Study on Mycobacterium ulcerans infection and carriage in Australian phalangeroid possums and assessment of a commercial DNA extraction kit’s effectiveness beyond its intended use

Abstract

Introduction and aim. Emerging infectious diseases (EIDs) have long posed significant threats to human health. A majority of EIDs constitutes of zoonotic diseases. Buruli ulcer (BU), caused by Mycobacterium ulcerans, is increasingly reported in Australia and other regions worldwide. It is an ulcerative disease of the skin and can affect subcutaneous tissue and bone. Australia is the only affluent country with significant transmission of BU. Despite its environmental origin, the mode of transmission and possible reservoirs have yet to be fully understood. Native Australian possums have been identified as potential reservoirs, shedding M. ulcerans DNA in their feces that genetically matches strains causing human disease. However, research on this host-pathogen relationship has not been updated since 2014, despite rising BU incidence. The aim of this study is to test for the presence of M. ulcerans in feces of common ringtail possums (CRPs, Pseudocheirus peregrinus) and common brushtail possums (CBPs, Trichosurus vulpecula) with and without Buruli-ulcer consistent skin disease (BCSD), originating from Australian BU endemic regions. This will be done by using a DNA extraction kit not specifically designed for fecal material (Qiagen DNeasy® Blood & Tissue kit), as previous studies have shown that fecal-specific kits do not always produce the highest DNA yield. The results of this research may offer valuable insight into the use of a DNA extraction kit not designed for fecal samples and help further assess the risk of human infection with M. ulcerans through contact with possums.

Materials and methods. Cadavers of 34 possums (both CRPs and CBPs) were collected from several veterinary clinics in the Mornington and Gippsland regions in Victoria, Australia. Possums with (n=12) and without (n=12) signs of BCSD (hereafter also referred to as ‘affected’ and ‘unaffected’) were chosen for further investigation. Fecal samples were collected from the distal colon and genomic DNA was extracted from these samples using the Qiagen DNeasy® Blood & Tissue kit. The samples were tested for the presence of M. ulcerans DNA by means of a conventional diagnostic polymerase chain reaction (PCR). The efficacy of PCR on feces was determined by using a spiked PCR with a positive control (M. ulcerans positive skin biopt from a CBP, confirmed through sequencing).

Results. While all the DNA extracts yielded sufficient amounts of DNA, genetic material of M. ulcerans was not detected in any of the fecal samples. Following the spiked PCR analysis, the positive control returned a negative result, indicating the likely presence of PCR inhibitors.

Discussion and conclusion. This study aimed to investigate the feasibility of using a commercial DNA extraction kit not specifically designed for fecal samples and to assess the potential role of CRPs and CBPs as reservoirs for M. ulcerans. Spiked samples suggested the presence of PCR inhibitors in fecal material, likely leading to false-negative results. The chosen DNA extraction kit might not be suitable for fecal material without additional steps to mitigate inhibition. Due to all the PCR results being negative, it is not possible to provide more insight into the prevalence of M. ulcerans in feces from affected and unaffected CRPs and CBPs in BU endemic regions in Australia. Further research is essential to determine the contemporary role of possums in the spread of M. ulcerans. The first step would be sample treatment prior to DNA extraction, and if still not successful, followed by use of a different DNA extraction kit or post-extraction cleanup steps, dilution series or use of feces-resistant polymerases. Understanding environmental reservoirs and transmission pathways of M. ulcerans remains critical for effective public health interventions.