Original Article     OPEN ACCESS

Quantitative real time PCR detection of Clostridium difficile growth inhibition

by probiotic organisms

Bryan L. Folkers, DO1, Craig Schuring, BS2, Michael Essmann, BS2, Bryan Larsen, PhD2, 3

1Department of Surgery, Mercy Medical Center, Des Moines, Iowa USA.
2Infectious Disease Research Laboratory, Des Moines University, Des Moines, Iowa, USA.
3Iowa Center for Translational and Clinical Research, Mercy Medical Center, Des Moines, Iowa USA.

Citation: Folkers BL, Schuring, C, Essmann M, Larsen B. Quantitative real time PCR detection of Clostridium difficile growth inhibition by probiotic organisms. North Am J Med Sci 2010; 2: 5-10.    
Doi: 10.4297/najms.2010.15
Availability: www.najms.org
ISSN: 1947 – 2714

Abstract
Background: Probiotic microorganisms are potential treatments for Clostridium difficile diarrheal disease (CDD) but better methods are needed to determine the relative potency of probiotic microorganisms against pathogenic organisms in mixed cultures. Aim: Quantify C. difficile in the presence of putative probiotic organisms using molecular methods to determine relative probiotic potency. Materials and Methods: C. difficile strains were cultivated anaerobically. Serial dilutions of Lactobacillus cultures or microbial mixtures from kefir were co-cultured with C. difficile for 48 hours.  Bacterial DNA was extracted and qPCR was used to measure C. difficile toxin A gene, on the basis of cycle threshold (Ct) number. Results: Strains of Lactobacillus (human and ATCC derived), and mixed cultures from commercial kefir were co-cultured with C. difficile. Lactobacillus and the microbial mixture from kefir were ranked in order of their potency in C. difficile growth inhibition. Conclusions: PCR allows facile quantification of C. difficle in the presence of other. The technique measures relative potency of over-the-counter probiotics and may predict human strains meriting probiotic status.

Keywords: Probiotic organisms, Clostridium difficile, in vitro testing, qPCR, probotic potency, antibiotic-associated diarrhea, Lactobacillus sp.

Correspondence to: Bryan L. Folkers, DO, 19 Kenwood Court, Beachwood, Ohio 44122, USA. Tel.: 216 2926800, Fax 216 2927775, Email:  Folkers311@yahoo.com