A microfluidic card-based electrochemical assay for the detection of sulfonamide resistance genes.

Most electroanalytical detection schemes for DNA markers require considerable time and effort from expert personnel to thoroughly follow the analysis and obtain reliable outcomes. This work aims to present an electrochemical assay performed inside a small card-based platform powered by microfluidic manipulation, requiring minimal human intervention and consumables. The assay couples a sample/signal dual amplification and DNA-modified magnetic particles for the detection of DNA amplification products. Particularly, the sul1 and sul4 genes involved in the resistance against sulfonamide antibiotics were analyzed. As recognized by the World Health Organization, antimicrobial resistance threatens global public health by hampering medication efficacy against infections. Consequently, analytical methods for the determination of such genes in environmental and clinical matrices are imperative. Herein, the resistance genes were extracted from E. coli cells and amplified using an enzyme-assisted isothermal amplification at 37 °C. The amplification products were analyzed in an easily-produced, low-cost, card-based set-up implementing a microfluidic system, demanding limited manual work and small sample volumes. The target amplicon was thus captured and isolated using versatile DNA-modified magnetic beads injected into the microchannel and exposed to the various reagents in a continuously controlled microfluidic flow. After the optimization of the efficiency of each phase of the assay, the platform achieved limits of detections of 44.2 pmol L-1 for sul1 and 48.5 pmol L-1 for sul4, and was able to detect down to ≥500-fold diluted amplification products of sul1 extracted from E. coli living cells in around 1 h, thus enabling numerous end-point analyses with a single amplification reaction.

A putative siderophore receptor of Gallibacterium anatis 12656-12 under Fur control also binds hemoglobin.

Pasteurellaceae family members obtain iron directly from host proteins or through siderophore-dependent mechanisms. Although Gallibacterum anatis expresses different virulence factors, its response to growth under iron restriction is unknown. G. anatis cultured in the presence of 2,2'-dipyridyl, up-expressed an approximately 65 kDa protein and repressed the expression of a 70 kDa protein. MALDI-TOF analysis of those proteins indicated homology with CirA (65 kDa), a protein involved in iron-siderophore acquisition in Mannheimia succinoproducens and a TonB-dependent receptor (70 kDa protein), a protein that binds chicken hemoglobin; however, G. anatis siderophore production was not detected by chromo azurol S (CAS)-BHI agar determination. This putative G. anatis siderophore receptor is under Fur control, but not the hemoglobin binding protein, as observed in G. anatis 12656-12 fur mutant (Ω fur 126.13) grown in the presence or not of 2,2'-dipyridyl. The addition of FeCl3 to the culture medium diminished the growth and biofilm production in approximately 30% and 35%, respectively, in the wild-type strain, but the growth of Ω fur 126.13 strain was not affected and biofilm production increased in 35%. G. anatis Ω fur 126.13 presented lower virulence when it was inoculated to 35-day-old chickens in comparison to the wild-type strain. The induction of more than one iron uptake mechanism could benefit pathogenic microorganisms such as Gallibacterium.

Importance of Cry Proteins in Biotechnology: Initially a Bioinsecticide, Now a Vaccine Adjuvant.

A hallmark of Bacillus thuringiensis bacteria is the formation of one or more parasporal crystal (Cry) proteins during sporulation. The toxicity of these proteins is highly specific to insect larvae, exerting lethal effects in different insect species but not in humans or other mammals. The aim of this review is to summarize previous findings on Bacillus thuringiensis, including the characteristics of the bacterium, its subsequent contribution to biotechnology as a bioinsecticide due to the presence of Cry proteins, and its potential application as an adjuvant. In several studies, Cry proteins have been administered together with specific antigens to immunize experimental animal models. The results have shown that these proteins can enhance immunogenicity by generating an adequate immune response capable of protecting the model against an experimental infectious challenge, whereas protection is decreased when the specific antigen is administered without the Cry protein. Therefore, based on previous results and the structural homology between Cry proteins, these molecules have arisen as potential adjuvants in the development of vaccines for both animals and humans. Finally, a model of the interaction of Cry proteins with different components of the immune response is proposed.

The Microbial Composition in Circumneutral Thermal Springs from Chignahuapan, Puebla, Mexico Reveals the Presence of Particular Sulfur-Oxidizing Bacterial and Viral Communities.

Terrestrial thermal springs are widely distributed globally, and these springs harbor a broad diversity of organisms of biotechnological interest. In Mexico, few studies exploring this kind of environment have been described. In this work, we explore the microbial community in Chignahuapan hot springs, which provides clues to understand these ecosystems' diversity. We assessed the diversity of the microorganism communities in a hot spring environment with a metagenomic shotgun approach. Besides identifying similarities and differences with other ecosystems, we achieved a systematic comparison against 11 metagenomic samples from diverse localities. The Chignahuapan hot springs show a particular prevalence of sulfur-oxidizing bacteria from the genera Rhodococcus, Thermomonas, Thiomonas, Acinetobacter, Sulfurovum, and Bacillus, highlighting those that are different from other recovered bacterial populations in circumneutral hot springs environments around the world. The co-occurrence analysis of the bacteria and viruses in these environments revealed that within the Rhodococcus, Thiomonas, Thermonas, and Bacillus genera, the Chignahuapan samples have specific species of bacteria with a particular abundance, such as Rhodococcus erytropholis. The viruses in the circumneutral hot springs present bacteriophages within the order Caudovirales (Siphoviridae, Myoviridae, and Podoviridae), but the family of Herelleviridae was the most abundant in Chignahuapan samples. Furthermore, viral auxiliary metabolic genes were identified, many of which contribute mainly to the metabolism of cofactors and vitamins as well as carbohydrate metabolism. Nevertheless, the viruses and bacteria present in the circumneutral environments contribute to the sulfur cycle. This work represents an exhaustive characterization of a community structure in samples collected from hot springs in Mexico and opens opportunities to identify organisms of biotechnological interest.

Analysis of Bacillus thuringiensis Population Dynamics and Its Interaction With Pseudomonas fluorescens in Soil.

BACKGROUND: Bacillus thuringiensis is the most successful biological control agent, however, studies so far have shown that B. thuringiensis is very sensitive to environmental factors such as soil moisture and pH. Ultraviolet light from the sun had been considered as the main limiting factor for its persistence in soil and it has recently been shown that the antagonism exerted by other native soil organisms, such as Pseudomonas fluorescens, is a determining factor in the persistence of this bacterium under in vitro culture conditions. OBJECTIVES: The aim of the present investigation was to analyze the population dynamics of B. thuringiensis and its interaction with P. fluorescens using microbiological and molecular methods in soil, under different conditions, and to determinate the effect of nutrients and moisture on its interaction. MATERIALS AND METHODS: The monitoring was performed by microbiological methods, such as viable count of bacteria, and molecular methods such as Polymerase Chain Reaction (PCR) and hybridization, using the direct extraction of DNA from populations of inoculated soil. RESULTS: The analysis of the interaction between B. thuringiensis and P. fluorescens in soil indicated that the disappearance of B. thuringiensis IPS82 is not dependent on the moisture but the composition of nutrients that may be affecting the secretion of toxic compounds in the environment of P. fluorescens. The results showed that the recovered cells were mostly spores and not vegetative cells in all proved treatments. The molecular methods were effective for monitoring bacterial population inoculated in soil. CONCLUSIONS: Bacillus thuringiensis is very sensitive to the interaction of P. fluorescens, however is capable to survive in soil due to its capacity of sporulate. Some of the cells in the form of spores germinated and folded slightly and remained in a constant cycle of sporulation and germination. This confirms that B. thuringiensis IPS82 can germinate, grow and sporulate in soil.