Burn Center

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Hajime Kanamori ◽  
Christian M. Parobek ◽  
Jonathan J. Juliano ◽  
David van Duin ◽  
Bruce A. Cairns ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Molecular Mechanisms ◽  
Enterobacter Cloacae ◽  
The United States ◽  
Multidrug Resistant ◽  
Control Measures ◽  
Content Type ◽  
Burn Center ◽  
Infection Control Measures ◽  
Carbapenem Resistant

ABSTRACT Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter cloacae has been recently recognized in the United States. Whole-genome sequencing (WGS) has become a useful tool for analysis of outbreaks and for determining transmission networks of multidrug-resistant organisms in health care settings, including carbapenem-resistant Enterobacteriaceae (CRE). We experienced a prolonged outbreak of CRE E. cloacae and K. pneumoniae over a 3-year period at a large academic burn center despite rigorous infection control measures. To understand the molecular mechanisms that sustained this outbreak, we investigated the CRE outbreak isolates by using WGS. Twenty-two clinical isolates of CRE, including E. cloacae (n = 15) and K. pneumoniae (n = 7), were sequenced and analyzed genetically. WGS revealed that this outbreak, which seemed epidemiologically unlinked, was in fact genetically linked over a prolonged period. Multiple mechanisms were found to account for the ongoing outbreak of KPC-3-producing E. cloacae and K. pneumoniae. This outbreak was primarily maintained by a clonal expansion of E. cloacae sequence type 114 (ST114) with distribution of multiple resistance determinants. Plasmid and transposon analyses suggested that the majority of bla KPC-3 was transmitted via an identical Tn4401b element on part of a common plasmid. WGS analysis demonstrated complex transmission dynamics within the burn center at levels of the strain and/or plasmid in association with a transposon, highlighting the versatility of KPC-producing Enterobacteriaceae in their ability to utilize multiple modes to resistance gene propagation.

scholarly journals The Gut Microbiota Is Associated with Clearance ofClostridium difficileInfection Independent of Adaptive Immunity

mSphere ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Jhansi L. Leslie ◽  
Kimberly C. Vendrov ◽  
Matthew L. Jenior ◽  
Vincent B. Young
Keyword(s):  
United States ◽  
Immune Response ◽  
Clostridium Difficile ◽  
Gut Microbiota ◽  
Adaptive Immunity ◽  
Adaptive Immune Response ◽  
The United States ◽  
Content Type ◽  
Adaptive Immune

ABSTRACTClostridium(Clostridioides)difficile, a Gram-positive, anaerobic bacterium, is the leading single cause of nosocomial infections in the United States. A major risk factor forClostridium difficileinfection (CDI) is prior exposure to antibiotics, as they increase susceptibility to CDI by altering the membership of the microbial community enabling colonization. The importance of the gut microbiota in providing protection from CDI is underscored by the reported 80 to 90% success rate of fecal microbial transplants in treating recurrent infections. Adaptive immunity, specifically humoral immunity, is also sufficient to protect from both acute and recurrent CDI. However, the role of the adaptive immune system in mediating clearance ofC. difficilehas yet to be resolved. Using murine models of CDI, we found that adaptive immunity is dispensable for clearance ofC. difficile. However, random forest analysis using only two members of the resident bacterial community correctly identified animals that would go on to clear the infection with 66.7% accuracy. These findings indicate that the indigenous gut microbiota independent of adaptive immunity facilitates clearance ofC. difficilefrom the murine gastrointestinal tract.IMPORTANCEClostridium difficileinfection is a major cause of morbidity and mortality in hospitalized patients in the United States. Currently, the role of the adaptive immune response in modulating levels ofC. difficilecolonization is unresolved. This work suggests that the indigenous gut microbiota is a main factor that promotes clearance ofC. difficilefrom the GI tract. Our results show that clearance ofC. difficilecan occur without contributions from the adaptive immune response. This study also has implications for the design of preclinical studies testing the efficacy of vaccines on clearance of bacterial pathogens, as inherent differences in the baseline community structure of animals may bias findings.

scholarly journals Antibiotic-Induced Alterations of the Murine Gut Microbiota and Subsequent Effects on Colonization Resistance against Clostridium difficile

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Alyxandria M. Schubert ◽  
Hamide Sinani ◽  
Patrick D. Schloss
Keyword(s):  
Clostridium Difficile ◽  
Gut Microbiota ◽  
Normal Structure ◽  
The United States ◽  
Context Dependency ◽  
Dependent Manner ◽  
Colonization Resistance ◽  
Bacterial Populations ◽  
Content Type ◽  
Antibiotic Perturbations

ABSTRACTPerturbations to the gut microbiota can result in a loss of colonization resistance against gastrointestinal pathogens such asClostridium difficile. AlthoughC. difficileinfection is commonly associated with antibiotic use, the precise alterations to the microbiota associated with this loss in function are unknown. We used a variety of antibiotic perturbations to generate a diverse array of gut microbiota structures, which were then challenged withC. difficilespores. Across these treatments we observed thatC. difficileresistance was never attributable to a single organism, but rather it was the result of multiple microbiota members interacting in a context-dependent manner. Using relative abundance data, we built a machine learning regression model to predict the levels ofC. difficilethat were found 24 h after challenging the perturbed communities. This model was able to explain 77.2% of the variation in the observed number ofC. difficileper gram of feces. This model revealed important bacterial populations within the microbiota, which correlation analysis alone did not detect. Specifically, we observed that populations associated with thePorphyromonadaceae,Lachnospiraceae,Lactobacillus, andAlistipeswere protective and populations associated withEscherichiaandStreptococcuswere associated with high levels of colonization. In addition, a population affiliated with theAkkermansiaindicated a strong context dependency on other members of the microbiota. Together, these results indicate that individual bacterial populations do not drive colonization resistance toC. difficile. Rather, multiple diverse assemblages act in concert to mediate colonization resistance.IMPORTANCEThe gastrointestinal tract harbors a complex community of bacteria, known as the microbiota, which plays an integral role preventing its colonization by gut pathogens. This resistance has been shown to be crucial for protection againstClostridium difficileinfections (CDI), which are the leading source of hospital-acquired infections in the United States. Antibiotics are a major risk factor for acquiring CDI due to their effect on the normal structure of the indigenous gut microbiota. We found that diverse antibiotic perturbations gave rise to altered communities that varied in their susceptibility toC. difficilecolonization. We found that multiple coexisting populations, not one specific population of bacteria, conferred resistance. By understanding the relationships betweenC. difficileand members of the microbiota, it will be possible to better manage this important infection.

scholarly journals Clostridium difficileModulates the Gut Microbiota by Inducing the Production of Indole, an Interkingdom Signaling and Antimicrobial Molecule

mSystems ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Charles Darkoh ◽  
Kimberly Plants-Paris ◽  
Dayna Bishoff ◽  
Herbert L. DuPont
Keyword(s):  
Gut Microbiota ◽  
Treatment Options ◽  
Intestinal Bacteria ◽  
Chemical Warfare ◽  
Accessory Gene ◽  
Content Type ◽  
Accessory Gene Regulator ◽  
Complex Interactions ◽  
Unique Markers ◽  
Insight Into

ABSTRACTClostridium(Clostridioides)difficileinfection (CDI) is associated with dysbiosis.C. difficilehas a characteristic propensity to persist and recur 1 to 4 weeks after treatment, but the mechanism is unknown. We hypothesized thatC. difficilemay persist by manipulating the intestinal microenvironment, thereby hampering gut microbiota reconstitution following antibiotic-mediated dysbiosis. By screening stools from CDI patients for unique markers, a metabolite identified to be indole by mass spectrometry and Fourier transform infrared spectroscopy was identified. The average fecal indole concentration detected in CDI patients (n= 216; mean, 1,684.0 ± 84.4 µM) was significantly higher than in stools of patients with non-C. difficilediarrhea (n = 204; mean, 762.8 ± 53.8 µM). Certain intestinal bacteria, but notC. difficile, produce indole, a potent antimicrobial antioxidant. Remarkably,C. difficileinduced other indole-producing gut microbes to produce increasing amounts of indole. Furthermore, aC. difficileaccessory gene regulator 1 quorum sensing system mutant cannot induce indole, but complementation of the mutant strain with the wild-type gene restored its ability to induce indole production. Indole tolerance assays indicated that the amount of indole required to inhibit growth of most gut-protective bacteria was within the range detected in the CDI stools. We think that a high indole level limits the growth of beneficial indole-sensitive bacteria in the colon and alters colonization resistance and this might allowC. difficileto proliferate and persist. Together, these results reveal a unique mechanism ofC. difficilepersistence and provide insight into complex interactions and chemical warfare among the gut microbiota.IMPORTANCEClostridiumdifficileinfection is the leading cause of hospital-acquired and antibiotic-associated diarrhea worldwide.C. difficileflourishes in the colon after the diversity of the beneficial and protective gut microbiota have been altered by antibiotic therapy.C. difficiletends to persist, as does dysbiosis, encouraging recurrence a few days to weeks after treatment, and this further complicates treatment options. Here, we show thatC. difficilemight persist by manipulating the indigenous microbiota to produce indole, a bioactive molecule that inhibits the growth and reconstitution of the protective gut microbiota during infection. This discovery may explain a unique strategyC. difficileuses to control other bacteria in the colon and provide insight into the complex interactions and chemical warfare among the gut microbiota.

scholarly journals Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis ofClostridium difficile

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Charles Darkoh ◽  
Chioma Odo ◽  
Herbert L. DuPont
Keyword(s):  
Health Care ◽  
Therapeutic Target ◽  
Treatment Options ◽  
Toxin Production ◽  
The United States ◽  
Multidrug Resistant ◽  
Accessory Gene ◽  
Ofclostridium Difficile ◽  
Accessory Gene Regulator ◽  
Toxin Regulation

ABSTRACTClostridium difficileinfection (CDI) is responsible for most of the definable cases of antibiotic- and hospital-associated diarrhea worldwide and is a frequent cause of morbidity and mortality in older patients.C. difficile, a multidrug-resistant anaerobic pathogen, causes disease by producing toxins A and B, which are controlled by an accessory gene regulator (Agr) quorum signaling system. SomeC. difficilestrains encode two Agr loci in their genomes, designatedagr1andagr2. Theagr1locus is present in all of theC. difficilestrains sequenced to date, whereas theagr2locus is present in a few strains. The functional roles ofagr1andagr2inC. difficiletoxin regulation and pathogenesis were unknown until now. Using allelic exchange, we deleted components of bothagrloci and examined the mutants for toxin production and virulence. The results showed that theagr1mutant cannot produce toxins A and B; toxin production can be restored by complementation with wild-typeagr1. Furthermore, theagr1mutant is able to colonize but unable to cause disease in a murine CDI model. These findings have profound implications for CDI treatment because we have uncovered a promising therapeutic target for the development of nonantibiotic drugs to treat this life-threatening emerging pathogen by targeting the toxins directly responsible for disease.IMPORTANCEWithin the last decade, the number of cases ofC. difficileinfections has been increasing exponentially in the United States, resulting in about 4.8 billion U.S. dollars in health care costs annually. As a multidrug-resistant, spore-forming, anaerobic pathogen,C. difficileoverpopulates the colon after the gut microbiota has been altered by antibiotic therapy. With increasing resistance to antibiotic treatment ofC. difficileinfections, patients are experiencing higher costs of health care and a lower quality of life as treatment options decrease. During infection,C. difficileproduces toxins A and B, which directly cause disease. As a result, the toxins have become promising nonantibiotic treatment targets. Here, we have identified a pathway responsible for activating the production of the toxins. This important finding opens up a unique therapeutic target for the development of a novel nonantibiotic therapy forC. difficileinfections.

scholarly journals Inflammasome Activation Contributes to Interleukin-23 Production in Response to Clostridium difficile

mBio ◽  
2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Carrie A. Cowardin ◽  
Sarah A. Kuehne ◽  
Erica L. Buonomo ◽  
Chelsea S. Marie ◽  
Nigel P. Minton ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Disease Severity ◽  
Tissue Damage ◽  
The United States ◽  
Host Cells ◽  
Inflammasome Activation ◽  
Content Type ◽  
Danger Signals ◽  
Interleukin 23 ◽  
Molecular Patterns

ABSTRACT  Clostridium difficileis the most common hospital-acquired pathogen, causing antibiotic-associated diarrhea in over 250,000 patients annually in the United States. Disease is primarily mediated by toxins A and B, which induce potent proinflammatory signaling in host cells and can activate an ASC-containing inflammasome. Recent findings suggest that the intensity of the host response to infection correlates with disease severity. Our lab has identified the proinflammatory cytokine interleukin-23 (IL-23) as a pathogenic mediator during C. difficile infection (CDI). The mechanisms by which C. difficile induces IL-23, however, are not well understood, and the role of toxins A and B in this process is unclear. Here, we show that toxins A and B alone are not sufficient for IL-23 production but synergistically increase the amount of IL-23 produced in response to MyD88-dependent danger signals, including pathogen-associated molecular patterns (PAMPs) and host-derived damage associated molecular patterns (DAMPs). Danger signals also enhanced the secretion of IL-1β in response to toxins A and B, and subsequent IL-1 receptor signaling accounted for the majority of the increase in IL-23 that occurred in the presence of the toxins. Inhibition of inflammasome activation in the presence of extracellular K+likewise decreased IL-23 production. Finally, we found that IL-1β was increased in the serum of patients with CDI, suggesting that this systemic response could influence downstream production of pathogenic IL-23. Identification of the synergy of danger signals with toxins A and B via inflammasome signaling represents a novel finding in the mechanistic understanding of C. difficile-induced inflammation.IMPORTANCEClostridium difficileis among the leading causes of death due to health care-associated infection, and factors determining disease severity are not well understood. C. difficile secretes toxins A and B, which cause inflammation and tissue damage, and recent findings suggest that some of this tissue damage may be due to an inappropriate host immune response. We have found that toxins A and B, in combination with both bacterium- and host-derived danger signals, can induce expression of the proinflammatory cytokines IL-1β and IL-23. Our results demonstrate that IL-1β signaling enhances IL-23 production and could lead to increased pathogenic inflammation during CDI.

Gram-positive bacteria secrete RNA aptamers to activate human STING for IL-1β release.

2021 ◽  
Author(s):  
Shivalee N Duduskar ◽  
Mohamed Ghait ◽  
Martin Westermann ◽  
Huijuan Guo ◽  
Anuradha Ramoji ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Rna Aptamers ◽  
Inflammasome Activation ◽  
Accessory Gene ◽  
Group B Streptococci ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Accessory Gene Regulator ◽  
New Perspective ◽  
Group B

Molecular mechanisms through which Gram-positive bacteria induce the canonical inflammasome are poorly understood. Here, we studied the effects of Group B streptococci (GBS) and Staphylococcus aureus (SA) on inflammasome activation in human macrophages. Dinucleotide binding small RNA aptamers released by SA and GBS were shown to trigger increased IL-1β generation by inflammasomes. The stimulator of interferon genes-STING as a central mediator of innate immune responses has been identified as the key target of pathogenic RNA. Multi-lamellar lipid bodies (MLBs) produced by SA function as vehicles for the RNA aptamers. Notably, expression of RNA aptamers is controlled by an accessory gene regulator quorum sensing system of the bacteria. These findings have been translated to patients with Gram-positive sepsis showing hallmarks of MLB-RNA-mediated inflammasome activation. Together our findings may provide a new perspective for the pathogenicity of Gram-positive bacterial infection in man.

scholarly journals Finding of Agr Phase Variants in Staphylococcus aureus

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Vishal Gor ◽  
Aya J. Takemura ◽  
Masami Nishitani ◽  
Masato Higashide ◽  
Veronica Medrano Romero ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Phase Variation ◽  
Accessory Gene ◽  
Content Type ◽  
Accessory Gene Regulator ◽  
Immune Mediated ◽  
A Minor ◽  
First Time ◽  
Phase Variants

ABSTRACT Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing accessory gene regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr-negative strains and screening for phenotypic reversion of hemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative methicillin-resistant S. aureus (MRSA) isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. We propose a model in which a minor fraction of Agr-negative S. aureus strains are phase variants that can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress. IMPORTANCE Staphylococcus aureus is responsible for a broad range of infections. This pathogen has a vast arsenal of virulence factors at its disposal, but avirulent strains are frequently isolated as the cause of clinical infections. These isolates have a mutated agr locus and have been believed to have no evolutionary future. Here we show that a fraction of Agr-negative strains can repair their mutated agr locus with mechanisms resembling phase variation. The agr revertants sustain an Agr OFF state as long as they exist as a minority but can activate their Agr system upon phagocytosis. These revertant cells might function as a cryptic insurance strategy to survive immune-mediated host stress that arises during infection.

scholarly journals Impact of agr Functionality on the Outcome of Patients with Methicillin-Susceptible Staphylococcus aureus Bacteremia

2021 ◽  
Author(s):  
Jeong Eun Lee ◽  
Shinwon Lee ◽  
Sohee Park ◽  
Soon O. Lee ◽  
Sun H. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Accessory Gene ◽  
Content Type ◽  
Accessory Gene Regulator ◽  
Staphylococcus Aureus Bacteremia ◽  
Mssa Infection

Few studies have examined the association between methicillin-susceptible Staphylococcus aureus (MSSA) infection and accessory gene regulator ( agr ) functionality. We evaluated the association between agr dysfunction and mortality in patients with MSSA bacteremia.

scholarly journals Impact of Oral Fidaxomicin Administration on the Intestinal Microbiota and Susceptibility to Clostridium difficile Colonization in Mice

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
N. J. Ajami ◽  
J. L. Cope ◽  
M. C. Wong ◽  
J. F. Petrosino ◽  
L. Chesnel
Keyword(s):  
Clostridium Difficile ◽  
Gut Microbiota ◽  
16S Rrna Gene Sequencing ◽  
Taxonomic Structure ◽  
Rrna Gene ◽  
Colonization Resistance ◽  
Content Type ◽  
Rrna Gene Sequencing ◽  
Hospital Acquired ◽  
Predetermined Time

ABSTRACT Clostridium difficile infection (CDI), a common cause of hospital-acquired infections, typically occurs after disruption of the normal gut microbiome by broad-spectrum antibiotics. Fidaxomicin is a narrow-spectrum antibiotic that demonstrates a reduced impact on the normal gut microbiota and is approved for the treatment of CDI. To further explore the benefits of this property, we used a murine model to examine the effects of fidaxomicin versus vancomycin on gut microbiota and susceptibility to C. difficile colonization while tracking microbiota recovery over time. Mice were exposed to fidaxomicin or vancomycin by oral gavage for 3 days and subsequently challenged with C. difficile spores at predetermined time points up to 21 days postexposure to antibiotics. Fecal samples were subsequently collected for analysis. Twenty-four hours postchallenge, mice were euthanized and the colon contents harvested. The microbiota was characterized using 16S rRNA gene sequencing. All fidaxomicin-exposed mice (except for one at day 8) were resistant to C. difficile colonization. However, 9 of 15 vancomycin-exposed mice were susceptible to C. difficile colonization until day 12. All vancomycin-exposed mice recovered colonization resistance by day 16. Bacterial diversity was similar prior to antibiotic exposure in both arms and decreased substantially after exposure. A shift in taxonomic structure and composition occurred after both exposures; however, the shift was greater in vancomycin-exposed than in fidaxomicin-exposed mice. In summary, compared with vancomycin, fidaxomicin exposure had less impact on microbiota composition, promoted faster microbial recovery, and had less impact on the loss of C. difficile colonization resistance.

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