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.

The Lack of the TetR-Like Repressor Gene BCG_2177c (Rv2160A) May Help Mycobacteria Overcome Intracellular Redox Stress and Survive Longer Inside Macrophages When Surrounded by a Lipid Environment.

Mycobacteria, like other microorganisms, survive under different environmental variations by expressing an efficient adaptive response, oriented by regulatory elements, such as transcriptional repressors of the TetR family. These repressors in mycobacteria also appear to be related to cholesterol metabolism. In this study, we have evaluated the effect of a fatty acid (oleic-palmitic-stearic)/cholesterol mixture on some phenotypic and genotypic characteristics of a tetR-mutant strain (BCG_2177c mutated gene) of M. bovis BCG, a homologous of Rv2160A of M. tuberculosis. In order to accomplish this, we have analyzed the global gene expression of this strain by RNA-seq and evaluated its neutral-lipid storage capacity and potential to infect macrophages. We have also determined the macrophage response by measuring some pro- and anti-inflammatory cytokine expressions. In comparison with wild-type microorganisms, we showed that the mutation in the BCG_2177c gene did not affect the growth of M. bovis BCG in the presence of lipids but it probably modified the structure/composition of its cell envelope. Compared to with dextrose, an overexpression of the transcriptome of the wild-type and mutant strains was observed when these mycobacteria were cultured in lipids, mainly at the exponential phase. Twelve putative intracellular redox balance maintenance genes and four others coding for putative transcriptional factors (including WhiB6 and three TetR-like) were the main elements repeatedly overexpressed when cultured in the presence of lipids. These genes belonged to the central part of what we called the "genetic lipid signature" for M. bovis BCG. We have also found that all these mycobacteria genotypic changes affected the outcome of BCG-infected macrophages, being the mutant strain most adapted to persist longer inside the host. This high persistence result was also confirmed when mutant-infected macrophages showed overexpression of the anti-inflammatory cytokine TGF-ß versus pro-inflammatory cytokines. In summary, the lack of this TetR-like repressor expression, within a lipid environment, may help mycobacteria overcome intracellular redox stress and survive longer inside their host.

cAMP Receptor Protein Positively Regulates the Expression of Genes Involved in the Biosynthesis of Klebsiella oxytoca Tilivalline Cytotoxin.

Klebsiella oxytoca is a resident of the human gut. However, certain K. oxytoca toxigenic strains exist that secrete the nonribosomal peptide tilivalline (TV) cytotoxin. TV is a pyrrolobenzodiazepine that causes antibiotic-associated hemorrhagic colitis (AAHC). The biosynthesis of TV is driven by enzymes encoded by the aroX and NRPS operons. In this study, we determined the effect of environmental signals such as carbon sources, osmolarity, and divalent cations on the transcription of both TV biosynthetic operons. Gene expression was enhanced when bacteria were cultivated in tryptone lactose broth. Glucose, high osmolarity, and depletion of calcium and magnesium diminished gene expression, whereas glycerol increased transcription of both TV biosynthetic operons. The cAMP receptor protein (CRP) is a major transcriptional regulator in bacteria that plays a key role in metabolic regulation. To investigate the role of CRP on the cytotoxicity of K. oxytoca, we compared levels of expression of TV biosynthetic operons and synthesis of TV in wild-type strain MIT 09-7231 and a Δcrp isogenic mutant. In summary, we found that CRP directly activates the transcription of the aroX and NRPS operons and that the absence of CRP reduced cytotoxicity of K. oxytoca on HeLa cells, due to a significant reduction in TV production. This study highlights the importance of the CRP protein in the regulation of virulence genes in enteric bacteria and broadens our knowledge on the regulatory mechanisms of the TV cytotoxin.

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.

Two Type VI Secretion Systems of Enterobacter cloacae Are Required for Bacterial Competition, Cell Adherence, and Intestinal Colonization.

Enterobacter cloacae has emerged as an opportunistic pathogen in healthcare-associated infections. Analysis of the genomic sequences of several E. cloacae strains revealed the presence of genes that code for expression of at least one type VI secretion system (T6SS). Here, we report that E. cloacae strain ATCC 13047 codes for two functional T6SS named T6SS-1 and T6SS-2. T6SS-1 and T6SS-2 were preferentially expressed in tryptic soy broth and tissue culture medium (DMEM), respectively. Mutants in T6SS-1-associated genes clpV1 and hcp1 significantly affected their ability of inter- and intra-bacterial killing indicating that T6SS-1 is required for bacterial competition. In addition, the Hcp effector protein was detected in supernatants of E. cloacae cultures and a functional T6SS-1 was required for the secretion of this protein. A clpV2 mutant was impaired in both biofilm formation and adherence to epithelial cells, supporting the notion that these phenotypes are T6SS-2 dependent. In vivo data strongly suggest that both T6SSs are required for intestinal colonization because single and double mutants in clpV1 and clpV2 genes were defective in gut colonization in mice. We conclude that the two T6SSs are involved in the pathogenesis scheme of E. cloacae with specialized functions in the interaction with other bacteria and with host cells.

Neonatal Sepsis Diagnosis Decision-Making Based on Artificial Neural Networks.

Neonatal sepsis remains difficult to diagnose due to its non-specific signs and symptoms. Traditional scoring systems help to discriminate between septic or not patients, but they do not consider every single patient particularity. Thus, the purpose of this study was to develop an early- and late-onset neonatal sepsis diagnosis model, based on clinical maternal and neonatal data from electronic records, at the time of clinical suspicion. A predictive model was obtained by training and validating an artificial Neural Networks (ANN) algorithm with a balanced dataset consisting of preterm and term non-septic or septic neonates (early- and late-onset), with negative and positive culture results, respectively, using 25 maternal and neonatal features. The outcome of the model was sepsis or not. The performance measures of the model, evaluated with an independent dataset, outperformed physician's diagnosis using the same features based on traditional scoring systems, with a 93.3% sensitivity, an 80.0% specificity, a 94.4% AUROC, and a regression coefficient of 0.974 between actual and simulated results. The model also performed well-relative to the state-of-the-art methods using similar maternal/neonatal variables. The top 10 factors estimating sepsis were maternal age, cervicovaginitis and neonatal: fever, apneas, platelet counts, gender, bradypnea, band cells, catheter use, and birth weight.

Molecular Characterization of SehB, a Type II Antitoxin of Salmonella enterica Serotype Typhimurium: Amino Acid Residues Involved in DNA-Binding, Homodimerization, Toxin Interaction, and Virulence.

Salmonella enterica serotype Typhimurium is a bacterium that causes gastroenteritis and diarrhea in humans. The genome of S. Typhimurium codes for diverse virulence factors, among which are the toxin-antitoxin (TA) systems. SehAB is a type II TA, where SehA is the toxin and SehB is the antitoxin. It was previously reported that the absence of the SehB antitoxin affects the growth of S. Typhimurium. In addition, the SehB antitoxin can interact directly with the SehA toxin neutralizing its toxic effect as well as repressing its own expression. We identified conserved residues on SehB homologous proteins. Point mutations were introduced at both N- and C-terminal of SehB antitoxin to analyze the effect of these changes on its transcription repressor function, on its ability to form homodimers and on the virulence of S. Typhimurium. All changes in amino acid residues at both the N- and C-terminal affected the repressor function of SehB antitoxin and they were required for DNA-binding activity. Mutations in the amino acid residues at the N-terminal showed a lower capacity for homodimer formation of the SehB protein. However, none of the SehB point mutants were affected in the interaction with the SehA toxin. In terms of virulence, the eight single-amino acid mutations were attenuated for virulence in the mouse model. In agreement with our results, the eight amino acid residues of SehB antitoxin were required for its repressor activity, affecting both homodimerization and DNA-binding activity, supporting the notion that both activities of SehB antitoxin are required to confer virulence to Salmonella enterica.

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.

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.

Hypoxia Is Not a Main Stress When Mycobacterium tuberculosis Is in a Dormancy-Like Long-Chain Fatty Acid Environment.

The capacity of Mycobacterium tuberculosis (Mtb) to sense, respond and adapt to a variable and hostile environment within the host makes it one of the most successful human pathogens. During different stages of infection, Mtb is surrounded by a plethora of lipid molecules and current evidence points out the relevance of fatty acids during the infectious process. In this study, we have compared the transcriptional response of Mtb to hypoxia in cultures supplemented with a mix of even long-chain fatty acids or dextrose as main carbon sources. Using RNA sequencing, we have identified differential expressed genes in early and late hypoxia, defined according to the in vitro Wayne and Hayes model, and compared the results with the exponential phase of growth in both carbon sources. We show that the number of genes over-expressed in the lipid medium was quite low in both, early and late hypoxia, relative to conditions including dextrose, with the exception of transcripts of stable and non-coding RNAs, which were more expressed in the fatty acid medium. We found that sigB and sigE were over-expressed in the early phase of hypoxia, confirming their pivotal role in early adaptation to low oxygen concentration independently of the carbon source. A drastic contrast was found with the transcriptional regulatory factors at early hypoxia. Only 2 transcriptional factors were over-expressed in early hypoxia in the lipid medium compared to 37 that were over-expressed in the dextrose medium. Instead of Rv0081, known to be the central regulator of hypoxia in dextrose, Rv2745c (ClgR), seems to play a main role in hypoxia in the fatty acid medium. The low level of genes associated to the stress-response during their adaptation to hypoxia in fatty acids, suggests that this lipid environment makes hypoxia a less stressful condition for the tubercle bacilli. Taken all together, these results indicate that the presence of lipid molecules shapes the metabolic response of Mtb to an adaptive state for different stresses within the host, including hypoxia. This fact could explain the success of Mtb to establish long-term survival during latent infection.

Comparative proteomic profiles reveal characteristic Mycobacterium tuberculosis proteins induced by cholesterol during dormancy conditions.

Cholesterol has been reported to play an important role during Mycobacterium tuberculosis infection and during its dormant state inside the host. We present the determination of proteomic profiles of M. tuberculosis H37Rv in the presence of cholesterol as the sole carbon source under exponential growth and in two in vitro dormancy phases (NRP1 and NRP2). Using 2D-PAGE, we detected that M. tuberculosis expressed a high diversity of proteins in both exponential and non-replicative phases. We also found that cholesterol was involved in the overexpression of some proteins related to sulfur metabolism (CysA2), electron transport (FixB), cell wall synthesis (Ald), iron storage (BfrB), protein synthesis (Tig and EF-Tu) and dormancy maintenance (HspX and TB 31.7). According to our results we propose that proteins Ald, BfrB, FadA5 and TB31.7 are likely to play a fundamental role during in vitro dormancy of M. tuberculosis in the presence of cholesterol, helping to counteract its intracellular hostile microenvironment.

In vitro activity of bedaquiline against rapidly growing nontuberculous mycobacteria.

Bedaquiline (BDQ) has been proven to be effective in the treatment of multidrug-resistant tuberculosis. We hypothesized that BDQ could be a potential agent to treat nontuberculous mycobacterial (NTM) infection. The objective of this study was to evaluate the in vitro activity of BDQ against rapidly growing mycobacteria by assessing the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against 18 NTM strains. For MIC determination we performed the resazurin microtitre assay broth dilution, and for the MBC the c.f.u. was determined. BDQ exhibited a strong inhibitory effect against most NTM tested; however, for some NTM strains the MBC was significantly higher than the MIC. A new finding is that Mycobacterium flavescens has a mutation in the gene atpE associated with natural resistance to BDQ. These preliminary promising results demonstrate that BDQ could be potentially useful for the treatment of NTM.

The sigma factor SigD of Mycobacterium tuberculosis putatively enhances gene expression of the septum site determining protein under stressful environments.

This work examined the expression of the septum site determining gene (ssd) of Mycobacterium tuberculosis CDC1551 and its ∆sigD mutant under different growing conditions. The results showed an up-regulation of ssd during stationary phase and starvation conditions, but not during in vitro dormancy, suggesting a putative role for SigD in the control of ssd expression mainly under lack-of-nutrients environments. Furthermore, we elucidated a putative link between ssd expression and cell elongation of bacilli at stationary phase. In addition, a -35 sigD consensus sequence was found for the ssd promoter region, reinforcing the putative regulation of ssd by SigD, and in turn, supporting this protein role during the adaptation of M. tuberculosis to some stressful environments.

Cholesterol plays a larger role during Mycobacterium tuberculosis in vitro dormancy and reactivation than previously suspected.

It is known that cholesterol plays a key role for Mycobacterium tuberculosis (Mtb) adaptation and survival within the host, thus contributing to the establishment of dormancy. It has been extensively demonstrated that fatty acids are the main energy source of Mtb during infection and dormancy, and it has been proposed that these molecules are implicated in reactivation of bacilli from a dormant state. We used in vitro models to analyze Mtb gene expression during dormancy and reactivation when fatty acids and cholesterol are the unique carbon source in the media. Our results suggest that cholesterol might function as a signal to trigger Mtb expression of some genes required for stress protection earlier than the one induced by fatty acids alone, indicating that cholesterol is very favorable for its development. This process is so conducive that cholesterol-adapted bacilli can reactivate their growth after NRP2 dormancy state even 10 min post ventilation. Thus, we hypothesize that cholesterol is not only involved in Mtb dormancy but that it also plays a critical role for favorable and almost immediate reactivation from an in vitro long-lasting dormant state induced by hypoxia.

Molecular epidemiology of Mycobacterium tuberculosis in Baja California, Mexico: A result of human migration?

The State of Baja California (BC) exhibits the highest incidence and prevalence rates of tuberculosis (TB), and multidrug-resistant TB (MDR-TB) in Mexico. However information about the circulation of M. tuberculosis lineages in BC and Mexico as a whole is limited. Here, we describe the genetic relationship and genetic diversity among M. tuberculosis clinical isolates (n=140) collected in BC between October 2009 and April 2011 with other regions of Mexico, the United States, and Latin America. All specimens were genotyped based on 24 mycobacterial interspersed repetitive units (MIRU)-variable number of tandem repeats (VNTR) loci. Population structure and minimum spanning tree (MST) analyses were used to assess the genetic diversity and distribution of BC isolates in comparison to USA and South America strains. Among the nine lineages observed, LAM, Haarlem and S were the most frequent identified in BC. Population structure analysis clustered most BC isolates (41%) into three distinctive groups that included strains from San Diego and South America, whereas other BC strains (22%) clustered with other Mexican strains. A subset of isolates (12%) seemed to be autochthonous of BC, while 25% were cosmopolitan and grouped into multiple clusters. It is highly likely that the TB genetic structure observed in BC is due to human migration. Additional studies are required to determine the mechanism involved in the phylogeographic distribution of M. tuberculosis in Mexico. Implementation of domestic molecular TB surveillance programs is required to better understand the molecular epidemiology of TB not only in the region but at the national level.

Identification of Mycobacterium tuberculosis in the cerebrospinal fluid of patients with meningitis using nested PCR.

Tuberculous meningitis (TBM) is the most severe form of tuberculosis. It is caused by Mycobacterium tuberculosis (M. tuberculosis; MT) and it is very difficult to diagnose. The symptoms are similar to other infectious neurological diseases, such as neurocysticercosis, neuroborreliosis, or herpes viral infection. The aim of this study was to identify tuberculosis (TB) in cases of meningitis with clinical and laboratory evidence suggestive of TBM, and to confirm our findings with molecular tests for TB infection. We recruited patients with neurological symptoms who were examined at the neurology services of Hospitals of Instituto Mexicano del Seguro Social (IMSS) in Mexico City. A total of 144 consecutive patients with suggestive infectious meningitis were initially included; 94 cases of meningitis with clinical and laboratory evidence suggestive of TBM were included, but only 50 of these cases fulfilled the criteria for probable TBM. As the controls, we included 50 cases of meningitis with clinical and laboratory evidence suggestive of non-TBM. Cerebrospinal fluid (CSF) was collected from all 100 patients (cases and controls) and tested for TB by multiplex and nested PCR analyses. Nested PCR detected 0.1 fg of M. tuberculosis DNA. TB infection was confirmed with molecular tests in 49 patients from the 50 cases suggestive of TBM and in 1 of the 50 non-TBM cases. The analysis exhibited a sensitivity of 98.0%, a specificity of 92.0%, a positive predictive value of 88.0% and a negative predictive value of 98.0%. The use CSF for the analyses proved to be effective for the rapid diagnosis of TBM using a developed system of multiplex and nested PCR analyses in patients presenting neurological symptoms.

Microbiological Quality and Occurrence of Nontuberculous Mycobacteria in Fresh-Squeezed Orange Juice Samples Purchased from Street Vendors in Mexico City.

Nontuberculous mycobacteria (NTM) are potentially pathogenic agents commonly found in a natural ecosystem. For this reason, food is considered another source of NTM transmission for humans. The aims of this study were to evaluate the microbiological quality and the occurrence of NTM in fresh-squeezed orange juice samples purchased from street vendors. All 102 samples analyzed were positive for aerobic mesophilic bacteria (AMB), with limits ranging from 1.8 to 6.2 log CFU/ml. A total of 55 (54%), 25 (25%), and 13 (13%) orange juice samples were positive for total coliforms (TC), fecal coliforms (FC), and Escherichia coli , respectively. TC, FC, and E. coli were present with limits ranging from <3 to >1,100 most probable number (MPN)/ml, <3 to 460 MPN/ml, and <3 to 11 MPN/ml, respectively. Six orange juice samples harbored NTM. These NTM were identified by using three molecular markers (hsp65, rrs, and rpoB genes) and corresponded to the fast-growing mycobacteria: Mycobacterium fortuitum (n = 3), Mycobacterium rhodesiae (n = 1), Mycobacterium obuense (n = 1), and a mixture of M. fortuitum and Mycobacterium mucogenicum in an additional sample (n = 1). No correlation was found between the presence NTM in orange juice samples with the presence and concentration of the indicator microorganisms (aerobic mesophilic bacteria, TC, and FC). Overall, these results suggest that fresh-squeezed orange juice might represent a vehicle for NTM transmission in humans. Therefore, prevention of contamination by humans (proper handling and washing of oranges) during juice preparation should be recommended.

Human multidrug-resistant Mycobacterium bovis infection in Mexico.

Here, we describe the molecular characterization of six human Mycobacterium bovis clinical isolates, including three multidrug resistant (MDR) strains, collected in Mexico through the National Survey on Tuberculosis Drug Resistance (ENTB-2008), a nationally representative survey conducted during 2008-2009 in nine states with a stratified cluster sampling design. The genetic background of bovine M. bovis strains identified in three different states of Mexico was studied in parallel to assess molecular relatedness of bovine and human strains. Additionally, resistance to first and second line anti-tuberculosis (TB) drugs and molecular identification of mutations conferring drug resistance was also performed. All strains were characterized by spoligotyping and 24-loci MIRU-VNTRs, and analyzed using the SITVIT2 (n = 112,000 strains) and SITVITBovis (n = 25,000 strains) proprietary databases of Institut Pasteur de la Guadeloupe. Furthermore, data from this study (n = 55 isolates), were also compared with genotypes recorded for M. bovis from USA (n = 203), Argentina (n = 726), as well as other isolates from Mexico (independent from the present study; n = 147), to determine any evidence for genetic relatedness between circulating M. bovis strains. The results showed that all human M. bovis cases were not genetically related between them or to any bovine strain. Interestingly, a high degree of genetic variability was observed among bovine strains. Several autochthonous and presumably imported strains were identified. The emergence of drug-resistant M. bovis is an important public health problem that jeopardizes the success of TB control programs in the region.