Phages, anti-CRISPR proteins, and drug-resistant bacteria: what do we know about this triad?

Phages are viruses that infect bacteria, relying on their genetic machinery to replicate. To survive the constant attack of phages, bacteria have developed diverse defense strategies to act against them. Nevertheless, phages rapidly co-evolve to overcome these barriers, resulting in a constant, and often surprising, molecular arms race. Thus, some phages have evolved protein inhibitors known as anti-CRISPRs (∼50-150 amino acids), which antagonize the bacterial CRISPR-Cas immune response. To date, around 45 anti-CRISPRs proteins with different mechanisms and structures have been discovered against the CRISPR-Cas type I and type II present in important animal and human pathogens such as Escherichia, Morganella, Klebsiella, Enterococcus, Pseudomonas, Staphylococcus, and Salmonella. Considering the alarming growth of antibiotic resistance, phage therapy, either alone or in combination with antibiotics, appears to be a promising alternative for the treatment of many bacterial infections. In this review, we illustrated the biological and clinical aspects of using phage therapy; furthermore, the CRISPR-Cas mechanism, and the interesting activity of anti-CRISPR proteins as a possible weapon to combat bacteria.

Crystal structure determination and dynamic studies of Mycobacterium tuberculosis Cytidine deaminase in complex with products.

Cytidine deaminase (CDA) is a key enzyme in the pyrimidine salvage pathway. It is involved in the hydrolytic deamination of cytidine or 2'-deoxycytidine to uridine or 2'-deoxyuridine, respectively. Here we report the crystal structures of Mycobacterium tuberculosis CDA (MtCDA) in complex with uridine (2.4 Å resolution) and deoxyuridine (1.9 Å resolution). Molecular dynamics (MD) simulation was performed to analyze the physically relevant motions involved in the protein-ligand recognition process, showing that structural flexibility of some protein regions are important to product binding. In addition, MD simulations allowed the analysis of the stability of tetrameric MtCDA structure. These findings open-up the possibility to use MtCDA as a target in future studies aiming to the rational design of new inhibitor of MtCDA-catalyzed chemical reaction with potential anti-proliferative activity on cell growth of M. tuberculosis, the major causative agent of tuberculosis.

Structural and functional analyses of Mycobacterium tuberculosis Rv3315c-encoded metal-dependent homotetrameric cytidine deaminase.

The emergence of drug-resistant strains of Mycobacterium tuberculosis, the causative agent of tuberculosis, has exacerbated the treatment and control of this disease. Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that recycles cytidine and 2'-deoxycytidine for uridine and 2'-deoxyuridine synthesis, respectively. A probable M. tuberculosis CDA-coding sequence (cdd, Rv3315c) was cloned, sequenced, expressed in Escherichia coli BL21(DE3), and purified to homogeneity. Mass spectrometry, N-terminal amino acid sequencing, gel filtration chromatography, and metal analysis of M. tuberculosis CDA (MtCDA) were carried out. These results and multiple sequence alignment demonstrate that MtCDA is a homotetrameric Zn(2+)-dependent metalloenzyme. Steady-state kinetic measurements yielded the following parameters: K(m)=1004 microM and k(cat)=4.8s(-1) for cytidine, and K(m)=1059 microM and k(cat)=3.5s(-1) for 2'-deoxycytidine. The pH dependence of k(cat) and k(cat)/K(M) for cytidine indicate that protonation of a single ionizable group with apparent pK(a) value of 4.3 abolishes activity, and protonation of a group with pK(a) value of 4.7 reduces binding. MtCDA was crystallized and crystal diffracted at 2.0 A resolution. Analysis of the crystallographic structure indicated the presence of a Zn(2+) coordinated by three conserved cysteines and the structure exhibits the canonical cytidine deaminase fold.

Characterization of Actinobacterial Communities from Arauca River Sediments (Colombia) Reveals Antimicrobial Potential Presented in Low Abundant Isolates.

INTRODUCTION: New strategies have been arisen to set a rapid and effective screening for selection of microorganism with bioactive potential. This study suggests that combination of physicochemical pretreatments and taxonomic dereplication of microbial collections through MALDI-TOF MS, facilitates the detection of low abundance actinobacteria with potential as a source of antimicrobial agents. MATERIAL AND METHODS: An unstudied microbial community from a tropical river sediment in Colombian Orinoquía is described, applying an extended cultivation strategy using physicochemical pretreatments, biological screenings and taxonomic dereplication through MALDI-TOF MS approach. RESULTS: Actinobacteria-like isolates (790) were growth and their antimicrobial activity was assessed against methicillin-resistant Staphylococcus aureus, Vancomycin-resistant Enterococcus faecium, extended-spectrum ß-lactamase Klebsiella pnumoniae, and clinical isolates of Cladosporium cladosporioides and Epicoccum nigrum. Seventy-eight isolates, belonging to the Streptomycetaceae family according to 16S rDNA analysis were found to have antimicrobial activity and were categorized as low abundance actinobacteria by MALDI-TOF MS. CONCLUSION: The results suggest that combination of physicochemical pretreatments and taxonomic dereplication of microbial collections through MALDI-TOF MS, facilitates the detection of low abundance actinobacteria with potential as a source of antimicrobial agents.

Concordance analysis between different methodologies used for identification of oral isolates of Candida species.

BACKGROUND: The yeasts species determination is fundamental not only for an accurate diagnosis but also for establishing a suitable patient treatment. We performed a concordance study of five methodologies for the species identification of oral isolates of Candida in Colombia. METHODS: Sixty-seven Candida isolates were tested by; API® 20C-AUX, Vitek®2 Compact, Vitek®MS, Microflex® and a molecular test (panfungal PCR and sequencing). The commercial cost and processing time of the samples was done by graphical analysis. RESULTS: Panfungal PCR differentiated 12 species of Candida, Vitek®MS and Microflex® methods identified 9 species, and API® 20C-AUX and Vitek®2 Compact methods identified 8 species each. Weighted Kappa (wK) showed a high agreement between Panfungal PCR, Vitek®MS, Microflex® and API® 20C-AUX (wK 0.62-0.93). The wK that involved the Vitek®2 Compact method presented moderate or good concordances compared with the other methods (wK 0.56-0.73). Methodologies based on MALDI TOF MS required 4 minutes to generate results and the Microflex® method had the lowest selling price. CONCLUSION: The methods evaluated showed high concordance in their results, being higher for the molecular methods and the methodologies based on MALDI TOF. The latter are faster and cheaper, presenting as promising alternatives for the routine identification of yeast species of the genus Candida.

INTRODUCCIÓN: La clasificación a nivel de especies de las levaduras del género Candida de origen clínico es fundamental para el diagnóstico y la instauración de un adecuado tratamiento para el paciente. Se realizó un estudio de concordancia de cinco metodologías usadas para la identificación de aislamientos orales de Candida spp en Colombia. MÉTODOS: Sesenta y siete aislamientos de Candida spp fueron identificados a nivel de especie utilizando; API® 20 C AUX' Vitek® 2 Compact, MALDI TOF (Vitek® MS y Microflex®) y una prueba molecular, PCR Panfungal y secuenciación. Un análisis del costo comercial y tiempo de procesamiento de las muestras por cada método fue realizado mediante el análisis gráfico de ambas variables. RESULTADOS: La PCR Panfungal y secuenciación diferenció 12 especies de Candida' los métodos Vitek® MS y Microflex® identificaron 9 especies y los métodos API® 20 C AUX y Vitek® 2 Compact identificaron 8 especies. El análisis de Kappa ponderado (wK) demostró una concordancia alta entre los métodos PCR Panfungal y secuenciación' Vitek® MS' Microflex® y API® 20 C AUX' concordancias agrupadas en las categorías buena y muy buena (wK 0.62 - 0.93); los Kp que involucraron el método Vitek® 2 Compact presentaron concordancias moderadas o buenas frente a los otros métodos (wK 0.56 - 0.73). Las metodologías basadas en MALDI TOF MS requirieron 4 minutos para generar un resultado y el método Microflex® fue el método que en nuestro medio presentó el menor precio de venta del servicio. CONCLUSIÓN: Los métodos evaluados presentaron una alta concordancia en sus resultados' siendo más alta para los métodos moleculares y las metodologías basadas en MALDI TOF MS; estas últimas son metodologías más rápidas, económicas y precisas, las cuales se presentan como alternativas prometedoras para la identificación rutinaria de especies de levaduras del género Candida.

Concordance analysis between different methodologies used for identification of oral isolates of Candida species / Análisis de concordancia de diferentes metodologías para la identificación de aislamientos orales de especies de Candida

Abstract Background: The yeasts species determination is fundamental not only for an accurate diagnosis but also for establishing a suitable patient treatment. We performed a concordance study of five methodologies for the species identification of oral isolates of Candida in Colombia. Methods: Sixty-seven Candida isolates were tested by; API® 20C-AUX, Vitek®2 Compact, Vitek®MS, Microflex® and a molecular test (panfungal PCR and sequencing). The commercial cost and processing time of the samples was done by graphical analysis. Results: Panfungal PCR differentiated 12 species of Candida, Vitek®MS and Microflex® methods identified 9 species, and API® 20C-AUX and Vitek®2 Compact methods identified 8 species each. Weighted Kappa (wK) showed a high agreement between Panfungal PCR, Vitek®MS, Microflex® and API® 20C-AUX (wK 0.62-0.93). The wK that involved the Vitek®2 Compact method presented moderate or good concordances compared with the other methods (wK 0.56-0.73). Methodologies based on MALDI TOF MS required 4 minutes to generate results and the Microflex® method had the lowest selling price. Conclusion: The methods evaluated showed high concordance in their results, being higher for the molecular methods and the methodologies based on MALDI TOF. The latter are faster and cheaper, presenting as promising alternatives for the routine identification of yeast species of the genus Candida.

Resumen Introducción: La clasificación a nivel de especies de las levaduras del género Candida de origen clínico es fundamental para el diagnóstico y la instauración de un adecuado tratamiento para el paciente. Se realizó un estudio de concordancia de cinco metodologías usadas para la identificación de aislamientos orales de Candida spp en Colombia. Métodos: Sesenta y siete aislamientos de Candida spp fueron identificados a nivel de especie utilizando; API® 20 C AUX‚ Vitek® 2 Compact, MALDI TOF (Vitek® MS y Microflex®) y una prueba molecular, PCR Panfungal y secuenciación. Un análisis del costo comercial y tiempo de procesamiento de las muestras por cada método fue realizado mediante el análisis gráfico de ambas variables. Resultados: La PCR Panfungal y secuenciación diferenció 12 especies de Candida‚ los métodos Vitek® MS y Microflex® identificaron 9 especies y los métodos API® 20 C AUX y Vitek® 2 Compact identificaron 8 especies. El análisis de Kappa ponderado (wK) demostró una concordancia alta entre los métodos PCR Panfungal y secuenciación‚ Vitek® MS‚ Microflex® y API® 20 C AUX‚ concordancias agrupadas en las categorías buena y muy buena (wK 0.62 - 0.93); los Kp que involucraron el método Vitek® 2 Compact presentaron concordancias moderadas o buenas frente a los otros métodos (wK 0.56 - 0.73). Las metodologías basadas en MALDI TOF MS requirieron 4 minutos para generar un resultado y el método Microflex® fue el método que en nuestro medio presentó el menor precio de venta del servicio. Conclusión: Los métodos evaluados presentaron una alta concordancia en sus resultados‚ siendo más alta para los métodos moleculares y las metodologías basadas en MALDI TOF MS; estas últimas son metodologías más rápidas, económicas y precisas, las cuales se presentan como alternativas prometedoras para la identificación rutinaria de especies de levaduras del género Candida.