Activity of Bacteriophage D29 Loaded on Nanoliposomes against Macrophages Infected with Mycobacterium tuberculosis.

The search for new antimicrobial agents is a continuous struggle, mainly because more and more cases of resistant strains are being reported. Mycobacterium tuberculosis (MTB) is the main microorganism responsible for millions of deaths worldwide. The development of new antimicrobial agents is generally aimed at finding strong interactions with one or more bacterial receptors. It has been proven that bacteriophages have the ability to adhere to specific and selective regions. However, their transport and administration must be carefully evaluated as an excess could prevent a positive response and the bacteriophages may be eliminated during their journey. With this in mind, the mycobacteriophage D29 was encapsulated in nanoliposomes, which made it possible to determine its antimicrobial activity during transport and its stability in the treatment of active and latent Mycobacterium tuberculosis. The antimicrobial activity, the cytotoxicity in macrophages and fibroblasts, as well as their infection and time-kill were evaluated. Phage nanoencapsulation showed efficient cell internalization to induce MTB clearance with values greater than 90%. Therefore, it was shown that nanotechnology is capable of assisting in the activity of degradation-sensitive compounds to achieve better therapy and evade the immune response against phages during treatment.

Growth-inhibitory effects of tris-(1,10-phenanthroline) iron (II) against Mycobacterium tuberculosis in vitro and in vivo.

Mycobacterium tuberculosis is the major etiological agent for tuberculosis (TB), which is the leading cause of single pathogen infection-related deaths worldwide. The End TB Strategy of the World Health Organization aimed to decrease the incidence of TB by 20% between 2015 and 2020, which was not achieved. Here, the growth-inhibitory effects of tris-(1,10-phenanthroline) iron (II) complex ([Fe(phen)3]2+), a known commercially available cheap chemical substance, were examined. The best in vitro results showed great activity with MIC ranging from 0.77 to 3.06 µM against clinical strains and at low pH (mimicking the granuloma) with MIC of 0.21 µM. Preliminary safety analysis revealed that the complex did not exhibit cytotoxic activity against different cell lines or mutagenic activity in vitro. The complex was orally bioavailable after 2 h of administration in vivo. Additionally, the results of the acute toxicity test revealed that the complex did not exert toxic effects in female BALB/c mice. The mechanism of action was performed using D29 mycobacteriophages where the treatment with different concentrations of the complex inhibited viral protein synthesis, which indicated that the anti-TB mechanisms of the complex involve protein synthesis inhibition. These findings suggested that [Fe(phen)3]2+ is a potential novel therapeutic for TB.

Antibacterial activity of high-dose nitric oxide against pulmonary Mycobacterium abscessus disease.

INTRODUCTION: Mycobacterium abscessus is an emerging pulmonary pathogen with limited treatment options. Nitric oxide (NO) demonstrates antibacterial activity against various bacterial species, including mycobacteria. In this study, we evaluated the effect of adjunctive inhaled NO therapy, using a novel NO generator, in a CF patient with pulmonary M. abscessus disease, and examined heterogeneity of response to NO in vitro. METHODS: In the compassionate-use treatment, a 24-year-old CF patient with pulmonary M. abscessus was treated with two courses of adjunctive intermittent NO, first at 160 p.p.m. for 21 days and subsequently by escalating the dose up to 240 p.p.m. for 8 days. Methemoglobin, pulmonary function, 6 min walk distance (6MWD), qualify of life and sputum microbiology were assessed. In vitro susceptibility tests were performed against patient's isolate and comparison clinical isolates and quantified by Hill's slopes calculated from time-kill curves. RESULTS: M. abscessus lung infection eradication was not achieved, but improvements in selected qualify of life domains, lung function and 6MWD were observed during the study. Inhaled NO was well tolerated at 160 p.p.m. Dosing at 240 p.p.m. was stopped due to adverse symptoms, although methemoglobin levels remained within safety thresholds. In vitro susceptibility tests showed a dose-dependent NO effect on M. abscessus susceptibility and significant heterogeneity in response between M. abscessus clinical isolates. The patient's isolate was found to be the least susceptible strain in vitro. CONCLUSION: These results demonstrate heterogeneity in M. abscessus susceptibility to NO and suggest that longer treatment regimens could be required to see the reduction or eradication of more resistant pulmonary strains.

Antimicrobial peptides against drug resistant Mycobacterium abscessus.

Mycobacterium abscessus (MAB) comprise rapidly growing, often multidrug-resistant (MDR), nontuberculous mycobacteria responsible for pulmonary and other infections in susceptible hosts. Antimicrobial peptides (APs) are natural and synthetic antimicrobials active against a range of microorganisms including mycobacteria. We evaluated APs activity against MAB reference and clinical strains. We observed minimal inhibitory concentrations of 1.6 to >50 µg/mL. Further work with the most active AP demonstrated protection of Acanthamoeba castellanii (AC) from killing by ingested MAB including MDR MAB strains. Antimicrobial peptides offer an attractive potential option for treatment of drug resistant treatment-refractory MAB.

Survival of pathogenic Mycobacterium abscessus subsp. massiliense in Acanthamoeba castellanii.

We used an amoeba model to study the intracellular growth and cytotoxicity of clinical strains of Mycobacterium abscessus subsp. massiliense (Mabsm) isolated from 2 patients (one with cystic fibrosis, the other one with idiopathic bronchiectasis) during the early (smooth colonies) and late stage (rough colonies) of chronic pulmonary infection. Acanthamoeba castellanii were infected with Mabsm (MOI 100) and samples collected every 24 h for 72 h. Results showed Mabsm is able to survive in trophozoites and persist in cysts for at least 7 days. Late Mabsm demonstrated higher cytotoxicity toward A. castellanii when compared to early strains. A. castellanii is a useful in vitro host model to study infection of Mabsm clinical isolates.

Application of aqueous two-phase micellar system to improve extraction of adenoviral particles from cell lysate.

Viral vectors are important in medical approaches, such as disease prevention and gene therapy, and their production depends on efficient prepurification steps. In the present study, an aqueous two-phase micellar system (ATPMS) was evaluated to extract human adenovirus type 5 particles from a cell lysate. Adenovirus was cultured in human embryonic kidney 293 (HEK-293) cells to a concentration of 1.4 × 1010 particles/mL. Cells were lysed, and the system formed by direct addition of Triton X-114 in a 23 full factorial design with center points. The systems were formed with Triton X-114 at a final concentration of 1.0, 6.0, and 11.0% (w/w), cell lysate pH of 6.0, 6.5, and 7.0, and incubation temperatures at 33, 35, and 37 °C. Adenovirus particles recovered from partition phases were measured by qPCR. The best system condition was with 11.0% (w/w) of Triton X-114, a cell lysate pH of 7.0, and an incubation temperature at 33 °C, yielding 3.51 × 1010 adenovirus particles/mL, which increased the initial adenovirus particles concentration by 2.3-fold, purifying it by 2.2-fold from the cell lysate, and removing cell debris. In conclusion, these results demonstrated that the use of an aqueous two-phase micellar system in the early steps of downstream processing could improve viral particle extraction from cultured cells while integrating clarification, concentration, and prepurification steps.

Application of aqueous two-phase micellar system to improve extraction of adenoviral particles from cell lysate

Viral vectors are important in medical approaches, such as disease prevention and gene therapy, and their production depends on efficient prepurification steps. In the present study, an aqueous two-phase micellar system (ATPMS) was evaluated to extract human adenovirus type 5 particles from a cell lysate. Adenovirus was cultured in human embryonic kidney 293 (HEK-293) cells to a concentration of 1.4 × 1010 particles/mL. Cells were lysed, and the system formed by direct addition of Triton X-114 in a 23 full factorial design with center points. The systems were formed with Triton X-114 at a final concentration of 1.0, 6.0, and 11.0% (w/w), cell lysate pH of 6.0, 6.5, and 7.0, and incubation temperatures at 33, 35, and 37 °C. Adenovirus particles recovered from partition phases were measured by qPCR. The best system condition was with 11.0% (w/w) of Triton X-114, a cell lysate pH of 7.0, and an incubation temperature at 33 °C, yielding 3.51 × 1010 adenovirus particles/mL, which increased the initial adenovirus particles concentration by 2.3-fold, purifying it by 2.2-fold from the cell lysate, and removing cell debris. In conclusion, these results demonstrated that the use of an aqueous two-phase micellar system in the early steps of downstream processing could improve viral particle extraction from cultured cells while integrating clarification, concentration, and prepurification steps.

Complete Genome Sequence of Linezolid-Susceptible Staphylococcus haemolyticus Sh29/312/L2, a Clonal Derivative of a Linezolid-Resistant Clinical Strain.

We report the whole-genome sequence (WGS) of an in vitro susceptible derivative revertant mutant from a bloodstream isolate involved in a nosocomial outbreak in Brazil. The WGS comprises 2.5 Mb with 2,500 protein-coding sequences, 16rRNA genes, and 60 tRNA genes.

Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP.

Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc(2) 155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages.

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting.

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Interventions for wound healing among diabetic patients infected with Staphylococcus aureus: a systematic review.

CONTEXT AND OBJECTIVE: Staphylococcus aureus is the most frequent agent isolated in diabetic foot infections and may be associated with changes to wound healing times. The aim of this study was to perform a systematic review of the literature, including studies that assessed the efficacy of any clinical or surgical intervention, as well as oral or topical therapy for diabetic ulcers infected with S. aureus. DESIGN AND SETTING: Systematic review with a search conducted in databases. METHODS: We conducted a systematic review with a comprehensive search in the Lilacs, SciELO, PubMed/Medline, Old Medline, Embase and Cochrane Library databases, for articles published from 1966 to 2010. The articles selected were limited to studies on diabetic patients with wounds infected with S. aureus for whom their healing was followed up, with the use of either antibiotics or experimental treatments. Animal studies and those that did not report the wound healing, as well as review articles, were excluded. RESULTS: Five studies that met the inclusion and exclusion criteria were analyzed. CONCLUSIONS: There are few studies reporting the healing of wounds infected with S. aureus in diabetic patients, although this is the most commonly found pathogen in this type of wound and it frequently consists of methicillin-resistant S. aureus (MRSA). There is insufficient evidence to support early use of broad-spectrum antibiotics against MRSA to promote healing of diabetic ulcers, since antibiotic resistance may develop from such treatment. This highlights the need for further studies on the subject.

Interventions for wound healing among diabetic patients infected with Staphylococcus aureus: a systematic review / Intervenções para a cicatrização de feridas de pacientes diabéticos infectados com Staphylococcus aureus: uma revisão sistemática

CONTEXT AND OBJECTIVE: Staphylococcus aureus is the most frequent agent isolated in diabetic foot infections and may be associated with changes to wound healing times. The aim of this study was to perform a systematic review of the literature, including studies that assessed the efficacy of any clinical or surgical intervention, as well as oral or topical therapy for diabetic ulcers infected with S. aureus. DESIGN AND SETTING: Systematic review with a search conducted in databases. METHODS: We conducted a systematic review with a comprehensive search in the Lilacs, SciELO, PubMed/Medline, Old Medline, Embase and Cochrane Library databases, for articles published from 1966 to 2010. The articles selected were limited to studies on diabetic patients with wounds infected with S. aureus for whom their healing was followed up, with the use of either antibiotics or experimental treatments. Animal studies and those that did not report the wound healing, as well as review articles, were excluded. RESULTS: Five studies that met the inclusion and exclusion criteria were analyzed. CONCLUSIONS: There are few studies reporting the healing of wounds infected with S. aureus in diabetic patients, although this is the most commonly found pathogen in this type of wound and it frequently consists of methicillin-resistant S. aureus (MRSA). There is insufficient evidence to support early use of broad-spectrum antibiotics against MRSA to promote healing of diabetic ulcers, since antibiotic resistance may develop from such treatment. This highlights the need for further studies on the subject.

CONTEXTO: Staphylococcus aureus é o agente mais frequentemente isolado nas infecções de pé em pacientes diabéticos e pode estar associado a mudança no tempo de cicatrização de feridas. O objetivo deste estudo foi realizar uma revisão sistemática da literatura, incluindo estudos que avaliaram a eficácia de qualquer intervenção clínica, cirúrgica, bem como terapia oral ou tópica para o tratamento de úlceras diabéticas infectadas com o S. aureus. TIPO DE ESTUDO E LOCAL: Revisão sistemática com busca realizada em bancos de dados. MÉTODOS: Realizamos uma revisão sistemática com uma busca abrangente nos bancos de dados Lilacs, SciELO, PubMed/Medline, Old Medline, Embase e no banco de dados da biblioteca Cochrane, publicados entre 1966 e 2010. Os artigos selecionados foram limitados aos estudos com feridas infectadas por S. aureus de pacientes diabéticos, que tiveram cicatrização relatada, quer pela utilização de antibióticos ou por substâncias experimentais. Foram excluídos os estudos com animais e os que não relataram a cicatrização das feridas, bem como artigos de revisão. RESULTADOS: Foram analisados cinco estudos que obedeceram aos critérios de inclusão e exclusão. CONCLUSÕES: Raros estudos relataram cicatrização de feridas infectadas com S. aureus em pacientes diabéticos, embora este seja o patógeno mais comumente encontrado neste tipo de ferida, sendo frequentemente resistente à meticilina MRSA (methicillin-resistant S. aureus). Não há evidências suficientes que suportem a utilização precoce de antibióticos de amplo espectro contra MRSA para promoção da cicatrização de úlceras diabéticas, uma vez que o desenvolvimento de resistência a antibióticos pode decorrer desse tipo de tratamento. Isso evidencia a necessidade de novos estudos sobre o assunto.

Desenvolvimento de micobacteriófago recombinante para detecção rápida de bacilos da tuberculose / Development of recombinant micobacteriophage for rapid detection of tubercle bacillus

O diagnóstico da tuberculose por métodos tradicionais é lento e laborioso. Por outro lado, os testes moleculares são rápidos, mas com custo elevado para países em desenvolvimento. Este projeto teve o objetivo de inserir no micobacteriófago D29 o gene que codifica a proteína verde fluorescente (eGFP) e estudar o fago recombinante na detecção rápida de bacilos da tuberculose. Para tanto, foi inserido o cassete Hsp60- eGFP no genoma do fago D29 por recombinação. Micobacteriófagos recombinantes purificados foram utilizados para infectar M. smegmatis mc2 155 e M. tuberculosis H37Rv durante um período de 1- 6h nas temperaturas de 30°C, 37°C e 42°C. Bactérias fluorescentes foram observadas em um período de 2h, mas em número reduzido, indicando que o micobacteriófago lisou às células rapidamente, dificultando a expressão da eGFP e visualização em microscópio de fluorescência. A deleção do gene LysA, foi efetuada a fim de aumentar o período de latência do fago. Não foi possível a purificação de fagos recombinantes, devido à baixa quantidade de recombinantes nos halos de inibição. Será necessário a redução da atividade o gene LysA e, provavelmente, de outros genes associados a lise celular a fim de aumentar a concentração de eGFP no interior da célula.

Classical biochemical methods for Mycobacterium tuberculosis identification are lengthy and time-consuming. On the other hand, molecular assays are rapid but expensive for developing countries. This project aimed to insert into the mycobacteriophage D29, the gene coding for the green fluorescent protein (eGFP) and use the recombineered phage to detect Mycobacterium tuberculosis rapidly and less costly. For that, the Hsp-eGFP cassette was inserted into D29 genome. Recombineered mycobacteriophages was purified and used to infect M. smegmatis mc2 155 and M. tuberculosis H37Rv from 1-6 hs at 30°C, 37°C and 42°C. Observation of fluorescent bacteria was difficult and only a small number of them were seen at 2 hs of infection. This indicated that recombineered bacteriophages were lysing cells rapidly. Deletion of LysA gene, was carried out to increase the time needed for bacterial lysing. it was not possible to purify mutant mycobacteriophages due to the low concentration of recombinant phages. We conclude that might be necessary the deletion of other genes such as LysB, a gene also involved in cell lysis and reduction LysA activity to increase the concentration of eGFP inside cells.

Metagenomic analysis of respiratory tract DNA viral communities in cystic fibrosis and non-cystic fibrosis individuals.

The human respiratory tract is constantly exposed to a wide variety of viruses, microbes and inorganic particulates from environmental air, water and food. Physical characteristics of inhaled particles and airway mucosal immunity determine which viruses and microbes will persist in the airways. Here we present the first metagenomic study of DNA viral communities in the airways of diseased and non-diseased individuals. We obtained sequences from sputum DNA viral communities in 5 individuals with cystic fibrosis (CF) and 5 individuals without the disease. Overall, diversity of viruses in the airways was low, with an average richness of 175 distinct viral genotypes. The majority of viral diversity was uncharacterized. CF phage communities were highly similar to each other, whereas Non-CF individuals had more distinct phage communities, which may reflect organisms in inhaled air. CF eukaryotic viral communities were dominated by a few viruses, including human herpesviruses and retroviruses. Functional metagenomics showed that all Non-CF viromes were similar, and that CF viromes were enriched in aromatic amino acid metabolism. The CF metagenomes occupied two different metabolic states, probably reflecting different disease states. There was one outlying CF virome which was characterized by an over-representation of Guanosine-5'-triphosphate,3'-diphosphate pyrophosphatase, an enzyme involved in the bacterial stringent response. Unique environments like the CF airway can drive functional adaptations, leading to shifts in metabolic profiles. These results have important clinical implications for CF, indicating that therapeutic measures may be more effective if used to change the respiratory environment, as opposed to shifting the taxonomic composition of resident microbiota.

In vitro synergic effect of beta-lapachone and isoniazid on the growth of Mycobacterium fortuitum and Mycobacterium smegmatis.

Nontuberculous mycobacteria are ubiquitous and saprophytic organisms that have been implicated in a wide spectrum of diseases due to an increasing number of immunocompromised patients. The natural resistance of atypical mycobacteria to classical antituberculous drugs has encouraged research into new chemotherapeutic agents and drug combinations. The aim of this study was to determine the in vitro antimycobacterial activities of (2)-lapachone alone and in combination with isoniazid against Mycobacterium fortuitum and Mycobacterium smegmatis via the Time-Kill Curve method. A 2 log10 CFU/mL reduction in the M. smegmatis culture was observed 72 h after adding (2)-lapachone at its minimum inhibitory concentration. This drug sterilised the culture in 120 h. For M. fortuitum, a reduction of 1.55 log10 CFU/mL occurred in 24 h, but regrowth was seen in contact with (2)-lapachone. Both microorganisms were resistant to isoniazid. Regrowth of M. fortuitum and M. smegmatis was observed at 48 h and 72 h, respectively. In combination, these two drugs had a bactericidal effect and sterilised both cultures in 96 h. These results are valuable because antibiotic-resistant bacteria are a major public health problem.

Bacteriophage: laboratorial diagnosis and phage therapy / Bacteriofagos: diagnóstico laboratorial e terapia fágica

Bacteriophages have been researched as a new alternative to antibiotics. These viruses inject their genetic material into bacteria and use their host machinery to multiply themselves. The research of bacteriophages in Brazil will certainly provide low-cost treatment of multidrug resistant bacteria, new microbiological diagnosis and advantages for the Brazilian food industry.

Bacteriófagos têm sido pesquisados como uma alternativa ao uso de antibióticos. Estes vírus infectam as bactérias e utilizam a maquinaria celular para multiplicar o próprio material genético. O estudo de bacteriófagos no Brasil levará ao desenvolvimento de tratamentos de baixo custo, novos testes diagnósticos e vantagens para a industria alimentícia.

In vitro synergic effect of beta-lapachone and isoniazid on the growth of Mycobacterium fortuitum and Mycobacterium smegmatis

Nontuberculous mycobacteria are ubiquitous and saprophytic organisms that have been implicated in a wide spectrum of diseases due to an increasing number of immunocompromised patients. The natural resistance of atypical mycobacteria to classical antituberculous drugs has encouraged research into new chemotherapeutic agents and drug combinations. The aim of this study was to determine the in vitro antimycobacterial activities of ²-lapachone alone and in combination with isoniazid against Mycobacterium fortuitum and Mycobacterium smegmatis via the Time-Kill Curve method. A 2 log10 CFU/mL reduction in the M. smegmatis culture was observed 72 h after adding ²-lapachone at its minimum inhibitory concentration. This drug sterilised the culture in 120 h. For M. fortuitum, a reduction of 1.55 log10 CFU/mL occurred in 24 h, but regrowth was seen in contact with ²-lapachone. Both microorganisms were resistant to isoniazid. Regrowth of M. fortuitum and M. smegmatis was observed at 48 h and 72 h, respectively. In combination, these two drugs had a bactericidal effect and sterilised both cultures in 96 h. These results are valuable because antibiotic-resistant bacteria are a major public health problem.

Bacteriophage: laboratorial diagnosis and phage therapy.

Bacteriophages have been researched as a new alternative to antibiotics. These viruses inject their genetic material into bacteria and use their host machinery to multiply themselves. The research of bacteriophages in Brazil will certainly provide low-cost treatment of multidrug resistant bacteria, new microbiological diagnosis and advantages for the Brazilian food industry.