Emphasis Type="Italic"Clostridium difficile/Emp

Authors: V C C Cheng W C Yam O T C Lam J L Y Tsang E Y F Tse G K H Siu J F W Chan H Tse K K W To J W M Tai P L Ho K Y Yuen

Publish Date: 2011/04/06

Volume: 30, Issue: 11, Pages: 1371-

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Abstract

We identified a predominant clone of Clostridium difficile PCR ribotype 002 which was associated with an increased sporulation frequency In 2009 3528 stool samples from 2440 patients were tested for toxigenic C difficile in a healthcare region in Hong Kong A total of 345 toxigenic strains from 307 133 patients were found Ribotype 002 was the predominant ribotype which constituted 35 samples from 29 94 patients The mean sporulation frequency of ribotype 002 was 202 which was significantly higher than that of the 56 randomly selected ribotypes other than 002 as concurrent controls 37 p  0001 Patients carrying toxigenic ribotype 002 were more frequently admitted from an elderly home p = 001 and received more βlactam antibiotics in the preceding 3 months compared with the controls p = 004 The identification of toxigenic ribotype 002 in 2009 was temporally related to a significant increase in both the incidence of toxigenic C difficile from 053 to 095 per 1000 admissions p  0001 and the rate of positive detection from 417 to 628 p  0001 between period 1 2004–2008 and period 2 2009 This finding should alert both the physician and the infection control team to the establishment of and possible outbreaks by ribotype 002 in our hospitals as in the case of ribotype 027Clostridium difficile is an anaerobic Grampositive sporeforming bacillus which causes gastrointestinal diseases ranging from antibioticassociated diarrhea to pseudomembranous colitis Though communityacquired infection can occur most cases are found in the hospital and longterm care facilities 1 The fecal colonization rate in ambulatory individuals is up to 2 2 3 and this increased to 15–30 among hospitalized patients due to the acquisition from healthcare workers and the hospital environment 4 About 15–60 of the colonized patients develop symptomatic diarrhea during hospitalization 5 6 7 which may increase the nosocomial transmission of C difficile with hospital outbreaks Recently a virulent strain identified as PCR ribotype 027 toxinotype III or North American pulsefield type 1 has emerged to cause severe colitis leading to a high mortality rate 8 9 10 The mechanism of increased virulence is still under investigation but it may be related to the 18bp deletion and singlebasepair deletion at position 117 in the toxin regulator gene tcdC leading to the hyperproduction of toxins A and B 11 12 In addition increased sporulation frequency of certain epidemic strains of C difficile PCR ribotype 027 may also contribute to its better survival and nosocomial spread 13 14 Sporulation occurs when the ability to maintain vegetative growth has failed and facilitates the transmission of C difficile in the healthcare setting as the spores remain infective and persist in the environment for many months 15A sporadic case of C difficile PCR ribotype 027 was reported in Hong Kong in 2008 16 In response to this emerging infectious agent further investigation was performed on all C difficile isolates in a healthcare region in Hong Kong We also conducted a retrospective review of the clinical and epidemiological data related to the different ribotypes of toxigenic C difficile isolated in 2009 The findings and their potential implications in disease transmission and infection control practice are discussedA surveillance program was conducted in a regional microbiology laboratory in Hong Kong The laboratory provided service to a healthcare network of five hospitals including one acute care university teaching hospital with 1400 beds and four chronic care hospitals with 110 to 524 beds The hospital network provided clinical service to a population of approximately 053 million people Between 1 January 2009 and 31 December 2009 stool specimens from hospitalized patients sent for culture and cytotoxin assay of C difficile were performed by our routine service protocol as usual but the toxigenic strains were further characterized by molecular tests The incidence of toxigenic C difficile per 1000 admissions and the rate of detection identified between period 1 2004 and 2008 and period 2 2009 were analyzed using databases of the laboratory information and hospital record systemLiquid or semisolid stool samples were tested within 24 h of receipt The samples were inoculated onto cycloserine–cefoxitin–fructose agar CCFA containing 4 proteose peptone 05 Na2HPO4 01 KH2PO4 001 MgSO4 02 NaCl 06 fructose 15 agar at pH 74 CM0601 Oxoid UK with added selective supplement containing 250 mg/L Dcycloserine 8 mg/mL cefoxitin SR0096 Oxoid UK and 7 horse blood Culture plates were incubated anaerobically at 35°C to 37°C for 48 h Obligatory anaerobic large Grampositive bacilli that were isolated from the CCFA and were susceptible to 5 μg of vancomycin were presumptively identified as C difficile The identification was confirmed by the Vitek® Anaerobe Identification Card ANI bioMérieux Inc USA Antimicrobial susceptibility testing against metronidazole vancomycin and ciprofloxacin were performed for selected strains using Etest strips according to the manufacturer’s instructions AB Biodisk SwedenApproximately 3 to 5 g of stool was suspended in 5 ml of phosphatebuffered saline PBS in a 12 dilution at a pH level of 7 The sample was centrifuged at 3000 rpm for 55 min to produce a supernatant which was subsequently transferred into eppendorf tubes and centrifuged at 13000 rpm for 15 min at 4°C to clarify the supernatant The supernatant was passed through a 022μmporesize membrane filter A toxinproducing C difficile strain UKEQAS QC 6109 was used as the positive control for the cell culture cytotoxicity neutralization assay CCCNA which was performed in sterile 96well plates coated with the HeLa cell line as previously described 16 In addition to the direct detection of cytotoxin from stool filtrates CCCNA was also performed on the stationaryphase culture supernatant of each C difficile isolate C difficile isolates were subcultured to brain heart infusion broth and incubated anaerobically for 96 h Culture supernatant was subjected to CCCNA as for stool filtrate with the same interpretation criteria as previously described 16DNA was extracted from C difficile colonies using alkaline lysis as described previously 17 Polymerase chain reaction PCR ribotyping was performed according to the method described by Bidet et al 18 After the electrophoresis of PCR products the phylogenetic tree was constructed using BioNumerics v60 software Applied Maths BelgiumslpA typing was performed for selected strains belonging to the same ribotype according to Joost et al 19 The purified PCR product was subjected to cycle sequencing by ABI BigDye terminator v11 Applied Biosystems Foster City USA and the resulting slpA sequence was compared to those in the NCBI database by using Nucleotide BLAST

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