Mycobacterium leprae's evolution and environmental adaptation.

Leprosy is an ancient disease caused by the acid-fast bacillus Mycobacterium leprae, also known as Hansen's bacillus. M. leprae is an obligate intracellular microorganism with a marked Schwann cell tropism and is the only human pathogen capable of invading the superficial peripheral nerves. The transmission mechanism of M. leprae is not fully understood; however, the nasal mucosa is accepted as main route of M. leprae entry to the human host. The complete sequencing and the comparative genome analysis show that M. leprae underwent a genome reductive evolution process, as result of lifestyle change and adaptation to different environments; some of lost genes are homologous to those of host cells. Thus, M. leprae reduced its genome size to 3.3 Mbp, contributing to obtain the lowest GC content (approximately 58%) among mycobacteria. The M. leprae genome contains 1614 open reading frames coding for functional proteins, and 1310 pseudogenes corresponding to 41% of the genome, approximately. Comparative analyses to different microorganisms showed that M. leprae possesses the highest content of pseudogenes among pathogenic and non-pathogenic bacteria and archaea. The pathogen adaptation into host cells, as the Schwann cells, brought about the reduction of the genome and induced multiple gene inactivation. The present review highlights the characteristics of genome's reductive evolution that M. leprae experiences in the genetic aspects compared with other pathogens. The possible mechanisms of pseudogenes formation are discussed.

Screening food-borne and zoonotic pathogens associated with livestock practices in the Sumapaz region, Cundinamarca, Colombia.

Hazardous practices regarding antibiotics misuse, unsanitary milking procedures, and the commercial sales of raw milk and unpasteurized dairy products are currently being practiced by livestock farmers in the Sumapaz region (Colombia). The purpose of this study was to screen for food-borne and zoonotic pathogens associated with local livestock practices. We evaluated 1098 cows from 46 livestock farms in the Sumapaz region that were selected by random. Of the total population of cattle, 962 animals (88%) were tested for bovine TB using a caudal-fold tuberculin test and 546 (50%) for brucellosis by a competitive ELISA. In the population tested, 23 cows were positive for Brucella sp. representing a 4.2% seroprevalence and no cases of bovine tuberculosis were found. In addition, food-borne contamination with Escherichia coli and Staphylococcus aureus was assessed together with antibiotic susceptibility for ten different antibiotics in milk samples from 16 livestock farms. We found that 12 of the farms (75%) were contaminated with these food-borne pathogens. Noteworthy, all of the isolated pathogenic strains were resistant to multiple antibiotics, primarily to oxytetracycline and erythromycin. Our findings suggest that livestock products could be a source of exposure to Brucella and multidrug-resistant E. coli and S. aureus strains as a result of unhygienic livestock practices in the Sumapaz region. Training in good farming practices is the key to improving safety in food production.

The ATPase activity of the mycobacterial plasma membrane is inhibited by the LL37-analogous peptide LLAP.

The emergence of multidrug-resistant Mycobacterium tuberculosis strains has led to the development of new antituberculous agents. In this context, antimicrobial targeting proteins to the cell membrane are interesting due to the avoidance of the plasma membrane permeation. Through this study, the antimicrobial activity, cellular toxicity, as well as the effect on the mycobacterial cell membrane ATPase activity of a cathelicidin-analogous peptide were assessed. By using bioinformatics analyses, a 15 amino acid LL37-analogous peptide called LLAP, which has the amino acid sequence: GRKSAKKIGKRAKRI, was designed to improve its helical structure and antibacterial activity compared to the native sequence. The LLAP peptide was synthesized, purified by RP-HPLC and its structural characteristics were determined by MALDI-TOF MS and circular dichroism. Compared to the native amino acid sequence, the minimum inhibitory concentration and cytotoxic activity of LLAP were 4.0 and 5.6-fold lower, respectively. In addition, the hemolytic activity of LLAP was lower than 1.1% and the cytotoxic activity of peptides was similar for both peptides. Interestingly, the LLAP peptide displayed approximately 50% inhibition of basal ATPase activity of the mycobacterial plasma membrane, which could in turn be associated with the impaired cell viability. The results suggest that LLAP could be considered as potential antimycobacterial compounds against cell membrane targeting ATPases. However, this antimycobacterial activity can be improved. It is expected further applications to be found for other antimicrobial peptides families based on the implemented methodology.

Identification of potential biomarkers to distinguish Mycobacterium colombiense from other mycobacterial species.

Mycobacterium avium complex (MAC) consists of 9 species of slow-growing mycobacteria with differing degrees of pathogenicity, host preference and environmental distribution. Mycobacterium colombiense is a novel member of MAC that is responsible for disseminated infections in HIV-infected patients in Colombia and lymphadenopathy cases in Europe. At present, methods to easily differentiate novel members of MAC are lacking. In this study, we identified possible biomarkers that are potentially useful for the detection of M. colombiense by PCR or chromatography. The Randomly Amplified Polymorphic DNA (RAPD) technique was used to amplify genomic fragments of M. colombiense CECT 3035 that were subsequently used in the development of a direct colony-specific PCR assay using specific primers. The designed primers amplified a 634-bp fragment of DNA from M. colombiense, which included a 450-bp genomic region that encodes a hypothetical protein of 149 amino acids that is exclusive to M. colombiense. Bioinformatic analyses revealed that this hypothetical protein had no signal peptide, active sites or functional domains to aid its identification or classification. In addition, using thin-layer chromatography, we identified a different profile of mycolates for M. colombiense strains. The test developed in this study has potential applications in the routine identification of M. colombiense and in molecular assays designed for the surveillance of MAC strains.

In silico identification and characterization of the ion transport specificity for P-type ATPases in the Mycobacterium tuberculosis complex.

BACKGROUND: P-type ATPases hydrolyze ATP and release energy that is used in the transport of ions against electrochemical gradients across plasma membranes, making these proteins essential for cell viability. Currently, the distribution and function of these ion transporters in mycobacteria are poorly understood. RESULTS: In this study, probabilistic profiles were constructed based on hidden Markov models to identify and classify P-type ATPases in the Mycobacterium tuberculosis complex (MTBC) according to the type of ion transported across the plasma membrane. Topology, hydrophobicity profiles and conserved motifs were analyzed to correlate amino acid sequences of P-type ATPases and ion transport specificity. Twelve candidate P-type ATPases annotated in the M. tuberculosis H37Rv proteome were identified in all members of the MTBC, and probabilistic profiles classified them into one of the following three groups: heavy metal cation transporters, alkaline and alkaline earth metal cation transporters, and the beta subunit of a prokaryotic potassium pump. Interestingly, counterparts of the non-catalytic beta subunits of Hydrogen/Potassium and Sodium/Potassium P-type ATPases were not found. CONCLUSIONS: The high content of heavy metal transporters found in the MTBC suggests that they could play an important role in the ability of M. tuberculosis to survive inside macrophages, where tubercle bacilli face high levels of toxic metals. Finally, the results obtained in this work provide a starting point for experimental studies that may elucidate the ion specificity of the MTBC P-type ATPases and their role in mycobacterial infections.

Effect of antimicrobial peptides on ATPase activity and proton pumping in plasma membrane vesicles obtained from mycobacteria.

The potential usefulness of antimicrobial peptides (AMPs) as antimycobacterial compounds has not been extensively explored. Although a myriad of studies on AMPs from different sources have been done, some of its mechanisms of action are still unknown. Maganins are of particular interest since they do not lyse non-dividing mammalian cells. In this work, AMPs with well-recognized activity against bacteria were synthesized, characterized, purified and their antimycobacterial activity and influence on ATPase activity in mycobacterial plasma membrane vesicles were assessed. Using bioinformatics tools, a magainin-I analog peptide (MIAP) with improved antimicrobial activity was designed. The influence of MIAP on proton (H(+)) pumping mediated by F(1)F(0)-ATPase in plasma membrane vesicles obtained from Mycobacterium tuberculosis was evaluated. We observed that the antimycobacterial activity of AMPs was low and variable. However, the activity of the designed peptide MIAP against M. tuberculosis was 2-fold higher in comparison to magainin-I. The basal ATPase activity of mycobacterial plasma membrane vesicles decreased approximately 24-30% in the presence of AMPs. On the other hand, the MIAP peptide completely abolished the F(1)F(0)-ATPase activity involved in H(+) pumping across M. tuberculosis plasma membranes vesicles at levels similar to the specific inhibitor N,N' dicyclohexylcarbodiimide. These finding suggest that AMPs can inhibit the H(+) pumping F(1)F(0)-ATPase of mycobacterial plasma membrane that potentially interferes the internal pH and viability of mycobacteria.

Electrochemical monitoring of the metabolic activity of mycobacteria in culture.

Mycobacterial metabolic activity is typically measured using time-consuming manual methods based on nutrient consumption, nucleic acid synthesis or reduction of tetrazolium salts. In this study, we propose much simpler electrochemical methods for continuous monitoring of the metabolic activity of mycobacteria in culture. Chronoamperometry and chronopotentiometry were used to detect metabolic activity of both slow-growing and fast-growing mycobacteria using a potentiostat with 2D-electrochemical cell. Electrochemical measurements were able to detect statistically significant differences in the metabolic activity of approximately 10(7) mycobacteria in different growth conditions, within less than 24 h of mycobacterial culture. The metabolic activity of mycobacteria measured by the used electrochemical methods correlated well with changes in general respiratory conditions within the cells as it was evaluated by different biochemical tests. Chronoamperometry and chronopotentiometry allowed measurement of mycobacterial metabolic activity without invasive chemical reactions, at minimal bacterial load and when metabolic response of mycobacteria occurs quickly. The proposed methodology is simple, rapid and cost-effective, and it is expected that both in vitro and in vivo metabolic activity of human mycobacterial pathogens as Mycobacterium tuberculosis can be measured when the implementation of this method to analyze virulent strains is adapted.

Detection and diversity evaluation of tetracycline resistance genes in grassland-based production systems in Colombia, South america.

Grassland-based production systems use ∼26% of land surface on earth. However, there are no evaluations of these systems as a source of antibiotic pollution. This study was conducted to evaluate the presence, diversity, and distribution of tetracycline resistance genes in the grasslands of the Colombian Andes, where administration of antibiotics to animals is limited to treat disease and growth promoters are not included in animals' diet. Animal (ruminal fluid and feces) and environmental (soil and water) samples were collected from different dairy cattle farms and evaluated by PCR for the genes tet(M), tet(O), tetB(P), tet(Q), tet(W), tet(S), tet(T), otr(A), which encode ribosomal protection proteins (RPPs), and the genes tet(A), tet(B), tet(D), tet(H), tet(J), and tet(Z), encoding efflux pumps. A wide distribution and high frequency for genes tet(W) and tet(Q) were found in both sample types. Genes tet(O) and tetB(P), detected in high frequencies in feces, were detected in low frequencies or not detected at all in the environment. Other genes encoding RPPs, such as tet(M), tet(S), and tet(T), were detected at very low frequencies and restricted distributions. Genes encoding efflux pumps were not common in this region, and only two of them, tet(B) and tet(Z), were detected. DGGE-PCR followed by comparative sequence analysis of tet(W) and tet(Q) showed that the sequences detected in animals did not differ from those coming from soil and water. Finally, the farms sampled in this study showed more than 50% similarity in relation to the tet genes detected. In conclusion, there was a remarkable presence of tet genes in these production systems and, although not all genes detected in animal reservoirs were detected in the environment, there is a predominant distribution of tet(W) and tet(Q) in both animal and environmental reservoirs. Sequence similarity analysis suggests the transmission of these genes from animals to the environment.

The virulence-associated two-component PhoP-PhoR system controls the biosynthesis of polyketide-derived lipids in Mycobacterium tuberculosis.

Two-component regulatory signal transduction systems are important elements of the adaptative response of prokaryotes to a variety of environmental stimuli. Disruption of PhoP-PhoR in Mycobacterium tuberculosis dramatically attenuates virulence, implying that this system directly and/or indirectly coordinates the expression of important virulence factors whose identity remains to be established. Interestingly, in knockingout the PhoP-PhoR two-component system in M. tuberculosis Mt103, dramatic changes in the colonial morphology, cording properties, and reactivity of the mutant strain to the basic dye neutral red, all intrinsic properties of tubercle bacilli known to correlate with virulence, were noted. Because deficiencies in the ability of the mutant to form serpentine cords and stain with the dye are likely the results of alterations of its cell envelope composition, we undertook to analyze the lipid content of phoP and phoP-phoR mutants constructed in two different strains of M. tuberculosis. Our results indicate that PhoP coordinately and positively regulates the synthesis of methyl-branched fatty acid-containing acyltrehaloses known to be restricted to pathogenic species of the M. tuberculosis complex, namely diacyltrehaloses, polyacyltrehaloses, and sulfolipids. Evidence is also provided that PhoP but not PhoR is required for the production of these lipids. This work represents an important step toward the functional characterization of PhoP-PhoR and the understanding of complex lipid synthesis in M. tuberculosis.