Cellulitis-related Rhodococcus equi in a cat harboring VAPA-type plasmid pattern.

Rhodococcus equi is a well-known intracellular facultative bacterium that is opportunistic in nature, and a contagious disease-causing agent of pyogranulomatous infections in humans and multihost animals. Feline rhodococcosis is an uncommon or unnoticed clinical condition, in which the organism is usually refractory to conventional antimicrobial therapy. The pathogenicity of the agent is intimately associated with plasmid-governed infectivity, which is attributed to the presence of plasmid-encoded virulence-associated proteins (Vap). Three host-adapted virulence plasmid types (VAPs) have been distinguished to date: pVAPA, pVAPB, and pVAPN, whose infections are related to equine, pig, and bovine or caprine origin, respectively, while humans are infected by all three VAP types. Most virulence studies with R. equi plasmid types in animals involve livestock species. Conversely, data on the pathogenicity and human relevance of the virulence plasmid profile of R. equi isolated from cats remains unclear. This report describes a case of cellulitis-related R. equi that harbors the pVAPA-type in a cat with cutaneous lesion. Long-term therapy of the cat using marbofloxacin, a broad-spectrum third-generation fluoroquinolone, resulted effectiveness. pVAPA is a host-adapted virulent type that has been associated predominantly with pulmonary foal infections. Our cat had a history of contact with other cats, livestock (including horses), and farm environment that could have favored the transmission of the pathogen. Besides no clear evidence of cat-to-humans transmission of the pathogen, the identification of R. equi harboring pVAPA-type in a cat with cutaneous abscessed lesion represent relevance in human health because this virulent type has been described in people worldwide with clinical rhodococcal disorders.

An Autobioluminescent Method for Evaluating In Vitro and In Vivo Growth of Rhodococcus equi.

A previously reported method for evaluating the intracellular growth of Rhodococcus equi using enhanced green fluorescent protein is unsuitable for the quantitative evaluation of the entire sample because the signal can be detected only in the excitation region. Therefore, we created an autobioluminescent R. equi using luciferase (luxABCDE). First, we connected luxABCDE to the functional promoter PaphII and introduced it into the chromosomes of ATCC33701 and ATCC33701_P-. Luminescence was detected in both transformants, and a correlation between the bacterial number and luminescence intensity in the logarithmic phase was observed, indicating that luxABCDE is functionally and quantitatively expressed in R. equi. The luminescence of ATCC33701 was significantly higher than that of ATCC33701_P- at 24 h after infection with J774A.1. Next, RNA-Seq analysis of ATCC33701 to search for endogenous high-expression promoters resulted in the upstream sequences of RS29370, RS41760, and vapA being selected as candidates. Luminescence was detected in each transformant expressing the luxABCDE using these upstream sequences. We examined the luminescence intensity by coexpressing the frp gene, an enhancer of the luciferase reaction, with luxABCDE. The luminescence intensity of the coexpressing transformant was significantly enhanced in J774A.1 compared with the non-coexpressing transformant. Finally, we examined the luminescence in vivo. The luminescence signals in the organs peaked on the third day following the administration of ATCC33701 derivatives in mice, but no luminescence signal was detected when the ATCC33701_P- derivative was administered. The autologous bioluminescent method described herein will enhance the in vitro and in vivo quantitative analysis of R. equi proliferation. IMPORTANCE We established an autologous bioluminescent strain of R. equi and a method to evaluate its proliferation in vitro and in vivo quantitatively. This method overcomes the weakness of the fluorescence detection system that only measures the site of excitation light irradiation. It is expected to be used as an in vitro and in vivo growth evaluation method with excellent quantitative properties. In addition, it was suggested that the selection of a promoter that expresses luxABCDE could produce a luminescence with high intensity. Although this method needs further improvement, such as creating transformants that can maintain high luminescence intensity regardless of environmental changes such as temperature fluctuations, it is possible to observe bacterial growth over time in mice without killing them. Therefore, this method can be used to not only evaluate the pathogenicity of various wild and gene-deficient strains but also to screen preventive and therapeutic methods such as vaccines.