An investigation of the subtype diversity of clinical isolates of Irish Clostridium difficile ribotypes 027 and 078 by repetitive-extragenic palindromic PCR.

A repetitive-extragenic palindromic PCR (rep-PCR) subtyping method (DiversiLab) in conjunction with ribotyping, toxinotyping and antimicrobial-susceptibility testing was used to detect subtypes within Clostridium difficile ribotypes 027 and 078. Clinical isolates of ribotypes 027 (toxinotype III) (n = 30) and 078 (toxinotype V) (n = 23) were provided by health-care facilities across the Republic of Ireland over 2 months in 2006 and 1 month in 2009. Ribotype 027 isolates were significantly more related to each other (9 different subtype profiles) when compared to ribotype 078 isolates (14 different profiles) (P = 0.001; cut-off >90 % similarity). Almost half of ribotype 078 isolates (45.5 %) showed no relationship to each other. The clonality of ribotype 027 isolates suggests effective adaptation to the human niche, whereas the considerable genetic diversity within ribotype 078 isolates suggests that they may have originated from a variety of sources. Subtyping correlated well with antimicrobial susceptibility, in particular clindamycin susceptibility for ribotype 027, but diverse antimicrobial-susceptibility profiles were seen in ribotype 078 isolates, even within a single health-care facility. Between 2006 and 2009, a change in the predominant subtype of ribotype 027 was seen, with the recent clone representing half of all ribotype 027 isolates studied. This strain exhibited 89 % similarity to a rep-PCR profile of the North American NAP-1 strain.

Update of Clostridium difficile infection due to PCR ribotype 027 in Europe, 2008.

Outbreaks of Clostridium difficile infections (CDI) with increased severity, high relapse rate and significant mortality have been related to the emergence of a new, hypervirulent C. difficile strain in North America and Europe. This emerging strain is referred to as PCR ribotype 027 (Type 027). Since 2005, individual countries have developed surveillance studies about the spread of type 027.C. difficile Type 027 has been reported in 16 European countries. It has been responsible for outbreaks in Belgium, Germany, Finland, France, Ireland, Luxembourg, The Netherlands, Switzerland and the United Kingdom (England, Wales, Northern Ireland and Scotland). It has also been detected in Austria, Denmark, Sweden, Norway, Hungary, Poland and Spain. Three countries experienced imported patients with CDI due to Type 027 who acquired the infection abroad.The antimicrobial resistance pattern is changing, and outbreaks due to clindamycin-resistant ermB positive Type 027 strains have occurred in three European countries. Ongoing epidemiological surveillance of cases of CDI, with periodic characterisation of the strains involved, is required to detect clustering of cases in time and space and to monitor the emergence of new, highly virulent clones.

Laboratory diagnosis of Clostridium difficile-associated disease in the Republic of Ireland: a survey of Irish microbiology laboratories.

The Health Protection Surveillance Centre (HPSC) established a group to produce national guidelines for Clostridium difficile in Ireland in 2006. A laboratory questionnaire was distributed to determine current C. difficile diagnostic practices. Twenty-nine out of 44 laboratories providing C. difficile diagnostic services to 34 hospitals responded. Twenty-five out of 29 (86%) laboratories processed specimens for C. difficile and four (13.8%) forwarded specimens to another laboratory. Sixteen laboratories (64%) processed specimens for other healthcare facilities. None routinely examined stool for C. difficile, seven (28%) examined specimens only when requested to do so and 18 (72%) used specific selection criteria, including testing all liquid stools (39%), all nosocomial diarrhoea (44%), specific clinical criteria (28%) and history of antibiotic therapy (22%). All tested stool directly for C. difficile toxin with a variety of enzyme immunoassays, with 24 (96%) detecting both toxin A and B and one detecting toxin A only. Three (12%) laboratories used cytotoxicity assays; none used polymerase chain reaction and six (24%) laboratories performed C. difficile culture but only under specific circumstances. Seven (28%) laboratories had isolates typed during outbreaks, but none had the facilities to do so on-site. The HPSC group will produce national recommendations for laboratory diagnosis, surveillance and management of C. difficile infection. Since there are marked differences in diagnostic practices throughout the country and no national reference laboratory, the implementation of these recommendations will have cost implications that will need to be addressed.

Update of Clostridium difficile-associated disease due to PCR ribotype 027 in Europe.

Recent outbreaks of Clostridium difficile-associated diarrhoea (CDAD) with increased severity, high relapse rate and significant mortality have been related to the emergence of a new, hypervirulent C. difficile strain in North America, Japan and Europe. Definitions have been proposed by the European Centre of Disease Prevention and Control (ECDC) to identify severe cases of CDAD and to differentiate community-acquired cases from nosocomial CDAD (http://www.ecdc.europa.eu/documents/pdf/Cl_dif_v2.pdf). CDAD is mainly known as a healthcare-associated disease, but it is also increasingly recognised as a community-associated disease. The emerging strain is referred to as North American pulsed-field type 1 (NAP1) and PCR ribotype 027. Since 2005, individual countries have developed surveillance studies to monitor the spread of this strain. C. difficile type 027 has caused outbreaks in England and Wales, Ireland, the Netherlands, Belgium, Luxembourg, and France, and has also been detected in Austria, Scotland, Switzerland, Poland and Denmark. Preliminary data indicated that type 027 was already present in historical isolates collected in Sweden between 1997 and 2001.

Isolation and characterisation of toxin A-negative, toxin B-positive Clostridium difficile in Dublin, Ireland.

Clostridium difficile is a major cause of infectious diarrhoea in hospitalised patients. Most pathogenic C. difficile strains produce two toxins, A and B; however, clinically relevant toxin A-negative, toxin B-positive (A- B+) strains of C. difficile that cause diarrhoea and colitis in humans have been isolated worldwide. The aims of this study were to isolate and characterise A- B+ strains from two university hospitals in Dublin, Ireland. Samples positive for C. difficile were identified daily by review of ELISA results and were cultured on selective media. Following culture, toxin-specific immunoassays, IMR-90 cytotoxicity assays and PCR were used to analyse consecutive C. difficile isolates from 93 patients. Using a toxin A-specific ELISA, 52 samples produced detectable toxin. All isolates were positive using a toxin A/B ELISA. Similarly, all isolates were positive with the cytoxicity assay, although variant cytopathic effects were observed in 41 cases. PCR amplification of the toxin A and toxin B genes revealed that 41 of the previous A- B+ strains had a c. 1.7-kb deletion in the 3'-end of the tcdA gene. Restriction enzyme analysis of these amplicons revealed the loss of polymorphic restriction sites. These 41 A- B+ isolates were designated toxinotype VIII by comparison with C. difficile strain 1470. PCR ribotyping revealed that all A- B+ isolates belonged to PCR-ribotype 017. A- B+ C. difficile isolates accounted for 44% of the isolates examined in this study, and appeared to be isolated more frequently in Dublin, Ireland, than reported rates for other countries.

Comparative phylogenomics of Clostridium difficile reveals clade specificity and microevolution of hypervirulent strains.

Clostridium difficile is the most frequent cause of nosocomial diarrhea worldwide, and recent reports suggested the emergence of a hypervirulent strain in North America and Europe. In this study, we applied comparative phylogenomics (whole-genome comparisons using DNA microarrays combined with Bayesian phylogenies) to model the phylogeny of C. difficile, including 75 diverse isolates comprising hypervirulent, toxin-variable, and animal strains. The analysis identified four distinct statistically supported clusters comprising a hypervirulent clade, a toxin A(-) B(+) clade, and two clades with human and animal isolates. Genetic differences among clades revealed several genetic islands relating to virulence and niche adaptation, including antibiotic resistance, motility, adhesion, and enteric metabolism. Only 19.7% of genes were shared by all strains, confirming that this enteric species readily undergoes genetic exchange. This study has provided insight into the possible origins of C. difficile and its evolution that may have implications in disease control strategies.

Detection of Enterobacter sakazakii in dried infant milk formula by cationic-magnetic-bead capture.

Enterobacter sakazakii has been associated with life-threatening infections in premature low-birth-weight infants. Contaminated infant milk formula (IMF) has been implicated in cases of E. sakazakii meningitis. Quick and sensitive methods to detect low-level contamination sporadically present in IMF preparations would positively contribute towards risk reduction across the infant formula food chain. Here we report on the development of a simple method, combining charged separation and growth on selective agar, to detect E. sakazakii in IMF. This protocol can reliably detect 1 to 5 CFU of E. sakazakii in 500 g of IMF in less than 24 h.

Greyhound meningoencephalitis: PCR-based detection methods highlight an absence of the most likely primary inducing agents.

Greyhound meningoencephalitis is currently classified as a breed-associated idiopathic central nervous system inflammatory disorder. The non-suppurative inflammatory response can be distinguished from the other breed-associated disorders based on histopathology and lesion topography, however the nature of the response primarily suggests a viral infection. In the present study PCR and RT-PCR technologies were employed on frozen cerebral tissue from confirmed cases of meningoencephalitis to target specific viruses and protozoa likely to be implicated and to exclude the presence of bacterial 16SrRNA. Secondly, degenerate primers were used to detect viruses of the herpesvirus and flavivirus families. In addition cerebral tissues were probed for West Nile Virus. Viral nucleic acid sequences to Borna disease virus, to louping ill, tick borne encephalitis, West Nile and other flaviviruses were not detected. Canine distemper virus was detected in one animal with 97% homology to strain A75/15. Degenerate PCR for herpesviruses detected viral amplification products in one animal with 90% homology to canine herpesvirus DNA polymerase gene. Protozoal amplification products were only detected in a single dog with pathological confirmation of a combination of lesions of greyhound meningoencephalitis and a protozoal encephalomyelitis. Neospora was confirmed with sequence homology to Austrian strain 1. Bacterial 16SrRNA was not detected. The present study supports previous observations that many of the known microbial causes of canine meningoencephalitis are not involved. Findings could reflect that the causal agent was not specifically targeted for detection, or that the agent is at undetectable levels or has been eliminated from brain tissue. The potential roles of genetics and of molecular mimicry also cannot be discounted.

Enterobacter sakazakii: an emerging pathogen in powdered infant formula.

Enterobacter sakazakii represents a significant risk to the health of neonates. This bacterium is an emerging opportunistic pathogen that is associated with rare but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in premature and full-term infants. Infants aged <28 days are considered to be most at risk. Feeding with powdered infant formula (PIF) has been epidemiologically implicated in several clinical cases. Infants should be exclusively breast-fed for the first 6 months of life, and those who are not should be provided with a suitable breast-milk substitute. PIF is not a sterile product; to reduce the risk of infection, the reconstitution of powdered formula should be undertaken by caregivers using good hygienic measures and in accordance with the product manufacturer's food safety guidelines.

Flow cytometric analysis of Clostridium difficile adherence to human intestinal epithelial cells.

Clostridium difficile is the most common cause of diarrhoea in hospitalised patients. Bacterial adherence to gut epithelial cells is a likely prerequisite to infection and toxin production. A novel flow cytometric method was developed for detecting adherence of C. difficile to human colonic and small intestinal epithelial cells (EC) and human intestinal cell lines. Small intestinal and colonic EC were isolated from biopsy specimens with mucolytic and chelating agents. Adherence of fluorochrome-labelled C. difficile to EC was measured by flow cytometry and was calculated as increase in median fluorescent intensity (deltaMFI). Cells with bacteria attached could be distinguished easily from cells alone or cells with unlabelled bacteria attached. Toxin-positive C. difficile adhered to colonic and small intestinal EC (deltaMFI mean 21.2 SD 16.7, n = 33 and 16.5 SD 20.7, n = 19 respectively). The toxin-negative strain also adhered to both epithelial cell types (deltaMFI 26.1 SD 32.5, n = 17 and 18.3 SD 31.3, n = 16). Adherence of toxin-positive C. difficile to the intestinal cell lines Caco-2 (deltaMFI 9.4 SD 4.4, n = 14) and HT29 (deltaMFI 8.1 SD 3.1, n = 12) was quantifiable, although at a significantly lower level than with primary colonic epithelial cells. Adherence of the toxin-negative strain was slightly lower, deltaMFI 6.5 SD 1.8, n = 9 with Caco-2 cells and deltaMFI 6.0 SD 2.0, n = 10 with HT29 cells. Adherence of C. difficile to epithelial cell lines was blocked with C. difficile antiserum, confirming specificity of adherence. In conclusion, flow cytometry is a useful approach to quantifying adherence of C. difficile to human colonic and small intestinal epithelial cells. Binding of toxin-negative as well as toxin-positive bacteria was detectable by this approach. Analysis of C. difficile adherence to target cells may have important implications for the understanding of the pathogenesis of C. difficile-related disease.