Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.
With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.
The human body harbors an estimated 10-100 trillion live microbial cells (bacteria, archaea fungi), which are primarily found in the gut, skin, and mucosal environments1. In a healthy state, these provide benefits to their host, including vitamin production, maturation of the immune system, stimulation of innate and adaptive immune responses to pathogens, regulation of fat metabolism, modulation of stress responses, and more, with an impact on growth and development, disease onset, and ageing2,3,4,5. The gut microbiota also evolves considerably throughout life. The most drastic evolution occurs during infancy and early childhood6, but significant changes also occur with age, including a decrease in Bifidobacterium abundance and an increase in Clostridium, Lactobacillus, Enterobacteriaceae, and Enterococcus species7. Lifestyle can further alter gut microbial composition leading to dysbiosis (loss of beneficial bacteria, overgrowth of opportunistic bacteria), resulting in various pathologies such as inflammatory bowel disease, diabetes, and obesity5, but also contributing to Alzheimer's and Parkinson's diseases8,9,10,11.
This realization has critically contributed to refining the concept of the gut-brain axis (GBA), where interactions between gut physiology (now including the microbes within it) and the nervous system are considered the main regulator of animal metabolism and physiological functions12. However, the precise role of microbiota in gut-brain signaling and the associated mechanisms of action are far from being fully understood13. With gut microbiota being a key determinant of healthy aging, how bacteria modulate the aging process has become a subject of intense research and controversy6,14,15.
With the demonstration that the roundworm Caenorhabditis elegans hosts a bonafide gut microbiota dominated-as in other species-by Bacteroidetes, Firmicutes, and Actinobacteria16,17,18,19,20, its rapid rise as an experimental platform to study host-gut commensal interactions21,22,23,24,25,26 has significantly expanded our investigative arsenal26,27,28,29. In particular, high-throughput experimental approaches available for C. elegans to study gene-diet, gene-drug, gene-pathogen, etc. interactions, can be adapted to rapidly explore how bacterial isolates and cocktails impact C. elegans health and aging.
The present protocol describes an experimental pipeline to screen at once arrays of bacterial isolates or mixtures set in multiwell plates for effects on C. elegans stress resistance as a proxy for health, which can be used to identify probiotics. It details how to grow large worm populations and handle bacterial arrays in 96- and 384-well plate formats before processing worms for automated stress resistance analysis using a fluorescence plate reader (Figure 1). The approach is based on label-free automated survival assays (LFASS)30 that exploit the phenomenon of death fluorescence31, whereby dying worms produce a burst of blue fluorescence that can be used to pinpoint the time of death. Blue fluorescence is emitted by glucosyl esters of anthranilic acid stored in C. elegans gut granules (a type of lysosome-related organelle), which burst when a necrotic cascade is triggered in the worm gut upon death31.
Figure 1: Experimental workflow for high-throughput screening of bacterial isolates with impact on C. elegans resistance to stress. (A) Timeline for worm and bacterial maintenance and assay setup. (B) 96-well bacterial plate array setup and handling. (C) 384-well worm plate setup. Please click here to view a larger version of this figure.
C. elegans offers many advantages for rapidly screening multiple experimental parameters at once, owing to its small size, transparency, fast development, short lifespan, inexpensiveness, and ease of handling. Its considerably simpler genome, body plan, nervous system, gut, and microbiome, yet complex and similar enough to humans, make it a powerful preclinical model, where mechanistic insight can be gained while testing for bioactive efficacy or toxicity. As interest is growing in developing microbial intervent…
The authors have nothing to disclose.
We thank the CGC Minnesota (Madison, USA, NIH – P40 OD010440) for providing worm strains and OP50 and Pr. Hinrich Schulenburg (CAU, Kiel, Germany) for providing all the environmental microbial isolates depicted here. This work was funded by a UKRI-BBSRC grant to AB (BB/S017127/1). JM is funded by a Lancaster University FHM PhD scholarship.
10 cm diameter plates (Non-vented) | Fisher Scientific | 10720052 | Venting is not necessary for bacterial cultures |
15 cm diameter plates (Vented) | Fisher Scientific | 168381 | |
384-well black, transparent flat bottom plates | Corning | 3712 or 3762 | Not essential to be sterile for fast stress assays |
6 cm diameter plates (Vented) | Fisher Scientific | 150288 | Venting is necessary for worm cultures to avoid hypoxia |
96-well transparent plates (Biolite) | Thermo | 130188 | |
Agar (<4% ash) | Sigma-Aldrich | 102218041 | Good quality agar is important for the structural integrity of the culture media, to avoid worm burrowing |
Agarose | Fisher Scientific | BP1356 | |
Avanti Centrifuge J-26 XP | Beckman coulter | ||
Bleach | Honeywell | 425044 | |
Calcium chloride | Sigma-Aldrich | C5080 | |
Centrifuge 5415 R | Eppendorf | ||
Centrifuge 5810 R | Eppendorf | ||
Cholesterol | Sigma-Aldrich | C8667 | |
LB agar | Difco | 240110 | |
LB broth | Invitrogen | 12795084 | |
LoBind tips | VWR | 732-1488 | Lo-bind reduce worm loss during transfers |
LoBind tubes | Eppendorf | 22431081 | |
Magnesium sulfate | Fisher Scientific | M/1100/53 | |
Plate reader- infinite M nano+ | Tecan | Monochromator setup enables fluorescence tuning but adequate filter-based setups may be used | |
Plate reader- Spark | Tecan | ||
Potassium phosphate monobasic | Honeywell | P0662 | |
Sodium chloride | Sigma-Aldrich | S/3160/63 | |
Stereomicroscope setup with transillumination base | Leica | MZ6, or M80 | Magnification from 0.6-0.8x up to 40-60x is necessary, as is a good quality transillumination base with a deformable, titable or slidable mirror to adjust contrast |
t-BHP (tert-Butyl hydroperoxide) | Sigma-Aldrich | 458139 | |
Transparent adhesive seals Nunc | Fisher Scientific | 101706871 | It is important that it is transparent and that it can tolerate the temperatures involved in the assays. |
Tryptophan | Sigma-Aldrich | 1278-7099 | |
Yeast extract | Fisher Scientific | BP1422 |