Survival of Campylobacter jejuni in Amoebae enhances subsequent invasion of mammalian cells

The ubiquitous unicellular eukaryote, Acanthamoeba, is known to play a role in the survival and dissemination of Campylobacter jejuni. C. jejuni is the leading cause of bacterial foodborne gastroenteritis world-wide and is a major public health problem. The ability of C. jejuni to interact and potentially invade epithelial cells is thought to be key for disease development in humans. We examined C. jejuni grown under standard laboratory conditions,11168HCBA with that harvested from within Acanthamoeba castellanii (11168HAC/CBA) or Acanthamoeba polyphaga (11168HAP/CBA), and compared their ability to invade different cell lines. C. jejuni harvested from within amoebae had a ~3.7-fold increase in invasiveness into T84 human epithelial cells and a striking ~11-fold increase for re-entry into A. castellanii cells. We also investigated the invasiveness and survivability of six diverse representative C. jejuni strains within Acanthamoeba spp., our results confirm that invasion and survivability is likely host cell dependent. Our survival assay data led us to conclude that Acanthamoeba spp. are a transient host for C. jejuni and that survival within amoebae pre-adapts C. jejuni and enhances subsequent cell invasion. This study provides new insight into C. jejuni interactions with amoebae and its increased invasiveness potential in mammalian hosts.

Anticonvulsant Activity of Elaesis guineensis (Jacq) Oil in Pentylenetetrazol-Induced Seizure in Drosophila melanogaster

Elaesis guineensis, a plant whose oil extract (palm kernel oil) is medicinal, is reported to treat a wide range of disorders, including seizures. However, the anticonvulsant activity of this oil extract has not been exhaustively studied. This study aimed at evaluating the anticonvulsant activity of Elaesis guineensis oil in pentylenetetrazol-induced seizure in Drosophila melanogaster (fruit-fly). Pentylenetetrazol (50 mg/5 g diet) was used to induce seizure in Drosophila melanogaster. Flies were exposed to different concentrations (0.5-5%) of the oil and phenytoin for 28 days in a survival assay to determine the safety in the fruit flies. Five replicate of fifty files each were exposed to diet containing the LC50 of phenytoin and other groups were exposed to different concentrations of the extract for 7 days. Seizure was then induced with Pentylenetetrazol. The Trikinetic system was used to monitor activity and the DAMSystem3 data collection program to collect, process and store data. The results showed that the extract increased the latency of seizures and improved survival in the flies and suggest that the extract possesses anticonvulsant properties.

Mutation in Plasmodium falciparum BTB/POZ domain of K13 protein confers artemisinin resistance

Partial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 ( pfk13 ) gene. Here, we carried out in vitro selection over a one-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA 0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13.

Effect of gold nanoparticles on the structure and neuroprotective function of protein L-isoaspartyl methyltransferase (PIMT)

Fibrillation of peptides and proteins is implicated in various neurodegenerative diseases and is a global concern. Aging leads to the formation of abnormal isoaspartate (isoAsp) residues from isomerization of normal aspartates in proteins, triggering fibril formation that leads to neurodegenerative diseases. Protein L-isoaspartyl methyltransferase (PIMT) is a repair enzyme which recognizes and converts altered isoAsp residues back to normal aspartate. Here we report the effect of gold nanoparticles (AuNPs) of different sizes on the structure and function of PIMT. Spherical AuNPs, viz. AuNS5, AuNS50 and AuNS100 (the number indicating the diameter in nm) stabilize PIMT, with AuNS100 exhibiting the best efficacy, as evident from various biophysical experiments. Isothermal titration calorimetry (ITC) revealed endothermic, but entropy driven mode of binding of PIMT with all the three AuNSs. Methyltransferase activity assay showed enhanced activity of PIMT in presence of all AuNSs, the maximum being with AuNS100. The efficacy of PIMT in presence of AuNS100 was further demonstrated by the reduction of fibrillation of Aβ42, the peptide that is implicated in Alzheimer’s disease. The enhancement of anti-fibrillation activity of PIMT with AuNS100 was confirmed from cell survival assay with PC12 derived neuronal cells against Aβ42 induced neurotoxicity.

Plasmodium falciparum phenotypic and genotypic resistance profile during the emergence of Piperaquine resistance in Northeastern Thailand

Malaria remains a public health problem in Thailand, especially along its borders where highly mobile populations can contribute to persistent transmission. This study aimed to determine resistant genotypes and phenotypes of 112 Plasmodium falciparum isolates from patients along the Thai-Cambodia border during 2013–2015. The majority of parasites harbored a pfmdr1-Y184F mutation. A single pfmdr1 copy number had CVIET haplotype of amino acids 72–76 of pfcrt and no pfcytb mutations. All isolates had a single pfk13 point mutation (R539T, R539I, or C580Y), and increased % survival in the ring-stage survival assay (except for R539I). Multiple copies of pfpm2 and pfcrt-F145I were detected in 2014 (12.8%) and increased to 30.4% in 2015. Parasites containing either multiple pfpm2 copies with and without pfcrt-F145I or a single pfpm2 copy with pfcrt-F145I exhibited elevated IC90 values of piperaquine. Collectively, the emergence of these resistance patterns in Thailand near Cambodia border mirrored the reports of dihydroartemisinin-piperaquine treatment failures in the adjacent province of Cambodia, Oddar Meanchey, suggesting a migration of parasites across the border. As malaria elimination efforts ramp up in Southeast Asia, host nations militaries and other groups in border regions need to coordinate the proposed interventions.

Toxicity of Azadirachta indica Hydroethanolic Leaf Extract in Adult Drosophila melanogaster (Harwich Strain)

Aim: This study is aimed at evaluating the toxic effect of A. indica hydroethanolic leaf extracts in D. melanogaster (fruit flies) by carrying out a survival study, locomotor, fecundity and biochemical assays. Place of study: This study was carried out in the Drosophila laboratory of Africa Centre of Excellence in Phytomedicine Research and Development (ACEPRD), University of Jos. Methods: Extraction of A. indica extract was carried using hydroethanolic solvent (70:30 v/v ethanol: water). Flies were treated with 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 250 mg, 500 mg and 5000 mg A. indica hydroethanolic leaf extracts per 10 g fly food for 7 days, to determine the lethal concentration (LC50). The survival assay was carried out for 28 days by treating flies with 5 mg, 10 mg, and 25 mg/10 g fly food of the extract. Young flies were treated with several concentrations of the extract for 7 days, to determine the effect of the extract on the fecundity and locomotion. Thereafter, flies exposed to the extracts for 7 days were immobilized, weighed, homogenized, and centrifuged. The supernatant was used to assay for acetylcholinesterase and catalase activities. The experiment was replicated 3 times and data was presented as mean ± SEM with statistical value at “P < 0.05” considered significant. Results: The percentage yield was calculated to be 12.7 % and the phytochemicals present in A. indica hydroethanolic leaf extract included alkaloids, flavonoids, saponins, tannins, steroids, phenols, and glycosides. The LC50 was determined to be 1499 mg/10 g diet and the result showed a dose-dependent significant decrease (P < 0.05) in the survival of the flies, when compared to the control group. Further results showed a non-significant decrease (P > 0.05) in the fecundity, as well as the locomotor, acetylcholinesterase, and catalase activities of the flies, compared to the control. Conclusion: This study concludes that A. indica hydroethanolic leaf extract, at certain concentrations, may not be safe for consumption as it showed some level of toxicity in D. melanogaster.

Synergistic Effect of Cefazolin Plus Fosfomycin Against Staphylococcus aureus in vitro and in vivo in an Experimental Galleria mellonella Model

Objectives: This study investigated the synergistic in vitro and in vivo activity of cefazolin plus fosfomycin against methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA) to provide the basis for a potential treatment alternative.Methods: Antimicrobial susceptibility and in vitro synergy tests were performed with five MSSA and five MRSA isolates using the broth microdilution and chequerboard assays, respectively. The in vivo efficacy of cefazolin plus fosfomycin for the treatment of MRSA infections was assessed using the Galleria mellonella survival assay.Results: Using fractional inhibitory concentration index (FICI), the evaluated combination of cefazolin plus fosfomycin showed synergistic in vitro activity against all MSSA and MRSA isolates tested. In addition, cefazolin susceptibility was recovered in all MRSA isolates except one fosfomycin-resistant strain when combined with fosfomycin at readily achievable concentrations. The G. mellonella survival assay demonstrated highly synergistic in vivo activity of cefazolin plus fosfomycin, resulting in a 44–52% reduction in mortality when compared to cefazolin-alone and fosfomycin-alone, respectively.Conclusion: If susceptibility to fosfomycin is either confirmed or can be assumed based on local resistance patterns, combination therapy with cefazolin plus fosfomycin could be a valuable treatment option for empirical as well as targeted therapy of S. aureus and MRSA infections. Future studies proving the clinical significance of this combination therapy are therefore warranted.

Plasmodium Falciparum Phenotypic and Genotypic Resistance Profile During the Emergence of Piperaquine Resistance From Royal Thai Army and Civilian patients in Northeastern Thailand

Malaria remains a public health problem in Thailand, especially along its borders where highly mobile populations can contribute to persistent transmission. This study aimed to determine resistant genotypes and phenotypes of 112 Plasmodium falciparum isolates from patients along the Thai-Cambodia border during 2013-2015. The majority of parasites harbored pfmdr1-Y184F mutation. A single pfmdr1 copy number, had CVIET haplotype of amino acids 72-76 of pfcrt and no pfcytb mutations. All isolates had a pfk13 point mutation (R539T, R539I, and C580Y), and increased ring-stage survival assay (except R539I). Multiple copies of pfpm2 and pfcrt-F145I were first detected in 2014 (12.8%) and increased to 30.4% in 2015. Parasites containing either multiple pfpm2 copies with and without pfcrt-F145I or a single pfpm2 copy with pfcrt-F145I exhibited the elevated IC90 values of piperaquine. Antimalarials were detected in 21 samples (18.8%) by mass spectrometer, and 12 samples (10.7%) by bioassay. Collectively, the emergence of these resistance patterns mirrored the reports of dihydroartemisinin-piperaquine treatment failures in the adjacent province of Cambodia, Oddar Meanchey, suggesting a migration of parasites across the border. As malaria elimination efforts ramp up in Southeast Asia, host nations militaries and other groups in border regions must be a focus for interventions

Protective Effect of Ethanol Extract of Legundi (Vitex trifolia L.) Leaves against Staphylococcus aureus in Drosophila Infection Model

Research to discover certain medicinal plants' antibacterial activity against Staphylococcus aureus was mostly performed in vitro. The purpose of this research was to investigate the antibacterial activity of ethanol extract of legundi leaves (EELL) against S. aureus using model organism Drosophila melanogaster. The extract was prepared by the maceration method using 70% ethanol. The antibacterial activities of EELL were determined by using fly survival assay and bacterial colony-forming assay. Fly survival assay was conducted to investigate the extracts' ability to enhance the survival of D. melanogaster (host) upon S. aureus infection. The results demonstrated that both EELL were able to increase the survival rate of the S. aureus-infected Drosophila. Furthermore, a colony-forming assay was carried out to determine the growth of bacteria in the host body that has been considered an important pathogenic factor for the host. The result found that the number of bacteria recovered from the EELL-treated infected flies was significantly lower than the ones obtained from the infected flies without any treatments. Overall, EELL protects the S. aureus-infected hosts, suggesting the potential antibacterial effect of EELL against S. aureus.