A novel experimental immunomodulatory therapy against Nocardia brasiliensis in a BALB/c murine model.

BACKGROUND: Mycetoma is recognized as a neglected tropical disease and there are still therapeutic challenges, especially in cases recalcitrant to standard therapy or with high risk of dissemination. Subcultures have been used previously to decrease the virulence of human pathogens. Previous reports have demonstrated that after carrying out 200 subcultures of Nocardia brasiliensis, a decrease in virulence was observed. AIM: To evaluate the effect of attenuated N. brasiliensis strains on the development of lesions in an established mycetoma infection. METHODS: Female 8-12-week-old BALB/c mice were injected with N. brasiliensis suspension to establish a mycetoma. Sixty mice were selected and divided into three groups: two of these groups were inoculated in the dorsum with N. brasiliensis subcultured 200 and 400 times, respectively, while the third group served as control. The thickness of each lesion was measured with calipers every week for 12 weeks. RESULTS: After 12 weeks, we observed that inoculation of 1 × 105 colony-forming units of attenuated N. brasiliensis strains was able to modify the natural history of the infection, with a decrease in the size of the lesions, particularly with P400, compared with the control group (P < 0.01). CONCLUSION: In this experimental evaluation of an immunomodulatory therapy with attenuated N. brasiliensis strains in a murine model, there was a greater stability in the size of the lesion over time in BALB/c mice inoculated with the P400 strain. This treatment could open the possibility of using the attenuated strain as immunomodulatory therapy in patients recalcitrant to standard therapy, with high risk of dissemination or who develop drug-related adverse effects.

Evaluation of the Combined Therapy of DA-7218, a New Oxazolidinone, and Trimethoprim/ Sulfamethoxazole in the Treatment of Experimental Actinomycetoma by Nocardia brasiliensis.

OBJECTIVES: Currently, for actinomycetoma, combined antimicrobial therapy is preferred to the use of a single compound. This is in order to provide a broader-spectrum coverage due to a combinatory or synergistic effect between the drugs, and to decrease the possibility of emergence of natural resistant strains. A new oxazolidinone pro-drug, DA-7218 [(R)-3-(4-(2-(2-methyltetrazol-5-yl)-pyridin-5-yl)-3-fluorophenyl)-2-oxo-5-oxazolidinyl) methyl-disodium-phosphate] (recently re-named TR-701), has shown very good in vitro and in vivo activities against several gram-positive bacteria including Nocardia spp. METHODS: In the present work we evaluated the effect of DA-7218 at two different doses, alone and combined with trimethoprim/ sulfamethoxazole (SXT), in an experimental Nocardia brasiliensis actinomycetoma murine model. We also included a negative and a positive control group (linezolid and saline solution respectively). RESULTS: At the end of the treatment period, we observed a clinically and statistically significant difference among the drug receiving groups (combined, alone and linezolid) and the control group (P=0.004). The difference was higher (P= 0.004) between the groups receiving DA-7218 (25mg/kg) alone or combined with SXT, and the control group (saline solution). CONCLUSIONS: In this work we proved that DA-7218 alone and combined with SXT is effective in the treatment of experimental actinomycetoma by Nocardia brasiliensis and that it could be potentially useful in the treatment of human actinomycetoma.

Effect of serial subculturing on the genetic composition and cytotoxic activity of Mycobacterium tuberculosis.

Continuous subculture has been observed to produce changes in the virulence of micro-organisms, e.g. rabies virus, poliovirus and Mycobacterium bovis BCG. The latter has been used as a vaccine for tuberculosis for the last 100 years; however, in some instances its efficacy has been observed to be very low. In order to determine whether similar changes can be produced in Mycobacterium tuberculosis, we selected four isolates, M. tuberculosis H37Rv, a Beijing strain (DR-689), and two more isolates with deletion of the phospholipase C locus (plcA-plcB-plcC ), and subjected them to serial culturing on Middlebrook 7H9 medium, with or without ox bile. After 100 passages, we performed RFLP-IS6110 analysis to determine whether genomic changes were produced. We also checked their genomic composition by microarray analysis. Changes in virulence were studied by measuring the cytotoxic effect of parental and subcultured isolates on a THP-1 macrophage monolayer. The most visible change was the change of position of an IS6110 band of approximately 1400 bp to approximately 1600 bp in the Beijing isolate subcultured in the ox bile medium. Analysis by microarray and PCR confirmation did not reveal any genomic changes. Cytotoxic activity was decreased in the isolates at levels close to that of BCG, and more consistently in those subcultured in the presence of ox bile.

In vitro activity of PNU-100766 (linezolid), a new oxazolidinone antimicrobial, against Nocardia brasiliensis.

The in vitro activity of a novel oxazolidinone, linezolid, was studied by comparing the activity of linezolid with those of amikacin, trimethoprim-sulfamethoxazole, and amoxicillin-clavulanic acid against 25 strains of Nocardia brasiliensis isolated from patients with mycetoma. All N. brasiliensis strains tested were sensitive to linezolid (MIC at which 90% of strains are inhibited [MIC(90)], 2 microg/ml; MIC(50), 1 microg/ml). This antimicrobial might constitute a good alternative for treatment of actinomycetoma.

Phospholipase region of Mycobacterium tuberculosis is a preferential locus for IS6110 transposition.

Enzymes with phospholipase C activity in Mycobacterium tuberculosis have been recently described. The three genes encoding these proteins, plcA, plcB, and plcC, are located at position 2351 of the genomic map of M. tuberculosis H37Rv and are arranged in tandem. We have previously described the presence of variations in the restriction fragment length polymorphism patterns of the plcA and plcB genes in M. tuberculosis clinical isolates. In the present work we investigated the origin of this polymorphism by sequence analysis of the phospholipase-encoding regions of 11 polymorphic M. tuberculosis clinical isolates. To do so, a long-PCR assay was used to amplify a 5,131-bp fragment that contains the plcA and plcB genes and part of the plcC gene. In the M. tuberculosis strains studied the production of an amplicon approximately 1,400 bp larger than anticipated was observed. Sequence analysis of the PCR products indicated the presence of a foreign sequence that corresponded to an IS6110 element. We observed insertion elements in the plcA, plcB, and plcC genes. One site in plcB had the highest incidence of transposition (5 out of 11 strains). In two strains the insertion element was found in plcA in the same nucleotide position. In all the cases, IS6110 was transposed in the same direction. The high level of transposition in the phospholipase region can lead to the excision of fragments of genomic DNA by recombination of neighboring IS6110 elements, as demonstrated by finding the deletion, in two strains, of a 2,837-bp fragment that included plcA and most of plcB. This can explain the negative results obtained by some authors when detecting the mtp40 sequence (plcA) by PCR. Given the high polymorphism in this region, the use of the mtp40 sequence as a genetic marker for M. tuberculosis sensu stricto is very restricted.

Dot blot assay for detection of antidiacyltrehalose antibodies in tuberculous patients.

A simple dot blot test with diacyltrehalose (DAT) as the antigen was developed to detect anti-DAT antibodies in tuberculous patients. To enhance antigen-antibody reaction detection, rabbit serum raised against human immunoglobulins was used prior to incubation with a protein A-colloidal gold complex. With the dot blot system, it was possible to obtain a sensitivity similar to that of enzyme-linked immunosorbent assay (ELISA) and a specificity of 97.14%, versus a specificity of 94.29% by the ELISA. We conclude that this simple and fast assay could be used in places where ELISA equipment is not easy available and that it might also be applicable with other Mycobacterium tuberculosis immunogenic antigens.

Distribution of a Nocardia brasiliensis catalase gene fragment in members of the genera Nocardia, Gordona, and Rhodococcus.

An immunodominant protein from Nocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis. Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system. The sequences showed homology to some eukaryotic catalases and to a bromoperoxidase-catalase from Streptomyces violaceus. Its identity as a catalase was confirmed by analysis of its enzymatic activity on H2O2 and by a double-staining method on a nondenaturing polyacrylamide gel with 3,3'-diaminobenzidine and ferricyanide; the result showed only catalase activity, but no peroxidase. By using one of the internal amino acid sequences and a consensus catalase motif (VGNNTP), we were able to design a PCR assay that generated a 500-bp PCR product. The amplicon was analyzed, and the nucleotide sequence was compared to the GenBank database with the observation of high homology to other bacterial and eukaryotic catalases. A PCR assay based on this target sequence was performed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in several N. brasiliensis strains isolated from mycetoma. The same assay was used to determine whether there were homologous sequences in several type strains from the genera Nocardia, Rhodococcus, Gordona, and Streptomyces. All of the N. brasiliensis strains presented a positive result but only some of the actinomycetes species tested were positive in the PCR assay. In order to confirm these findings, genomic DNA was subjected to Southern blot analysis. A 1.7-kbp band was observed in the N. brasiliensis strains, and bands of different molecular weight were observed in cross-reacting actinomycetes. Sequence analysis of the amplicons of selected actinomycetes showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria.

Analysis of genetic polymorphism in the phospholipase region of Mycobacterium tuberculosis.

mtp40 was originally identified as a short genomic region that was found in strains of Mycobacterium tuberculosis but not in Mycobacterium bovis. Subsequent studies have revealed that the sequence is part of the mpcA gene, which encodes a phospholipase C. To investigate further the distribution of the mtp40 sequence, we analyzed strains of the M. tuberculosis complex by PCR and were able to amplify the mtp40 sequence in 90 of 94 strains of M. tuberculosis and in 2 strains of Mycobacterium microti but not in M. bovis or M. bovis BCG. Based on this, we developed a dot blot assay using genomic DNA which allows M. bovis to be distinguished from the majority of M. tuberculosis strains. We also probed Southern blots of 140 clinical isolates of M. tuberculosis to determine the frequency of strains lacking mtp40. This revealed an unexpected polymorphism in the phospholipase region. Two fragments were detected in 57% of samples. The expected fragment of 0.75 kbp corresponds to the region of mpcA containing mtp40. A 2.1-kbp fragment was observed to belong to a recently discovered second phospholipase gene, mpcB. In addition, some strains appeared to lack both genes, while others showed only the presence of mpcA. A few strains had additional bands, suggesting the existence of other homologs to the two phospholipase genes. We also detected the insertion of IS6110 in the mpcA coding region of one strain. The absence of these genes in some clinical isolates raises questions about their function during infection and in the development of tuberculosis disease in humans.

Development of an enzyme-linked immunosorbent assay (ELISA) combined with a streptavidin-biotin and enzyme amplification method to detect anti-2,3-di-o-acyltrehalose (DAT) antibodies in patients with tuberculosis.

IgG antibodies against the 2,3-di-o-acyltrehalose glycolipid of Mycobacterium tuberculosis were determined in a set of 49 sera from patients with pulmonary tuberculosis and 65 negative control subjects. We compared a conventional ELISA method using a beta-galactosidase anti-human IgG conjugate developed with ONPG, with an amplification ELISA system constituted of an anti-human IgG biotinylated conjugate, a streptavidin-alkaline phosphatase complex, and NADP as a substrate. The resulting NAD was measured by using a redox enzymatic recycling system of alcohol dehydrogenase, diaphorase and iodonitrotetrazolium as chromogen. With specificity set at 92.31% in both methods, we obtained a sensitivity of 42.86% in the conventional method and a sensitivity of 61.22% in the amplified method. We conclude that by using a more sensitive method we can detect cases that otherwise could be identified as false negatives.

Isolation and purification of two immunodominant antigens from Nocardia brasiliensis.

Two immunogenic proteins from a crude extract of Nocardia brasiliensis were purified to homogeneity. A 61-kDa protein (P61) was isolated from a 50% ammonium sulfate precipitate in two steps. Initially, P61 was obtained by electroelution in a 10% nondenatured preparative polyacrylamide gel electrophoresis (PAGE). In a second step, the eluate from the nondenatured gel was run in a 12% sodium dodecyl sulfate (SDS) preparative polyacrylamide gel. After elution, a single band was demonstrated by SDS-PAGE and Western blot (immunoblot). Also, a 24-kDa immunogenic protein (P24) was isolated by gel filtration in a Sephadex G-100 column and then by electroelution in a 12% nondenatured polyacrylamide gel. In a previous paper, we showed by Western blot assays that these proteins are recognized by the sera of mycetoma patients and not by sera from mycobacterial-infected or healthy individuals. We consider these proteins to be good candidates for the study of the host-parasite relationship in nocardial infections. The possible clinical application of these purified antigens in a serological diagnosis is discussed.