ABSTRACT
Clostridium difficile infection is the leading cause of healthcare-associated diarrhoea in Europe and North America. During infection, C. difficile produces two key virulence determinants, toxin A and toxin B. Experiments with purified toxins have indicated that toxin A alone is able to evoke the symptoms of C. difficile infection, but toxin B is unable to do so unless it is mixed with toxin A or there is prior damage to the gut mucosa. However, a recent study indicated that toxin B is essential for C. difficile virulence and that a strain producing toxin A alone was avirulent. This creates a paradox over the individual importance of toxin A and toxin B. Here we show that isogenic mutants of C. difficile producing either toxin A or toxin B alone can cause fulminant disease in the hamster model of infection. By using a gene knockout system to inactivate the toxin genes permanently, we found that C. difficile producing either one or both toxins showed cytotoxic activity in vitro that translated directly into virulence in vivo. Furthermore, by constructing the first ever double-mutant strain of C. difficile, in which both toxin genes were inactivated, we were able to completely attenuate virulence. Our findings re-establish the importance of both toxin A and toxin B and highlight the need to continue to consider both toxins in the development of diagnostic tests and effective countermeasures against C. difficile.
Subject(s)
Bacterial Toxins/metabolism , Clostridioides difficile/metabolism , Clostridioides difficile/pathogenicity , Clostridium Infections/microbiology , Enterotoxins/metabolism , Animals , Antibodies, Neutralizing , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/genetics , Chlorocebus aethiops , Clostridioides difficile/classification , Clostridioides difficile/genetics , Cricetinae , Disease Models, Animal , Enterotoxins/antagonists & inhibitors , Enterotoxins/genetics , Gene Deletion , HT29 Cells , Humans , Neutralization Tests , Vero Cells , Virulence/geneticsABSTRACT
Comparative analysis of the Clostridium difficile BI/NAP1/027 strain R20291 and ClosTron-derived ermB mutants in the hamster infection model are compromised by the clindamycin susceptibility of the parent. Mutants can appear more virulent. We have rectified this anomaly by genome engineering. The variant created (CRG20291) represents an ideal control strain for virulence assays of ClosTron mutants.
Subject(s)
Clostridioides difficile/genetics , Cricetulus/genetics , Disease Models, Animal , Enterocolitis, Pseudomembranous/microbiology , Genes, Synthetic , Genome, Bacterial , Methyltransferases/genetics , Animals , Anti-Bacterial Agents/pharmacology , Clindamycin/pharmacology , Clostridioides difficile/drug effects , Clostridioides difficile/pathogenicity , Cricetulus/microbiology , Enterocolitis, Pseudomembranous/drug therapy , Enterocolitis, Pseudomembranous/mortality , Enterocolitis, Pseudomembranous/pathology , Gene Expression , Genetic Engineering , Humans , Microbial Sensitivity Tests , Mutation , Survival Analysis , VirulenceABSTRACT
Point mutations conferring resistance to fluoroquinolones were introduced in the gyr genes of the reference strain Clostridium difficile 630. Only mutants with the substitution Thr-82âIle in GyrA, which characterizes the hypervirulent epidemic clone III/027/NAP1, were resistant to all fluoroquinolones tested. The absence of a fitness cost in vitro for the most frequent mutations detected in resistant clinical isolates suggests that resistance will be maintained even in the absence of antibiotic pressure.
Subject(s)
Clostridioides difficile/drug effects , Clostridioides difficile/genetics , Drug Resistance, Bacterial/genetics , Fluoroquinolones/pharmacology , Anti-Bacterial Agents/pharmacology , DNA Gyrase/genetics , Point Mutation/geneticsABSTRACT
The role of the protein TcdC in pathogenicity of the bacterium Clostridium difficile is currently unclear: conflicting reports suggest it is either a negative regulator of toxin production or, on the other hand, has no effect on virulence at all. We exploit a theoretical approach by taking what is known about the network of proteins surrounding toxin production by C. difficile and translating this into a mathematical model. From there it is possible to investigate a range of possible interactions (using numerical and asymptotic analyses), identifying properties of TcdC which would make it a realistic candidate as a toxin inhibitor. Our findings imply that if TcdC is really an inhibitor of toxin production then TcdC production should be at least as fast as that of the protein TcdR and TcdC should remain in the cells throughout growth. These are experimentally-testable hypotheses and are equally applicable to alternative candidates for toxin production inhibition.
Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/metabolism , Bacterial Toxins/biosynthesis , Clostridioides difficile/metabolism , Clostridioides difficile/pathogenicity , Enterotoxins/biosynthesis , Models, Biological , Repressor Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Clostridioides difficile/genetics , Enterotoxins/genetics , Gene Regulatory Networks , Genes, Bacterial , Genomic Islands , Humans , Mathematical Concepts , Nonlinear Dynamics , Repressor Proteins/genetics , Virulence/genetics , Virulence/physiologyABSTRACT
Germination, the process by which dormant endospores return to vegetative growth, is a critical process in the life cycle of the notorious pathogen Clostridium botulinum. Crucial is the degradation by hydrolytic enzymes of an inner peptidoglycan spore layer termed the cortex. Two mechanistically different systems of cortex lysis exist in spores of Clostridium species. C. botulinum ATCC 3502 harbours the Bacillus-like system of SleB, CwlJ and YpeB cortex lytic enzymes (CLEs). Through the construction of insertional gene knockout mutants in the sleB, cwlJ and ypeB genes of C. botulinum ATCC 3502 and the production of spores of each mutant strain, the effect on germination was assessed. This study demonstrates a reduced germination efficiency in spores carrying mutations in either sleB or ypeB with an approximate 2-fold reduction in heat resistant colony forming units (CFU/OD600) when plated on rich media. This reduction could be restored to wild-type levels by removing the spore coat and plating on media supplemented with lysozyme. It was observed that cwlJ spores displayed a similar germination efficiency as wild-type spores (P > 0.05). An optimal germinant commixture was identified to include a combination of l-alanine with sodium bicarbonate as it resulted in a 32% drop in OD600, while the additional incorporation of l-lactate resulted in a 57% decrease. Studies of the germination efficiency of spores prepared from all three CLE mutants was performed by monitoring the associated decrease in optical density but a germination defect was not observed in any of the CLE mutant strains. This was likely due to the lack of specificity of this particular assay. Taken together, these data indicate that functional copies of SleB and YpeB, but not CwlJ are required for the optimal germination of the spores of C. botulinum ATCC 3502.
Subject(s)
Bacterial Proteins/metabolism , Clostridium botulinum/growth & development , Genes, Bacterial , Hydrolases/metabolism , Spores, Bacterial/growth & development , Bacterial Proteins/genetics , Cell Wall/metabolism , Gene Knockout Techniques , Hydrolases/genetics , Mutagenesis, Insertional , Peptidoglycan/metabolism , Spores, Bacterial/geneticsABSTRACT
Clostridium difficile infection is the main cause of healthcare-acquired diarrhea in the developed world. In addition to the main virulence factors toxin A and B, epidemic, PCR Ribotype 027 strains, such as R20291, produce a third toxin, CDT. To develop effective medical countermeasures, it is important to understand the importance of each toxin. Accordingly, we created all possible combinations of isogenic toxin mutants of R20291 and assessed their virulence. We demonstrated that either toxin A or toxin B alone can cause fulminant disease in the hamster infection model and present tantalizing data that C. difficile toxin may also contribute to virulence.
Subject(s)
ADP Ribose Transferases/physiology , Bacterial Proteins/physiology , Clostridioides difficile/pathogenicity , Clostridium Infections/microbiology , Enterotoxins/physiology , ADP Ribose Transferases/genetics , Animals , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Cell Death , Clostridioides difficile/genetics , Clostridium Infections/pathology , Cricetinae , Enterotoxins/genetics , Female , HT29 Cells , Humans , Mesocricetus , Virulence/genetics , Virulence/physiologyABSTRACT
Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms.
Subject(s)
Chromosomes, Bacterial , Plasmids , Transformation, Bacterial , Alcohol Dehydrogenase/genetics , Alcohol Dehydrogenase/metabolism , Bacteriophage lambda/genetics , Base Sequence , Clostridium acetobutylicum/genetics , DNA/chemistry , Genetic Markers , Genome, Viral , Molecular Sequence Data , Orotate Phosphoribosyltransferase/genetics , Promoter Regions, GeneticABSTRACT
Clostridium difficile causes a potentially fatal diarrheal disease through the production of its principal virulence factors, toxin A and toxin B. The tcdC gene is thought to encode a negative regulator of toxin production. Therefore, increased toxin production, and hence increased virulence, is often inferred in strains with an aberrant tcdC genotype. This report describes the first allele exchange system for precise genetic manipulation of C. difficile, using the codA gene of Escherichia coli as a heterologous counterselection marker. It was used to systematically restore the Δ117 frameshift mutation and the 18-nucleotide deletion that occur naturally in the tcdC gene of C. difficile R20291 (PCR ribotype 027). In addition, the naturally intact tcdC gene of C. difficile 630 (PCR ribotype 012) was deleted and then subsequently restored with a silent nucleotide substitution, or "watermark," so the resulting strain was distinguishable from the wild type. Intriguingly, there was no association between the tcdC genotype and toxin production in either C. difficile R20291 or C. difficile 630. Therefore, an aberrant tcdC genotype does not provide a broadly applicable rationale for the perceived notion that PCR ribotype 027 strains are "high-level" toxin producers. This may well explain why several studies have reported that an aberrant tcdC gene does not predict increased toxin production or, indeed, increased virulence.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Toxins/biosynthesis , Clostridioides difficile/genetics , Clostridioides difficile/pathogenicity , Repressor Proteins/genetics , Repressor Proteins/metabolism , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Escherichia coli/genetics , Frameshift Mutation , Gene Deletion , Genetics, Microbial/methods , Genotype , Molecular Sequence Data , Mutagenesis, Insertional , Recombination, Genetic , Sequence Analysis, DNAABSTRACT
BACKGROUND: Clostridium difficile is the major cause of antibiotic associated diarrhoea and in recent years its increased prevalence has been linked to the emergence of hypervirulent clones such as the PCR-ribotype 027. Characteristically, C. difficile infection (CDI) occurs after treatment with broad-spectrum antibiotics, which disrupt the normal gut microflora and allow C. difficile to flourish. One of the relatively unique features of C. difficile is its ability to ferment tyrosine to para-cresol via the intermediate para-hydroxyphenylacetate (p-HPA). P-cresol is a phenolic compound with bacteriostatic properties which C. difficile can tolerate and may provide the organism with a competitive advantage over other gut microflora, enabling it to proliferate and cause CDI. It has been proposed that the hpdBCA operon, rarely found in other gut microflora, encodes the enzymes responsible for the conversion of p-HPA to p-cresol. RESULTS: We show that the PCR-ribotype 027 strain R20291 quantitatively produced more p-cresol in-vitro and was significantly more tolerant to p-cresol than the sequenced strain 630 (PCR-ribotype 012). Tyrosine conversion to p-HPA was only observed under certain conditions. We constructed gene inactivation mutants in the hpdBCA operon in strains R20291 and 630Δerm which curtails their ability to produce p-cresol, confirming the role of these genes in p-cresol production. The mutants were equally able to tolerate p-cresol compared to the respective parent strains, suggesting that tolerance to p-cresol is not linked to its production. CONCLUSIONS: C. difficile converts tyrosine to p-cresol, utilising the hpdBCA operon in C. difficile strains 630 and R20291. The hypervirulent strain R20291 exhibits increased production of and tolerance to p-cresol, which may be a contributory factor to the virulence of this strain and other hypervirulent PCR-ribotype 027 strains.
Subject(s)
Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/toxicity , Clostridioides difficile/drug effects , Clostridioides difficile/metabolism , Cresols/metabolism , Cresols/toxicity , Drug Resistance, Bacterial , Gene Knockout Techniques , Genes, Bacterial , Humans , Metabolic Networks and Pathways/genetics , Operon , Tyrosine/metabolismABSTRACT
The impact of Clostridium difficile-associated disease (CDAD) in healthcare settings throughout the developed world is considerable in terms of mortality, morbidity, and disease management. The incidence of CDAD has risen dramatically since the turn of this century, concomitant with the emergence of so-called hypervirulent strains which are thought to cause a more severe disease, higher relapse rates, and increased mortality. Pre-eminent amongst hypervirulent strains are those belonging to ribotype 027, which were first reported in Canada in 2003 and shortly thereafter in the UK. Since its arrival in Europe, it has spread rapidly and has now been reported in 16 member states and Switzerland. The physiological factors responsible for the rapid emergence of hypervirulent C. difficile strains remain unclear. It is known that they produce a binary toxin (CDT) in addition to toxins A and B, that they are resistant to fluoroquinolones due to mutations in gyrA, and that they are resistant to erythromycin. Representative strains have been suggested to produce more toxin A and B in the 'laboratory flask' (most likely due to a frameshift mutation in the repressor gene tcdC), to be more prolific in terms of spore formation, and also exhibit increased adherence to human intestinal epithelial cells due to altered surface proteins. However, the contribution of these and other as yet unidentified factors to the rapid spread of certain C. difficile variants (e.g., ribotypes 027 and 078) remains unclear at present. The advent of ClosTron technology means that it is now possible to construct genetically stable isogenic mutants of C. difficile and carry out reverse genetic studies to elucidate the role of specific gene loci in causing disease. The identification of virulence factors using this approach should help lead to the rational development of therapeutic countermeasures against CDAD.
Subject(s)
Clostridioides difficile/pathogenicity , Enterocolitis, Pseudomembranous/epidemiology , Evolution, Molecular , Virulence Factors/genetics , Clostridioides difficile/genetics , Clostridioides difficile/metabolism , Enterocolitis, Pseudomembranous/microbiology , Humans , Introns/genetics , Mutation , Ribotyping , Virulence , Virulence Factors/metabolismABSTRACT
Understanding the molecular basis of Clostridium difficile infection is a prerequisite to the development of effective countermeasures. Although there are methods for constructing gene-specific mutants of C. difficile, currently there is no effective method for generating libraries of random mutants. In this study, we developed a novel mariner-based transposon system for in vivo random mutagenesis of C. difficile R20291, the BI/NAP1/027 epidemic strain at the center of the C. difficile outbreaks in Stoke Mandeville, United Kingdom, in 2003 to 2004 and 2004 to 2005. Transposition occurred at a frequency of 4.5 (+/-0.4) x 10(-4) per cell to give stable insertions at random genomic loci, which were defined only by the nucleotide sequence TA. Furthermore, mutants with just a single transposon insertion were generated in an overwhelming majority (98.3% in this study). Phenotypic screening of a C. difficile R20291 random mutant library yielded a sporulation/germination-defective clone with an insertion in the germination-specific protease gene cspBA and an auxotroph with an insertion in the pyrimidine biosynthesis gene pyrB. These results validate our mariner-based transposon system for use in forward genetic studies of C. difficile.
Subject(s)
Clostridioides difficile/genetics , DNA Transposable Elements , DNA-Binding Proteins/genetics , Mutagenesis, Insertional/methods , Transposases/genetics , Base Sequence , Chromosome Mapping , Clostridioides difficile/pathogenicity , Clostridioides difficile/physiology , DNA Primers/genetics , DNA, Bacterial/genetics , Enterocolitis, Pseudomembranous/microbiology , Gene Library , Genes, Bacterial , Humans , Phenotype , Plasmids/geneticsABSTRACT
A number of poultry probiotics contain bacterial spores. In this study, orally administered spores of Bacillus subtilis germinated in the gastrointestinal (GI) tracts of chicks. Furthermore, 20 h after spores were administered, vegetative cells outnumbered spores throughout the GI tract. This demonstrates that spore-based probiotics may function in this host through metabolically active mechanisms.
Subject(s)
Bacillus subtilis/growth & development , Food Microbiology , Gastrointestinal Tract/microbiology , Probiotics , Administration, Oral , Animal Feed/microbiology , Animals , Bacillus subtilis/genetics , Chickens , Colony Count, Microbial , RNA, Bacterial/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Spores, Bacterial/genetics , Spores, Bacterial/growth & development , Time FactorsABSTRACT
Progress in exploiting clostridial genome information has been severely impeded by a general lack of effective methods for the directed inactivation of specific genes. Those few mutants that have been generated have been almost exclusively derived by single crossover integration of a replication-deficient or defective plasmid by homologous recombination. The mutants created are therefore unstable. Here we have adapted a mutagenesis system based on the mobile group II intron from the ltrB gene of Lactococcus lactis (Ll.ltrB) to function in clostridial hosts. Integrants are readily selected on the basis of acquisition of resistance to erythromycin, and are generated from start to finish in as little as 10 to 14 days. Unlike single crossover plasmid integrants, the mutants are extremely stable. The system has been used to make 6 mutants of Clostridium acetobutylicum and 5 of Clostridium difficile, exceeding the number of published mutants ever generated in these species. Genes have also been inactivated for the first time in Clostridium botulinum and Clostridium sporogenes, suggesting the system will be universally applicable to the genus. The procedure is highly efficient and reproducible, and should revolutionize functional genomic studies in clostridia.
Subject(s)
Clostridium/genetics , Mutagenesis, Insertional/genetics , Recombination, Genetic , Bacterial Proteins/genetics , Base Sequence , Conjugation, Genetic/genetics , Gene Deletion , Introns , Isopropyl Thiogalactoside/genetics , Lactococcus lactis/genetics , Mutagenesis, Insertional/methods , Plasmids/genetics , Promoter Regions, Genetic , RNA-Directed DNA Polymerase/genetics , Retroelements/geneticsABSTRACT
Mutant strains of Clostridium botulinum ATCC 3502 were generated using the ClosTron in four genes (CBO1789, CBO1790, CBO3048, CBO3145) identified as encoding α/ß-type SASP homologues. The spores of mutant strains in which CBO1789 or CBO1790 was inactivated demonstrated a significant increase in sensitivity to the damaging agent nitrous acid (P<0.01), a phenotype that was partially restored to wild-type in complementation studies. In contrast to nitrous acid, the spores of the CBO1789 and CBO1790 mutants showed no change in their resistance to formaldehyde and hydrogen peroxide (P>0.05), two other chemicals commonly used as components of disinfection regimes. These data indicate that the SASPs CBO1789 or CBO1790 play a significant role in resistance to nitrous acid, but not in resistance to formaldehyde or hydrogen peroxide.
Subject(s)
Clostridium botulinum/drug effects , Disinfectants/pharmacology , Formaldehyde/pharmacology , Hydrogen Peroxide/pharmacology , Nitrous Acid/pharmacology , Spores, Bacterial/drug effects , Bacterial Proteins/genetics , Botulism/microbiology , Botulism/prevention & control , Clostridium botulinum/genetics , Clostridium botulinum/metabolism , Drug Resistance, Bacterial/genetics , Foodborne Diseases/microbiology , Foodborne Diseases/prevention & control , Gene Knockout Techniques , Spores, Bacterial/genetics , Spores, Bacterial/metabolismABSTRACT
BACKGROUND: Clostridium acetobutylicum represents a paradigm chassis for the industrial production of the biofuel biobutanol and a focus for metabolic engineering. We have previously developed procedures for the creation of in-frame, marker-less deletion mutants in the pathogen Clostridium difficile based on the use of pyrE and codA genes as counter selection markers. In the current study we sought to test their suitability for use in C. acetobutylicum. RESULTS: Both systems readily allowed the isolation of in-frame deletions of the C. acetobutylicum ATCC 824 spo0A and the cac824I genes, leading to a sporulation minus phenotype and improved transformation, respectively. The pyrE-based system was additionally used to inactivate a putative glycogen synthase (CA_C2239, glgA) and the pSOL1 amylase gene (CA_P0168, amyP), leading to lack of production of granulose and amylase, respectively. Their isolation provided the opportunity to make use of one of the key pyrE system advantages, the ability to rapidly complement mutations at appropriate gene dosages in the genome. In both cases, their phenotypes were restored in terms of production of granulose (glgA) and amylase (amyP). Genome re-sequencing of the ATCC 824 COSMIC consortium laboratory strain used revealed the presence of 177 SNVs and 49 Indels, including a 4916-bp deletion in the pSOL1 megaplasmid. A total of 175 SNVs and 48 Indels were subsequently shown to be present in an 824 strain re-acquired (Nov 2011) from the ATCC and are, therefore, most likely errors in the published genome sequence, NC_003030 (chromosome) and NC_001988 (pSOL1). CONCLUSIONS: The codA or pyrE counter selection markers appear equally effective in isolating deletion mutants, but there is considerable merit in using a pyrE mutant as the host as, through the use of ACE (Allele-Coupled Exchange) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Our study also revealed a surprising number of errors in the ATCC 824 genome sequence, while at the same time emphasising the need to re-sequence commonly used laboratory strains.
ABSTRACT
The LexA regulated SOS network is a bacterial response to DNA damage of metabolic or environmental origin. In Clostridium difficile, a nosocomial pathogen causing a range of intestinal diseases, the in-silico deduced LexA network included the core SOS genes involved in the DNA repair and genes involved in various other biological functions that vary among different ribotypes. Here we describe the construction and characterization of a lexA ClosTron mutant in C. difficile R20291 strain. The mutation of lexA caused inhibition of cell division resulting in a filamentous phenotype. The lexA mutant also showed decreased sporulation, a reduction in swimming motility, greater sensitivity to metronidazole, and increased biofilm formation. Changes in the regulation of toxin A, but not toxin B, were observed in the lexA mutant in the presence of sub-inhibitory concentrations of levofloxacin. C. difficile LexA is, therefore, not only a regulator of DNA damage but also controls many biological functions associated with virulence.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biofilms/growth & development , Clostridioides difficile/physiology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Anti-Bacterial Agents/pharmacology , Bacterial Toxins/metabolism , Biofilms/drug effects , Cell Division , Clostridioides difficile/drug effects , Clostridioides difficile/genetics , Enterotoxins/metabolism , Gene Expression Regulation, Bacterial/drug effects , Levofloxacin/pharmacology , Mutation , SOS Response, Genetics , Spores, Bacterial/drug effects , Spores, Bacterial/growth & developmentABSTRACT
Sophisticated genetic tools to modify essential biological processes at the molecular level are pivotal in elucidating the molecular pathogenesis of Clostridium difficile, a major cause of healthcare associated disease. Here we have developed an efficient procedure for making precise alterations to the C. difficile genome by pyrE-based allelic exchange. The robustness and reliability of the method was demonstrated through the creation of in-frame deletions in three genes (spo0A, cwp84, and mtlD) in the non-epidemic strain 630Δerm and two genes (spo0A and cwp84) in the epidemic PCR Ribotype 027 strain, R20291. The system is reliant on the initial creation of a pyrE deletion mutant, using Allele Coupled Exchange (ACE), that is auxotrophic for uracil and resistant to fluoroorotic acid (FOA). This enables the subsequent modification of target genes by allelic exchange using a heterologous pyrE allele from Clostridium sporogenes as a counter-/negative-selection marker in the presence of FOA. Following modification of the target gene, the strain created is rapidly returned to uracil prototrophy using ACE, allowing mutant phenotypes to be characterised in a PyrE proficient background. Crucially, wild-type copies of the inactivated gene may be introduced into the genome using ACE concomitant with correction of the pyrE allele. This allows complementation studies to be undertaken at an appropriate gene dosage, as opposed to the use of multicopy autonomous plasmids. The rapidity of the 'correction' method (5-7 days) makes pyrE(-) strains attractive hosts for mutagenesis studies.
Subject(s)
Bacterial Proteins/genetics , Clostridioides difficile/genetics , Genetic Vectors/genetics , Genome, Bacterial/genetics , Alleles , Clostridioides difficile/growth & development , DNA, Bacterial/genetics , Genetic Complementation Test , Phenotype , Sequence DeletionABSTRACT
Clostridium difficile is a major cause of healthcare-associated infection and inflicts a considerable financial burden on healthcare systems worldwide. Disease symptoms range from self-limiting diarrhoea to fatal pseudomembranous colitis. Whilst C. difficile has two major virulence factors, toxin A and B, it is generally accepted that other virulence components of the bacterium contribute to disease. C. difficile colonises the gut of humans and animals and hence the processes of adherence and colonisation are essential for disease onset. Previously it has been suggested that flagella might be implicated in colonisation. Here we tested this hypothesis by comparing flagellated parental strains to strains in which flagella genes were inactivated using ClosTron technology. Our focus was on a UK-outbreak, PCR-ribotype 027 (B1/NAP1) strain, R20291. We compared the flagellated wild-type to a mutant with a paralyzed flagellum and also to mutants (fliC, fliD and flgE) that no longer produce flagella in vitro and in vivo. Our results with R20291 provide the first strong evidence that by disabling the motor of the flagellum, the structural components of the flagellum rather than active motility, is needed for adherence and colonisation of the intestinal epithelium during infection. Comparison to published data on 630Δerm and our own data on that strain revealed major differences between the strains: the R20291 flagellar mutants adhered less than the parental strain in vitro, whereas we saw the opposite in 630Δerm. We also showed that flagella and motility are not needed for successful colonisation in vivo using strain 630Δerm. Finally we demonstrated that in strain R20291, flagella do play a role in colonisation and adherence and that there are striking differences between C. difficile strains. The latter emphasises the overriding need to characterize more than just one strain before drawing general conclusions concerning specific mechanisms of pathogenesis.
Subject(s)
Clostridioides difficile/pathogenicity , Enterocolitis, Pseudomembranous/microbiology , Flagella/physiology , Bacterial Toxins/metabolism , Clostridioides difficile/classification , Clostridioides difficile/metabolism , Enterocolitis, Pseudomembranous/epidemiology , Enterocolitis, Pseudomembranous/physiopathology , Humans , Intestinal Mucosa/microbiology , Species SpecificityABSTRACT
Clostridium difficile spores play a pivotal role in the transmission of infectious diarrhoea, but in order to cause disease spores must complete germination and return to vegetative cell growth. While the mechanisms of spore germination are well understood in Bacillus, knowledge of C. difficile germination remains limited. Previous studies have shown that bile salts and amino acids play an important role in regulating the germination response of C. difficile spores. Taurocholate, in combination with glycine, can stimulate germination, whereas chenodeoxycholate has been shown to inhibit spore germination in a C. difficile clinical isolate. Our recent studies of C. difficile sporulation characteristics have since pointed to substantial diversity among different clinical isolates. Consequently, in this study we investigated how the germination characteristics of different C. difficile isolates vary in response to bile salts. By analysing 29 isolates, including 16 belonging to the BI/NAP1/027 type, we show that considerable diversity exists in both the rate and extent of C. difficile germination in response to rich medium containing both taurocholate and glycine. Strikingly, we also show that although a potent inhibitor of germination for some isolates, chenodeoxycholate does not inhibit the germination, or outgrowth, of all C. difficile strains. Finally, we provide evidence that components of rich media may induce the germination of C. difficile spores, even in the absence of taurocholate. Taken together, these data suggest that the mechanisms of C. difficile spore germination in response to bile salts are complex and require further study. Furthermore, we stress the importance of studying multiple isolates in the future when analysing the nutrients or chemicals that either stimulate or inhibit C. difficile spore germination.
Subject(s)
Bile Acids and Salts/pharmacology , Clostridioides difficile/metabolism , Spores, Bacterial/drug effects , Spores, Bacterial/metabolism , Chenodeoxycholic Acid/pharmacology , Gastrointestinal Agents/pharmacology , Glycine/chemistry , Species Specificity , Stem Cells , Taurocholic Acid/pharmacology , Time FactorsABSTRACT
Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and a major burden to healthcare services worldwide. In recent years, C. difficile strains belonging to the BI/NAP1/027 type have become highly represented among clinical isolates. These so-called 'hypervirulent' strains are associated with outbreaks of increased disease severity, higher relapse rates and an expanded repertoire of antibiotic resistance. Spores, formed during sporulation, play a pivotal role in disease transmission and it has been suggested that BI/NAP1/027 strains are more prolific in terms of sporulation in vitro than 'non-epidemic' C. difficile types. Work in our laboratory has since provided credible evidence to the contrary suggesting that the strain-to-strain variation in C. difficile sporulation characteristics is not type-associated. However, the BI/NAP1/027 type is still widely stated to have an increased rate of sporulation. In this study, we analysed the sporulation rates of 53 C. difficile strains, the largest sample size used to-date in such a study, including 28 BI/NAP1/027 isolates. Our data confirm that significant variation exists in the rate at which different C. difficile strains form spores. However, we clearly show that the sporulation rate of the BI/NAP1/027 type was no higher than that of non-BI/NAP1/027 strains. In addition, we observed substantial variation in sporulation characteristics within the BI/NAP1/027 type. This work highlights the danger of assuming that all strains of one type behave similarly without studying adequate sample sizes. Furthermore, we stress the need for more rigorous experimental procedures in order to quantify C. difficile sporulation more accurately in the future.