Antimycobacterial Activity of Hedeoma drummondii against Mycobacterium tuberculosis and Non-Tuberculous Mycobacteria.

Tuberculosis (TB) remains a major health problem worldwide, and the emergence of multi-resistant strains to first-line drugs has become the biggest obstacle to its treatment. On the other hand, the incidence of non-tuberculous mycobacteria (NTM) in humans has increased remarkably in recent years. The search for new and better treatments against mycobacterial infections is a constant at the global level. Hence, in this study, we propose to investigate the antimycobacterial effect of the extracts and major compounds of Hedeoma drummondii against clinical isolates of Mycobacterium tuberculosis and non-tuberculous mycobacteria: M. abscessus, M. fortuitum, M. intracellulare, and M. gordonae. To determine the antimycobacterial activity, a microdilution assay was used to establish the minimum inhibitory concentration (MIC) of the different strains of Mycobacterium. The methanolic extract presented the best activity against M. tuberculosis, inhibiting ten of the twelve strains analyzed at a concentration < 2500 µg/mL; meanwhile, the hexanic extract presented the best activity against non-tuberculous mycobacteria (NTM) by inhibiting eight of the ten strains studied at ≤625 µg/mL. Moreover, there is a strong positive correlation between the antimycobacterial activity of pulegone and the hexanic extract against non-tuberculous strains, so this compound could serve as a predictability marker against these types of microorganisms.

Genetic Diversity of Mycobacterium tuberculosis Isolates From an Amerindian Population in Chiapas, México.

This is the first report of the genetic diversity of the Mycobacterium tuberculosis complex isolates found in a Mexican-Amerindian setting. In this study, we analyzed isolates collected from the Highlands region of Chiapas, Mexico, by using spoligotyping and whole-genome sequencing analyses. Seventy-three M. tuberculosis isolates were analyzed initially by spoligotyping; no new spoligotypes were identified. Nineteen percent of the isolates were identified as SIT53 (T1) (n = 14), followed by SIT42 (14%, n = 10, LAM9) and SIT119 (11%; n = 8, X1). SIT53, SIT42, and orphan isolates (16.4%, n = 12) constituted about 50% of the isolates studied and were subjected to whole-genome sequencing (WGS) analysis. Most SIT53 (10/12) isolates belonged to the Euro-American sub-lineage 4.8. Most SIT42 isolates (4/7) as .well as most orphan isolates (5/8) belonged to the lineage 4.3.3 LAM group. By comparing the single-nucleotide polymorphism (SNP) patterns of the SIT53 isolates, we found one clone (<7 SNPs) and four clustered isolates (<15 SNPs). In isolates from the SIT42 and orphan groups, we did not find any clones or clusters. This work demonstrates the success of sub-lineage 4.8 to predominate in Mexico and confirms the dominion of sub-lineage 4.3.3 in Central and South America.

Kerion celsi caused by Trichophyton tonsurans in an adult.

Tinea capitis is an uncommon infection in adults, and predominantly affects women and the elderly with hormonal disorders and immunosuppression. Clinical features are often polymorphic and atypical. A kerion celsi case in an elderly female patient with type 2 diabetes mellitus and menopause is presented. The diagnosis was established by direct examination and the isolation of Trichophyton tonsurans in culture and typified by microculture. Treatment with prednisone and itraconazol was very successful. Recognizing the presentation of tinea capitis in adults will help clinicians to avoid delay in the diagnosis, awareness of the risk factors and provide early treatment to minimize sequelae of the disease.

Mycobacterium leprae Infection in a Wild Nine-Banded Armadillo, Nuevo León, Mexico.

Nine-banded armadillos (Dasypus novemcinctus) are naturally infected with Mycobacterium leprae and are implicated in the zoonotic transmission of leprosy in the United States. In Mexico, the existence of such a reservoir remains to be characterized. We describe a wild armadillo infected by M. leprae in the state of Nuevo León, Mexico.

Case Report: Coral Reef Pathogen Aspergillus sydowii Causing Black Grain Mycetoma.

Mycetoma is an infrequent subcutaneous infection caused by true fungi (eumycetoma) or aerobic actinomycetes (actinomycetoma). We report the case of a 62-year-old man with eumycetoma involving the left foot and ankle. Skin biopsy revealed black-brown grains, and in culture, a white colony fungus grew at day 8. Molecular sequencing using ITS1-ITS4 primers identified the species as Aspergillus sydowii. The patient was treated with itraconazole 200 mg twice daily and terbinafine 250 mg daily for 8 months, with complete response and no recurrence after 2.5 years of follow-up. Aspergillus sydowii is a saprotrophic fungus that rarely causes skin or nail disease. No cases of eumycetoma caused by this agent have been previously reported. As its geographic distribution continues to expand, it may increasingly be recognized as a cause of human disease.

Ex vivo infection of murine precision-cut lung tissue slices with Mycobacterium abscessus: a model to study antimycobacterial agents.

BACKGROUND: Multidrug-resistant infections due to Mycobacterium abscessus often require complex and prolonged regimens for treatment. Here, we report the evaluation of a new ex vivo antimicrobial susceptibility testing model using organotypic cultures of murine precision-cut lung slices, an experimental model in which metabolic activity, and all the usual cell types of the organ are found while the tissue architecture and the interactions between the different cells are maintained. METHODS: Precision cut lung slices (PCLS) were prepared from the lungs of wild type BALB/c mice using the Krumdieck® tissue slicer. Lung tissue slices were ex vivo infected with the virulent M. abscessus strain L948. Then, we tested the antimicrobial activity of two drugs: imipenem (4, 16 and 64 µg/mL) and tigecycline (0.25, 1 and 4 µg/mL), at 12, 24 and 48 h. Afterwards, CFUs were determined plating on blood agar to measure the surviving intracellular bacteria. The viability of PCLS was assessed by Alamar Blue assay and corroborated using histopathological analysis. RESULTS: PCLS were successfully infected with a virulent strain of M. abscessus as demonstrated by CFUs and detailed histopathological analysis. The time-course infection, including tissue damage, parallels in vivo findings reported in genetically modified murine models for M. abscessus infection. Tigecycline showed a bactericidal effect at 48 h that achieved a reduction of > 4log10 CFU/mL against the intracellular mycobacteria, while imipenem showed a bacteriostatic effect. CONCLUSIONS: The use of this new organotypic ex vivo model provides the opportunity to test new drugs against M. abscessus, decreasing the use of costly and tedious animal models.

Antimycobacterial Activity of Laurinterol and Aplysin from Laurencia johnstonii.

Marine environments represent a great opportunity for the discovery of compounds with a wide spectrum of bioactive properties. Due to their large variety and functions derived from natural selection, marine natural products may allow the identification of novel drugs based not only on newly discovered bioactive metabolites but also on already known compounds not yet thoroughly investigated. Since drug resistance has caused an increase in infections by Mycobacterium tuberculosis and nontuberculous mycobacteria, the re-evaluation of known bioactive metabolites has been suggested as a good approach to addressing this problem. In this sense, this study presents an evaluation of the in vitro effect of laurinterol and aplysin, two brominated sesquiterpenes isolated from Laurencia johnstonii, against nine M. tuberculosis strains and six nontuberculous mycobacteria (NTM). Laurinterol exhibited good antimycobacterial activity, especially against nontuberculous mycobacteria, being remarkable its effect against Mycobacterium abscessus, with minimum inhibitory concentration (MIC) values lower than those of the reference drug imipenem. This study provides further evidence for the antimycobacterial activity of some sesquiterpenes from L. johnstonii, which can be considered interesting lead compounds for the discovery of novel molecules to treat NTM infections.

Complete Genome Sequences of Mycobacterium tuberculosis Isolates Subjected to 200 Continuous Passages.

We report 6 draft genome sequences corresponding to Mycobacterium tuberculosis H37Rv and M. tuberculosis DR689, a Beijing isolate, plus their counterparts subjected to 200 continuous passages in Middlebrook 7H9 broth, either alone or with ox bile.

Climate, soil type, and geographic distribution of actinomycetoma cases in Northeast Mexico: A cross-sectional study.

BACKGROUND: Mycetoma is a chronic, granulomatous infection of subcutaneous tissue, that may involve deep structures and bone. It can be caused by bacteria (actinomycetoma) or fungi (eumycetoma). There is an epidemiological association between mycetoma and the environment, including rainfall, temperature and humidity but there are still many knowledge gaps in the identification of the natural habitat of actinomycetes, their primary reservoir, and their precise geographical distribution. Knowing the potential distribution of this infection and its ecological niche in endemic areas is relevant to determine disease management strategies and etiological agent habitat or reservoirs. METHODOLOGY/PRINCIPAL FINDINGS: This was an ambispective descriptive study of 31 patients with actinomycetoma. We determined the biophysical characteristics including temperature, precipitation, soil type, vegetation, etiological agents, and mapped actinomycetoma cases in Northeast Mexico. We identified two disease cluster areas. One in Nuevo Leon, with a predominantly kastanozems soil type, with a mean annual temperature of 22°, and a mean annual precipitation of 585.2 mm. Herein, mycetoma cases were produced by Actinomadura pelletieri, Actinomadura madurae, Nocardia brasiliensis, and Nocardia spp. The second cluster was in San Luis Potosí, where lithosols soil type predominates, with a mean annual temperature of 23.5° and a mean annual precipitation of 635.4 mm. In this area, all the cases were caused by N. brasiliensis. A. madurae cases were identified in rendzinas, kastanozems, vertisols, and lithosols soils, and A. pelletieri cases in xerosols, kastanozems, and rendzinas soils. Previous thorn trauma with Acacia or Prosopis plants was referred by 35.4% of subjects. In these states, the presence of thorny plants, such as Acacia spp., Prosopis spp., Senegalia greggi, Vachellia farnesiana and Vachellia rigidula, are common. CONCLUSIONS/SIGNIFICANCE: Mapping this neglected tropical infection aids in the detection of disease cluster areas, the development of public health strategies for early diagnosis and disease prediction models; this paves the way for more ecological niche etiological agent research.

Clinical characteristics and treatment of actinomycetoma in northeast Mexico: A case series.

BACKGROUND: Mycetoma is a neglected tropical disease characterized by nodules, scars, abscesses, and fistulae that drain serous or purulent material containing the etiological agent. Mycetoma may be caused by true fungi (eumycetoma) or filamentous aerobic bacteria (actinomycetoma). Mycetoma is more frequent in the so-called mycetoma belt (latitude 15° south and 30° north around the Tropic of Cancer), especially in Sudan, Nigeria, Somalia, India, Mexico, and Venezuela. The introduction of new antibiotics with fewer side effects, broader susceptibility profiles, and different administration routes has made information on actinomycetoma treatment and outcomes necessary. The objective of this report was to provide an update on clinical, therapeutic, and outcome data for patients with actinomycetoma attending a reference center in northeast Mexico. METHODOLOGY/PRINCIPAL FINDINGS: This was a retrospective, cross-sectional, descriptive study of 31 patients (male to female ratio 3.4:1) diagnosed with actinomycetoma by direct grain examination, histopathology, culture, or serology from January 2009 to September 2018. Most lesions were caused by Nocardia brasiliensis (83.9%) followed by Actinomadura madurae (12.9%) and Actinomadura pelletieri (3.2%). About 50% of patients had bone involvement, and the right leg was the most commonly affected region in 38.7% of cases. Farmers/agriculture workers were most commonly affected, representing 41.9% of patients. The most commonly used treatment regimen was the Welsh regimen (35.5% of cases), a combination of trimethoprim/sulfamethoxazole (TMP/SMX) plus amikacin, which had a 90% cure rate, followed by TMP/SMX plus amoxicillin/clavulanic acid in 19.4% of cases with a cure rate of 100%. In our setting, 28 (90.3%) patients were completely cured and three (9.7%) were lost to follow-up. Four patients required multiple antibiotic regimens due to recurrences and adverse effects. CONCLUSIONS/SIGNIFICANCE: In our sample, actinomycetoma was predominantly caused by N. brasiliensis. Most cases responded well to therapy with a combination of TMP/SMX with amikacin or TMP/SMX and amoxicillin/clavulanic acid. Four patients required multiple antibiotics and intrahospital care.

British Red Squirrels Remain the Only Known Wild Rodent Host for Leprosy Bacilli.

Eurasian red squirrels (Sciurus vulgaris) in the British Isles are the most recently discovered animal reservoir for the leprosy bacteria Mycobacterium leprae and Mycobacterium lepromatosis. Initial data suggest that prevalence of leprosy infection is variable and often low in different squirrel populations. Nothing is known about the presence of leprosy bacilli in other wild squirrel species despite two others (Siberian chipmunk [Tamias sibiricus], and Thirteen-lined ground squirrel [Ictidomys tridecemlineatus]) having been reported to be susceptible to experimental infection with M. leprae. Rats, a food-source in some countries where human leprosy occurs, have been suggested as potential reservoirs for leprosy bacilli, but no evidence supporting this hypothesis is currently available. We screened 301 squirrel samples covering four species [96 Eurasian red squirrels, 67 Eastern gray squirrels (Sciurus carolinensis), 35 Siberian chipmunks, and 103 Pallas's squirrels (Callosciurus erythraeus)] from Europe and 72 Mexican white-throated woodrats (Neotoma albigula) for the presence of M. leprae and M. lepromatosis using validated PCR protocols. No DNA from leprosy bacilli was detected in any of the samples tested. Given our sample-size, the pathogen should have been detected if the prevalence and/or bacillary load in the populations investigated were similar to those found for British red squirrels.

Evaluation of the intracellular activity of drugs against Mycobacterium abscessus using a THP-1 macrophage model.

We evaluated the antimicrobial effectiveness against M. abscessus in a THP-1 cell line model. No intracellular activity was observed when using amikacin or imipenem. A bacteriostatic effect was observed for cefoxitin, clarithromycin and azithromycin. Tigecycline showed the best antibacterial effect by decreasing the intracellular growth up to bactericidal level.

Phylogenomics and antimicrobial resistance of the leprosy bacillus Mycobacterium leprae.

Leprosy is a chronic human disease caused by the yet-uncultured pathogen Mycobacterium leprae. Although readily curable with multidrug therapy (MDT), over 200,000 new cases are still reported annually. Here, we obtain M. leprae genome sequences from DNA extracted directly from patients' skin biopsies using a customized protocol. Comparative and phylogenetic analysis of 154 genomes from 25 countries provides insight into evolution and antimicrobial resistance, uncovering lineages and phylogeographic trends, with the most ancestral strains linked to the Far East. In addition to known MDT-resistance mutations, we detect other mutations associated with antibiotic resistance, and retrace a potential stepwise emergence of extensive drug resistance in the pre-MDT era. Some of the previously undescribed mutations occur in genes that are apparently subject to positive selection, and two of these (ribD, fadD9) are restricted to drug-resistant strains. Finally, nonsense mutations in the nth excision repair gene are associated with greater sequence diversity and drug resistance.

Phylogenomics and antimicrobial resistance of the leprosy bacillus Mycobacterium leprae

Leprosy is a chronic human disease caused by the yet-uncultured pathogen Mycobacterium leprae. Although readily curable with multidrug therapy (MDT), over 200,000 new cases are still reported annually. Here, we obtain M. leprae genome sequences from DNA extracted directly from patients' skin biopsies using a customized protocol. Comparative and phylogenetic analysis of 154 genomes from 25 countries provides insight into evolution and antimicrobial resistance, uncovering lineages and phylogeographic trends, with the most ancestral strains linked to the Far East. In addition to known MDT-resistance mutations, we detect other mutations associated with antibiotic resistance, and retrace a potential stepwise emergence of extensive drug resistance in the pre-MDT era. Some of the previously undescribed mutations occur in genes that are apparently subject to positive selection, and two of these (ribD, fadD9) are restricted to drug-resistant strains. Finally, nonsense mutations in the nth excision repair gene are associated with greater sequence diversity and drug resistance.

Modeling tuberculosis pathogenesis through ex vivo lung tissue infection.

Tuberculosis (TB) is one of the top 10 causes of death worldwide. Several in vitro and in vivo experimental models have been used to study TB pathogenesis and induction of immune response during Mycobacterium tuberculosis infection. Precision cut lung tissue slices (PCLTS) is an experimental model, in which all the usual cell types of the organ are found, the tissue architecture and the interactions amongst the different cells are maintained. PCLTS in good physiological conditions, monitored by MTT assay and histology, were infected with either virulent Mycobacterium tuberculosis strain H37Rv or the TB vaccine strain Mycobacterium bovis BCG. Histological analysis showed that bacilli infecting lung tissue slices were observed in the alveolar septa, alveolar light spaces, near to type II pneumocytes, and inside macrophages. Mycobacterial infection of PCLTS induced TNF-α production, which is consistent with previous M. tuberculosis in vitro and in vivo studies. This is the first report of using PCLTS as a system to study M. tuberculosis infection. The PCLTS model provides a useful tool to evaluate the innate immune responses and other aspects during the early stages of mycobacterial infection.

Detection of anti-HspX antibodies and HspX protein in patient sera for the identification of recent latent infection by Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a pathogen causing tuberculosis (TB) a spectrum of disease including acute and asymptomatic latent stages. Identifying and treating latently-infected patients constitutes one of the most important impediments to TB control efforts. Those individuals can remain undiagnosed for decades serving as potential reservoirs for disease reactivation. Tests for the accurate diagnosis of latent infection currently are unavailable. HspX protein (α-crystallin), encoded by Rv2031c gene, is produced in vitro by M. tuberculosis during stationary growth phase and hypoxic or acidic culture conditions. In this study, using standard, and Luminex xMAP® bead capture ELISA, respectively, we report on detection of anti-HspX IgG and IgM antibodies and HspX protein in sera from acute and latent TB patients. For the antibody screen, levels of IgG and IgM antibodies were similar between non-infected and active TB patients; however, individuals classified into the group with latent TB showed higher values of anti-HspX IgM (p = 0.003) compared to active TB patients. Using the bead capture antigen detection assay, HspX protein was detected in sera from 56.5% of putative latent cases (p< 0.050) compared to the background median with an average of 9,900 pg/ml and a range of 1,000 to 36,000 pg/ml. Thus, presence of anti-HspX IgM antibodies and HspX protein in sera may be markers of latent TB.