Integrated processes (HPSE+scCO2) to prepare sterilized alginate-gelatine-based aerogel.

Biopolymers application in biomedical areas has been limited due to the physicochemical degradation that occurs using conventional processing/sterilization methods (e.g., steam heat, γ-radiation, ethylene oxide). Aiming to avoid/minimize degradation and preserve their properties, supercritical carbon dioxide (scCO2) has been proposed as an alternative sterilization method for such materials. ScCO2 can simultaneously be used as a drying method to produce aerogels (i) and sterilize them (ii). However, a solvent exchange is required to prepare the alcogel from hydrogel, achievable through high-pressure solvent exchange (HPSE) (iii). This study integrated three processes: HPSE, scCO2 drying, and sterilization to prepare alginate-gelatine sterilized aerogels. Two scCO2 sterilization methods were tested. Results showed that sterilization did not compromise the aerogels' chemical, thermal and swelling properties. Conversely, Young's Modulus increased, and BET surface area decreased, due to the structural changes caused by the fast pressurization/depressurization rates applied during sterilization. Regarding the sterilization efficiency, results showed a reduction in contamination throughout the process, achieving a SAL of 10-4. The sterilized aerogels were non-cytotoxic in vitro and showed improved wound-healing properties. The innovative integrated process produced decontaminated/sterile and ready-to-use aerogels reducing process time by 75 %, from 2 days up to 12 h without compromising the aerogel's properties.

Drinking Water Microbiota, Entero-Mammary Pathways, and Breast Cancer: Focus on Nontuberculous Mycobacteria.

The prospect of drinking water serving as a conduit for gut bacteria, artificially selected by disinfection strategies and a lack of monitoring at the point of use, is concerning. Certain opportunistic pathogens, notably some nontuberculous mycobacteria (NTM), often exceed coliform bacteria levels in drinking water, posing safety risks. NTM and other microbiota resist chlorination and thrive in plumbing systems. When inhaled, opportunistic NTM can infect the lungs of immunocompromised or chronically ill patients and the elderly, primarily postmenopausal women. When ingested with drinking water, NTM often survive stomach acidity, reach the intestines, and migrate to other organs using immune cells as vehicles, potentially colonizing tumor tissue, including in breast cancer. The link between the microbiome and cancer is not new, yet the recognition of intratumoral microbiomes is a recent development. Breast cancer risk rises with age, and NTM infections have emerged as a concern among breast cancer patients. In addition to studies hinting at a potential association between chronic NTM infections and lung cancer, NTM have also been detected in breast tumors at levels higher than normal adjacent tissue. Evaluating the risks of continued ingestion of contaminated drinking water is paramount, especially given the ability of various bacteria to migrate from the gut to breast tissue via entero-mammary pathways. This underscores a pressing need to revise water safety monitoring guidelines and delve into hormonal factors, including addressing the disproportionate impact of NTM infections and breast cancer on women and examining the potential health risks posed by the cryptic and unchecked microbiota from drinking water.

Effect of ScCO2 on the decontamination of PECs-based cryogels: A comparison with H2O steam and H2O2 nebulization methods.

Biopolymers present ideal properties to be used in wound dressing solutions. By mixing two oppositely charged macromolecules it is possible to form polyelectrolyte complex (PEC) based cryogels using lyophilization. Their application in the biomedical field is limited due to their sterilization requirements, as conventional methods compromise their physicochemical properties. ScCO2 appears as an alternative method for decontamination. This work assessed several cryogel PEC formulations, chitosan-pectin, gelatine-xanthan gum and alginate-gelatine. PEC formation was confirmed by FTIR and rheological analysis. While steam sterilization compromised cryogels' chemical and morphological properties, decontamination with scCO2 proved to be a promising method for decontamination of PEC-cryogels, because, similarly to what is observed with hydrogen peroxide, it does not compromise their physicochemical properties.

Footprints of a microbial toxin from the gut microbiome to mesencephalic mitochondria.

OBJECTIVE: Idiopathic Parkinson's disease (PD) is characterised by alpha-synuclein (aSyn) aggregation and death of dopaminergic neurons in the midbrain. Recent evidence posits that PD may initiate in the gut by microbes or their toxins that promote chronic gut inflammation that will ultimately impact the brain. In this work, we sought to demonstrate that the effects of the microbial toxin ß-N-methylamino-L-alanine (BMAA) in the gut may trigger some PD cases, which is especially worrying as this toxin is present in certain foods but not routinely monitored by public health authorities. DESIGN: To test the hypothesis, we treated wild-type mice, primary neuronal cultures, cell lines and isolated mitochondria with BMAA, and analysed its impact on gut microbiota composition, barrier permeability, inflammation and aSyn aggregation as well as in brain inflammation, dopaminergic neuronal loss and motor behaviour. To further examine the key role of mitochondria, we also determined the specific effects of BMAA on mitochondrial function and on inflammasome activation. RESULTS: BMAA induced extensive depletion of segmented filamentous bacteria (SFB) that regulate gut immunity, thus triggering gut dysbiosis, immune cell migration, increased intestinal inflammation, loss of barrier integrity and caudo-rostral progression of aSyn. Additionally, BMAA induced in vitro and in vivo mitochondrial dysfunction with cardiolipin exposure and consequent activation of neuronal innate immunity. These events primed neuroinflammation, dopaminergic neuronal loss and motor deficits. CONCLUSION: Taken together, our results demonstrate that chronic exposure to dietary BMAA can trigger a chain of events that recapitulate the evolution of the PD pathology from the gut to the brain, which is consistent with 'gut-first' PD.

A review on urinary tract infections diagnostic methods: Laboratory-based and point-of-care approaches.

Urinary tract infections (UTIs) are among the most common infectious diseases worldwide. This type of infections can be healthcare-associated or community-acquired and affects millions of people every year. Different diagnostic procedures are available to detect pathogens in urine and they can be divided into two main categories: laboratory-based and point-of-care (POC) detection techniques. Traditional methodologies are often time-consuming, thus, achieving a rapid and accurate identification of pathogens is a challenging feature that has been pursued by many research groups and companies operating in this area. The purpose of this review is to compare and highlight advantages and disadvantages of the traditional and currently most used detection methods, as well as the emerging POC approaches and the relevant advances in on-site detection of pathogens´ mechanisms, suitable to be adapted to UTI diagnosis. Lately, the commercially available UTI self-testing kits and devices are helping in the diagnosis of urinary infections as patients or care givers are able to perform the test, easily and comfortably at home and, upon the result, decide when to attend an appointment/Urgent Health Care Unit.

Improved diabetic wound healing by LFcinB is associated with relevant changes in the skin immune response and microbiota.

Bovine lactoferricin (LFcinB) has antimicrobial and immunomodulatory properties; however, the effects on diabetic wound healing remain poorly understood. The wound healing potential of LFcinB was investigated with in vitro, ex vivo, and in vivo models. Cell migration and proliferation were tested on keratinocytes and on porcine ears. A type 1 diabetic mouse model was also used to evaluate wound healing kinetics, bacterial diversity patterns, and the effect of LFcinB on oxidative stress, macrophage phenotype, angiogenesis, and collagen deposition. LFcinB increased keratinocyte migration in vitro (p < 0.05) and ex vivo (p < 0.001) and improved wound healing in diabetic mice (p < 0.05), though not in normoglycemic control mice. In diabetic mouse wounds, LFcinB treatment led to the eradication of Bacillus pumilus, a decrease in Staphylococcus aureus, and an increase in the Staphylococcus xylosus prevalence. LFcinB increased angiogenesis in diabetic mice (p < 0.01), but this was decreased in control mice (p < 0.05). LFcinB improved collagen deposition in both diabetic and control mice (p < 0.05). Both oxidative stress and the M1-to-M2 macrophage ratios were decreased in LFcinB-treated wounds of diabetic animals (p < 0.001 and p < 0.05, respectively) compared with saline, suggesting a downregulation of inflammation in diabetic wounds. In conclusion, LFcinB treatment demonstrated noticeable positive effects on diabetic wound healing.

A genuine mycobacterial thermophile: Mycobacterium hassiacum growth, survival and GpgS stability at near-pasteurization temperatures.

Mycobacterium hassiacum is so far the most thermophilic among mycobacteria as it grows optimally at 50 °C and up to 65 °C in a glycerol-based medium, as verified in this study. Since this and other nontuberculous mycobacteria (NTM) thrive in diverse natural and artificial environments, from where they may access and infect humans, we deemed essential to probe M. hassiacum resistance to heat, a strategy routinely used to control microbial growth in water-supply systems, as well as in the food and drink industries. In addition to possibly being a threat in its own right in rare occasions, M. hassiacum is also a good surrogate for studying other NTM species more often associated with opportunistic infection, namely Mycobacterium avium and Mycobacterium abscessus as well as their strictly pathogenic counterparts Mycobacterium tuberculosis and Mycobacterium leprae. In this regard, this thermophilic species is likely to be useful as a source of stable proteins that may provide more detailed structures of potential drug targets. Here, we investigate M. hassiacum growth at near-pasteurization temperatures and at different pHs and also characterize its thermostable glucosyl-3-phosphoglycerate synthase (GpgS), an enzyme considered essential for M. tuberculosis growth and associated with both nitrogen starvation and thermal stress in different NTM species.

The structural characterization of a glucosylglycerate hydrolase provides insights into the molecular mechanism of mycobacterial recovery from nitrogen starvation.

Bacteria are challenged to adapt to environmental variations in order to survive. Under nutritional stress, several bacteria are able to slow down their metabolism into a nonreplicating state and wait for favourable conditions. It is almost universal that bacteria accumulate carbon stores to survive during this nonreplicating state and to fuel rapid proliferation when the growth-limiting stress disappears. Mycobacteria are exceedingly successful in their ability to become dormant under harsh circumstances and to be able to resume growth when conditions are favourable. Rapidly growing mycobacteria accumulate glucosylglycerate under nitrogen-limiting conditions and quickly mobilize it when nitrogen availability is restored. The depletion of intracellular glucosyl-glycerate levels in Mycolicibacterium hassiacum (basonym Mycobacterium hassiacum) was associated with the up-regulation of the gene coding for glucosylglycerate hydrolase (GgH), an enzyme that is able to hydrolyse glucosylglycerate to glycerate and glucose, a source of readily available energy. Highly conserved among unrelated phyla, GgH is likely to be involved in bacterial reactivation following nitrogen starvation, which in addition to other factors driving mycobacterial recovery may also provide an opportunity for therapeutic intervention, especially in the serious infections caused by some emerging opportunistic pathogens of this group, such as Mycobacteroides abscessus (basonym Mycobacterium abscessus). Using a combination of biochemical methods and hybrid structural approaches, the oligomeric organization of M. hassiacum GgH was determined and molecular determinants of its substrate binding and specificity were unveiled.

High-Quality Draft Genome Sequences of Rare Nontuberculous Mycobacteria Isolated from Surfaces of a Hospital.

Nontuberculous mycobacteria (NTM), some of which had multidrug-resistant profiles, were isolated from a tertiary care hospital setting. Although most NTM are nonpathogenic, contamination of hospital surfaces by these opportunistic pathogens poses a health risk to vulnerable inpatients. These high-quality NTM draft genomes are fundamental for future genetic and epidemiological studies.

Studies of antimicrobial resistance in rare mycobacteria from a nosocomial environment.

BACKGROUND: Nontuberculous mycobacteria (NTM) are ubiquitous in nature and recognized agents of opportunistic infection, which is often aggravated by their intrinsic resistance to antimicrobials, poorly defined therapeutic strategies and by the lack of new drugs. However, evaluation of their prevalence in anthropogenic environments and the associated antimicrobial resistance profiles have been neglected. In this work, we sought to determine minimal inhibitory concentrations of 25 antimicrobials against 5 NTM isolates recovered from a tertiary-care hospital surfaces. Antimicrobial susceptibilities of 5 other Corynebacterineae isolated from the same hospital were also determined for their potential clinical relevance. RESULTS: Our phylogenetic study with each of the NTM isolates confirm they belong to Mycobacterium obuense, Mycobacterium mucogenicum and Mycobacterium paragordonae species, the latter initially misidentified as strains of M. gordonae, a species frequently isolated from patients with NTM disease in Portugal. In contrast to other strains, the M. obuense and M. mucogenicum examined here were resistant to several of the CLSI-recommended drugs, suggestive of multidrug-resistant profiles. Surprisingly, M. obuense was susceptible to vancomycin. Their genomes were sequenced allowing detection of gene erm (erythromycin resistance methylase) in M. obuense, explaining its resistance to clarithromycin. Remarkably, and unlike other strains of the genus, the Corynebacterium isolates were highly resistant to penicillin, ciprofloxacin and linezolid. CONCLUSIONS: This study highlights the importance of implementing effective measures to screen, accurately identify and control viable NTM and closely related bacteria in hospital settings. Our report on the occurrence of rare NTM species with antibiotic susceptibility profiles that are distinct from those of the corresponding Type strains, along with unexpected resistance mechanisms detected seem to suggest that resistance may be more common than previously thought and also a potential threat to frail and otherwise vulnerable inpatients.

Hand hygiene management among nurses: collective health challenges.

OBJECTIVE: To describe the determining factors in hand hygiene management among nurses and identify associated collective health challenges. METHOD: Cross-sectional descriptive study. Data were collected using a questionnaire that was applied in four internal medicine units of a hospital of reference in Portugal. RESULTS: The sample was composed of 50 nurses aged 26 to 55 years (mean age of 34.88 years); 80% were women, 58% had a Bachelor's degree, and had 5-30 years of nursing practice (X̄ =11.94;±5.92). The vast majority of nurses (90%) reported complying with the existing recommendations on hand hygiene in pre-established moments. However, none of the nurses were able to identify all the moments for hand hygiene using water and soap or alcohol-based handrub. CONCLUSION: This study shows that continuous training, adequate materials/structures in the units, and redesigned administration/supervision practices are determining factors to achieve higher levels of adherence to hand hygiene among nurses, as well as increased quality and safety in care delivery, which is a current collective health challenge.

Hand hygiene management among nurses: collective health challenges / Gestión del procedimiento de la higiene de las manos por enfermeros: desafíos para la salud colectiva / Gestão do procedimento higiene das mãos por enfermeiros: desafios para saúde coletiva

ABSTRACT Objective: To describe the determining factors in hand hygiene management among nurses and identify associated collective health challenges. Method: Cross-sectional descriptive study. Data were collected using a questionnaire that was applied in four internal medicine units of a hospital of reference in Portugal. Results: The sample was composed of 50 nurses aged 26 to 55 years (mean age of 34.88 years); 80% were women, 58% had a Bachelor's degree, and had 5-30 years of nursing practice (X̄ =11.94;±5.92). The vast majority of nurses (90%) reported complying with the existing recommendations on hand hygiene in pre-established moments. However, none of the nurses were able to identify all the moments for hand hygiene using water and soap or alcohol-based handrub. Conclusion: This study shows that continuous training, adequate materials/structures in the units, and redesigned administration/supervision practices are determining factors to achieve higher levels of adherence to hand hygiene among nurses, as well as increased quality and safety in care delivery, which is a current collective health challenge.

RESUMEN Objetivo: Describir los factores determinantes en la gestión de la higiene de las manos por enfermeros e identificar los desafíos inherentes para la salud colectiva. Método: Estudio descriptivo-transversal. Los datos se recogieron mediante un cuestionario en cuatro unidades de medicina interna de un hospital de referencia en Portugal. Resultados: se incluyó a 50 enfermeros con una edad media de 34,88 años (entre los 26 y 55 años), el 80 % del sexo femenino, el 58 % son licenciados y desempeñan sus funciones de 5 a 30 años (X̄ = 11.94; ± 5.92). De ellos, el 90 % considera que respeta las recomendaciones existentes sobre higiene de las manos en los momentos estipulados para tal efecto. Sin embargo, los enfermeros no identificaron ninguno de los momentos para realizar este procedimiento, con agua y jabón o solución antiséptica de base alcohólica. Conclusión: Las contribuciones como formación continua, adecuación de los materiales / estructuras de las unidades y reconstrucción de prácticas por gestores / supervisores emergen en este trabajo como factores determinantes para alcanzar niveles superiores de adhesión a la higiene de las manos por enfermeros, de calidad y seguridad, un desafío actual para la salud pública.

RESUMO Objectivo: Descrever fatores determinantes na gestão da higiene das mãos por Enfermeiros e identificar desafios inerentes para a saúde coletiva. Método: Estudo descritivo-transversal. Dados coletados por questionário em quatro unidades de medicina interna de um hospital de referência em Portugal. Resultados: incluídos 50 enfermeiros, idade média 34,88 anos (entre os 26 e 55 anos), 80% sexo feminino, 58% são licenciados e exercem funções entre 5-30 anos (X̄ =11.94;±5.92). Destes, 90% considera respeitar as recomendações existentes sobre higiene das mãos nos momentos estipulados para o efeito. Porém, nenhum dos momentos para a realização deste procedimento, com água e sabão ou solução antissética de base alcoólica, foi identificado por todos os enfermeiros. Conclusão: Contribuições como formação contínua, adequação dos materiais/estruturas das unidades e reconstrução de práticas por gestores/supervisores emergem neste trabalho como fatores determinantes para atingir níveis superiores de adesão à higiene das mãos por enfermeiros, de qualidade e segurança, desafio atual para a saúde pública.

Production, crystallization and structure determination of a mycobacterial glucosylglycerate hydrolase.

Glucosylglycerate hydrolase is highly conserved among rapidly growing mycobacteria and has been found to be involved in recovery from nitrogen starvation by promoting the rapid mobilization of the glucosylglycerate that accumulates under these conditions. Here, the production, crystallization and structure determination of glucosylglycerate hydrolase from Mycobacterium hassiacum using two-wavelength anomalous diffraction of selenomethionine-substituted crystals are described. The monoclinic (space group P21) crystals diffracted to ∼2.0 Šresolution at a synchrotron-radiation source and contained four molecules in the asymmetric unit, corresponding to a Matthews coefficient of 3.07 Å3 Da-1 and a solvent content of 59.9%. The quality of the experimental phases allowed the automated building of 1677 of the 1792 residues in the asymmetric unit.

Hospital microbial surface colonization revealed during monitoring of Klebsiella spp., Pseudomonas aeruginosa, and non-tuberculous mycobacteria.

Hospital environmental conditions, human occupancy, and the characteristics of the equipment influence the survival of microbial communities and raise a concern with regard to nosocomial infections. The objective of the present work was to use the monitoring of Pseudomonas aeruginosa, Klebsiella spp. and non-tuberculous mycobacteria as a strategy to improve knowledge on microbial colonization of non-critical equipment and surfaces, in a tertiary hospital from Central Portugal. A 3-month microbiological survey was performed in a district teaching hospital. A total of 173 samples were obtained from the wards Hematology, Urology, Medicine, and Renal Transplants, and 102 presumptive strains recovered. Per sampling, Pseudomonas Isolation agar showed 42.8 to 73.3% of presumptive P. aeruginosa colonies and MacConkey agar recovered mostly Staphylococcus. Most of the colonies recovered in Middlebrook 7H10-PANTA belonged to the genus Methylobacterium. Taps and WC shower curtains carry high bacterial species diversity. The Redundancy Analysis grouped the samples in those mostly handled by patients, and those mostly handled by healthcare staff or of mixed use. This study shows that the preferential users of the space and equipment seem to be important contributors to the microbial community. The most recovered genus was Methylobacterium, known as colonizer of the water distribution system therefore, it is possible that the water points and biofilms in taps also contribute as dispersion hotspots.

Glucosylglycerate metabolism, bioversatility and mycobacterial survival.

Despite the progressive decline in tuberculosis mortality, strains resistant to our dated antibiotics remain a global threat, as are the emerging nontuberculous mycobacteria, ubiquitous in natural and human environments. This pressing situation boosted by debilitated immune systems, chronic illness and the aged population calls for efficient strategies to fight these successful organisms, and identifying pathways critical for their survival is a crucial step towards this goal. In this context, the glycoside glucosylglycerate (GG) has been implicated in the adaptation of mycobacteria to nitrogen starvation and to thermal stress, and the key gene for GG synthesis has been considered essential for Mycobacterium tuberculosis growth. The many organisms we now know to have genes for GG metabolism opened new exciting avenues of research into its functions, hinting for example at hypothetical roles as an inter-cellular messenger among bacteria and in microbe-plant interactions, or at key roles in the global nitrogen cycle beyond what cyanobacteria and mycobacteria have taught us so far. Indeed, the insights into GG biology gained over the last decade have changed the perception of GG from a rare polysaccharide constituent to a widespread molecule with multiple functions and biosynthetic origins. It is now possible to build upon this knowledge and further explore its physiological importance in both pathogenic and environmentally relevant microorganisms. In particular, the vital roles of GG and of its important derivative the mycobacterial methylglucose lipopolysaccharide (MGLP) discussed here are now evident, making their metabolic links attractive targets for the development of new urgently needed antimycobacterial therapies.

The looming tide of nontuberculous mycobacterial infections in Portugal and Brazil.

Nontuberculous mycobacteria (NTM) are widely disseminated in the environment and an emerging cause of infectious diseases worldwide. Their remarkable natural resistance to disinfectants and antibiotics and an ability to survive under low-nutrient conditions allows NTM to colonize and persist in man-made environments such as household and hospital water distribution systems. This overlap between human and NTM environments afforded new opportunities for human exposure, and for expression of their often neglected and underestimated pathogenic potential. Some risk factors predisposing to NTM disease have been identified and are mainly associated with immune fragilities of the human host. However, infections in apparently immunocompetent persons are also increasingly reported. The purpose of this review is to bring attention to this emerging health problem in Portugal and Brazil and to emphasize the urgent need for increased surveillance and more comprehensive epidemiological data in both countries, where such information is scarce and seriously thwarts the adoption of proper preventive strategies and therapeutic options.

Mycobacterium hassiacum recovers from nitrogen starvation with up-regulation of a novel glucosylglycerate hydrolase and depletion of the accumulated glucosylglycerate.

Some microorganisms accumulate glucosylglycerate (GG) during growth under nitrogen deprivation. However, the molecular mechanisms underlying the role of GG and the regulation of its levels in the nitrogen stress response are elusive. Since GG is required for biosynthesis of mycobacterial methylglucose lipopolysaccharides (MGLP) we examined the molecular mechanisms linking replenishment of assimilable nitrogen to nitrogen-starved M. hassiacum with depletion of GG accumulated during nitrogen deficiency. To probe the involvement of a newly identified glycoside hydrolase in GG depletion, we produced the mycobacterial enzyme recombinantly and confirmed the specific hydrolysis of GG (GG hydrolase, GgH) in vitro. We have also observed a pronounced up-regulation of GgH mRNA in response to the nitrogen shock, which positively correlates with GG depletion in vivo and growth stimulation, implicating GgH in the recovery process. Since GgH orthologs seem to be absent from most slowly-growing mycobacteria including M. tuberculosis, the disclosure of the GgH function allows reconfiguration of the MGLP pathway in rapidly-growing species and accommodation of this possible regulatory step. This new link between GG metabolism, MGLP biosynthesis and recovery from nitrogen stress furthers our knowledge on the mycobacterial strategies to endure a frequent stress faced in some environments and during long-term infection.

The molecular biology of mycobacterial trehalose in the quest for advanced tuberculosis therapies.

Trehalose is a natural glucose disaccharide identified in the 19th century in fungi and insect cocoons, and later across the three domains of life. In members of the genus Mycobacterium, which includes the tuberculosis (TB) pathogen and over 160 species of nontuberculous mycobacteria (NTM), many of which are opportunistic pathogens, trehalose has been an important focus of research over the last 60 years. It is a crucial player in the assembly and architecture of the remarkable mycobacterial cell envelope as an element of unique highly antigenic glycolipids, namely trehalose dimycolate ('cord factor'). Free trehalose has been detected in the mycobacterial cytoplasm and occasionally in oligosaccharides with unknown function. TB and NTM infection statistics and death toll, the decline in immune responses in the aging population, human immunodeficiency virus/AIDS or other debilitating conditions, and the proliferation of strains with different levels of resistance to the dated drugs in use, all merge into a serious public-health threat urging more effective vaccines, efficient diagnostic tools and new drugs. This review deals with the latest findings on mycobacterial trehalose biosynthesis, catabolism, processing and recycling, as well with the ongoing quest for novel trehalose-related mechanisms to be targeted by novel TB therapeutics. In this context, the drug-discovery pipeline has recently included new lead compounds directed toward trehalose-related targets highlighting the potential of these pathways to stem the tide of rising drug resistance.

A new bacterial hydrolase specific for the compatible solutes α-D-mannopyranosyl-(1→2)-D-glycerate and α-D-glucopyranosyl-(1→2)-D-glycerate.

The accumulation patterns and biosynthesis of compatible solutes in hyper/thermophiles have been extensively studied. However, there is little information available on their hydrolysis, leading us to search for enzymes for this activity. From the analysis of the genomes of several microorganisms known to accumulate α-D-mannopyranosyl-(1→2)-D-glycerate (mannosylglycerate, MG) or α-D-glucopyranosyl-(1→2)-D-glycerate (glucosylglycerate, GG) we were able to identify a likely candidate gene for the hydrolysis of these molecules. The Thermus thermophilus HB27 homologue encoded a putative enzyme with motifs of the GH63 and GH37 families of glycoside hydrolases. We expressed the gene from this thermophilic bacterium and from Rubrobacter radiotolerans, and confirmed that the recombinant enzymes, here designated mannosylglycerate hydrolase (MgH), specifically hydrolysed MG (or GG) to mannose (or glucose) and glycerate. Both enzymes were highly stable and maximally active at temperatures close to each organisms' optimal growth temperatures (half-lives of 15.4 ± 0.5 h at 55 °C and 16.1 ± 0.4 h at 70 °C) but at low pH (4.0-4.5). Cations were not required for their activity and each enzyme exhibited Michaelis-Menten kinetics at 50 °C and 70 °C, respectively, with comparable catalytic efficiencies towards MG and GG. Herein, we purified and characterized a novel and highly specific MG- and GG-hydrolyzing enzyme that represent an attractive tool for development of enzymatic assays for quantification of these solutes, which seem to be more prevalent in microorganisms than initially suspected.

Genome sequence of Mycobacterium hassiacum DSM 44199, a rare source of heat-stable mycobacterial proteins.

Mycobacterium hassiacum is a rapidly growing mycobacterium isolated from human urine and so far the most thermophilic among mycobacterial species. Its thermotolerance and phylogenetic relationship to M. tuberculosis render its proteins attractive tools for crystallization and structure-guided drug design. We report the draft genome sequence of M. hassiacum DSM 44199.