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.

The Dual Role of the Immune Response in Reproductive Organs During Zika Virus Infection.

Zika virus is a mosquito-borne viral disease that emerged as a significant health problem in the Americas after an epidemic in 2015. Especially concerning are cases where Zika is linked to the development of brain abnormalities in newborns. Unlike other flaviviruses, Zika can be transmitted sexually, increasing the potential for intraspecies infection. Several reports show that the virus can persist for months in the testis of males after clearance of viremia, and that females are highly susceptible to infection via sexual transmission. The most common route of sexual transmission is male-to-female, which suggests that the mechanism driving persistence of Zika in the testis is essential for dissemination. The immune system plays an essential role in Zika infection. In females, a robust response inhibits the virus to control the infection. In males, however, the immunological response to Zika infection correlates with viral persistence. Thus, the immune system may have a dual role in sexually transmitted pathogenesis. The mechanism by which the immune system allows the virus to enter an immune-privileged site while continuing to disseminate is unclear. In this mini-review, we highlight advances in our knowledge of sexually transmitted Zika virus pathogenesis and the possible mechanisms mounted by the immune system that control or exacerbate the 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.

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.

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.

Occurrence of potentially pathogenic nontuberculous mycobacteria in Mexican household potable water: a pilot study.

BACKGROUND: Nontuberculous mycobacteria (NTM) are environmental opportunistic pathogens found in natural and human-engineered waters, including drinking water distribution systems and household plumbing. This pilot study examined the frequency of occurrence of NTM in household potable water samples in Mexico City. Potable water samples were collected from the "main house faucet" and kitchen faucet. The presence of aerobic-mesophilic bacteria (AMB), total coliforms (TC), fecal coliforms (FC) and NTM species were determined. Mycobacteria species were identified by PCR restriction enzyme pattern analysis (PRA) of the 65-kDa heat shock protein gene (hsp65) and sequencing of the hypervariable region 2 (V2) of the 16S rRNA gene and of the rpoB gene. RESULTS: AMB (<100 CFU/ml) were present in 118 out of 120 samples; only two samples were outside guidelines ranges (>100 CFU/ml). TC and FC were detected in four and one samples, respectively. NTM species were recovered from 16% samples (19/120) and included M. mucogenicum (nine), M. porcinum (three), M. avium (three), M. gordonae (one), M. cosmeticum (one), M. fortuitum (one), and Mycobacterium sp (one). All household water samples that contained NTM complied with the standards required to grade the water as "good quality" potable water. CONCLUSION: Household potable water may be a potential source of NTM infection in Mexico City.

Evaluation of the cell growth of mycobacteria using Mycobacterium smegmatis mc2 155 as a representative species.

The study of the in vitro cell growth of mycobacteria still remains a fastidious, difficult, and time-consuming procedure. In addition, assessing mycobacterial growth in the laboratory is often complicated by cell aggregation and slow growth-rate. We now report that the use of a stainless steel spring in the culture led to an absence of large cell clumps, to a decrease of dead cells in the exponential phase and to growth of a more homogeneous population of large cells. We also report that flow cytometry is a rapid, simple and reliable approach to monitor mycobacterial cell growth and viability. Here, we monitored Mycobacterium smegmatis cellular growth by optical density, dry cell mass, and colony forming units; in addition, viability, cell size and granularity profiles were analyzed by flow cytometry, and cell morphology by electron microscopy. Cultures monitored by flow cytometry may lead to a better understanding of the physiology of mycobacteria. Moreover, this methodology may aid in characterizing the cell growth of other fastidious species of microorganisms.

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.