Nocardia brasiliensis: in vitro and in vivo growth response to steroid sex hormones

As actinomycetoma is more frequent in males than in females, the possibility that hormones might modify theNocardia brasiliensis growth and the course of experimental actinomycetoma was explored. FiveN. brasiliensis strains were grown on Sabouraud agar containing estradiol, progesterone or testosterone, in 3 different concentrations. Colony diameters were measured weekly for 7 weeks.N. brasiliensis strains were also grown in Sabouraud broth containing hormones. Glucose concentration was measured weekly for 6 weeks. Finally, experimental actinomycetoma was produced in male and female hormone-treated mice. Invasion rate, plantar pad diameter and positive retrocultures were assessed. In vitro experiments showed that progesterone and testosterone inhibitN. brasiliensis growth, manifested by lower colony diameters and greater glucose concentrations. In vivo experiments demonstrated that estradiol limits actinomycetoma development. Progesterone and testosterone induced greater diameters of inoculated plantar pads and greater invasion rates with greater positive culture numbers than estradiol. Results partially explain the resistance of females to actinomycetoma.     

In Vivo Activity of the Benzothiazinones PBTZ169 and BTZ043 against Nocardia brasiliensis

Background

Mycetoma is a neglected, chronic, and deforming infectious disease caused by fungi and actinomycetes. In Mexico, N. brasiliensis is the predominant etiologic agent. Therapeutic alternatives are necessary because the current drug regimens have several disadvantages. Benzothiazinones (BTZ) are a new class of candidate drugs that inhibit decaprenyl-phosphoribose-epimerase (DprE1), an essential enzyme involved in the cell wall biosynthesis of Corynebacterineae.

Methodology/Principal findings

In this study, the in vitro activity of the next generation BTZ, PBTZ169, was tested against thirty Nocardia brasiliensis isolates. The MIC₅₀ and MIC₉₀ values for PBTZ169 were 0.0075 and 0.03 μg/mL, respectively. Because Nocardia is a potential intracellular bacterium, a THP-1 macrophage monolayer was infected with N. brasiliensis HUJEG-1 and then treated with PBTZ169, resulting in a decrease in the number of colony-forming units (CFUs) at a concentration of 0.25X the in vitro value. The in vivo activity was evaluated after infecting female BALB/c mice in the right hind food-pad. After 6 weeks, treatment was initiated with PBTZ169 and its activity was compared with the first generation compound, BTZ043. Both BTZ compounds were administered at 100 mg/kg twice daily by gavage, and sulfamethoxazole/trimethoprim (SXT), at 100 mg/kg sulfamethoxazole, was used as a positive control. After 22 weeks of therapy, only PBTZ169 and SXT displayed statistically significant activity.

Conclusion

These results indicate that DprE1 inhibitors may be useful for treating infections of Nocardia and may therefore be active against other actinomycetoma agents. We must test combinations of these compounds with other antimicrobial agents, such as linezolid, tedizolid or SXT, that have good to excellent in vivo activity, as well as new DprE1 inhibitors that can achieve higher plasma levels.     

A note on the isolation of pathogenic aerobic actinomycetes from sudanese soils

A preliminary investigation of two methods of isolating pathogenic aerobic actinomycetes from a group of Sudanese soils was undertaken. By one method,Nocardia asteroides was isolated from 3 out of 4 soils. By the second method, two strains ofN. brasiliensis and three ofActinomadura madurae were recovered from 4 out of 10 soils. Both procedures seem to be advantageous for isolating pathogenic aerobic actinomycetes from soils.     

Physiological and pathogenic characteristics of Nocardia brasiliensis isolated from human mycetomas

A previous analysis of the physiological properties of Nocardia brasiliensis strains isolated from soil of Tucumán proves that non-pathogenic strains have a different behaviour pattern from the pathogenic strains.In the present paper, 16 Nocardia brasiliensis strains isolated from human mycetomas were studied in the same way. The object is to determine if any of the Nocardia brasiliensis present in soil can produce human mycetomas.The macro and micromorphological, biochemical (17 tests), physiological (4 tests) and pathological characteristics were determined for each of the strains. Experimental pathogenicity was determined using albino Swiss mice by inoculation into the footpads.The strains of Nocardia brasiliensis that cause human mycetomas have the same physiological pattern and experimental pathogenicity as the virulent strains present in soil.     

Immune Response to Nocardia brasiliensis Extracellular Antigens in Patients with Mycetoma

The ability of culture-filtrate proteins to induce a cellular immune response in infected mice and humans was investigated. A crude extract culture filtrate of Nocardia brasiliensis (CFA) and five semi-purified CFA fractions (P1, P2, P3, P4, P5) were used to stimulate BALB/c mice spleen-cell cultures. The animals were divided into three groups: the first group was infected with 1 × 10⁷ CFU of N. brasiliensis in the footpad, the second group was immunized with heat-killed bacteria, and the third was injected with sterile saline. IFN-γ, IL-1α, and IL-4 concentrations were determined in culture supernatants. Protein fractions eliciting IFN-γ production in mice, as well as the CFA, were used to stimulate IFN-γ production and in vitro cell proliferation assays with peripheral blood mononuclear cells of patients with actinomycetoma by N. brasiliensis, individuals with pulmonary tuberculosis, and healthy controls. In mice, CFA and three of the protein fractions (P3, P4 and P5) induced significant IFN-γ production in the infected group. In humans, only the CFA-induced IFN-γ production and cell proliferation in the group of patients with actinomycetoma. There was no stimulation in tuberculosis patients nor healthy controls. These results suggest that some culture-filtrate antigens are recognized by patients with active actinomycetoma and do not cross-react with M. tuberculosis antigens, being therefore potential candidates to develop a diagnostic test.     

Mycetoma: Experience of 482 Cases in a Single Center in Mexico

Mycetoma is a chronic granulomatous disease. It is classified into eumycetoma caused by fungi and actinomycetoma due to filamentous actinomycetes. Mycetoma can be found in geographic areas in close proximity to the Tropic of Cancer. Mexico is one of the countries in which this disease is highly endemic. In this retrospective study we report epidemiologic, clinical and microbiologic data of mycetoma observed in the General Hospital of Mexico in a 33 year-period (1980 to 2013). A total of 482 cases were included which were clinical and microbiology confirmed. Four hundred and forty four cases (92.11%) were actinomycetomas and 38 cases (7.88%) were eumycetomas. Most patients were agricultural workers; there was a male predominance with a sex ratio of 3∶1. The mean age was 34.5 years old (most ranged from 21 to 40 years). The main affected localization was lower and upper limbs (70.74% and 14.52% respectively). Most of the patients came from humid tropical areas (Morelos, Guerrero and Hidalgo were the regions commonly reported). The main clinical presentation was as tumor-like soft tissue swelling with draining sinuses (97.1%). Grains were observed in all the cases. The principal causative agents for actinomycetoma were: Nocardia brasiliensis (78.21%) and Actinomadura madurae (8.7%); meanwhile, for eumycetomas: Madurella mycetomatis and Scedosporium boydii (synonym: Pseudallescheria boydii) were identified. This is a single-center, with long-follow up, cross-sectional study that allows determining the prevalence and characteristics of mycetoma in different regions of Mexico.     

Actinomycete infections in humans--a review.

Diseases caused by pathogenic aerobic and facultatively anaerobic actinomycetes differ considerably with respect to their etiology, pathogenesis, clinical appearance and epidemiology. Facultatively anaerobic (fermentative) actinomycetes may not only be involved etiologically in the three classical forms of cervicofacial, thoracic and abdominal actinomycoses, but also in infections of the female genital organs, the eye, the tissue adjacent to dental implantation elements and tooth extraction wounds. The species distribution of the fermentative actinomycetes isolated from these conditions varied to a certain, but characteristic, extent, as did the concomitant actinomycotic flora. The sex ratio reported for human Actinomyces infections (male:female = 3:1) appeared to be restricted to actinomycotic abscesses and empyemas. The prevailing pathogenic, obligately aerobic actinomycete species in Germany was found to be Nocardia farcinica followed by Nocardia asteroides. The comparatively high incidence of N. farcinica infections was chiefly due to the occurrence of nosocomial postoperative wound infections by this pathogen observed in two German hospitals. Besides surgical treatment, immunosuppressive treatment appeared to be the most common factor predisposing for nocardiosis. Recent observations strongly suggested that the spectrum of human nocardial infections in Germany has been changing, as regards the overall incidence, the prevalence of N. farcinica, the sex ratio, the mean age of patients, as well as the role of N. farcinica as a possibly important nosocomial pathogen.     

Mycetomas in Iran: a review article

Mycetomas are the subcutaneous and relatively rare chronic pustular infections. The etiologic agents of mycetomas are a group of saprophytic fungi and actinomycetes living in soil. We retrospectively discussed the overall prevalence of mycetomas and the prevalence of infective agents in Iran between 1972 and 2005. Seventy-six cases of mycetomas have been reported from various geographical locations in Iran during 33 years. Analysis of the records revealed that 84.5% were actinomycetoma and only 15.5% were eumycetoma. Disease mainly has been seen in foot, and the male to female ratio was 2:1. Mycetomas were abundant among farmers in rural areas of Iran. The commonest agents of mycetomas were Nocardia asteroids, Actinomadura madura (actinomycetoma) and Allesheria boydii (eumycetoma). The peak age of onset was between 31 and 51 years.     

Marine actinomycetes: An ongoing source of novel bioactive metabolites

Actinomycetes are virtually unlimited sources of novel compounds with many therapeutic applications and hold a prominent position due to their diversity and proven ability to produce novel bioactive compounds. There are more than 22,000 known microbial secondary metabolites, 70% of which are produced by actinomycetes, 20% from fungi, 7% from Bacillus spp. and 1–2% by other bacteria. Among the actinomycetes, streptomycetes group are considered economically important because out of the approximately more than 10,000 known antibiotics, 50–55% are produced by this genus. The ecological role of actinomycetes in the marine ecosystem is largely neglected and various assumptions meant there was little incentive to isolate marine strains for search and discovery of new drugs. The search for and discovery of rare and new actinomycetes is of significant interest to drug discovery due to a growing need for the development of new and potent therapeutic agents. Modern molecular technologies are adding strength to the target-directed search for detection and isolation of bioactive actinomycetes, and continued development of improved cultivation methods and molecular technologies for accessing the marine environment promises to provide access to this significant new source of chemical diversity with novel/rare actinomycetes including new species of previously reported actinomycetes.     

Antagonistic Activity of <Emphasis Type="Italic">Nocardia brasiliensis</Emphasis> PTCC 1422 Against Isolated Enterobacteriaceae from Urinary Tract Infections

The main drawback of current antibiotic therapies is the emergence and rapid increase in antibiotic resistance. Nocardiae are aerobic, Gram-positive, catalase-positive, non-motile actinomycetes. Nocardia brasiliensis was reported as antibiotic producer. The purpose of the study was to determine antibacterial activity of N. brasiliensis PTCC 1422 against isolated Enterobacteriaceae from urinary tract infections (UTIs). The common bacteria from UTIs were isolated from hospital samples. Antimicrobial susceptibility test was performed for the isolated pathogens using Kirby–Bauer disk diffusion method according to clinical and Laboratory Standards Institute guideline. Antagonistic activity of N. brasiliensis PTCC 1422 was examined with well diffusion methods. Supernatant of N. brasiliensis PTCC 1422 by submerged culture was analyzed with gas chromatography–mass spectrometry. Isolated strains included Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Proteus mirabilis. The most common pathogen isolated was E. coli (72.5 %). Bacterial isolates revealed the presence of high levels of antimicrobial resistances to ceftriaxone and low levels of resistance to cephalexin. Supernatant of N. brasiliensis PTCC 1422 showed antibacterial activity against all of the isolated microorganisms in well diffusion method. The antibiotic resistance among the uropathogens is an evolving process, so a routine surveillance to monitor the etiologic agents of UTI and the resistance pattern should be carried out timely to choose the most effective empirical treatment by the physicians. Our present investigation indicates that the substances present in the N. brasiliensis PTCC 1422 could be used to inhibit the growth of human pathogen. Antibacterial resistance among bacterial uropathogen is an evolving process. Therefore, in the field on the need of re-evaluation of empirical treatment of UTIs, our present. The study has demonstrated that N. brasiliensis PTCC 1422 has a high potential for the treatment of UTIs.     

Actinomycetoma in Arm Disseminated to Lung with Grains of <Emphasis Type="Italic">Nocardia brasiliensis</Emphasis> with Peripheral Filaments

Actinomycetomas represent 97.8% of mycetomas in Mexico, where 86.6% are produced by Nocardia brasiliensis. We report a case of actinomycetoma in the arm by Nocardia brasiliensis disseminated to lung. Uncommon grains were observed which present outside peripheral filaments and also numerous filaments loosing the grains. These characteristics of the grains are due probably because for the long treatment with antibiotics of the patient. In situ antibiotic action against the microcolonies is discussed.     

Cytokine production and lymphocyte proliferation in patients with Nocardia brasiliensis actinomycetoma

IFN-γ, TNF-α, IL-4, IL10 and IL-12 concentrations in the supernatant of peripheral blood mononuclear cell (PBMC) cultures and the in vitro proliferation of PBMC were studied in 25 patients with actinomycetoma caused by Nocardia brasiliensisand in 10 healthy controls from endemic zones. Cell cultures were stimulated by a N. brasiliensiscrude cytoplasmic antigen (NB) and five semi-purified protein fractions (NB2, NB4, NB6, NB8, and NB10) separated by isoelectric. Phytohemagglutinin (PHA) and purified protein derivative (PPD) of Mycobacterium tuberculosiswere used as control antigens. Skin tests were performed by injecting 0.1 ml of candidin and PPD intradermally (ID). Patients showed a poor response to tuberculin, while their response to candidin was more than two fold greater than that observed in the controls. Cell proliferation showed no statistically significant differences in either group. IFN-γ production was higher in the healthy controls than in the patients, whereas TNF-α secretion was slightly higher in the patients’ cultures. IL-4 was detected in the patients’ cultures but not in the controls. IL-10 and IL-12 were present at low concentrations in both groups. These results suggest that patients with actinomycetoma show normal antigen recognition, but with low IFN-γ production, and higher concentrations of IL-4, IL-10 and TNF-α in the patients’ PBMC cultures, indicating that they probably have a Th2 type of immune response.     

Relationships between the physiological characteristics and pathogenicity of Nocardia brasiliensis

The analysis of 19 physiological properties of 28 strains of Nocardia brasiliensis isolated from soil reveal differences which could be of great value in distinguishing pathogenic from nonpathogenic strains.     

Phosphosite-specific regulation of the oxidative-stress response of Paracoccidioides brasiliensis: a shotgun phosphoproteomic analysis

Paracoccidioides brasiliensis, a thermally dimorphic fungus, is the causative agent of paracoccidioidomycosis, a systemic mycosis that is widespread in Latin America. This fungus is a facultative intracellular pathogen able to survive and replicate inside non-activated macrophages. Therefore, the survival of P. brasiliensis inside the host depends on the ability to adapt to oxidative stress induced by immune cells, especially alveolar macrophages. For several years, reactive oxygen species (ROS) were only associated with pathological processes. Currently, a plethora of roles for ROS in cell signaling have emerged. We have previously reported that low ROS concentrations cause cell proliferation in the human pathogenic fungus P. brasiliensis. In the present report, we investigated the influence of phosphorylation events in that process. Using a mass spectrometry-based approach, we mapped 440 phosphorylation sites in 230 P. brasiliensis proteins and showed that phosphorylation at different sites determines fungal responses to oxidative stress, which are regulated by phosphatases and kinases activities. Furthermore, we present additional evidence for a functional two-component signal transduction system in P. brasiliensis. These findings will help us to understand the phosphorylation events involved in the oxidative stress response.     

The mycoses of Oceania

A review of the mycoses of Oceania, defined as the Pacific islands of Melanesia, Micronesia and Polynesia, has revealed the occurrence of cutaneous, subcutaneous and systemic mycoses.Tinea versicolor is the most prevalent of the cutaneous mycotic infections. Dermatophyte infections are also common. The etiologic agents incriminated have beenEpidermophyton floccosum, Microsporum canis, members of theMicrosporum fulvum-gypseum complex,Trichophyton concentricum, T. mentagrophytes, T. rubrum, T. tonsurans andT. verrucosum. Only one case of tinea nigra has been reported and that was in Hawaii. Among the fungi that cause subcutaneous mycoses onlyAllescheria boydii, Nocardia brasiliensis, Madurella mycetomi andSporothrix schenckii have been encountered to date. Among the systemic mycoses, actinomycosis, cryptococcosis and histoplasmosis have been found to be present. The geographic distribution of these actinomycotic and mycotic infections is provided along with data on soil isolation records and histoplasmin sensitivity surveys.Paper presented at the Symposium on Microbiology, Canadian Medical Expedition to Easter Island, Montreal, April 26–28, 1971     

Actinomycetes in Karstic caves of northern Spain (Altamira and Tito Bustillo).

A variety of isolation procedures were carried out to study the involvement of bacteria in the colonisation and biodeterioration of Spanish caves with paleolithic rock art (Altamira and Tito Bustillo). The applied techniques mainly aimed to isolate heterotrophic bacteria such as streptomycetes, nocardioform and coryneform actinomycetes, and other gram-positive and gram-negative bacteria. The results demonstrated that actinomycetes were the most abundant gram-positive bacteria in the caves. Actinomycetes revealed a great taxonomic diversity with the predominant isolates belonging to the genus Streptomyces. Members of the genera Nocardia, Rhodococcus, Nocardioides, Amycolatopsis, Saccharothrix, Brevibacterium, Microbacterium, and coccoid actinomycetes (family Micrococcaceae) were also found.     

Genome based analysis of type-I polyketide synthase and nonribosomal peptide synthetase gene clusters in seven strains of five representative Nocardia species

Background

Actinobacteria of the genus Nocardia usually live in soil or water and play saprophytic roles, but they also opportunistically infect the respiratory system, skin, and other organs of humans and animals. Primarily because of the clinical importance of the strains, some Nocardia genomes have been sequenced, and genome sequences have accumulated. Genome sizes of Nocardia strains are similar to those of Streptomyces strains, the producers of most antibiotics. In the present work, we compared secondary metabolite biosynthesis gene clusters of type-I polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS) among genomes of representative Nocardia species/strains based on domain organization and amino acid sequence homology.

Results

Draft genome sequences of Nocardia asteroides NBRC 15531^T, Nocardia otitidiscaviarum IFM 11049, Nocardia brasiliensis NBRC 14402^T, and N. brasiliensis IFM 10847 were read and compared with published complete genome sequences of Nocardia farcinica IFM 10152, Nocardia cyriacigeorgica GUH-2, and N. brasiliensis HUJEG-1. Genome sizes are as follows: N. farcinica, 6.0 Mb; N. cyriacigeorgica, 6.2 Mb; N. asteroides, 7.0 Mb; N. otitidiscaviarum, 7.8 Mb; and N. brasiliensis, 8.9 - 9.4 Mb. Predicted numbers of PKS-I, NRPS, and PKS-I/NRPS hybrid clusters ranged between 4–11, 7–13, and 1–6, respectively, depending on strains, and tended to increase with increasing genome size. Domain and module structures of representative or unique clusters are discussed in the text.

Conclusion

We conclude the following: 1) genomes of Nocardia strains carry as many PKS-I and NRPS gene clusters as those of Streptomyces strains, 2) the number of PKS-I and NRPS gene clusters in Nocardia strains varies substantially depending on species, and N. brasiliensis strains carry the largest numbers of clusters among the species studied, 3) the seven Nocardia strains studied in the present work have seven common PKS-I and/or NRPS clusters, some of whose products are yet to be studied, and 4) different N. brasiliensis strains have some different gene clusters of PKS-I/NRPS, although the rest of the clusters are common within the N. brasiliensis strains. Genome sequencing suggested that Nocardia strains are highly promising resources in the search of novel secondary metabolites.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-323) contains supplementary material, which is available to authorized users.     

Nocardia brasiliensis: from microbe to human and experimental infections

Nocardia brasiliensis is a Gram-positive bacterium that lives as a saprophyte in soil. In this article the physical properties, chemical composition and taxonomic position of this species is reviewed. Human infections and an experimental model of actinomycetoma in BALB/c mice as well as the host-immune response is described.     

Antifungal Resistance Mechanisms in Dermatophytes

Although fungi do not cause outbreaks or pandemics, the incidence of severe systemic fungal infections has increased significantly, mainly because of the explosive growth in the number of patients with compromised immune system. Thus, drug resistance in pathogenic fungi, including dermatophytes, is gaining importance. The molecular aspects involved in the resistance of dermatophytes to marketed antifungals and other cytotoxic drugs, such as modifications of target enzymes, over-expression of genes encoding ATP-binding cassette (ABC) transporters and stress-response-related proteins are reviewed. Emphasis is placed on the mechanisms used by dermatophytes to overcome the inhibitory action of terbinafine and survival in the host environment. The relevance of identifying new molecular targets, of expanding the understanding about the molecular mechanisms of resistance and of using this information to design new drugs or to modify those that have become ineffective is also discussed.