Dissertation Title: Rapid Identification of Rhodococcus equi by a PCR Assay Targeting the choE Gene
Abstract
Rhodococcus equi is a commonly known bacteria that affects horses and individuals with weak immunity. This bacterium causes severe pyogranulomatous pneumonia and other life-threatening infections, such as lung diseases, amongst the above population. Recent research has indicated that Rhodococcus equi also infects other domestic animals, including cattle and pigs. Rhodococcus equi is usually found in soil, specifically those that are enriched with domestic and wild animal fecal matter. The common method of identifying Rhodococcus equi bacteria is through culturing and subsequent phenotypic analysis. However, accurate identification of this bacterium is crucial for effective treatment but has been challenging with traditional methods because they lead to misclassification due to similarities with other bacteria, thus increasing the need to adapt a more reliable identification method. This study introduces a new method using Polymerase Chain Reaction (PCR) to identify Rhodococcus equi by targeting the choE gene, which is essential for the bacterium’s virulence. This new method is considered effective and accurate as it differentiated Rhodococcus equi from other bacteria, such as actinomycetes, which were incorrectly identified by traditional methods.
Samples Materials
The materials used in this study were bacterial strains. The total number of bacterial strains used in this study was 132, composed of clinical and nonclinical strains. Clinical strains comprised 32 strains from pneumonic foals and 49 strains. Nonclinical strains comprised 46 strains from soil and three from foal feces. Moreover, the study also included non-R. Equi actinomycete species as control strains sample comprised three Rhodococcus equi reference strains, strains 1053(5), ATCC 6939, and ATCC 33701 (28).
Methodology
The study extracted bacterial genomic DNA from all the collected samples for examination. The collected bacterial genomic DNA was then prepared by suspending one sample in 50 ul of distilled water and adding an equal amount of Instagene matrix. The mixture was heated for ten minutes at eighty Degrees Celsius. The study team tested their prepared bacterial genomic DNA of 132 Rhodococcus equi samples using their new PCR method. Supernatants were used in the study for PCR amplification and all the tested strains were amplified at least three times, hence enabling precise identification of R. equi. The choE-based PCR assay produced the expected 959-bp amplicon exclusively in R. equi isolates. Sequencing a variable region of the 16S RNA gene from 20 PCR-positive isolates confirmed the assay’s specificity.
Results
The PCR method successfully identified R. equi in the collected study samples. For instance, Dietzia maris, Mycobacterium peregrinum, and Staphylococcus epidermidis samples that were initially identified as a species of Rhodococcus equi by traditional identification methods tested negative for the ChoE gene. Also, 16S RNA gene sequence analysis indicated that they originate from other species. Conversely, all non-R. Equi isolates, except Brevibacterium sterolicum ATCC 21387, were negative by the choE-based PCR. The 16S RNA gene sequence comparison suggested that ATCC 21387 is an R. equi isolate.
The research study has revealed that the PCR assay developed using choE DNA is a reliable tool for the diagnosis of pathogenic R. equi, as opposed to other identification tests. The sample test adopted in the study has shown that the PCR assay’s method is a reliable diagnostic tool in confirming Rhodococcus equi infections, providing an opportunity for accurate treatment of both immunocompromised and infected horses.
Also, the PCR method can be described as effective since it is fast and accurate in diagnosis; this is helpful to human and veterinary medicine because controlling the spread of infections is easier. This method could also assist in enhancing the environment as it can be applied to test soil and many other environments for the R. equi pathogen; this, in turn, will assist in the early identification of the pathogen and the prevention of its spread to humans and our animals. The study also points out the drawbacks of any other molecular method, including the detection of the vapA gene or 16S rDNA sequencing, which is due to the specificity and sensitivity in the case of different R. equi strains. PCR assay, targeted on the choE gene, demonstrates the scale of enhancing the process of R. Equi diagnosing and curing. From the findings of the study, it is clear that this method is better than the traditional diagnostic methods of detecting this pathogen because it is effective, efficient, and fast. Also, the use of choE-based PCR assay offers a rapid, sensitive, and specific approach to identifying R. equi, which is imperative for correctly diagnosing and treating the diseases protruding from this pathogen.