The effect of disinfectants of sub-optimal concentrations on the expressions of toxin gene tcdB and sporulation genes spo0A and sigH in Clostridium Difficile

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The effect of disinfectants of sub-optimal concentrations on the expressions of toxin gene tcdB and sporulation genes spo0A and sigH in Clostridium Difficile

 

Author: Fung, Sze Nga
Title: The effect of disinfectants of sub-optimal concentrations on the expressions of toxin gene tcdB and sporulation genes spo0A and sigH in Clostridium Difficile
Degree: M.Sc.
Year: 2016
Subject: Clostridium difficile.
Disinfection and disinfectants.
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Health Technology and Informatics
Pages: xv, 70 pages : color illustrations
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2864142
URI: http://theses.lib.polyu.edu.hk/handle/200/8433
Abstract: Introduction: C. difficile is an anaerobic, Gram-positive and spore-forming bacillus causing antibiotic-associated diarrhoea and pseudo-membranous colitis, most noticeably in the healthcare settings. Two of the virulence factors of C. difficile include toxin production and sporulation. To control C. difficile infection, it is essential to eradicate the highly infectious spores of C. difficile that are shed into the environment. One of the common disinfectants used in healthcare settings is Clorox. Other disinfectants used include Cidex OPA and Virkon S. Various studies have investigated toxin production and spore formation of C. difficile when grown in the presence of sub-lethal concentrations of antibiotics. However not many studies have investigated the effect of sub-optimal concentrations of disinfectants on toxin gene and sporulation-associated gene expressions of C. difficile. Aims: This study aimed to investigate the effect of sub-optimal concentrations of three disinfectants, Clorox, Cidex OPA and Virkon S, on the expressions of tcdB, spo0A and sigH. This study also aimed to investigate the association between tcdB, spo0A and sigH expressions under disinfectants of sub-optimal concentrations. Methods: The minimum inhibitory concentrations (MIC) of three disinfectants Clorox, Cidex OPA and Virkon S for two pure strains of C. difficile of PCR ribotypes 002 and 027 were determined. Each disinfectant was then prepared in 0.5 and 0.25 of its MIC. C. difficile was then subjected to the sub-optimal disinfectant challenge. The expressions of tcdB, sigH and spo0A were measured by relative qualification method using rpoB gene as a reference gene in reverse-transcription quantitative PCR subsequently, with comparison to a negative control that received no disinfectant treatment.
Results: For both PCR ribotypes 002 and 027, there was no significant difference (p>0.05) in sigH expressions between samples receiving sub-optimal concentrations of disinfectants and water. However, there was significant difference (p<0.05) in spo0A expressions between samples receiving Virkon and water for both PCR ribotypes 002 and 027. Significant difference (p<0.05) in spo0A expressions also existed between samples receiving sub-optimal concentrations of Cidex and water for PCR ribotype 027. Moreover, there was significant difference (p<0.05) in tcdB expressions between samples receiving sub-optimal concentrations of Clorox and water for PCR ribotype 027. Lastly, there was no significant association (p>0.05) between the expressions of tcdB, sigH and spo0A under disinfectants of sub-optimal concentrations. Conclusion: Sub-optimal concentrations of Clorox did not affect the expressions of spo0A and sigH for both PCR ribotypes but repressed tcdB expression for PCR ribotype 027. Sub-optimal concentrations of Cidex did not enhance the expressions of sigH and tcdB for both PCR ribotypes but enhanced spo0A expression for ribotype 027. Virkon was unable to inhibit growth of C. difficile for both PCR ribotypes even at concentration recommended by the manufacturer. It enhanced the expression of spo0A but did not affect the expressions of sigH and tcdB for both PCR ribotypes. From the result, Clorox was the best disinfectant among the three disinfectants tested since sub-optimal concentrations of it did not stimulate the expression of sporulation-associated gene and it also repressed the expression of toxin gene of C. difficile. Virkon was not suitable in disinfecting C. difficile since it did not inhibit growth and it enhanced the expression of sporulation-associated gene of C. difficile.

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