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ABSTRACT: 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 01/2012; 7(2):e32381. · 4.09 Impact Factor
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ABSTRACT: Clostridium difficile is a leading cause of healthcare-associated diarrhoea. In recent years, certain C. difficile types have become highly represented among clinical isolates and are associated with outbreaks of increased disease severity, higher relapse rates and an expanded repertoire of antibiotic resistance. Endospores, produced during sporulation, play a pivotal role in infection and disease transmission and it has been suggested in the literature that these so-called 'hypervirulent' C. difficile types are more prolific in terms of sporulation in vitro. However, work in our laboratory has provided evidence to the contrary suggesting that although there is significant strain-to-strain variation in C. difficile sporulation characteristics this variation does not appear to be type-associated. On analysis of the literature, it is apparent that the methods used to quantify sporulation in previous studies have varied greatly and sample sizes have remained small. The conflicting data in the literature may, therefore, not necessarily be generally representative of C. difficile sporulation. Instead, these inconsistencies may reflect differences in the experimental design of each study. In this review, the need for further investigations of C. difficile sporulation rates is highlighted. Specifically, the advantages and disadvantages of the different experimental approaches previously used are discussed and a standard set of principles for measuring C. difficile sporulation in the future is proposed.
Journal of microbiological methods 08/2011; 87(2):133-8. · 2.43 Impact Factor
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ABSTRACT: 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.
PLoS ONE 01/2011; 6(9):e24894. · 4.09 Impact Factor
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ABSTRACT: Endospore production is vital for the spread of Clostridium difficile infection. However, in order to cause disease, these spores must germinate and return to vegetative cell growth. Knowledge of germination is therefore important, with potential practical implications for routine cleaning, outbreak management and potentially in the design of new therapeutics. Germination has been well studied in Bacillus, but until recently there had been few studies reported in C. difficile. The role of bile salts as germinants for C. difficile spores has now been described in some detail, which improves our understanding of how C. difficile spores interact with their environment following ingestion by susceptible individuals. Furthermore, with the aid of novel genetic tools, it has now become possible to study the germination of C. difficile spores using both a forward and reverse genetics approach. Significant progress is beginning to be made in the study of this important aspect of C. difficile disease.
Research in Microbiology 11/2010; 161(9):730-4. · 2.76 Impact Factor
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ABSTRACT: Clostridium difficile causes diarrhoeal diseases ranging from asymptomatic carriage to a fulminant, relapsing, and potentially fatal colitis. Endospore production plays a vital role in transmission of infection, and in order to cause disease these spores must then germinate and return to vegetative cell growth. Type BI/NAP1/027 strains of C. difficile have recently become highly represented among clinical isolates and are associated with increased disease severity. It has also been suggested that these 'epidemic' types generally sporulate more prolifically than 'non-epidemic' strains, although the few existing reports are inconclusive and encompass only a small number of isolates. In order to better understand any differences in sporulation rates between epidemic and non-epidemic C. difficile types, we analysed these characteristics using 14 C. difficile clinical isolates of a variety of types. Sporulation rates varied greatly between individual BI/NAP1/027 isolates, but this variation did not appear to be type-associated. Furthermore, a number of BI/NAP1/027 spores appeared to form colonies with a lower frequency than specific non-BI/NAP1/027 strains. The data suggest that (i) careful experimental design is required in order to accurately quantify sporulation; and (ii) current evidence cannot link differences in sporulation rates with the disease severity of the BI/NAP1/027 type.
Anaerobe 10/2010; 16(6):618-22. · 2.41 Impact Factor
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ABSTRACT: Clostridium difficile is the major cause of infectious diarrhea and a major burden to health care services. The ability of this organism to form endospores plays a pivotal role in infection and disease transmission. Spores are highly resistant to many forms of disinfection and thus are able to persist on hospital surfaces and disseminate infection. In order to cause disease, the spores must germinate and the organism must grow vegetatively. Spore germination in Bacillus is well understood, and genes important for this process have recently been identified in Clostridium perfringens; however, little is known about C. difficile. Apparent homologues of the spore cortex lytic enzyme genes cwlJ and sleB (Bacillus subtilis) and sleC (C. perfringens) are present in the C. difficile genome, and we describe inactivation of these homologues in C. difficile 630Delta erm and a B1/NAP1/027 clinical isolate. Spores of a sleC mutant were unable to form colonies when germination was induced with taurocholate, although decoated sleC spores formed the same number of heat-resistant colonies as the parental control, even in the absence of germinants. This suggests that sleC is absolutely required for conversion of spores to vegetative cells, in contrast to CD3563 (a cwlJ/sleB homologue), inactivation of which had no effect on germination and outgrowth of C. difficile spores under the same conditions. The B1/NAP1/027 strain R20291 was found to sporulate more slowly and produce fewer spores than 630Delta erm. Furthermore, fewer R20291 spores germinated, indicating that there are differences in both sporulation and germination between these epidemic and nonepidemic C. difficile isolates.
Journal of bacteriology 11/2009; 192(3):657-64. · 3.94 Impact Factor
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ABSTRACT: Continuous paravertebral nerve blocks can provide effective postoperative analgesia after abdominal and thoracic surgery. While offering a number of advantages compared with thoracic epidural analgesia, access to the paravertebral space using a classic approach is not always easily accomplished and/or possible. In this regard, continuous paravertebral blockade via a percutaneous intercostal approach may theoretically serve as an alternative approach to the paravertebral space.
One hundred ten patients undergoing major abdominal, thoracic, or retroperitoneal procedures had preoperative placement of unilateral or bilateral paravertebral catheter(s) via an intercostal approach. At a point 8 cm lateral to the midline a 5 cm, 18 G Tuohy needle was advanced with the needle tip angled 45 degrees cephalad and 60 degrees medial to the sagittal plane to come in contact with the lower third of the rib. The needle was "walked-off" the inferior border of the rib while maintaining its orientation and advanced a further 5 to 6 mm under the rib to lie in the subcostal groove. After injection of 5 mL ropivacaine 0.5%, a catheter was advanced medially the estimated distance to the paravertebral space. Postoperatively 0.2% ropivacaine was continuously infused at 10 mL/h in each catheter with hourly boluses of 5 mL available for breakthrough pain.
Median pain scores averaged 2 on a scale of 0-10 and patient-controlled analgesia hydromorphone consumption averaged only 1.69 mg for the first 24 h postoperatively. There were no clinically significant complications of the technique.
The intercostally placed paravertebral catheter provides postoperative analgesia after major surgery of the chest, abdomen, or retroperitoneum.
Anesthesia and analgesia 07/2008; 107(1):339-41. · 3.08 Impact Factor
