ANTIBACTERIAL EFFECT AND PHYTOCHEMICAL ANALYSIS OF THE SHOOT SYSTEM OF RUBUS CANESCENS DC. GROWING IN LEBANON

The misuse of antibiotics followed by improved fitness of resistant strains of infectious microorganisms hindered the efficacy of many known antimicrobial agents, and fueled research for the discovery of novel remedies. The current study aims at assessing the antimicrobial activity and understanding the mechanism of action of Rubus canescens DC. growing wild in Lebanon, as well as qualitatively determining its phytochemical profile. The antibacterial activity, MIC, and MBC of the extracts were evaluated by two-fold dilution. Time-kill curves were plotted to assess the bactericidal activity of the R. canescens DC. extracts against the growth of microorganisms, and TEM images were collected to confirm such effect. Overall, the extracts exhibited good antibacterial activity against MRSA and E. coli but not against S. pneumoniae and K. pneumoniae as determined by measuring the inhibition zones in plate-diffusion assays. TEM images of treated microorganisms revealed that the R. canescens DC. extracts induced irreversible deformations and damage to the cell membranes of the microorganisms leading to the leakage of cytoplasmic components and eventual cell death. Analysis of Time-Kill curves indicated that the extracts induced 100% killing of the test microorganisms within 10-18 h at the respective MBC. Finally, qualitative phytochemical analysis was conducted to decipher the active ingredients in the plant extracts.The current study reports the first data on the antimicrobial activity of different parts of R. canescens DC. Such promising data opens new avenues for broader assessment of the pharmacological profile of the scarcely investigated R. canescens DC

Isolation and characterization of novel phages in treating multidrug resistant Klebsiella pneumoniae using zebrafish larvae model

As time progresses, Klebsiella pneumoniae is becoming more resistant to antibiotics, thus rendering them ineffective. Phages have the potential to replace the antibiotics. In this study, two phages, Klebsiella virus UPM2146 and UPM1705, which were able to lyse K. pneumoniae ATCC BAA-2146 and ATCC BAA-1705, respectively were isolated from a polluted lake. These phages had a titer of 1012 PFU/ml and 107 PFU/ml, respectively. Transmission electron micrographs showed that both phages belong to the order Caudoviriales. Klebsiella virus UPM2146 had an adsorption period of 2 min, a latent period of 20 min, a rise period of 5 min, and a burst size of 20 PFU/bacteria. UPM1705 had an adsorption period of 2 min, a latent period of 75 min, a rise period of 45 min, and a burst size of 298 PFU/bacteria. Turbidity assay at multiplicities-of-infection (MOI) of 0.02, 0.2, and 2 indicated that Klebsiella virus UPM2146 and UPM1705 were able to lyse their hosts at 60 min and 180 min, respectively. Furthermore, spot and efficiency-ofplating (EOP) tests indicated that Klebsiella virus UPM2146 had a narrow host-range lysing 22.72% of the K. pneumoniae species tested but UPM1705 was only specific to its host. Klebsiella virus UPM2146 was selected for further analyses. Whole genomic sequencing revealed that Klebsiella virus UPM2146 has a double-stranded DNA genome of 160,795 bp with 214 putative open-reading-frames (ORF). Klebsiella virus UPM2146 is lytic, and lacking toxin and integrase genes. Phylogenetic tree analysis classified Klebsiella virus UPM2146 within the new Ackermannviridae family. The zebrafish larvae model was used to test the efficacy of Klebsiella virus UPM2146 in lysing K. pneumoniae ATCC BAA-2146. The larvae were able to survive up to 24 hours after 30 min exposure to the host bacteria. The appearance of a curved spine resembling a hook indicated dead larvae. Upon treatment with Klebsiella virus UPM2146, the zebrafish larvae was able to survive up to 10 hours. Therefore, Klebsiella virus UPM2146 has the potential to be used in phage therapy

Klebsiella virus UPM2146 lyses multiple drug-resistant Klebsiella pneumoniae in vitro and in vivo.

Klebsiella pneumoniae are opportunistic bacteria found in the gut. In recent years they have been associated with nosocomial infections. The increased incidence of multiple drug-resistant K. pneumoniae makes it necessary to find new alternatives to treat the disease. In this study, phage UPM2146 was isolated from a polluted lake which can lyse its host K. pneumoniae ATCC BAA-2146. Observation from TEM shows that UPM2146 belongs to Caudoviriales (Order) based on morphological appearance. Whole genome analysis of UPM2146 showed that its genome comprises 160,795 bp encoding for 214 putative open reading frames (ORFs). Phylogenetic analysis revealed that the phage belongs to Ackermannviridae (Family) under the Caudoviriales. UPM2146 produces clear plaques with high titers of 1010 PFU/ml. The phage has an adsorption period of 4 min, latent period of 20 min, rise period of 5 min, and releases approximately 20 PFU/ bacteria at Multiplicity of Infection (MOI) of 0.001. UPM2146 has a narrow host-range and can lyse 5 out of 22 K. pneumoniae isolates (22.72%) based on spot test and efficiency of plating (EOP). The zebrafish larvae model was used to test the efficacy of UPM2146 in lysing its host. Based on colony forming unit counts, UPM2146 was able to completely lyse its host at 10 hours onwards. Moreover, we show that the phage is safe to be used in the treatment against K. pneumoniae infections in the zebrafish model