Isolamento e identificação de micobacterias e microrganismos indicadores de contaminação em águas tratadas provenientes do sistema de abstecimento público de Araraquara-SP

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Isolamento e identificação das micobactérias em águas tratadas provenientes do sistema de abastecimento de Araraquara-SP

Water quality is very important for the health and the population welfare, and the public supply system must provide water quality and suffi cient quantity for the entire population. Water treatment stations, are the main way to obtain water quality. When this doesn’t occur, several problems can affect the population, in this case, using water with poor quality is a constant risk of emergence causing various diseases. The elimination of microorganisms in treated water reduces competition, encouraging the multiplication of chlorine resistant bacteria as Mycobacterium genus frequently isolated from treated and chlorinated water. Considering the lack of indication from examinations of mycobacteria routine laboratory for quality control of drinking water and other human uses, the objective was to verify the presence isolate and identify the environmental mycobacteria in the system water source surface of Araraquara - SP. We analyzed 40 water samples, distributed as follows: ten water gross collected at Station Water Treatment Plant (WTP), harvested after ten fi ltration; ten collected in the reservoir after chlorination and ten in the network distribution. Were recovered 43 isolates of mycobacteria. All isolates were subjected to PCR-PRA. The mycobacteria were identifi ed as M. lentifl avum, M. parafortuitum, M. genavense, M. gordonae, M. fortuitum, M. confl uent, M. duvalii, M. avium subspecies paratuberculosis and M. szulgai. With these results, was concluded that water is an important source of environmental mycobacteria probably related to several human diseases, suggesting the carrying out continuous monitoring of the microorganisms in the system drinking water.

Heterogeneity of clinical and environmental isolates of Mycobacterium fortuitum using repetitive element sequence-based PCR: municipal water an unlikely source of community-acquired infections

M. fortuitum is a rapidly growing mycobacterium associated with community-acquired and nosocomial wound, soft tissue, and pulmonary infections. It has been postulated that water has been the source of infection especially in the hospital setting. The aim of this study was to determine if municipal water may be the source of community-acquired or nosocomial infections in the Brisbane area. Between 2007 and 2009, 20 strains of M. fortuitum were recovered from municipal water and 53 patients’ isolates were submitted to the reference laboratory. A wide variation in strain types was identified using repetitive element sequence-based PCR, with 13 clusters of ≥2 indistinguishable isolates, and 28 patterns consisting of individual isolates. The clusters could be grouped into seven similar groups (>95% similarity). Municipal water and clinical isolates collected during the same time period and from the same geographical area consisted of different strain types, making municipal water an unlikely source of sporadic human infection.

Interaction between Mycobacterium avium subsp. paratuberculosis and environmental protozoa

Background: Interactions between Mycobacterium avium subsp. paratuberculosis (Map) and free-living protozoa in water are likely to occur in nature. The potential impact of ingestion of Map by two naturally occurring Acanthamoeba spp. on this pathogen's survival and chlorine resistance was investigated. Results: Between 4.6 and 9.1% of spiked populations of three Map strains (NCTC 8578, B2 and ATCC 19698), which had been added at a multiplicity of infection of 10: 1, were ingested by Acanthamoeba castellanii CCAP 1501/1B and A. polyphaga CCAP 1501/3B during co-culture for 3 h at 25 C. Map cells were observed to be present within the vacuoles of the amoebae by acid-fast staining. During extended co-culture of Map NCTC 8578 at 25 degrees C for 24 d with both A. castellanii and A. polyphaga Map numbers did not change significantly during the first 7 days of incubation, however a 1-1.5 log(10) increase in Map numbers was observed between days 7 and 24 within both Acanthamoeba spp. Ingested Map cells were shown to be more resistant to chlorine inactivation than free Map. Exposure to 2 mu g/ml chlorine for 30 min resulted in a log(10) reduction of 0.94 in ingested Map but a log(10) reduction of 1.73 in free Map (p <0.001). Conclusion: This study demonstrated that ingestion of Map by and survival and multiplication of Map within Acanthamoeba spp. is possible, and that Map cells ingested by amoebae are more resistant to inactivation by chlorine than free Map cells. These findings have implications with respect to the efficacy of chlorination applied to Map infected surface waters.

Effect of environmental mycobacteria (Mycobacterium avium) on immunity induced by a DNA vaccine (DNAhsp65) against tuberculosis

The efficacy of BCG vaccine (attenuated Mycobacterium bovis) against pulmonary tuberculosis varies enormously among different populations. The prevailing hypothesis attributes this variation to interactions between the vaccine and mycobacteria common in the environment. Studies have revealed that most protective antigens expressed by the antituberculous vaccine are conserved in M. avium, supporting the hypothesis that exposure to environmental mycobacteria generates a cross-reactive immune response that interferes with BCG efficacy. In this study we investigated the effect of a prior exposure to heat-killed M. avium on the immune response and the protective efficacy induced by a genetic vaccine pVAXhsp65 (hsp65 gene from M. leprae inserted in pVAX vector) against experimental tuberculosis. To evaluate the effect on the immune response, female BALB/c mice were initially injected with distinct doses (0.08×106, 4×106, and 200×10 6) of heat-killed M. avium by subcutaneous route. Three weeks later, the animals were immunized with 3 doses of DNAhsp65 by intramuscular route (100μg/15 days apart). Control groups received only M. avium, vaccine (pVAXhsp65), vector (pVAX) or saline solution. Cytokine production and antibody levels were determined by ELISA. To evaluate the effect on the protective efficacy, animals were initially sensitized with 200×106 heat-killed CFU of M. avium by subcutaneous route and then immunized with 3 doses of pVAXhsp65 (100μg/15 days apart) by intramuscular route. Control groups were injected with saline, pVAX (4 doses), pVAXhsp65 (4 doses), M. avium or M. avium plus pVAX (3 doses). Fifteen days after last DNA dose, the animals were infected with 1×104 viable CFU of H37Rv M. tuberculosis by intratracheal route. Thirty days after challenge, the animals were sacrificed and the bacterial burden was determined by counting the number of CFU in the lungs. Lung histological sections were also analyzed. Splenic cells from primed animals produced more IL-5 but less IFN-gamma than non-primed ones. Also, prior contact with M. avium determined higher production of IgG1 and IgG2a anti-hsp65 antibodies in comparison to control groups. However, this higher immune response did not decrease the bacterial burden in the lungs. In addition, prior sensitization with M. avium decreased the parenchyma preservation observed in the group immunized only with pVaxhsp65. These results indicate that environmental mycobacteria can interfere with immunity and protective efficacy induced by DNAhsp65.

Clinical significance of Mycobacterium asiaticum isolates in Queensland, Australia

Mycobacterium asiaticum was first reported as a cause of human disease in 1982, with only a few cases in the literature to date. This study aims to review the clinical significance of M. asiaticum isolates in Queensland, Australia. A retrospective review (1989 to 2008) of patients with M. asiaticum isolates was conducted. Data were collected through the Queensland TB Control Centre database. Disease was defined in accordance with the American Thoracic Society criteria. Twenty-four patients (13 female) had a positive culture of M. asiaticum, many residing around the Tropic of Capricorn. M. asiaticum was responsible for pulmonary disease (n = 2), childhood lymphadenitis (n = 1), olecranon bursitis (n = 1), 6 cases of possible pulmonary disease, and 2 possible wound infections. Chronic lung disease was a risk factor for pulmonary infection, and wounds/lacerations were a risk factor for extrapulmonary disease. Extrapulmonary disease responded to local measures. Pulmonary disease responded to ethambutol-isoniazid-rifampin plus pyrazinamide for the first 2 months in one patient, and amikacin-azithromycin-minocycline in another patient. While M. asiaticum is rare in Queensland, there appears to be an environmental niche. Although often a colonizer, it can be a cause of pulmonary and extrapulmonary disease. Treatment of pulmonary disease remains challenging. Extrapulmonary disease does not mandate specific nontuberculous mycobacterium (NTM) treatment.

Mycobacterium lentiflavum in Drinking Water Supplies, Australia

Mycobacterium lentiflavum, a slow-growing nontuberculous mycobacterium, is a rare cause of human disease. It has been isolated from environmental samples worldwide. To assess the clinical significance of M. lentiflavum isolates reported to the Queensland Tuberculosis Control Centre, Australia, during 2001-2008, we explored the genotypic similarity and geographic relationship between isolates from humans and potable water in the Brisbane metropolitan area. A total of 47 isolates from 36 patients were reported; 4 patients had clinically significant disease. M. lentiflavum was cultured from 13 of 206 drinking water sites. These sites overlapped geographically with home addresses of the patients who had clinically significant disease. Automated repetitive sequence-based PCR genotyping showed a dominant environmental clone closely related to clinical strains. This finding suggests potable water as a possible source of M. lentiflavum infection in humans.

Mycobacterium abscessus infection and potable water

Mycobacterium abscessus is a rapidly growing mycobacteria responsible for progressive pulmonary disease, soft tissue and wound infections, and can contaminate clinical specimens. Nontuberculous mycobacteria (NTM) are generally considered environmental organisms though M. abscessus has not featured frequently in environmental studies, particularly those examining potable water. In a study of Brisbane potable water, M. abscessus was isolate from ten different locations. The incidence of disease due to M. abscessus has been increasing in Queensland. Aim: To compare genotypically the M. abscessus isolates obtained from water to those obtained from human clinical specimens. Methods: From a study of Brisbane potable water between 2007 and 2009, ten isolates confirmed as M. abscessus were recovered. In addition, one strain was isolated from a rainwater tank of a patient with disease due to M. avium, and another from the swimming pool of a patient with M. intracellulare disease. A random sample of 74 clinical isolates referred to the QLD Mycobacterial reference laboratory during the same time period was available for comparison using repPCR strain typing (Diversilab). Results: The drinking water isolates formed two distinct strain patterns (A and B) that shared >90% similarity. The tankwater isolate (pattern C) shared >85% similarity with the potable water isolates, but the pool isolate (D) was distinctly different. Fifty-three clinical isolates clustered tightly (>95% similarity) with the Group A potable water isolates, 4 patients with Group B. Thirteen patient isolates clustered with the Rainwater tank isolate. One patient matched the pool isolate. There were a further 3 patient isolates that were unrelated to the water isolates. No differences were found between strain types in terms of geographic origin, gender, age, or site/type of infection. Conclusion: The high degree of similarity between strains of M. abscessus from potable water and strains causing infection in humans from the same area, strengthens the possibility that drinking water may be a source of infection in these patients.

Mycobacterium tuberculosis nucleoid-associated DNA-binding protein H-NS binds with high-affinity to the Holliday junction and inhibits strand exchange promoted by RecA protein

A number of studies have shown that the structure and composition of bacterial nucleoid influences many a processes related to DNA metabolism. The nucleoid-associated proteins modulate not only the DNA conformation but also regulate the DNA metabolic processes such as replication, recombination, repair and transcription. Understanding of how these processes occur in the context of Mycobacterium tuberculosis nucleoid is of considerable medical importance because the nucleoid structure may be constantly remodeled in response to environmental signals and/or growth conditions. Many studies have concluded that Escherichia coli H-NS binds to DNA in a sequence-independent manner, with a preference for A-/T-rich tracts in curved DNA; however, recent studies have identified the existence of medium- and low-affinity binding sites in the vicinity of the curved DNA. Here, we show that the M. tuberculosis H-NS protein binds in a more structure-specific manner to DNA replication and repair intermediates, but displays lower affinity for double-stranded DNA with relatively higher GC content. Notably, M. tuberculosis H-NS was able to bind Holliday junction (HJ), the central recombination intermediate, with substantially higher affinity and inhibited the three-strand exchange promoted by its cognate RecA. Likewise, E. coli H-NS was able to bind the HJ and suppress DNA strand exchange promoted by E. coli RecA, although much less efficiently compared to M. tuberculosis H-NS. Our results provide new insights into a previously unrecognized function of H-NS protein, with implications for blocking the genome integration of horizontally transferred genes by homologous and/or homeologous recombination.

A full-length bifunctional protein involved in c-di-GMP turnover is required for long-term survival under nutrient starvation in Mycobacterium smegmatis

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) plays an important role in a variety of cellular functions, including biofilm formation, alterations in the cell surface, host colonization and regulation of bacterial flagellar motility, which enable bacteria to survive changing environmental conditions. The cellular level of c-di-GMP is regulated by a balance between opposing activities of diguanylate cyclases (DGCs) and cognate phosphodiesterases (PDE-As). Here, we report the presence and importance of a protein, MSDGC-1 (an orthologue of Rv1354c in Mycobacterium tuberculosis), involved in c-di-GMP turnover in Mycobacterium smegmatis. MSDGC-1 is a multidomain protein, having GAF, GGDEF and EAL domains arranged in tandem, and exhibits both c-di-GMP synthesis and degradation activities. Most other proteins containing GGDEF and EAL domains have been demonstrated to have either DGC or PDE-A activity. Unlike other bacteria, which harbour several copies of the protein involved in c-di-GMP turnover, M. smegmatis has a single genomic copy, deletion of which severely affects long-term survival under conditions of nutrient starvation. Overexpression of MSDGC-1 alters the colony morphology and growth profile of M. smegmatis. In order to gain insights into the regulation of the c-di-GMP level, we cloned individual domains and tested their activities. We observed a loss of activity in the separated domains, indicating the importance of full-length MSDGC-1 for controlling bifunctionality.

Mycobacterium tuberculosis RsdA provides a conformational rationale for selective regulation of sigma-factor activity by proteolysis

The relative levels of different sigma factors dictate the expression profile of a bacterium. Extracytoplasmic function sigma factors synchronize the transcriptional profile with environmental conditions. The cellular concentration of free extracytoplasmic function sigma factors is regulated by the localization of this protein in a sigma/anti-sigma complex. Anti-sigma factors are multi-domain proteins with a receptor to sense environmental stimuli and a conserved anti-sigma domain (ASD) that binds a sigma factor. Here we describe the structure of Mycobacterium tuberculosis anti-sigma(D) (RsdA) in complex with the -35 promoter binding domain of sigma(D) (sigma(D)(4)). We note distinct conformational features that enable the release of sigma(D) by the selective proteolysis of the ASD in RsdA. The structural and biochemical features of the sigma(D)/RsdA complex provide a basis to reconcile diverse regulatory mechanisms that govern sigma/anti-sigma interactions despite high overall structural similarity. Multiple regulatory mechanisms embedded in an ASD scaffold thus provide an elegant route to rapidly re-engineer the expression profile of a bacterium in response to an environmental stimulus.

The adenylyl cyclase Rv2212 modifies the proteome and infectivity of Mycobacterium bovis BCG

All organisms have the capacity to sense and respond to environmental changes. These signals often involve the use of second messengers such as cyclic adenosine monophosphate (cAMP). This second messenger is widely distributed among organisms and coordinates gene expression related with pathogenesis, virulence, and environmental adaptation. Genomic analysis in Mycobacterium tuberculosis has identified 16 adenylyl cyclases (AC) and one phosphodiesterase, which produce and degrade cAMP, respectively. To date, ten AC have been biochemically characterized and only one (Rv0386) has been found to be important during murine infection with M. tuberculosis. Here, we investigated the impact of hsp60-driven Rv2212 gene expression in Mycobacterium bovis Bacillus Calmette-Guerin (BCG) during growth in vitro, and during macrophage and mice infection. We found that hsp60-driven expression of Rv2212 resulted in an increased capacity of replication in murine macrophages but an attenuated phenotype in lungs and spleen when administered intravenously in mice. Furthermore, this strain displayed an altered proteome mainly affecting proteins associated with stress conditions (bfrB, groEL-2, DnaK) that could contribute to the attenuated phenotype observed in mice.

Novel Functions of (p)ppGpp and Cyclic di-GMP in Mycobacterial Physiology Revealed by Phenotype Microarray Analysis of Wild-Type and Isogenic Strains of Mycobacterium smegmatis

The bacterial second messengers (p)ppGpp and bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, a Pseudomonas aeruginosa strain lacking (p) ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whether Mycobacterium smegmatis strains lacking the gene for either (p)ppGpp synthesis (Delta rel(Msm)) or c-di-GMP synthesis (Delta dcpA) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the Delta rel(Msm) and Delta dcpA strains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls of M. smegmatis. Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids in M. smegmatis.

Crystallographic studies of the extracytoplasmic function sigma factor sigma(J) from Mycobacterium tuberculosis

Mycobacterium tuberculosis has multiple sigma factors which enable the bacterium to reprogram its transcriptional machinery under diverse environmental conditions. sigma(J), an extracytoplasmic function sigma factor, is upregulated in late stationary phase cultures and during human macrophage infection. sigma(J) governs the cellular response to hydrogen peroxide-mediated oxidative stress. sigma(J) differs from other canonical sigma factors owing to the presence of a SnoaL_2 domain at the C-terminus. sigma(J) crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 133.85, c = 75.08 angstrom. Diffraction data were collected to 2.16 angstrom resolution on the BM14 beamline at the European Synchrotron Radiation Facility (ESRF).

The two-component signalling networks of Mycobacterium tuberculosis display extensive cross-talk in vitro

Two-component systems (TCSs), which contain paired sensor kinase and response regulator proteins, form the primary apparatus for sensing and responding to environmental cues in bacteria. TCSs are thought to be highly specific, displaying minimal cross-talk, primarily due to the co-evolution of the participating proteins. To assess the level of cross-talk between the TCSs of Mycobacterium tuberculosis, we mapped the complete interactome of the M. tuberculosis TCSs using phosphotransfer profiling. Surprisingly, we found extensive crosstalk among the M. tuberculosis TCSs, significantly more than that in the TCSs in Escherichia coli or Caulobacter crescentus, thereby offering an alternate to specificity paradigm in TCS signalling. Nearly half of the interactions we detected were significant novel cross-interactions, unravelling a potentially complex signalling landscape. We classified the TCSs into specific `one-to-one' and promiscuous `one-to-many' and `many-to-one' circuits. Using mathematical modelling, we deduced that the promiscuous signalling observed can explain several currently confounding observations about M. tuberculosis TCSs. Our findings suggest an alternative paradigm of bacterial signalling with significant cross-talk between TCSs yielding potentially complex signalling landscapes.