Design of a Multi-Epitope Vaccine against Tuberculosis from Mycobacterium tuberculosis PE_PGRS49 and PE_PGRS56 Proteins by Reverse Vaccinology.

Tuberculosis is a disease caused by Mycobacterium tuberculosis, representing the second leading cause of death by an infectious agent worldwide. The available vaccine against this disease has insufficient coverage and variable efficacy, accounting for a high number of cases worldwide. In fact, an estimated third of the world's population has a latent infection. Therefore, developing new vaccines is crucial to preventing it. In this study, the highly antigenic PE_PGRS49 and PE_PGRS56 proteins were analyzed. These proteins were used for predicting T- and B-cell epitopes and for human leukocyte antigen (HLA) protein binding efficiency. Epitopes GGAGGNGSLSS, FAGAGGQGGLGG, GIGGGTQSATGLG (PE_PGRS49), and GTGWNGGKGDTG (PE_PGRS56) were selected based on their best physicochemical, antigenic, non-allergenic, and non-toxic properties and coupled to HLA I and HLA II structures for in silico assays. A construct with an adjuvant (RS09) plus each epitope joined by GPGPG linkers was designed, and the stability of the HLA-coupled construct was further evaluated by molecular dynamics simulations. Although experimental and in vivo studies are still necessary to ensure its protective effect against the disease, this study shows that the vaccine construct is dynamically stable and potentially effective against tuberculosis.

Organization and Characterization of the Promoter Elements of the rRNA Operons in the Slow-Growing Pathogen Mycobacterium kumamotonense.

The slow-growing, nontuberculous mycobacterium Mycobacterium kumamotonense possesses two rRNA operons, rrnA and rrnB, located downstream from the murA and tyrS genes, respectively. Here, we report the sequence and organization of the promoter regions of these two rrn operons. In the rrnA operon, transcription can be initiated from the two promoters, named P1 rrnA and PCL1, while in rrnB, transcription can only start from one, called P1 rrnB. Both rrn operons show a similar organization to the one described in Mycobacterium celatum and Mycobacterium smegmatis. Furthermore, by qRT-PCR analyses of the products generated from each promoter, we report that stress conditions such as starvation, hypoxia, and cellular infection affect the contribution of each operon to the synthesis of pre-rRNA. It was found that the products from the PCL1 promoter of rrnA play a pivotal role in rRNA synthesis during all stress conditions. Interestingly, the main participation of the products of transcription from the P1 promoter of rrnB was found during hypoxic conditions at the NRP1 phase. These results provide novel insights into pre-rRNA synthesis in mycobacteria, as well as the potential ability of M. kumamotonense to produce latent infections.

Occurrence of Nontuberculous Mycobacteria, Salmonella, Listeria monocytogenes, and Staphylococcus aureus in Artisanal Unpasteurized Cheeses in the State of Michoacan, Mexico.

ABSTRACT: This study investigated the presence of nontuberculous mycobacteria (NTM) for the first time in two types of unpasteurized fresh cheese produced in the state of Michoacan, Mexico. We tested for this pathogen, along with the others, to broaden the study of microbiological quality in 60 samples of cheese, 30 fresh and 30 Adobera, which were collected from six artisanal cheese factories (ACFs). The hygienic conditions of these establishments and the practices of cheese manufacture were generally poor. Although Mycobacterium bovis was not detected, four cheese samples harbored NTM isolates. The four NTM isolates were identified using three molecular markers (hsp65, rrs, and rpoB genes) that corresponded to Mycolicibacterium fortuitum (n = 3) and Mycolicibacterium mageritense (n = 1). All 60 cheese samples analyzed had unsatisfactory microbiological quality according to the Mexican Official Guideline. Regarding fresh cheeses, all 30 samples analyzed were positive for aerobic mesophilic bacteria, total coliforms, fecal coliforms, and yeasts and molds. Escherichia coli and Staphylococcus aureus were present in 23 and 21 samples, respectively. Listeria monocytogenes was identified in a sample and was isolated from a bulk milk tank in the same ACF. With regard to Adobera cheeses, all samples were positive for aerobic mesophilic bacteria, total coliforms, fecal coliforms, yeasts and molds, and S. aureus. E. coli was isolated from 28 samples. Salmonella was isolated from a sample and from a wooden shovel used in the manufacture of the cheeses in the same ACF. Thus, the consumption of unpasteurized fresh cheese may represent a public health risk. Because of this, health authorities should enforce the legislation that forbids the processing of cheese with unpasteurized milk and encourage producers to follow good manufacturing practices from original ingredients, through the production process of the cheese, to its sale to assure a safe product.

Short Disordered Epitope of CRTAM Ig-Like V Domain as a Potential Target for Blocking Antibodies.

Class-I Restricted T Cell-Associated Molecule (CRTAM) is a protein that is expressed after T cell activation. The interaction of CRTAM with its ligand, nectin-like 2 (Necl2), is required for the efficient production of IL-17, IL-22, and IFNγ by murine CD4 T cells, and it plays a role in optimal CD8 T and NK cell cytotoxicity. CRTAM promotes the pro-inflammatory cytokine profile; therefore, it may take part in the immunopathology of autoimmune diseases such as diabetes type 1 or colitis. Thus, antibodies that block the interaction between CRTAM and Necl2 would be useful for controlling the production of these inflammatory cytokines. In this work, using bioinformatics predictions, we identified three short disordered epitopes (sDE1-3) that are located in the Ig-like domains of murine CRTAM and are conserved in mammalian species. We performed a structural analysis by molecular dynamics simulations of sDE1 (QHPALKSSKY, Ig-like V), sDE2 (QRNGEKSVVK, Ig-like C1), and sDE3 (CSTERSKKPPPQI, Ig-like C1). sDE1, which is located within a loop of the contact interface of the heterotypic interaction with Nectl2, undergoes an order-disorder transition. On the contrary, even though sDE2 and sDE3 are flexible and also located within loops, they do not undergo order-disorder transitions. We evaluated the immunogenicity of sDE1 and sDE3 through the expression of these epitopes in chimeric L1 virus-like particles. We confirmed that sDE1 induces polyclonal antibodies that recognize the native folding of CRTAM expressed in activated murine CD4 T cells. In contrast, sDE3 induces polyclonal antibodies that recognize the recombinant protein hCRTAM-Fc, but not the native CRTAM. Thus, in this study, an exposed disordered epitope in the Ig-like V domain of CRTAM was identified as a potential site for therapeutic antibodies.

Bacteriological quality of bottled water obtained from Mexico City small water purification plants: Incidence and identification of potentially pathogenic nontuberculous mycobacteria species.

The aim of this study was to determine the bacteriological quality of bottled water samples obtained from small purification plants located in Mexico City and to identify potentially pathogenic nontuberculous mycobacteria (NTM) species found in these samples. All 111 samples analyzed were positive for aerobic mesophilic bacteria (AMB) and 46 (41.4%) did not comply with Mexico's Official Guidelines. Sixty-nine (62.1%) and 23 (20.7%) water samples were positive for total coliforms (TC) and fecal coliforms (FC), respectively. A total of 81 (72.9%) of the water samples exceeded the maximum allowed limit stipulated in the guideline. Thirty-three (29.7%) of the purified water samples were positive for NTM, being recovered a total of 40 isolates. These NTM isolates were identified using three molecular markers (hsp65, rrs and rpoB genes) which corresponded to the fast-growing mycobacteria M. chelonae (n = 12), M. porcinum (n = 8), M. senegalense (n = 5), M. abscessus (n = 4), M. septicum (n = 4), M. wolinskyi (n = 3), M. mucogenicum (n = 2), M. fortuitum (n = 1) and M. sp. (n = 1). In seven purified water samples, two different NTM species were isolated simultaneously. Overall, these results showed that most of the purified bottled water samples analyzed in this study had unsatisfactory microbiological quality and some harbored NTM associated with illness. Our data could hasten health authorities to intensify efforts in the routine monitoring of activities in the purified bottled water industry in order to supply safe and healthy water to the public.

Identification and biofilm development by a new fungal keratitis aetiologic agent.

BACKGROUND: In recent years, human keratitis caused by fungal plant pathogens has become more common. Biofilm is a structure that confers adaptations and virulence to fungi in keratitis. Neoscytalidium spp. are phytopathogenic and recently have been recognised as a human pathogen, using biofilm formation as a virulence factor. OBJECTIVES: The aim of this study was isolation, identification (at the species level) and characterisation of a new fungal keratitis agent. PATIENTS/METHODS: The fungus was isolated from a 67-year-old male patient with a corneal ulcer. Biofilm formation and structure were evaluated by colorimetric methods and microscopy. To identify the fungus, morphological characteristics were examined and a phylogenetic analysis was performed. RESULTS AND CONCLUSIONS: We report the identification of a fungus, a member of the genus Neoscytalidium which is associated with human keratitis. Phylogenetic analysis and morphological observations on conidiogenous cells, which occur only in arthric chains in aerial mycelium and the coelomycetous synasexual morph is absent, identified a new species, Neoscytalidium oculus sp. nov. The fungus formed biofilm at a concentration of 1 × 106  conidia/mL, during 96 hours of incubation at 37°C, and also manifested haemolysis and melanin production. This is the first report in Latin America of a new species of Neoscytalidium from a clinical isolate has been identified.

Genetic diversity of drug and multidrug-resistant Mycobacterium tuberculosis circulating in Veracruz, Mexico.

BACKGROUND: Mexico is one of the most important contributors of drug and multidrug-resistant tuberculosis in Latin America; however, knowledge of the genetic diversity of drug-resistant tuberculosis isolates is limited. METHODS: In this study, the genetic structure of 112 Mycobacterium tuberculosis strains from the southeastern Mexico was determined by spoligotyping and 24-loci MIRU-VNTRs. FINDINGS: The results show eight major lineages, the most of which was T1 (24%), followed by LAM (16%) and H (15%). A total of 29 (25%) isolates were identified as orphan. The most abundant SITs were SIT53/T1 and SIT42/LAM9 with 10 isolates each and SIT50/H3 with eight isolates. Fifty-two spoligotype patterns, twenty-seven clusters and ten clonal complexes were observed, demonstrating an important genetic diversity of drug and multidrug-resistant tuberculosis isolates in circulation and transmission level of these aggravated forms of tuberculosis. Being defined as orphan or as part of an orphan cluster, was a risk factor for multidrug resistant-tuberculosis (OR 2.5, IC 1.05-5.86 and OR 3.3, IC 1-11.03, respectively). Multiple correspondence analyses showed association of some clusters and SITs with specific geographical locations. CONCLUSIONS: Our study provides one of the most detailed description of the genetic structure of drug and multidrug-resistant tuberculosis strains in southeast Mexico, establishing for the first time a baseline of the genotypes observed in resistant isolates circulating, however further studies are required to better elucidate the genetic structure of tuberculosis in region and the factors that could be participating in their dispersion.

Microbiological Quality of Ready-to-Eat Vegetables Collected in Mexico City: Occurrence of Aerobic-Mesophilic Bacteria, Fecal Coliforms, and Potentially Pathogenic Nontuberculous Mycobacteria.

The aims of this study were to evaluate the microbiological quality and the occurrence of nontuberculous mycobacteria (NTM) in a variety of salads and sprouts from supermarkets and street vendors in Mexico City. Aerobic-mesophilic bacteria (AMB) were present in 100% of RTE-salads samples; 59% of samples were outside guidelines range (>5.17 log10 CFU per g). Although fecal coliforms (FC) were present in 32% of samples, only 8% of them exceeded the permissible limit (100 MPN/g). Regarding the 100 RTE-sprouts, all samples were also positive for AMB and total coliforms (TC) and 69% for FC. Seven NTM species were recovered from 7 salad samples; they included three M. fortuitum, two M. chelonae, one M. mucogenicum, and one M. sp. Twelve RTE-sprouts samples harbored NTM, which were identified as M. porcinum (five), M. abscessus (two), M. gordonae (two), M. mucogenicum (two), and M. avium complex (one). Most RTE-salads and RTE-sprouts had unsatisfactory microbiological quality and some harbored NTM associated with illness. No correlation between the presence of coliforms and NTM was found. Overall, these results suggest that RTE-salads and RTE-sprouts might function as vehicles for NTM transmission in humans; hence, proper handling and treatment before consumption of such products might be recommendable.