Development of In Vitro Assays with the Canine Hookworm Uncinaria stenocephala and Assessment of Natural Plant Products for Anti-Parasitic Activity.

Enteric helminth infection is an increasing concern in companion animals due to reports of resistance to commonly used anthelmintic drugs. Thus, the assessment of new therapeutic options such as bioactive dietary additives is of high importance. Here, we adapted egg hatch, larval migration, and larval motility assays to screen extracts of several natural ingredients against the canine hookworm Uncinaria stenocephala, a prevalent parasite of dogs in northern Europe. Egg hatch and larval migration assays were established showing that the anthelmintic drugs levamisole and albendazole had strong anti-parasitic activity against U. stenocephala, validating the use of these assays for the assessment of novel anti-parasitic substances. Subsequently, we identified that extracts from the seaweed Saccharina latissima, but not extracts from grape seed or chicory, significantly inhibited both hatching and larval migration. Finally, we showed that α-linolenic acid, a putative anti-parasitic compound from S. latissima, also exhibited anti-parasitic activity. Collectively, our results established a platform for the screening for anthelmintic resistance or novel drug candidates against U. stenocephala and highlighted the potential use of seaweed extracts as a functional food component to help control hookworm infection in dogs.

Identification of compounds responsible for the anthelmintic effects of chicory (Cichorium intybus) by molecular networking and bio-guided fractionation.

Increasing resistance towards anthelmintic drugs has necessitated the search for alternative treatments for the control of gastrointestinal nematode parasites. Animals fed on chicory (Cichorium intybus L.), a temperate (pasture) crop, have reduced parasite burdens, hence making C. intybus a potentially useful source for novel anthelmintic compounds or a diet-based preventive/therapeutic option. Here, we utilized in vitro bioassays with the parasitic nematode Ascaris suum and molecular networking techniques with five chicory cultivars to identify putative active compounds. Network analysis predicted sesquiterpene lactones (SL) as the most likely group of anthelmintic compounds. Further bioassay-guided fractionation supported these predictions, and isolation of pure compounds demonstrated that the SL 8-deoxylactucin (8-DOL) is the compound most strongly associated with anti-parasitic activity. Furthermore, we showed that 8-DOL acts in a synergistic combination with other SL to exert the anti-parasitic effects. Finally, we established that chicory-derived extracts also showed activity against two ruminant nematodes (Teladorsagia circumcincta and Cooperia oncophora) in in vitro assays. Collectively, our results confirm the anti-parasitic activity of chicory against a range of nematodes, and pave the way for targeted extraction of active compounds or selective breeding of specific cultivars to optimize its future use in human and veterinary medicine.

Adaptive responses to cefotaxime treatment in ESBL-producing Escherichia coli and the possible use of significantly regulated pathways as novel secondary targets.

OBJECTIVES: The aim of the study was to determine how ESBL-producing Escherichia coli change the expression of metabolic and biosynthesis genes when adapting to inhibitory concentrations of cefotaxime. Secondly, it was investigated whether significantly regulated pathways constitute putative secondary targets that can be used to combat the resistant bacteria. METHODS: Strains of E. coli MG1655 encoding blaCTX-M-1 from an IncI1 plasmid and from the chromosome were challenged with cefotaxime corresponding to inhibitory concentrations, and transcriptional patterns were compared with growth without or with very low concentrations of cefotaxime by RNA sequencing. Significantly regulated pathways were inhibited with suitable inhibitors, or genes encoding the enzymes of the regulated pathways were knocked out. The ability of the bacteria to grow in the presence of cefotaxime was determined. Chequerboard assays were utilized to confirm synergies between treatments. RESULTS: Genes belonging to 16 different functional gene classes were significantly regulated. Protein and peptidoglycan syntheses were up-regulated and low concentrations of chloramphenicol or d-cycloserine, targeting these systems, strongly reduced the MIC of cefotaxime (>32-fold). Inhibition and/or mutations in other genes that were significantly regulated, belonging to energy synthesis, purine synthesis, proline uptake or potassium uptake, also rendered the resistant bacteria more susceptible to cefotaxime. CONCLUSIONS: The results show that ESBL-producing E. coli adapt to treatment with cefotaxime by changing their gene expression patterns and furthermore that targeting regulated adaptive pathways may be a suitable way to identify targets for drugs that will specifically inhibit the resistant bacteria.