Susceptibility of selected multi-drug resistant clinical isolates to leaves of Carpolobia lutea

Background: The decline in the production of new effective antibiotics coupled with the constantly evolving antimicrobial resistance remains a public health concern.This study thus evaluated the antibacterial and antifungal effects of the ethanolic, n-hexane and hot aqueous extracts of Carpolobia lutea leaves.Methods: The extracts were tested using agar well diffusion method against selected clinical isolates: Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, Staphylococcus aureus and Candida albicans. Antibiogram profile of the isolates were deduced by disc diffusion method.Results: Multi-drug resistance was confirmed in all bacteria with a notable pandrug resistance in Pseudomonas aeruginosa. Ofloxacin, Erythromycin and Gentamicin were effective on two or three organisms, notably on Salmonella typhi and Escherichia coli. The preliminary antibacterial assay marked the efficacy ofthe ethanol and n-hexane extracts except on E. coli, with zero activity for hot water extracts at the stock concentration (200 mg/ml). Pseudomonas aeruginosa and Candida albicans were susceptible to lesser concentrations of the ethanol extracts at 5 mg/ml and 25 mg/ml respectively. None of the isolates showed sensitivity to lesser concentrations of n-hexane extract. Carpolobia lutea leaves proved to be effective over the use of antibiotics in inhibiting the activity of Pseudomonas aeruginosa which was resistant to the latter. The Minimum Inhibitory concentration of the ethanolic extract was considerably low (≤5mg/ml for P. aeruginosa and 25mg/ml for C. albicans). However, there was no Minimum Bactericidal concentration for the extracts against the clinical isolates.Conclusion: Carpolobia lutea shelters bioactive components with pharmacological potentials that could show efficiency in the treatment of bacterial infections.Keywords: Carpolobia lutea, antifungal, antibacterial, multi-drug resistanc

Engineering chimaeric recombinases for HIV-1 proviral DNA excision

‘Cutting out’ HIV-1 proviral DNA could potentially cure a person of the infection. Genome editing approaches have been proffered for eradicating the provirus in infected persons by activating all latent viral reservoirs for further antiretroviral therapy or for the excision of the proviral DNA from memory T- cells. Previous approaches to do this have used nuclease-based tools or reprogrammed tyrosine recombinases; the former presenting unpredictable therapeutic outcomes and the latter, lengthy design time for newer tool variants if viral mutability erodes their effectiveness. Unlike nuclease-based tools that only cut DNA and rely on host-mediated repair mechanisms, chimaeric recombinases (CRs) cut DNA and carry the inherent ability to re-ligate cut ends at the cleavage site. The modular domain architecture of small serine recombinases can be redesigned to mediate site-specific recombination on non-cognate sites, by replacing the C-terminal DNA binding domains (DBDs) of serine recombinases with programmable DBDs such as Zinc Finger (ZF) proteins, TAL effector proteins and CRISPR-dCas9. For HIV-1 proviral DNA excision, CR requirement for the interaction of two recombinase-bound sites, and the lack of necessity for host cell-encoded factors should maximize the fidelity and efficiency of provirus removal. In this work, the engineering and characterization of CRs with the specificity to recognize and promote site-specific recombination at highly conserved regions within the HIV-1 proviral DNA is explored. This research provides a solid proof-of-concept for the use of CRs to target divergent novel target sequences, expanding their applicability for applied genome editing and wider biotechnological applications

High fidelity one‐pot DNA assembly using orthogonal serine integrases

Background: Large serine integrases (LSIs, derived from temperate phages) have been adapted for use in a multipart DNA assembly process in vitro, called serine integrase recombinational assembly (SIRA). The versatility, efficiency, and fidelity of SIRA is limited by lack of a sufficient number of LSIs whose activities have been characterized in vitro. Methods and Major Results: In this report, we compared the activities in vitro of 10 orthogonal LSIs to explore their suitability for multiplex SIRA reactions. We found that Bxb1, ϕR4, and TG1 integrases were the most active among the set we studied, but several others were also usable. As proof of principle, we demonstrated high-efficiency one-pot assembly of six DNA fragments (made by PCR) into a 7.5 kb plasmid that expresses the enzymes of the β-carotenoid pathway in Escherichia coli, using six different LSIs. We further showed that a combined approach using a few highly active LSIs, each acting on multiple pairs of att sites with distinct central dinucleotides, can be used to scale up “poly-part” gene assembly and editing. Conclusions and Implications: We conclude that use of multiple orthogonal integrases may be the most predictable, efficient, and programmable approach for SIRA and other in vitro applications

Antimicrobial Activities of Bryophyllum pinnatum on Some Selected Clinical Isolates.

Ethanol, methanol and aqueous extracts of the leaves of Bryophyllum pinnatum obtained through cold maceration, were screened for their antibacterial activities against selected multi-drug resistant bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi Staphylococcus aureus) using the agar well diffusion method. Broth dilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts at concentrations ranging from 100mg/ml to 25mg/ml. The ethanol extract was the most reactive while the aqueous extract showed lesser antibacterial activity. Ofloxacin was the most effective antibiotic in the antibiotic susceptibility profiling of the test organisms. It was however evident that the ethanol extract of Bryophyllum pinnatum has higher antibacterial efficacy on the test organisms than Ofloxacin. Bacteriostatic and -cidal activities were exhibited by the plant extracts against the organisms ranging from 25 – 100 × 103 μg/ml. Succinctly, Bryophyllum pinnatum possesses biologically active constituents with explorable pharmacological potentials. Keywords: Antimicrobial, Bryophyllum pinnatum, Extraction solvents, minimum inhibitory concentration (MIC), minimum bactericidal concentratio