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1. Lipoproteins of bacterial pathogens.

Kovacs-Simon A, Titball RW, Michell SL.

Room 416, School of Biosciences, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter, Devon, United Kingdom EX4 4QD.

Bacterial lipoproteins are a set of membrane proteins with many different functions. Due to this broad-ranging functionality, these proteins have a considerable significance in many phenomena, from cellular physiology through cell division and virulence. Here we give a general overview of lipoprotein biogenesis and highlight examples of the roles of lipoproteins in bacterial disease caused by a selection of medically relevant Gram-negative and Gram-positive pathogens: Mycobacterium tuberculosis, Streptococcus pneumoniae, Borrelia burgdorferi, and Neisseria meningitidis. Lipoproteins have been shown to play key roles in adhesion to host cells, modulation of inflammatory processes, and translocation of virulence factors into host cells. As such, a number of lipoproteins have been shown to be potential vaccines. This review provides a summary of some of the reported roles of lipoproteins and of how this knowledge has been exploited in some cases for the generation of novel countermeasures to bacterial diseases.

Infect Immun. 2011 Feb;79(2):548-61. doi: 10.1128/IAI.00682-10. Epub 2010 Oct 25
PMID: 20974828

2. Molecular basis of toxicity of Clostridium perfringens epsilon toxin.

Bokori-Brown M, Savva CG, Fernandes da Costa SP, Naylor CE, Basak AK, Titball RW.

Biosciences, College of Life and Environmental Sciences, University of Exeter, UK Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, London, UK.

Clostridium perfringens ε-toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated with enteritis and enterotoxaemia in goats, calves and foals. It is considered to be a potential biowarfare or bioterrorism agent by the US Government Centers for Disease Control and Prevention. The relatively inactive 32.9 kDa prototoxin is converted to active mature toxin by proteolytic cleavage, either by digestive proteases of the host, such as trypsin and chymotrypsin, or by C.perfringens λ-protease. In vivo, the toxin appears to target the brain and kidneys, but relatively few cell lines are susceptible to the toxin, and most work has been carried out using Madin-Darby canine kidney (MDCK) cells. The binding of ε-toxin to MDCK cells and rat synaptosomal membranes is associated with the formation of a stable, high molecular weight complex. The crystal structure of ε-toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin-2 from Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these toxins, ε-toxin appears to form heptameric pores in target cell membranes. The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells.

FEBS J. 2011 Dec;278(23):4589-601. doi: 10.1111/j.1742-4658.2011.08140.x. Epub 2011 May 19.
PMID: 21518257

3. Reconsidering the Sporulation Characteristics of Hypervirulent Clostridium difficile BI/NAP1/027

DA Burns*, D Heeg*, ST Cartman and NP Minton (joint first authorship)

Clostridia Research Group, School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom

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.

2011;6(9):e24894. doi: 10.1371/journal.pone.0024894. Epub 2011 Sep 15
PMID: 21949780

4. Spores of Clostridium difficile clinical isolates display a diverse germination response to bile salts.

D Heeg
*, DA Burns*, ST Cartman and NP Minton
*joint first author

Clostridia Research Group, School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom

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.

PLoS One. 2012;7(2):e32381. doi: 10.1371/journal.pone.0032381. Epub 2012 Feb 22

PMID: 22384234

5. Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC genotype and toxin production.

ST Cartman, ML Kelly, D Heeg, JT Heap, and NP Minton

Clostridia Research Group, School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom

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.

Appl Environ Microbiol. 2012 Jul;78(13):4683-90. doi: 10.1128/AEM.00249-12. Epub 2012 Apr 20
PMID: 22522680

6. Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation.

Kirk DG, Dahlsten E, Zhang Z, Korkeala H, Lindström M.

Department of Food Hygiene and Environmental Health, Centre of Excellence in Microbial Food Safety Research, Faculty of Veterinary Medicine, University of Helsinki, Finland.

A key survival mechanism of Clostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organism Bacillus subtilis, nothing is known about these mechanisms in C. botulinum. Using the ClosTron gene-knockout tool, sigK, encoding late-stage (stage IV) sporulation sigma factor K in B. subtilis, was disrupted in C. botulinum ATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast, sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis of sigK in the parent strain revealed expression at the late log growth phase in the parent strain. Analysis of spo0A, encoding the sporulation master switch, in the sigK mutant and the parent showed significantly reduced relative levels of spo0A expression in the sigK mutant compared to the parent strain. Similarly, sigF showed significantly lower relative transcription levels in the sigK mutant than the parent strain, suggesting that the sporulation pathway was blocked in the sigK mutant at an early stage. We conclude that σ(K) is essential for early-stage sporulation in C. botulinum ATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organism B. subtilis. Understanding the sporulation mechanism of C. botulinum provides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.

Appl Environ Microbiol. 2012 Jul;78(13):4590-6. doi: 10.1128/AEM.00304-12. Epub 2012 Apr 27
PMID: 22544236

7. Effect of iron limitation and fur gene inactivation on the transcriptional profile of the strict anaerobe Clostridium acetobutylicum.

Vasileva D, Janssen H, Hönicke D, Ehrenreich A, Bahl H.

Abteilung Mikrobiologie, Institut für Biowissenschaften, Universität Rostock, Albert-Einstein-Str. 3, D-18051 Rostock, Germany.

Iron is a nutrient of critical importance for the strict anaerobe Clostridium acetobutylicum, as it is involved in numerous basic cellular functions and metabolic pathways. A gene encoding a putative ferric uptake regulator (Fur) has been identified in the genome of C. acetobutylicum. In this work, we inactivated the fur gene by using insertional mutagenesis. The resultant mutant showed a slow-growing phenotype and enhanced sensitivity to oxidative stress, but essentially no dramatic change in its fermentation pattern. A unique feature of its physiology was the overflowing production of riboflavin. To gain further insights into the role of the Fur protein and the mechanisms for establishment of iron balance in C. acetobutylicum, we characterized and compared the gene-expression profile of the fur mutant and the iron-limitation stimulon of the parental strain. Not surprisingly, a repertoire of iron-transport systems was upregulated in both microarray datasets, suggesting that they are regulated by Fur according to the availability of iron. In addition, iron limitation and inactivation of fur affected the expression of several genes involved in energy metabolism. Among them, two genes, encoding a lactate dehydrogenase and a flavodoxin, were highly induced. In order to support the function of the latter, the ribDBAH operon responsible for riboflavin biosynthesis was also upregulated significantly. Furthermore, the iron-starvation response of C. acetobutylicum involved transcriptional modifications that were not detected in the fur mutant, suggesting that there exist additional mechanisms for adaptation to low-iron environments. Collectively, these results demonstrate that the strict anaerobe C. acetobutylicum senses and responds to availability of iron on multiple levels using a sophisticated system, and that Fur plays an important role in this process.

Microbiology, 2012 Jul;158(Pt 7):1918-29. doi: 10.1099/mic.0.056978-0. Epub 2012 May 3

PMID: 22556358

8. Analysis of metronidazole susceptibility in different Clostridium difficile PCR-ribotypes
Moura I, Spigaglia P, Barbanti F, Mastrantonio P,

Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy

Objectives: Susceptibility to metronidazole was investigated in 81 Clostridium difficile strains, belonging to 9 different PCR-ribotypes, by three different laboratory methods.
Methods: Minimum inhibitory concentrations (MICs) of 81 C. difficile clinical isolates were determined by Etest, agar dilution method (ADM) and agar incorporation method (AIM). Twenty selected strains were also subjected to sub-inhibitory concentrations of metronidazole and the MIC heterogeneity was analysed in colonies from each strain that showed increased values before and after exposure to the antibiotic, using ADM and AIM.
Results: Overall, the MICs obtained by Etest were lower compared to those obtained using ADM and AIM, determining discrepancies in the categorization, as susceptible or with reduced susceptibility, of some strains. Reduced susceptibility to metronidazole was observed by both ADM and AIM, with higher MIC values by AIM, in isolates belonging to PCR-ribotypes 001 and 010. An increase of MIC values after exposure to metronidazole was observed for strains belonging to these PCR-ribotypes by Etest and ADM, but not by AIM. In particular, MICs of colonies from strains belonging to either PCR-ribotype 001 or 010 were less heterogenic by AIM compared to ADM, suggesting a better ability of AIM in detecting strains with reduced susceptibility.
Conclusions: These results suggest that the presence of C. difficile sub-populations with reduced susceptibility to metronidazole in the human intestine may be one of the several factors responsible for reduced antibiotic efficacy in vivo. The possibility that higher MICs may have often gone unnoticed underlines the importance of choosing the best method for MICs determination and the necessity to monitor C. difficile susceptibility to this antibiotic.

J Antimicrob Chemother. 2013 Feb;68(2):362-5. doi: 10.1093/jac/dks420. Epub 2012 Oct 26
PMID: 23104495

9. Two-Component Signal Transduction System CBO0787/CBO0786 Represses Transcription from Botulinum Neurotoxin Promoters in Clostridium botulinum ATCC 3502

Zhen Zhang, Hannu Korkeala, Elias Dahlsten, Elina Sahala, John T. Heap, Nigel P. Minton, Miia Lindström.

Department of Food Hygiene and Environmental Health, Centre of Excellence in Microbial Food Safety Research, Faculty of Veterinary Medicine, University of Helsinki, Finland

Blocking neurotransmission, botulinum neurotoxin is the most poisonous biological substance known to mankind. Despite its infamy as the scourge of the food industry, the neurotoxin is increasingly used as a pharmaceutical to treat an expanding range of muscle disorders. Whilst neurotoxin expression by the spore-forming bacterium Clostridium botulinum appears tightly regulated, to date only positive regulatory elements, such as the alternative sigma factor BotR, have been implicated in this control. The identification of negative regulators has proven to be elusive. Here, we show that the two-component signal transduction system CBO0787/CBO0786 negatively regulates botulinum neurotoxin expression. Single insertional inactivation of cbo0787 encoding a sensor histidine kinase, or of cbo0786 encoding a response regulator, resulted in significantly elevated neurotoxin gene expression levels and increased neurotoxin production. Recombinant CBO0786 regulator was shown to bind to the conserved -10 site of the core promoters of the ha and ntnh-botA operons, which encode the toxin structural and accessory proteins. Increasing concentration of CBO0786 inhibited BotR-directed transcription from the ha and ntnh-botA promoters, demonstrating direct transcriptional repression of the ha and ntnh-botA operons by CBO0786. Thus, we propose that CBO0786 represses neurotoxin gene expression by blocking BotR-directed transcription from the neurotoxin promoters. This is the first evidence of a negative regulator controlling botulinum neurotoxin production. Understanding the neurotoxin regulatory mechanisms is a major target of the food and pharmaceutical industries alike.

PLoS Pathog. 2013 Mar;9(3):e1003252. doi: 10.1371/journal.ppat.1003252. Epub 2013 Mar 28

10. A two-component system controls the expression of genes encoding CBM6-containing proteins in response to straw in Clostridium cellulolyticum.

Hamza Celik , Jean-Charles Blouzard , Birgit Voigt , Dörte Becher , Valentine Trotter, Henri-Pierre Fierobe , Chantal Tardif , Sandrine Pagés , Pascale de Philip

Laboratoire de Chimie Bactérienne, IMM-CNRS-UMR7283, University of Aix-Marseille, Marseille, France

The composition of the cellulosomes (multi enzymatic complexes involved in the degradation of plant cell wall polysaccharides) produced by Clostridium cellulolyticum differs according to the growth substrate. In particular, the expression of a cluster of 14 hemicellulase-encoding genes (called xyl-doc) seems to be induced by the presence of straw and not of cellulose. Genes encoding a putative two-component regulation system (XydS/R) were found upstream of xyldoc.
First evidence for the involvement of the response regulator, XydR, part of this two-component system, in the expression of xyl-doc genes was given by the analysis of the cellulosomes produced by a regulator overproducing strain when grown on cellulose. Nano-LC MS/MS analysis allowed the detection of the products of all xyl-doc genes and of the
product of the gene at locus Ccel_1656 predicted to bear a carbohydrate binding domain targeting hemicellulose. RT-PCR experiments further demonstrated that the regulation occurs at the transcriptional level and that all xyl-doc genes are transcriptionally linked. mRNA quantification in a regulator knock-out strain and in its complemented derivative confirmed the involvement of the regulator in the expression of xyl-doc genes and of the gene at locus Ccel_1656 in response to straw.
Electrophoretic mobility shift assays using the purified regulator further demonstrated that the regulator binds to DNA regions located upstream of the first gene of the xyl-doc gene cluster and upstream of the gene at locus Ccel_1656.

PLoS One. 2013;8(2):e56063. doi: 10.1371/journal.pone.0056063. Epub 2013 Feb 13
PMID: 23418511

11. Molecular architecture and functional analysis of NetB, a pore-forming toxin from Clostridium perfringens.

Savva CG*, Fernandes da Costa SP*, Bokori-Brown M, Naylor CE, Cole AR, Moss DS, Titball RW, Basak AK. (* joint first authorship)

Biosciences, College of Life and Environmental Sciences, University of Exeter, UK Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, London, UK.

NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore-form of NetB solved to 3.9Å. The heptameric assembly shares structural homology to the Staphylococcal α-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerisation and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology.

J Biol Chem. 2013 Feb 1;288(5):3512-22. doi: 10.1074/jbc.M112.430223. Epub 2012 Dec 13
PMID: 23239883

12. Plasmid-borne type E neurotoxin gene clusters in Clostridium botulinum strains.

Zhang Z, Hintsa H, Chen Y, Korkeala H, Lindström M.

Department of Food Hygiene and Environmental Health, Centre of Excellence in Microbial Food Safety Research, Faculty of Veterinary Medicine, University of Helsinki, Finland.

A collection of 36 Clostridium botulinum type E strains was examined by pulsed-field gel electrophoresis (PFGE) and Southern hybridization with probes targeted to botE and orfX1 in the neurotoxin gene cluster. Three strains were found to encode neurotoxin subtype E1 gene clusters in large plasmids of about 146 kb in size.
Appl Environ Microbiol. 2013 Jun;79(12):3856-9. doi: 10.1128/AEM.00080-13. Epub 2013 Apr 5.
PMID: 23563942

13. Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies

Bokori-Brown M, Kokkinidou MC, Savva CG, Fernandes da Costa SP, Naylor CE, Cole AR, Moss DS, Basak AK, Titball RW.

Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter EX4 4QD, United Kingdom.

Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.
Protein Sci. 2013 May 22 (5):650-9. doi: 10.1002/pro.2250. Epub 2013 Apr 8
PMID: 23504825

14. Protection against avian necrotic enteritis after immunisation with NetB genetic or formaldehyde toxoids.

Fernandes da Costa SP, Mot D, Bokori-Brown M, Savva CG, Basak AK, Van Immerseel F, Titball RW.

College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, United Kingdom.

NetB (necrotic enteritis toxin B) is a recently identified β-pore-forming toxin produced by Clostridium perfringens. This toxin has been shown to play a major role in avian necrotic enteritis. In recent years, a dramatic increase in necrotic enteritis has been observed, especially in countries where the use of antimicrobial growth promoters in animal feedstuffs has been banned. The aim of this work was to determine whether immunisation with a NetB toxoid would provide protection against necrotic enteritis. The immunisation of poultry with a formaldehyde NetB toxoid or with a NetB genetic toxoid (W262A) resulted in the induction of antibody responses against NetB and provided partial protection against disease.

Vaccine. 2013 May 29 pii: S0264-410X(13)00665-8. doi: 10.1016/j.vaccine.2013.05.063. [Epub ahead of print]
PMID: 23727000

15. Multiple factors modulate biofilm formation by the anaerobic pathogen Clostridium difficile.

Ðapa T, Leuzzi R, Ng YK, Baban ST, Adamo R, Kuehne SA, Scarselli M, Minton NP, Serruto D, Unnikrishnan M.

Novartis Vaccines and Diagnostics, Siena, Italy.


Bacteria within biofilms are protected from multiple stresses, including immune responses and antimicrobial agents. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Although biofilms have been well studied for several gut pathogens, little is known about biofilm formation by anaerobic gut species. The obligate anaerobe Clostridium difficile causes C. difficile infection (CDI), a major health care-associated problem primarily due to the high incidence of recurring infections. C. difficile colonizes the gut when the normal intestinal microflora is disrupted by antimicrobial agents; however, the factors or processes involved in gut colonization during infection remain unclear. We demonstrate that clinical C. difficile strains, i.e., strain 630 and the hypervirulent strain R20291, form structured biofilms in vitro, with R20291 accumulating substantially more biofilm. Microscopic and biochemical analyses show multiple layers of bacteria encased in a biofilm matrix containing proteins, DNA, and polysaccharide. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella, and a putative quorum-sensing regulator, LuxS, are all required for maximal biofilm formation by C. difficile. Interestingly, a mutant in Spo0A, a transcription factor that controls spore formation, was defective for biofilm formation, indicating a possible link between sporulation and biofilm formation. Furthermore, we demonstrate that bacteria in clostridial biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.

J Bacteriol. 2013 Feb;195(3):545-55. doi: 10.1128/JB.01980-12. Epub 2012 Nov 21.
PMID: 23175653

16. Alternative sigma factor SigK has a role in stress tolerance of group I Clostridium botulinum ATCC 3502.

Dahlsten E, Kirk D, Lindström M, Korkeala H.
Department of Food Hygiene and Environmental Health, University of Helsinki, Helsinki, Finland.


The role of the alternative sigma factor SigK in cold and osmotic stress tolerance of Clostridium botulinum ATCC 3502 was demonstrated by induction of sigK after temperature downshift and exposure to hyperosmotic conditions and by impaired growth of the sigK mutants under the respective conditions

Appl Environ Microbiol. 2013 Jun;79(12):3867-9. doi: 10.1128/AEM.04036-12. Epub 2013 Apr 5
PMID: 23563953

17. Clostridium difficile erm(B)-containing elements and the burden on the in vitro fitness.

Wasels F, Spigaglia P, Barbanti F, Mastrantonio P.

Istituto Superiore di Sanità


In Clostridium difficile, resistance to the macrolide-lincosamide-streptogramin B group of antibiotics generally relies on erm(B) genes. In this study, we investigated elements with a genetic organisation different from Tn5398, the mobilizable non-conjugative element identified in C. difficile strain 630. Our results suggested that the elements most frequently found in strains isolated during the European Surveillance 2005 were related to Tn6194, the conjugative transposon recently detected in different C. difficile types, including PCR-ribotype 027. We characterized a Tn6194-like and a novel element rarely found in clinical isolates. A burden on the in vitro fitness of C. difficile was observed after the acquisition of these elements as well as of Tn5398.

J Med Microbiol. 2013 Sep;62(Pt 9):1461-7. doi: 10.1099/jmm.0.057117-0. Epub 2013 Jun 5

18.Biofilm formation by Clostridium difficile.

Dapa T, Unnikrishnan M.

Novartis Vaccines and Diagnostics; Siena, Italy.


Clostridium difficile infection (CDI) is a major healthcare-associated disease worldwide. Recurring infections and increasing antibiotic resistance have complicated treatment of CDI. While C. difficile spores were shown to be responsible for transmission and persistence of CDI, other factors such as gut colonization and formation of bacterial communities in the gut may also contribute to pathogenesis and persistence, but have not been well investigated. Recently, we reported that important clinical C. difficile strains are able to form composite biofilms in vitro. C. difficile biofilm formation is a complex process modulated by several different factors, including cell surface components and regulators. We also reported that bacteria within biofilms are more resistant to high concentrations of vancomycin, the antibiotic of choice for treatment of CDI. Here we summarize our recent findings and discuss the implications of biofilm formation by this anaerobic gut pathogen in disease pathogenesis and treatment.

Gut Microbes. 2013 Jul 25;4(5). [Epub ahead of print