Atividade de nanoformulações de Melaleuca alternifolia e terpinen-4-ol em isolados de Rhodococcus equi / Melaleuca alternifolia activity in nanoformulations and terpinen-4-ol against Rhodococcus equi isolates

Rhodococcus equi é o agente etiológico da rodococose equina, importante doença respiratória de potros. Especialmente na última década, a emergência de cepas resistentes aos antimicrobianos empregados no tratamento da rodococose tem sido relatada. Nesse sentido, há a necessidade de estudos envolvendo terapias alternativas e novas tecnologias, incluindo o uso de plantas medicinais e nanotecnologia. Neste trabalho utilizou-se Melaleuca alternifolia nas seguintes formulações: óleo livre, nanocápsula, nanoemulsão e a combinação de óleo livre com nanocápsula e com nanoemulsão, além do seu composto majoritário, terpinen-4-ol, a fim de verificar a atividade antimicrobiana frente a isolados de R. equi de diferentes origens. Utilizou-se o método de microdiluição em caldo na determinação das concentrações inibitória mínima (CIM) e bactericida mínima (CBM) das diferentes formulações frente aos isolados (n=24). Verificou-se baixo potencial para atividade antibacteriana de M. alternifolia na formulação de óleo livre. Todavia, essa atividade foi potencializada quando se incorporou o óleo essencial às nanoformulações. O composto terpinen-4-ol demonstrou potencial atividade antibacteriana quando incorporado ao óleo essencial e quando utilizado isoladamente. Verificou-se que tanto M. alternifolia quanto terpinen-4-ol testados possuem atividade antimicrobiana contra isolados de R. equi, sugerindo seu emprego em estudos avaliando seu potencial para o tratamento da rodococose.

Rhodococcus equi causes rodococose in horses, characterized by bronchopneumonia in foals. Due to reports of antimicrobial resistance, it is important to develop studies involving alternative therapies and new technologies, including the use of medicinal plants and nanotechnology. In this work, the plant Melaleuca alternifolia in oil free formulations, nanocapsule, nanoemulsion and the combination of free and nanocapsule oil nanoemulsion, besides its major compound, terpinen-4-ol, were used in order to verify antimicrobial activity against isolates of R. equi. The broth microdilution method was employed to determine the minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations of different formulations against 24 isolates. There was low antibacterial activity of M. alternifolia in oil free formulation; however, it was observed that the activity was enhanced when incorporated as essential oil the nanoformulations. The major compound, terpinen-4-ol, showed bactericidal and bacteriostatic activity when used alone. It is suggested that M. alternifolia, in association with nanocarriers systems, as well as terpinen -4-ol, presents potential for future studies concerning the equine rodococosis therapy.

Genome and proteome analysis of phage E3 infecting the soil-borne actinomycete Rhodococcus equi.

We report on the characterization and genomic analysis of bacteriophage E3 isolated from soil and propagating in Rhodococcus equi strains. Phage E3 has a circular genome of 142 563 bp and is the first Myoviridae reported for the genus Rhodococcus and for a non-mycobacterial actinomycete. Phylogenetic analyses placed E3 in a distinct Myoviridae clade together with Mycobacterium phages Bxz1 and Myrna. The highly syntenic genomes of this myoviridal group comprise vertically evolving core phage modules flanked by hyperplastic regions specific to each phage and rich in horizontally acquired DNA. The hyperplastic regions contain numerous tRNA genes in the mycobacteriophages which are absent in E3, possibly reflecting bacterial host-specific translation-related phage fitness constraints associated with rate-limiting tRNAs. A structural proteome analysis identified 28 E3 polypeptides, including 15 not previously known to be virion-associated proteins. The E3 genome and comparative analysis provide insight into short-term genome evolution and adaptive plasticity in tailed phages from the environmental microbiome.

Isolation and characterization of soilborne virulent bacteriophages infecting the pathogen Rhodococcus equi.

AIMS: To isolate and characterize a diversity of bacteriophages (phages) that infect the soilborne pathogen Rhodococcus equi. METHODS AND RESULTS: Twenty-seven phages were isolated from soil samples from geographically distinct locations using a range of R. equi bacterial strains, including clinical isolates. On the basis of host range, genomic DNA restriction profiles and virion protein profiles, the diversity of these phages was extensive, with phages being divided into 16 groupings. CONCLUSIONS: Based on a range of criteria, these phages could be divided into 16 distinct groupings. The majority of the phages recovered from soil were Siphoviridae, adding to the limited number of Siphoviridae described to date for R. equi. One grouping consisted of phages belonging to the Myoviridae. SIGNIFICANCE AND IMPACT OF THE STUDY: This represents the first study looking at the diversity of phages infecting the pathogen R. equi, including the first Myoviridae to be isolated and characterized for the genus Rhodococcus and for the nonmycobacterial actinomycetes. Given their diverse host range, including clinical isolates, this collection of phages offers the potential for the development of phage cocktails for use as a therapeutic or alternatively in the biocontrol of this pathogen in reservoirs of infection relating to animal husbandry.

Genome sequence and characterization of a Rhodococcus equi phage REQ1.

Rhodococcus equi is a pathogenic member of the Actinobacteria responsible for causing serious infections in equines. A novel Siphoviridae bacteriophage (REQ1) lytic in R. equi was isolated and characterized. The genome size of REQ1 is 51,342 bp, and its sequence shares 7 % similarity to other DNA sequence in GenBank. Putative open reading frames were identified, and their functions were identified based on their predicted amino acid similarities. REQ1 phage has a modular genome, a feature common in double-stranded DNA phages.

Genomic and functional analyses of Rhodococcus equi phages ReqiPepy6, ReqiPoco6, ReqiPine5, and ReqiDocB7.

The isolation and results of genomic and functional analyses of Rhodococcus equi phages ReqiPepy6, ReqiDocB7, ReqiPine5, and ReqiPoco6 (hereafter referred to as Pepy6, DocB7, Pine5, and Poco6, respectively) are reported. Two phages, Pepy6 and Poco6, more than 75% identical, exhibited genome organization and protein sequence likeness to Lactococcus lactis phage 1706 and clostridial prophage elements. An unusually high fraction, 27%, of Pepy6 and Poco6 proteins were predicted to possess at least one transmembrane domain, a value much higher than the average of 8.5% transmembrane domain-containing proteins determined from a data set of 36,324 phage protein entries. Genome organization and protein sequence comparisons place phage Pine5 as the first nonmycobacteriophage member of the large Rosebush cluster. DocB7, which had the broadest host range among the four isolates, was not closely related to any phage or prophage in the database, and only 23 of 105 predicted encoded proteins could be assigned a functional annotation. Because of the relationship of Rhodococcus to Mycobacterium, it was anticipated that these phages should exhibit some of the features characteristic of mycobacteriophages. Traits that were identified as shared by the Rhodococcus phages and mycobacteriophages include the prevalent long-tailed morphology and the presence of genes encoding LysB-like mycolate-hydrolyzing lysis proteins. Application of DocB7 lysates to soils amended with a host strain of R. equi reduced recoverable bacterial CFU, suggesting that phage may be useful in limiting R. equi load in the environment while foals are susceptible to infection.

Interaction of virulent and non-virulent Rhodococcus equi human isolates with phagocytes, fibroblast- and epithelial-derived cells.

Rhodococcus equi is a facultative, intracellular, Gram-positive coccobacillus, increasingly reported in pneumonia of AIDS-infected patients. We investigated killing resistance properties of human R. equi virulent and avirulent human strains. Avirulent beta-lactam-susceptible strains had lower intracellular colony forming units after 45 min incubation in murine macrophages J774 and human monocyte-macrophage TPH-1 than those of virulent strains. Only virulent beta-lactam-resistant strains persisted within macrophages for at least 18 min only. A beta-lactam-resistant mutant was obtained from a beta-lactam-susceptible strain after selection in a penicillin G-containing culture medium. This mutant strain, like the natural virulent strains, persisted within macrophages, harboured cell-associated appendages, produced phage-like particles and induced, after its intravenous inoculation, a chronic infection in BALB/c nude mice. Supernatant culture of virulent strains transferred partial macrophage-killing resistance properties to avirulent strains. The same supernatant was toxic for L-929, HeLa and Vero cell cultures. These supernatant effects were heat-inactivated, trypsin-inactivated and did not seem to be linked to phage-like particle presence. These data argue that virulence, beta-lactam-resistance, and macrophage-killing resistance are associated in human R. equi isolates. Moreover, only virulent strains produced uncharacterized toxic factors.

Isolation of Rhodococcus equi from the abscess of a goat in Trinidad

Rhodococcus equi was first islolated from infected foals by Magnusson in 1923. On a review of R. equi infection in humans and animals, Prescott showed that only two cases of R. equi infection have been reported in goats. According to him, the organism has a tendency to cause liver abscesses, possibly following penetration from the intestinal tract. It is not clear to us how the animal acquired the infection. There is no information about close contact with horses in the farm or origin in England (AU)