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Hybrid antimicrobials containing an antibacterial linked to a multidrug resistance (MDR) pump inhibitor make up a promising new class of agents for countering efflux-mediated bacterial drug resistance. This study explores the effects of varying the relative orientation of the antibacterial and efflux pump inhibitor components in three isomeric hybrids (SS14, SS14-M, and SS14-P) which link the antibacterial alkaloid and known substrate for the NorA MDR pump berberine to different positions on INF55 (5-nitro-2-phenylindole), an inhibitor of NorA. The MICs for all three hybrids against wild-type, NorA-knockout, and NorA-overexpressing Staphylococcus aureus cells were found to be similar (9.4 to 40.2 μM), indicating that these compounds are not effectively effluxed by NorA. The three hybrids were also found to have similar curing effects in a Caenorhabditis elegans live infection model. Each hybrid was shown to accumulate in S. aureus cells to a greater extent than either berberine or berberine in the presence of INF55, and the uptake kinetics of SS14 were found to differ from those of SS14-M and SS14-P. The effects on the uptake and efflux of the NorA substrate ethidium bromide into S. aureus cells in the presence or absence of the hybrids were used to confirm MDR inhibition by the hybrids. MDR-inhibitory activity was confirmed for SS14-M and SS14-P but not for SS14. Molecular dynamics simulations revealed that SS14 prefers to adopt a conformation that is not prevalent in either SS14-M or SS14-P, which may explain why some properties of SS14 diverge from those of its two isomers. In summary, subtle repositioning of the pump-blocking INF55 moiety in berberine-INF55 hybrids was found to have a minimal effect on their antibacterial activities but to significantly alter their effects on MDR pumps.

In bacteria, multidrug-resistance pumps (MDRs) confer resistance to chemically unrelated amphipathic toxins. A major challenge in developing efficacious antibiotics is identifying antimicrobial compounds that are not rapidly pumped out of bacterial cells. The plant antimicrobial berberine, the active component of the medicinal plants echinacea and golden seal, is a cation that is readily extruded by bacterial MDRs, thereby rendering it relatively ineffective as a therapeutic agent. However, inhibition of MDR efflux causes a substantial increase in berberine antimicrobial activity, suggesting that berberine and potentially many other compounds could be more efficacious if an effective MDR pump inhibitor could be identified. Here we show that covalently linking berberine to INF 55 , an inhibitor of Major Facilitator MDRs, results in a highly effective antimicrobial that readily accumulates in bacteria. The hybrid molecule showed good efficacy in a Caenorhabditis elegans model of enterococcal infection, curing worms of the pathogen.

The alarming increase of antibiotic-resistant bacterial pathogens points to the need for novel therapeutic approaches to combat infection. To discover novel antimicrobials, we devised a screen to identify compounds that promoted the survival of the model laboratory nematode Caenorhabditis elegans infected with the human opportunistic pathogen Enterococcus faecalis . E. faecalis colonizes the nematode intestinal tract, forming a persistent lethal infection. Infected nematodes were rescued by antibiotic treatment in a dose-dependent manner, and antibiotic treatment markedly reduced the number of bacteria colonizing the nematode intestine. To facilitate high throughput screening of compound libraries, we adapted a previously developed agar-based C. elegans - E. faecalis infection assay so that it could be carried out in liquid medium in standard 96-well microtiter plates. We used this simple infection system to screen 6,000 synthetic compounds and 1,136 natural product extracts. We identified 16 compounds and 9 extracts that promoted nematode survival. Some of the compounds and extracts inhibited E. faecalis growth in vitro , but, in contrast to traditional antibiotics, the in vivo effective dose of many of these compounds was significantly lower than the minimum inhibitory concentration needed to prevent the growth of E. faecalis in vitro . Moreover, many of the compounds and extracts had little or no affect on in vitro bacterial growth. Our findings indicate that the whole-animal C. elegans screen identifies not only traditional antibiotics, but also compounds that target bacterial virulence or stimulate host defense.

Although the opportunistic bacterial pathogen Enterococcus faecium is a leading source of nosocomial infections, it appears to lack many of the overt virulence factors produced by other bacterial pathogens, and the underlying mechanism of pathogenesis is not clear. Using E. faecium -mediated killing of the nematode worm Caenorhabditis elegans as an indicator of toxicity, we determined that E. faecium produces hydrogen peroxide at levels that cause cellular damage. We identified E. faecium transposon insertion mutants with altered C. elegans killing activity, and these mutants were altered in hydrogen peroxide production. Mutation of an NADH oxidase-encoding gene eliminated nearly all NADH oxidase activity and reduced hydrogen peroxide production. Mutation of an NADH peroxidase-encoding gene resulted in the enhanced accumulation of hydrogen peroxide. E. faecium is able to produce hydrogen peroxide by using glycerol-3-phosphate oxidase, and addition of glycerol to the culture medium enhanced the killing of C. elegans . Conversely, addition of glucose, which leads to the down-regulation of glycerol metabolism, prevented both C. elegans killing and hydrogen peroxide production. Lastly, detoxification of hydrogen peroxide either by exogenously added catalase or by a C. elegans transgenic strain overproducing catalase prevented E. faecium -mediated killing. These results suggest that hydrogen peroxide produced by E. faecium has cytotoxic effects and highlight the utility of C. elegans pathogenicity models for identifying bacterial virulence factors.

The nematode Caenorhabditis elegans is a unique whole animal model system for identifying small molecules with in vivo anti-infective properties. C. elegans can be infected with a broad range of human pathogens, including Enterococcus faecalis, an important human nosocomial pathogen with a mortality rate of up to 37% that is increasingly acquiring resistance to antibiotics. Here, we describe an automated, high throughput screen of 37,200 compounds and natural product extracts for those that enhance survival of C. elegans infected with E. faecalis. The screen uses a robot to accurately dispense live, infected animals into 384-well plates, and automated microscopy and image analysis to generate quantitative, high content data. We identified 28 compounds and extracts that were not previously reported to have antimicrobial properties, including 6 structural classes that cure infected C. elegans animals but do not affect the growth of the pathogen in vitro, thus acting by a mechanism of action distinct from antibiotics currently in clinical use. Our versatile and robust screening system can be easily adapted for other whole animal assays to probe a broad range of biological processes.

The nematode Caenorhabditis elegans shows a high degree of conservation of molecular pathways related to human disease, yet is only 1-mm long and can be considered as a microorganism. Because of the development of a simple but systematic RNA-interference (RNAi) methodology, C. elegans is the only metazoan in which the impact of "knocking-down" nearly every gene in the genome can be analyzed in a whole living animal. Both functional genomic studies and chemical screens can be carried out using C. elegans in vivo screens in a context that preserves intact cell-to-cell communication, neuroendocrine signaling, and every aspect of the animal's metabolism necessary to survive and reproduce in lab conditions. This feature enables studies that are impossible to undertake in cell-culture-based screens. Although genome-wide RNAi screens and limited small-molecule screens have been successfully performed in C. elegans, they are typically extremely labor-intensive. Furthermore, technical limitations have precluded quantitative measurements and time-resolved analyses.In this chapter, we provide detailed protocols to carry out automated high-throughput whole-animal RNAi and chemical screens. We describe methods to perform screens in solid and liquid media, in 96 and 384-well format, respectively. We describe the use of automated handling, sorting, and microscopy of worms. Finally, we give information about worm-adapted image analysis tools to quantify phenotypes. The technology presented here facilitates large-scale C. elegans genetic and chemical screens and it is expected to help shed light on relevant biological areas.

The nematode Caenorhabditis elegans shows a high degree of conservation of molecular pathways related to human disease, yet is only 1-mm long and can be considered as a microorganism. Because of the development of a simple but systematic RNA-interference (RNAi) methodology, C. elegans is the only metazoan in which the impact of "knocking-down" nearly every gene in the genome can be analyzed in a whole living animal. Both functional genomic studies and chemical screens can be carried out using C. elegans in vivo screens in a context that preserves intact cell-to-cell communication, neuroendocrine signaling, and every aspect of the animal's metabolism necessary to survive and reproduce in lab conditions. This feature enables studies that are impossible to undertake in cell-culture-based screens. Although genome-wide RNAi screens and limited small-molecule screens have been successfully performed in C. elegans, they are typically extremely labor-intensive. Furthermore, technical limitations have precluded quantitative measurements and time-resolved analyses.In this chapter, we provide detailed protocols to carry out automated high-throughput whole-animal RNAi and chemical screens. We describe methods to perform screens in solid and liquid media, in 96 and 384-well format, respectively. We describe the use of automated handling, sorting, and microscopy of worms. Finally, we give information about worm-adapted image analysis tools to quantify phenotypes. The technology presented here facilitates large-scale C. elegans genetic and chemical screens and it is expected to help shed light on relevant biological areas.

There is an urgent need for the development of new antifungal agents. A facile in vivo model that evaluates libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery. We show that Candida albicans, as well as other Candida species, are ingested by Caenorhabditis elegans and establish a persistent lethal infection in the C. elegans intestinal track. Importantly, key components of Candida pathogenesis in mammals, such as filament formation, are also involved in nematode killing. We devised a Candida-mediated C. elegans assay that allows high-throughput in vivo screening of chemical libraries for antifungal activities, while synchronously screening against toxic compounds. The assay is performed in liquid media using standard 96-well plate technology and allows the study of C. albicans in non-planktonic form. A screen of 1,266 compounds with known pharmaceutical activities identified 15 (∼1.2%) that prolonged survival of C. albicans-infected nematodes and inhibited in vivo filamentation of C. albicans. Two compounds identified in the screen, caffeic acid phenethyl ester, a major active component of honeybee propolis, and the fluoroquinolone agent enoxacin exhibited antifungal activity in a murine model of candidiasis. The whole-animal C. elegans assay may help to study the molecular basis of C. albicans pathogenesis and identify antifungal compounds that most likely would not be identified by in vitro screens that target fungal growth. Compounds identified in the screen that affect the virulence of Candida in vivo can potentially be used as “probe compounds” and may have antifungal activity against other fungi., Author Summary Candida spp. are among the most significant causes of nosocomial infections, and disseminated candidiasis continues to have an attributable mortality rate of over 25%. For this reason, we have developed a liquid media assay using the model nematode Caenorhabditis elegans as a model organism for Candida infection. The worms are infected on solid media lawns and then moved to pathogen-free liquid media. Unless antifungal compounds are added to the wells, the majority of worms die within 3–4 d. This model is similar to the infection process in humans, in that Candida cells are able to produce filaments, which are essential for the infection process in humans. We used this pathogen model to create a semi-automated, high-throughput screen using C. elegans to evaluate the antifungal effectiveness of many types of chemical compounds. Through this process, we have identified three compounds that we show have varying degrees of antifungal activity in C. elegans, in vitro, and in mice.