Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. ...Many C. difficile strains representing multiple ribotypes develop two colony morphotypes, termed rough and smooth, but the biological implications of this phenomenon have not been explored. Here, we examine the molecular basis and physiological relevance of the distinct colony morphotypes produced by this bacterium. We show that C. difficile reversibly differentiates into rough and smooth colony morphologies and that bacteria derived from the isolates display discrete motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein colony morphology regulators RST (CmrRST), which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of flagella and type IV pili, suggesting a novel mechanism of cell migration in C. difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes the formation of elongated bacteria arranged in bundled chains, which may contribute to bacterial migration on surfaces. In a hamster model of acute C. difficile disease, the CmrRST system is required for disease development. Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting that the intestinal environment impacts the proportion of CmrRST-expressing C. difficile. Our findings indicate that C. difficile employs phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis.
Bacteria that reside in the gastrointestinal tract of healthy humans are essential for our health, sustenance and well-being. About 50-60% of those bacteria have the ability to produce resilient ...spores that are important for the life cycle in the gut and for host-to-host transmission. A genomic signature for sporulation in the human intestine was recently described, which spans both commensals and pathogens such as Clostridioides difficile and contains several genes of unknown function. We report on the characterization of a signature gene, CD25890, which, as we show is involved in the control of sporulation initiation in C. difficile under certain nutritional conditions. Spo0A is the main regulatory protein controlling entry into sporulation and we show that an in-frame deletion of CD25890 results in increased expression of spo0A per cell and increased sporulation. The effect of CD25890 on spo0A is likely indirect and mediated through repression of the sinRR´ operon. Deletion of the CD25890 gene, however, does not alter the expression of the genes coding for the cytotoxins or the genes involved in biofilm formation. Our results suggest that CD25890 acts to modulate sporulation in response to the nutrients present in the environment.
Abstract Since data about Clostridium difficile infection in sub-Saharan Africa are scarce, we determined its epidemiology and risk factors in a cross-sectional study in Eikwe, a rural community in ...Ghana. We tested stool samples from 176 hospitalized patients with diarrhoea and from 131 asymptomatic non-hospitalized individuals for C. difficile and some other enteric pathogens. The overall prevalence rate of C. difficile was 4.9% with ribotype 084 being predominant. With 75% of the isolates, a high rate of nontoxigenic strains was present in symptomatic patients, most of whom had no other identified enteric pathogens. All strains were susceptible against metronidazole and vancomycin, respectively. Data on lifestyle and medical history showed that age <5 years (p = 0.004), and use of ceftriaxone (p = 0.023) were the most important risk factors for C. difficile carriage status. Although our data suggest that C. difficile is currently not a major cause of diarrhoea in this setting, the epidemiology of C. difficile in sub-Saharan Africa awaits further investigation.
Abstract Clostridioides difficile (formerly Clostridium difficile ) is a major nosocomial pathogen with an increasing number of community-acquired infections causing symptoms from mild diarrhea to ...life-threatening colitis. The pathogenicity of C. difficile is considered to be mainly associated with the production of genome-encoded toxins A and B. In addition, some strains also encode and express the binary toxin CDT. However; a large number of non-toxigenic C. difficile strains have been isolated from the human gut and the environment. In this study, we characterized the growth behavior, motility and fermentation product formation of 17 different C. difficile isolates comprising five different major genomic clades and five different toxin inventories in relation to the C. difficile model strains 630Δ erm and R20291. Within 33 determined fermentation products, we identified two yet undescribed products (5-methylhexanoate and 4-(methylthio)-butanoate) of C. difficile . Our data revealed major differences in the fermentation products obtained after growth in a medium containing casamino acids and glucose as carbon and energy source. While the metabolism of branched chain amino acids remained comparable in all isolates, the aromatic amino acid uptake and metabolism and the central carbon metabolism-associated fermentation pathways varied strongly between the isolates. The patterns obtained followed neither the classification of the clades nor the ribotyping patterns nor the toxin distribution. As the toxin formation is strongly connected to the metabolism, our data allow an improved differentiation of C. difficile strains. The observed metabolic flexibility provides the optimal basis for the adaption in the course of infection and to changing conditions in different environments including the human gut.
Clostridioides difficile infections (CDI) have emerged over the past decade causing symptoms that
range from mild, antibiotic-associated diarrhea (AAD) to life-threatening toxic megacolon. In this ...study, we describe a multiple and isochronal (mixed) CDI caused by the isolates DSM 27638, DSM 27639 and DSM 27640 that already initially showed different morphotypes on solid media.
The three isolates belonging to the ribotypes (RT) 012 (DSM 27639) and 027 (DSM 27638 and DSM 27640)
were phenotypically characterized and high quality closed genome sequences were generated. The genomes were compared with seven reference strains including three strains of the RT 027, two of the RT 017, and one of the RT 078 as well as a multi-resistant RT 012 strain. The analysis of horizontal gene transfer events revealed gene acquisition incidents that sort the strains within the time line of the spread of their RTs within Germany. We could show as well that horizontal gene transfer between the members of different RTs occurred within this multiple infection. In addition, acquisition and exchange of virulence-related features including antibiotic resistance genes were observed. Analysis of the two genomes assigned to RT 027 revealed three single nucleotide polymorphisms (SNPs) and apparently a regional genome modification within the flagellar switch that regulates the fli operon.
Our findings show that (i) evolutionary events based on horizontal gene transfer occur within an ongoing
CDI and contribute to the adaptation of the species by the introduction of new genes into the genomes, (ii) within a multiple infection of a single patient the exchange of genetic material was responsible for a much higher genome variation than the observed SNPs.
Significant increases in potential microbial translocation, especially along the oral–gut axis, have been identified in many immune-related and inflammatory diseases, such as inflammatory bowel ...disease, colorectal cancer, rheumatoid arthritis, and liver cirrhosis, for which we currently have no cure or long-term treatment options. Recent advances in computational and experimental omics approaches now enable strain tracking, functional profiling, and strain isolation in unprecedented detail, which has the potential to elucidate the causes and consequences of microbial translocation. In this review, we discuss current evidence for the detection of bacterial translocation, examine different translocation axes with a primary focus on the oral–gut axis, and outline currently known translocation mechanisms and how they adversely affect the host in disease. Finally, we conclude with an overview of state-of-the-art computational and experimental tools for strain tracking and highlight the required next steps to elucidate the role of bacterial translocation in human health.
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•Microbial communities and strains have adapted to specific body sites.•Increased bacterial translocation occurs in diseases, such as IBD and CRC.•Causes and consequences of bacterial translocation in disease are largely unknown.•Strain tracking is critical to detect and understand bacterial translocation.
is a pathogenic bacterium that infects the human colon to cause diarrheal disease. Growth of the bacterium is known to be dependent on certain bile acids, oxygen levels, and nutrient availability in ...the intestine, but how the environmental pH can influence
is mostly unknown. Previous studies indicated that
modulates the intestinal pH, and prospective cohort studies have found a strong association between a more alkaline fecal pH and
infection. Based on these data, we hypothesized that
physiology can be affected by various pH conditions. In this study, we investigated the impact of a range of pH conditions on
to assess potential effects on growth, sporulation, motility, and toxin production in the strains 630Δ
and R20291. We observed pH-dependent differences in sporulation rate, spore morphology, and viability. Sporulation frequency was lowest under acidic conditions, and differences in cell morphology were apparent at low pH. In alkaline environments,
sporulation was greater for strain 630Δ
, whereas R20291 produced relatively high levels of spores in a broad range of pH conditions. Rapid changes in pH during exponential growth impacted sporulation similarly among the strains. Furthermore, we observed an increase in
motility with increases in pH, and strain-dependent differences in toxin production under acidic conditions. The data demonstrate that pH is an important parameter that affects
physiology and may reveal relevant insights into the growth and dissemination of this pathogen.
is an anaerobic bacterium that causes gastrointestinal disease.
forms dormant spores which can survive harsh environmental conditions, allowing their spread to new hosts. In this study, we determine how intestinally relevant pH conditions impact
physiology in the two divergent strains, 630Δ
and R20291. Our data demonstrate that low pH conditions reduce
growth, sporulation, and motility. However, toxin production and spore morphology were differentially impacted in the two strains at low pH. In addition, we observed that alkaline environments reduce
growth, but increase cell motility. When pH was adjusted rapidly during growth, we observed similar impacts on both strains. This study provides new insights into the phenotypic diversity of
grown under diverse pH conditions present in the intestinal tract, and demonstrates similarities and differences in the pH responses of different
isolates.
The ability of the anaerobic gastrointestinal pathogen Clostridioides difficile to survive outside the host relies on the formation of dormant endospores. Spore formation is contingent on the ...activation of a conserved transcription factor, Spo0A, by phosphorylation. Multiple kinases and phosphatases regulate Spo0A activity in other spore-forming organisms; however, these factors are not well conserved in C. difficile. Previously, we discovered that deletion of a predicted histidine kinase, CD1492, increases sporulation, indicating that CD1492 inhibits C. difficile spore formation. In this study, we investigate the functions of additional predicted orphan histidine kinases CD2492, CD1579, and CD1949, which are hypothesized to regulate Spo0A phosphorylation. Disruption of CD2492 also increased sporulation frequency, similarly to the
mutant and in contrast to a previous study. A
mutant phenocopied the sporulation and gene expression patterns of the single mutants, suggesting that these proteins function in the same genetic pathway to repress sporulation. Deletion of CD1579 variably increased sporulation frequency; however, knockdown of
expression did not influence sporulation. We provide evidence that CD1492, CD2492, and CD1579 function as phosphatases, as mutation of the conserved histidine residue for phosphate transfer abolished CD2492 function, and expression of the
or
histidine site-directed mutants or the wild-type
allele in a parent strain resulted in a dominant-negative hypersporulation phenotype. Altogether, at least three predicted histidine kinases, CD1492, CD2492, and CD1579 (herein, PtpA, PtpB and PtpC), repress C. difficile sporulation initiation by regulating activity of Spo0A.
The formation of inactive spores is critical for the long-term survival of the gastrointestinal pathogen Clostridioides difficile. The onset of sporulation is controlled by the master regulator of sporulation, Spo0A, which is activated by phosphorylation. Multiple kinases and phosphatases control Spo0A phosphorylation; however, this regulatory pathway is not defined in C. difficile. We show that two predicted histidine kinase proteins, CD1492 (PtpA) and CD2492 (PtpB), function in the same regulatory pathway to repress sporulation by preventing Spo0A phosphorylation. We show that another predicted histidine kinase protein, CD1579 (PtpC), also represses sporulation and present evidence that a fourth predicted histidine kinase protein, CD1949, does not impact sporulation. These results support the idea that C. difficile inhibits sporulation initiation through multiple phosphatases.
Summary
Clostridium (Clostridioides) difficile is a gastrointestinal pathogen that colonizes the intestinal tract of mammals and can cause severe diarrheal disease. Although C. difficile growth is ...confined to the intestinal tract, our understanding of the specific metabolites and host factors that are important for the growth of the bacterium is limited. In other enteric pathogens, the membrane‐derived metabolite, ethanolamine (EA), is utilized as a nutrient source and can function as a signal to initiate the production of virulence factors. In this study, we investigated the effects of ethanolamine and the role of the predicted ethanolamine gene cluster (CD1907‐CD1925) on C. difficile growth. Using targeted mutagenesis, we disrupted genes within the eut cluster and assessed their roles in ethanolamine utilization, and the impact of eut disruption on the outcome of infection in a hamster model of disease. Our results indicate that the eut gene cluster is required for the growth of C. difficile on ethanolamine as a primary nutrient source. Further, the inability to utilize ethanolamine resulted in greater virulence and a shorter time to morbidity in the animal model. Overall, these data suggest that ethanolamine is an important nutrient source within the host and that, in contrast to other intestinal pathogens, the metabolism of ethanolamine by C. difficile can delay the onset of disease.
is an anaerobic gastrointestinal pathogen that spreads through the environment as dormant spores. To survive, replicate, and sporulate in the host intestine,
must adapt to a variety of conditions in ...its environment, including changes in pH, the availability of metabolites, host immune factors, and a diverse array of other species. Prior studies showed that changes in intestinal conditions, such as pH, can affect
toxin production, spore formation, and cell survival. However, little is understood about the specific genes and pathways that facilitate environmental adaptation and lead to changes in
cell outcomes. In this study, we investigated two genes,
and
that are differentially regulated by pH to determine if they impact
growth and sporulation. Using deletion mutants, we examined the effects of both genes (herein
and
) on sporulation frequency, toxin production, and antimicrobial resistance. We determined that SmrR is a repressor of
that responds to pH and suppresses sporulation and toxin production through regulation of the SmrT transporter. Further, we showed that SmrT confers resistance to erythromycin and lincomycin, establishing a connection between the regulation of sporulation and antimicrobial resistance.IMPORTANCE
is a mammalian pathogen that colonizes the large intestine and produces toxins that lead to severe diarrheal disease.
is a major threat to public health due to its intrinsic resistance to antimicrobials and its ability to form dormant spores that are easily spread from host to host. In this study, we examined the contribution of two genes,
and
, on sporulation, toxin production, and antimicrobial resistance. Our results indicate that SmrR represses
expression, while production of SmrT increases spore and toxin production, as well as resistance to antibiotics.
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