Targeted Expression to Liver of an antimiR-33 Sponge as a Gene Therapy Strategy against Hypercholesterolemia: In Vitro Study.

Atherosclerosis is the leading cause of cardiovascular diseases in Mexico and worldwide. The membrane transporters ABCA1 and ABCG1 are involved in the reverse transport of cholesterol and stimulate the HDL synthesis in hepatocytes, therefore the deficiency of these transporters promotes the acceleration of atherosclerosis. MicroRNA-33 (miR-33) plays an important role in lipid metabolism and exerts a negative regulation on the transporters ABCA1 and ABCG1. It is known that by inhibiting the function of miR-33 with antisense RNA, HDL levels increase and atherogenic risk decreases. Therefore, in this work, a genetic construct, pPEPCK-antimiR-33-IRES2-EGFP, containing a specific antimiR-33 sponge with two binding sites for miR-33 governed under the PEPCK promoter was designed, constructed, and characterized, the identity of which was confirmed by enzymatic restriction, PCR, and sequencing. Hep G2 and Hek 293 FT cell lines, as well as a mouse hepatocyte primary cell culture were transfected with this plasmid construction showing expression specificity of the PEPCK promoter in hepatic cells. An analysis of the relative expression of miR-33 target messengers showed that the antimiR-33 sponge indirectly induces the expression of its target messengers (ABCA1 and ABCG1). This strategy could open new specific therapeutic options for hypercholesterolemia and atherosclerosis, by blocking the miR-33 specifically in hepatocytes.

Differential expression of miRNA-146a and miRNA-155 in gastritis induced by Helicobacter pylori infection in paediatric patients, adults, and an animal model.

BACKGROUND: Helicobacter pylori is a major aetiologic agent associated with gastritis. H. pylori infections increase the expression of the Toll-like receptor (TLR), which in turn modulates the expression of microRNA (miRNA)-146a and miRNA-155. The objective of this study was to compare the expression of miRNA-146a and miRNA-155 in gastric lesions of paediatric and adult patients with different pathologies and in Mongolian gerbils (Meriones unguiculatus) infected with H. pylori 26,695. METHODS: Quantification of miRNA expression was performed by quantitative real-time polymerase chain reaction (qRT-PCR) of paraffin-embedded gastric lesions of children with or without an infection (n = 25), adults with follicular gastritis and metaplasia (n = 32) and eight-week-old M. unguiculatus males (Hsd:MON) infected with H. pylori 26,695 for 0, 3, 6, 12 and 18 months (n = 25). The genes RNU48 and RNU6 were used as endogenous controls for data normalization. Statistical analyses were performed using Kruskal-Wallis, Mann-Whitney, ANOVA and Student's t-test. RESULTS: The expression of miRNA-146a and miRNA-155 in infected children increased by 247.6- and 79.4-fold (on average), respectively, compared to that observed in the control group. However, these results were not significant (p = 0.12 and p = 0.07 respectively). In some children a gradual increase in expression was observed, while in others, expression was very high. Additionally, the expression levels of miRNA-146a and miRNA-155 increased by an average of 21.7- and 62-fold, respectively, in adult patients with follicular gastritis when compared to those of the controls. In M. unguiculatus infected with H. pylori 26,695, the expression of both miRNAs increased as the infection progressed. CONCLUSION: This is the first report to show differences in the expression of miRNA-146a and miRNA-155 in paediatric and adult patients with gastritis who were infected with H. pylori. In addition, in M. unguiculatus infected with H. pylori, miRNA expression was associated with the progression of infection and the ability of the bacteria to adapt to the host.

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.

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.

Infection of Fetal Membranes with Mycobacterium tuberculosis and Tissue Processing to Isolate RNA for Expression Analysis.

This chapter outlines the methodology employed to infect the chorionic and amniotic membranes with Mycobacterium tuberculosis during pregnancy. Particularly, congenital tuberculosis, a rare and serious condition associated with cases in neonates and reactivation of latent tuberculosis in pregnant mothers, is interesting to study. Understanding the mechanisms of infection and the response of fetal membranes is crucial for developing effective treatments in these cases, which will promote better neonatal and maternal health in situations of tuberculosis during pregnancy. Establishing a standardized infection model in the chorioamniotic membranes is imperative, followed by a treatment protocol for isolating both cellular and mycobacterial RNA. This will enable the expression analysis during the maternal-fetal interface interaction with M. tuberculosis. The proposed methodology might be invaluable for qRT-PCR, microarrays, and sequencing research.

Phenotypic and Genotypic Drug Resistance of Mycobacterium tuberculosis Strains Isolated from HIV-Infected Patients from a Third-Level Public Hospital in Mexico.

BACKGROUND: Drug-resistant tuberculosis (TB) is associated with higher mortality rates in patients with human immunodeficiency virus (HIV). In Mexico, the number of deaths due to TB among the HIV-positive population has tripled in recent years. METHODS: Ninety-three Mycobacterium tuberculosis strains isolated from the same number of HIV-infected patients treated in a public hospital in Mexico City were studied to determine the drug resistance to first- and second-line anti-TB drugs and to identify the mutations associated with the resistance. RESULTS: Of the 93 patients, 82.7% were new TB cases, 86% were male, and 73% had extrapulmonary TB. Most patients (94%) with a CD4 T-lymphocyte count <350 cells/mm3 were associated with extrapulmonary TB (p <0.0001), whilst most patients (78%) with a CD4 T-lymphocyte count >350 cells/mm3 were associated with pulmonary TB (p = 0.0011). Eighty-two strains were pan-susceptible, four mono-resistant, four poly-resistant, two multidrug-resistant, and one was extensively drug-resistant. In the rifampicin-resistant strains, rpoB S531L was the mutation most frequently identified, whereas the inhA C15T and katG S315T1 mutations were present in isoniazid-resistant strains. The extensively drug-resistant strain also contained the mutation gyrA D94A. CONCLUSIONS: These data highlight the need to promptly diagnose the drug resistance of M. tuberculosis among all HIV-infected patients by systematically offering access to first- and second-line drug susceptibility testing and to tailor the treatment regimen based on the resistance patterns to reduce the number of deaths in HIV-infected patients.

Genetic Diversity of Mycobacterium tuberculosis Strains Isolated from HIV-Infected Patients in Mexico.

There has been very limited investigation regarding the genetic diversity of Mycobacterium tuberculosis (MTb) strains isolated from human immunodeficiency virus (HIV)-infected patients in Mexico. In this study, we isolated 93 MTb strains from pulmonary and extrapulmonary samples of HIV-infected patients treated in a public hospital in Mexico City to evaluate the genetic diversity using spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) typing (based on 24 loci). The cohort comprised 80 male and 13 female individuals. There was a positive correlation between a high HIV viral load (>100,000 copies) and extrapulmonary tuberculosis (TB) (r = 0.306, p = 0.008). Lineage 4 was the most frequent lineage (79 strains). In this lineage, we found the H clade (n = 24), including the Haarlem, H3, and H1 families; the T clade (n = 22), including T1 and T2; the X clade (n = 15), including X1 and X3; the LAM clade (n = 14), including LAM1, LAM2, LAM3, LAM6, and LAM9; the S clade (n = 2); Uganda (n = 1); and Ghana (n = 1). We also found 12 strains in the EAI clade belonging to lineage 1, including the EAI2-Manila and EAI5 families. Interestingly, we identified one strain belonging to the Beijing family, which is part of lineage 2. One strain could not be identified. This study reports high genetic diversity among MTb strains, highlighting the need for a molecular epidemiological surveillance system that can help to monitor the spread of these strains, leading to more appropriate measures for TB control in HIV-infected patients.

Presence of Indicator Bacteria and Occurrence of Potentially Pathogenic Nontuberculous Mycobacteria Species in Packaged Ice Cubes in Central Mexico.

This study aimed to determine the bacteriological quality and presence of diarrheagenic Escherichia coli pathotypes (DEP) and nontuberculous mycobacteria (NTM) species in 85 packaged ice samples from 12 different states of central Mexico. Three samples had a pH of 9.8 and therefore fell outside of the acceptable range for pH. All samples were positive for aerobic-mesophilic bacteria, with limits ranging from 1 to 3.47 log CFU/mL. In total, 35, 11, and 3 ice samples were positive for total coliforms (TC), fecal coliforms (FC), and E. coli, respectively. In the samples, the TC concentration ranged from <1.1 to >23 MPN/100 mL and from <1.1 to 23 MPN/100 mL for FC and E. coli. In total, 38 (44.7%) ice samples were outside of Mexico's official guidelines. None of the 12 E. coli strains isolated from the three ice samples belonged to DEP. NTM were recovered from 20 ice samples and included M. neoaurum (n = 7), M. porcinum (n = 2), M. flavescens (n = 2), M. fortuitum (n = 1), M. abscessus (n = 1), M. senegalense (n = 1), M. conceptionense (n = 1), and M. sp. (n = 1). In the remaining four samples, two NTM were isolated simultaneously. Thus, we recommend that producers should evaluate the microbiological quality of purified water used as a raw material as well as that of the final product, the ice should be packed in thick bags to avoid stretching and tearing during transportation or storage to prevent environmental contamination of ice, personnel involved in the production, and handling of ice should be trained in relative hygiene matters and how ice-machines should be cleaned and disinfected and the implementation of hazard analysis and critical control points must be applied throughout the chain of production. Finally, regular inspection by the authorities is also of great importance. These recommendations can be applied in different countries with low microbiological quality packaged ice.

The CpxRA two-component system represses gene expression of the heat-labile toxin of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) strains produce at least one of two types of enterotoxins: the heat-labile (LT) and heat-stable (ST) toxins, which are responsible for the watery secretory diarrhoea that is a hallmark of the human ETEC infection. One regulatory system that controls the transcription of virulence genes in pathogenic bacteria is the CpxRA two-component system (TCS). We reported that the eltAB bicistronic operon, which encodes for the A and B subunits of LT, was repressed for the CpxRA TCS by direct binding of CpxR-P from -12 to +6 bp with respect to the transcription start site of eltAB. Moreover, the Cpx-response activation down-regulated the transcription of eltAB genes, and this negative effect was CpxRA-dependent. Our data show that CpxRA TCS is a negative regulator of the LT, one of the main virulence determinants of ETEC.

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 Two-Component System CpxRA Represses Salmonella Pathogenicity Island 2 by Directly Acting on the ssrAB Regulatory Operon.

The acquisition of Salmonella pathogenicity island 2 (SPI-2) conferred on Salmonella the ability to survive and replicate within host cells. The ssrAB bicistronic operon, located in SPI-2, encodes the SsrAB two-component system (TCS), which is the central positive regulator that induces the expression of SPI-2 genes as well as other genes located outside this island. On the other hand, CpxRA is a two-component system that regulates expression of virulence genes in many bacteria in response to different stimuli that perturb the cell envelope. We previously reported that the CpxRA system represses the expression of SPI-1 and SPI-2 genes under SPI-1-inducing conditions by decreasing the stability of the SPI-1 regulator HilD. Here, we show that under SPI-2-inducing conditions, which mimic the intracellular environment, CpxRA represses the expression of SPI-2 genes by the direct action of phosphorylated CpxR (CpxR-P) on the ssrAB regulatory operon. CpxR-P recognized two sites located proximal and distal from the promoter located upstream of ssrA. Consistently, we found that CpxRA reduces the replication of Salmonella enterica serovar Typhimurium inside murine macrophages. Therefore, our results reveal CpxRA as an additional regulator involved in the intracellular lifestyle of Salmonella, which in turn adds a new layer to the intricate regulatory network controlling the expression of Salmonella virulence genes. IMPORTANCE SPI-2 encodes a type III secretion system (T3SS) that is a hallmark for the species Salmonella enterica, which is essential for the survival and replication within macrophages. Expression of SPI-2 genes is positively controlled by the two-component system SsrAB. Here, we determined a regulatory mechanism involved in controlling the overgrowth of Salmonella inside macrophages. In this mechanism, CpxRA, a two-component system that is activated by extracytoplasmic stress, directly represses expression of the ssrAB regulatory operon; as a consequence, expression of SsrAB target genes is decreased. Our findings reveal a novel mechanism involved in the intracellular lifestyle of Salmonella, which is expected to sense perturbations in the bacterial envelope that Salmonella faces inside host cells, as the synthesis of the T3SS-2 itself.

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.

Validation and Selection of New Reference Genes for RT-qPCR Analysis in Pediatric Glioma of Different Grades.

Gliomas are heterogeneous, solid, and intracranial tumors that originate from glial cells. Malignant cells from the tumor undergo metabolic alterations to obtain the energy required for proliferation and the invasion of the cerebral parenchyma. The alterations in the expression of the genes related to the metabolic pathways can be detected in biopsies of gliomas of different CNS WHO grades. In this study, we evaluated the expression of 16 candidate reference genes in the HMC3 microglia cell line. Then, statistical algorithms such as BestKeeper, the comparative ΔCT method, geNorm, NormFinder, and RefFinder were applied to obtain the genes most suitable to be considered as references for measuring the levels of expression in glioma samples. The results show that PKM and TPI1 are two novel genes suitable for genic expression studies on gliomas. Finally, we analyzed the expression of genes involved in metabolic pathways in clinical samples of brain gliomas of different CNS WHO grades. RT-qPCR analysis showed that in CNS WHO grade 3 and 4 gliomas, the expression levels of HK1, PFKM, GAPDH, G6PD, PGD1, IDH1, FASN, ACACA, and ELOVL2 were higher than those of CNS WHO grade 1 and 2 glioma biopsies. Hence, our results suggest that reference genes from metabolic pathways have different expression profiles depending on the stratification of gliomas and constitute a potential model for studying the development of this type of tumor and the search for molecular targets to treat gliomas.

First insights into the genetic diversity of Mycobacterium tuberculosis isolates from HIV-infected Mexican patients and mutations causing multidrug resistance.

BACKGROUND: The prevalence of infections with Mycobacterium tuberculosis (MTb) and nontuberculous mycobacteria (NTM) species in HIV-infected patients in Mexico is unknown. The aims of this study were to determine the frequency of MTb and NTM species in HIV-infected patients from Mexico City, to evaluate the genotypic diversity of the Mycobacterium tuberculosis complex strains, to determine their drug resistance profiles by colorimetric microplate Alamar Blue assay (MABA), and finally, to detect mutations present in katG, rpoB and inhA genes, resulting in isoniazid (INH) and rifampin (RIF) resistance. RESULTS: Of the 67 mycobacterial strains isolated, 48 were identified as MTb, 9 as M. bovis, 9 as M. avium and 1 as M. intracellulare. IS6110-RFLP of 48 MTb strains showed 27 profiles. Spoligotyping of the 48 MTb strains yielded 21 patterns, and 9 M. bovis strains produced 7 patterns. Eleven new spoligotypes patterns were found. A total of 40 patterns were produced from the 48 MTb strains when MIRU-VNTR was performed. Nineteen (39.6%) MTb strains were resistant to one or more drugs. One (2.1%) multidrug-resistant (MDR) strain was identified. A novel mutation was identified in a RIF-resistant strain, GAG --> TCG (Glu --> Ser) at codon 469 of rpoB gene. CONCLUSIONS: This is the first molecular analysis of mycobacteria isolated from HIV-infected patients in Mexico, which describe the prevalence of different mycobacterial species in this population. A high genetic diversity of MTb strains was identified. New spoligotypes and MIRU-VNTR patterns as well as a novel mutation associated to RIF-resistance were found. This information will facilitate the tracking of different mycobacterial species in HIV-infected individuals, and monitoring the spread of these microorganisms, leading to more appropriate measures for tuberculosis control.

Differential expression of dnaA and dosR genes among members of the Mycobacterium tuberculosis complex under oxic and hypoxic conditions.

Major differences regarding the pathology and host immune response of the Beijing and Canettii genotypes of Mycobacterium tuberculosis have been reported; however, studies on the genetic expression of these genotypes during in vitro dormancy are scarce. This study examined the expression of five cell-cycle-related genes and two dormancy-related genes in M. canettii, M. tuberculosis H37Rv, and M. tuberculosis Beijing during the Wayne model of dormancy. The results showed that under hypoxic conditions the three tuberculosis genotypes were able to transcribe genes involved in DNA replication and cellular division. In addition, dosR was found to be up-regulated in M. tuberculosis Beijing during the exponential growth phase but down-regulated under hypoxic conditions. In this genotype, the replication-related gene dnaA was also strongly down-regulated. These latter two findings suggest that, compared to M. tuberculosis H37Rv and M. canettii, the Beijing genotype has a lower capacity to synthesize dosR, hspX, and dnaA mRNAs during in vitro dormancy.

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.

par genes in Mycobacterium bovis and Mycobacterium smegmatis are arranged in an operon transcribed from "SigGC" promoters.

BACKGROUND: The ParA/Soj and ParB/Spo0J proteins, and the cis-acting parS site, participate actively in chromosome segregation and cell cycle progression. Genes homologous to parA and parB, and two putative parS copies, have been identified in the Mycobacterium bovis BCG and Mycobacterium smegmatis chromosomes. As in Mycobacterium tuberculosis, the parA and parB genes in these two non-pathogenic mycobacteria are located near the chromosomal origin of replication. The present work focused on the determination of the transcriptional organisation of the ~6 Kb orf60K-parB region of M. bovis BCG and M. smegmatis by primer extension, transcriptional fusions to the green fluorescence protein (GFP) and quantitative RT-PCR. RESULTS: The parAB genes were arranged in an operon. However, we also found promoters upstream of each one of these genes. Seven putative promoter sequences were identified in the orf60K-parB region of M. bovis BCG, whilst four were identified in the homologous region of M. smegmatis, one upstream of each open reading frame (ORF).Real-time PCR assays showed that in M. smegmatis, mRNA-parA and mRNA-parB levels decreased between the exponential and stationary phases. In M. bovis BCG, mRNA-parA levels also decreased between the exponential and stationary phases. However, parB expression was higher than parA expression and remained almost unchanged along the growth curve. CONCLUSION: The majority of the proposed promoter regions had features characteristic of Mycobacterium promoters previously denoted as Group D. The -10 hexamer of a strong E. coli sigma70-like promoter, located upstream of gidB of M. bovis BCG, overlapped with a putative parS sequence, suggesting that the transcription from this promoter might be regulated by the binding of ParB to parS.

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.