Table 1 Suggestions for research on low-biomass microbiomes considering philosophical principles

1. Rationale for the study. It is legitimate to challenge dogma but published evidence that led to the dogma in the first place should not be ignored but incorporated and explained. If such published data does not support the hypothesis, and there is no good reason to question the quality without making ad hoc auxiliary assumptions, strongly consider if the project has merit. Microbiology is an old discipline that dates back more than 100 years, and the more traditional methodologies such as culture and microscopy are, in many cases, legitimate approaches to determine the presence of microbial communities. Immunological assays represent complementary tools to functionally test the presence of microorganisms. Only because a technology is new and exciting does not necessarily mean it is superior to the traditional approaches.

2. Use appropriate methodology and try to extend beyond descriptive studies that are mere verifications. DNA- or RNA-based next-generation technologies, as powerful as they are to study complex microbial communities, have immense limitations for the study of low-biomass samples due to contamination issues. DNA-based methods are also not suitable to establish sterility as they do not assess viability. Classical approaches, such as culture, and microscopy to detect nonculturable microbes, are better suited to establish the presence of microbes as they are less susceptible to contamination. With the right microbiological expertise, it is not hard to establish the existence of entire microbiomes. Sequencing can be used as a follow-up after the presence of microbes is clearly established to determine which microbes are there. Other techniques, such as microscopy using selective labels for bacteria or specific taxa, should also be employed. For all techniques, aseptic sampling remains a major challenge, and appropriate contamination controls must be employed. Admittedly, initial studies in any field are often exploratory studies that are descriptive in nature and represent verifications. Subsequent studies should, to be truly insightful, go beyond this and try to provide explanatory and mechanistic insight in accordance with IBE.

3. Study the broader biological context. It is imperative for future studies to strictly differentiate between viable symbiotic microbiomes/microbes, pathogens, and microbial metabolites/products, as they would differ in their functionality, biological effects on the host, and in the experimental approaches by which they would have to be studied. Extend purely descriptive investigations with experiments that allow a broader interpretation of the findings considering the overall biology, ecology, and evolution of the biological system. For example, if bacterial sequences are detected in utero, are the same bacterial strains detectable in the infant gut? If there are bacteria detected in breast milk, determine if they really have an evolved role in establishing the infant’s gut microbiome, or are they just reflective of the mother’s skin microbiota or the infant’s oral microbiota inoculating the breast milk [54]? Are bacteria in the brain not prevented by the immune response they would elicit? Can the expected immune response be detected? Consider various angles of evidence, especially those that provide explanatory and mechanistic information, in accordance with IBE.

4. Conduct studies that involve genuine tests of falsifiable hypotheses. Make efforts to design studies that test hypotheses through “risky” predictions that are genuine tests of conditions that are prohibited by the theory. As a minimum, hypotheses must be falsifiable and refutable, and they should be rejected if proven false without using ad hoc auxiliary assumptions to make the data fit the theory.

5. Avoid hype. It is legitimate to challenge dogma, but it requires “extraordinary evidence to back up an extraordinary claim” [52]. Experimental and technical limitations of any study should be acknowledged and unsubstantiated claims about the significance of the data avoided. Be especially careful with unsubstantiated claims on mainstream media as journalists tend to exaggerate the importance of scientific findings and remain critical and balanced with statements on social media.