Inactivation of spores of Clostridium difficilewith chlorine dioxide (SanDes®) Background Clostridium difficile (CD) is a spore forming bacteria that normally is present in the intestine
of 1-3% of human beings. When treated with antibiotics the presence of CD is 10-20%. CD is
a bacteria that can cause diarrhoea1. CD has most likely a low infective dose and is in high
degree faeces and orally infectious via direct and indirect contact2,3. When CD bacteria is
discovered in faeces approximately 10% consists of spores. The spores may live for months in
the environment around a patient, in a contaminated hospital room and may infect a patient
newly admitted to hospital with CD, if the patient is treated with antibiotics2. Infection may
also take place from an infected patient to another patient and cause colony build-up in
fellow-patients within the same care department, which makes it to a nosocomial intestine
In Sweden is spread of CD a fast growing problem within the health care. The ways for
contamination are difficult to substantiate but most likely the contamination takes place via
indirect contagion, for instance via the hands of personal, gloves or other objects4. In 1979 in
Sweden 86 cases were diagnosed with a steady increase noticed that grew to 533 cases in
19842. The diagnostics was thereafter centralized causing lack of continuous national
statistics. The problem grew and January 1, 1995, the Swedish Institute for Infectious Disease
Control (SMI) initiated a study to determine the number of incidents. The study showed that
more than 6000 patients in Sweden were CD positive. In 1955 there were in Sweden as an
average 50 cases per 100 000 inhabitants and year. In Örebro the number of incidents was
higher than the national average, showing 60 cases per 100 000 inhabitants and year. Half of
the infected cases in Örebro were initiated in hospital5.
In 2004 a CD study was performed in the Örebro county. At that time the average number of
cases was 5,3 for 1000 persons hospitalized. The yearly number of incidents in the county
were 97 per 100 000 inhabitants, an increase since 1995. Of the 372 patients 208 patients
(78%) were hospital related. Of these 36 patients (17%) were suspected to have been
During the fall of 2005 an increase of CD cases was reported from Canada. The number of
cases per 100 000 inhabitants had increased from 102 cases during 1991-1992 to 210 cases
2002 and 866 cases in 2003. A new strain of CD, not reported earlier in connection to hospital
SanDes is chlorine dioxide and has unlike chlorine no recognized undesired side-effects.
Chlorine dioxide is known as a very effective means for inactivation of bacteria. It is used for
disinfection of drinking water. Chlorine dioxide has a documented effect against Clostridium perfringens spores8. and for the moment there is no functioning means against Clostridium difficile spores. For this reason we are interested in investigating if SanDes has effect for
inactivation of Clostridium difficile spores.
To find out whether SanDes can inactivate Clostridium difficile spores.
The test was performed January 14-23, 2008, at the Microbiological unit in Örebro by the
nurse Maria Sjöberg and biomedical analyzer Josefin Andersson. Physician Torbjörn Norén
had the overall responsibility for the project
Nutritious clear soup (PY-substance preproduced in anaerobe environment for 24 hours) was
added to the test bacteria Clostridium difficile 1067. This mixture was left in anaerobe
Two different types of plates for cultivation have been used, FAAAP and TCCFA. These
were preproduced (i.e. kept in anaerobe environment) for 3 days before being used.
After 6 days in anaerobe environment the amount of spores/vegetative cells was determined
with phase contrast microscope in bürker chamber.
20 tubes were filled with 0,1 mL of the Clostridium difficile soup and 4 tubes were filled with
staphylococcus (for control). To half of all the tubes PBS 0,9 mL was added and to the
remaining tubes EtOH 95% was added. All tubes chock treated with alcohol were left for 1
hour for maximum amount of spores. 6 PBS tubes and 6 EtOH 95% tubes were centrifuged
for 40 minutes 3000G4000 rpm. The centrifuged tubes were drained. To every test tube (see
table 1) different concentrations of SanDes, except the 0 test, were added for 60 seconds
treatment, after which 10 and 300 microliters were cultivated on the two plates.
The result shows that at not centrifuged and exposed to 200-800 ppm of SanDes a moderate
Log10 reduction between 0,7 and 2.9 (Table 1) was achieved while with 1500 ppm of SanDes
a total inactivation of spores was achieved.
Xinix inactivation of Clostridium difficile spores Inactivation of C. difficile spores in solution 1 min Xinix 200 ppm 40 0 ppm Table 1.The table shows that exposed to 200-800 ppm of SanDes a moderate Log10 reduction between 0,7 and 2.9 was achieved while with 1500 ppm of SanDes a total inactivation of spores was achieved. Comments
The centrifuged tubes also cause a certain inactivation but with a randomized variation
probably because of organic ”packing” in pellets and worse access for SanDes. These results
are not presented. The FAAAP plates showed best function in this test.
Importance and applicability
The result will have great importance for the elimination of Clostridium difficile spores. The
areas for use can be surfaces in hospital rooms and means for hand disinfection.
As we have proven inactivation of spores we are proceeding with application for disinfection
of surfaces in health care environment and eventually use for hand disinfection. Project
description for this tests are in progress.
Abbreviations PY-medium = ”Peptone yeast”-medium with added cystein. FAAAP = fastidious anaerobe agar (FAA) is a medium for primary isolation of demanding
anaerobic microorganisms. Contains 5% horse blood.
TCCFA = taurocholate cycloserine-cefoxitin-fructose-agar. A selective and differentiating
medium that stimulates germination of Clostridium difficile spores. Cycloserine and cefoxitin
inhibit growth of most aerobic, anaerobic, and facultative anaerobic gram-positive and gram-
negative bacteria. Taurochol acid separates C. difficile from lecitina positive clortides.
Clostridium difficile 1067: Epidemic strain B1/NAP1/027 toxinotype III that just now is
spreading in North america and in Europe.
PBS: Phosphate buffered saline solution List of references
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Clostridium difficile - en "ny" patogen på frammarsch. epidAKTUELLT
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Socialstyrelsen. Att förebygga vårdrelaterade infektioner : ett kunskapsunderlag. Stockholm: Socialstyrelsen; 2006.
Karlstrom O, Fryklund B, Tullus K, Burman LG. Samhällsförvärvad C difficile-associerad diarré en realitet. Färre recept och mera tvål. Lakartidningen 1997;94(23):2187-92.
Norén T, Åkerlund T, Bäck E, Sjöberg L, Persson I, Alriksson I, et al. Molecular epidemiology of hospital-associated and community-acquired Clostridium difficile infection in a Swedish county. J Clin Microbiol 2004;42(8):3635-43.
Warny M, Pepin J, Fang A, Killgore G, Thompson A, Brazier J, et al. Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet 2005;366(9491):1079-84.
Foegeding PM, Hemstapat V, Giesbrecht FG. Chlorine Dioxide Inactivation of Bacillus and Clostridium Spores. Journal of Food Science 1986;51(1):197-201.
MINUTES OF BOARD OF TRUSTEES REGULAR MEETING DISTRICT NO. 537 RICHLAND COMMUNITY COLLEGE ONE COLLEGE PARK – DECATUR, ILLINOIS 62521 January 15, 2008 CONVENING OF THE MEETING Call to Order The regular meeting was called to order at 5:31 p.m. Tuesday, January 15, 2008, in Conference Room A/B of the College by Chairman Prince. Chairman Prince also recited the College Visi
Speranta IACOB medic specialist gastroenterolog, doctor in stiinte medicale Data si locul nasterii: 8 aprilie 1977, Bucuresti, Romania Telefon/Fax: +40213180455 E-mail : [email protected] Etape medicale si universitare: 1995-2001: UMF “Carol Davila”, Bucuresti, Facultatea de Medicină Generală 2001 – 2002 - Medic rezident pediatrie, Clinica de Pediatrie, Institutul Clini