Molecular and Susceptibility Analysis of Toxigenic Clostridium difficile Obtained from Adult Patients Suspected of CDI in Trinidad

Lemar Wayne Blake^1*, Patrick Eberechi Akpaka¹, Adesh Ramsubhag², Reneasha Kevi-Ann Steffi Hitlal-Blake³

¹Microbiology & Pathology Unit, Department of Para-Clinical Sciences, The University of the West Indies, St. Augustine, Trinidad & Tobago.
²Department of Life Sciences, The University of The West Indies, St. Augustine, Trinidad and Tobago.
³Department of Medical Sciences, The University of the West Indies, Mona, Jamaica.

DOI: 10.4236/ojmm.2015.51006                

Abstract

Clostridium difficile is a Gram positive rod-shaped bacterium that produces two major toxins, A and B. The detection of the organism and its toxins has been widely carried out using specialized Enzyme-Linked Immunosorbent Assay (ELISA) kits; however these generally have been unsuccessful in identifying all Clostridium difficile positive samples. In this study, fifteen clinically symptomatic patients from three of the five major regional hospitals in Trinidad were investigated for Clostridium difficile infections. Stool samples were assessed by ELISA and cultured isolates were characterized using agar dilution antibiotic sensitivity assays, conventional Polymerase Chain Reaction (PCR), DNA sequencing and phylogenetic analysis of toxin A and B genes. All 15 patient stool samples and isolates were positive for toxigenic Clostridium difficile via ELISA and PCR respectively. All isolates were positive for the housekeeping tpi and Toxin B genes by PCR but only three of these were positive for the Toxin A gene. The Toxin B gene sequences showed 100% similarity levels among isolates while the Toxin A gene sequences showed 99% similarity among isolates. Phylogenetic analysis showed that the strains isolated in Trinidad most likely belonged to the same strain/group. Agar dilution sensitivity tests showed highest susceptibility to Pipercillin/Tazobactam and Meropenem (87%) and the highest resistance was seen with Cefotaxime in 93%. These results indicate that similar virulent strains of C. difficile are present in the Trinidad population and that pathogenic strains are more likely to be susceptible to Pipercillin/Tazobactam and Meropenem.

Keywords

Clostridium difficile Infection (CDI), Toxigenic Clostridium difficile, Toxin A Gene, Toxin B Gene, Housekeeping Gene (tpi), Antibiotic Susceptibility Testing, Phylogenetic, Sequencing

Conflicts of Interest

The authors declare no conflicts of interest.

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