**4. Conclusion: Why should 'microbiomology' be on the benchtop of the medical microbiologist?**

In this experiment we applied PCR TTGE to the study of microbiome dynamics in skin and tissues during a cardiac surgery procedure. We present the results obtained for 5 patients in a culture-independent approach and in 4 patients in a culture-dependent approach. Our results enable us to describe the bacteria involved in surgical site colonization during the procedure. The bacteria detected belonged to OTUs previously described in the skin microbiome (Grice et al., 2009). Gram-positive bacteria belonged to *Bacilli* and *Clostridia* in the phylum *Firmicutes*, and to *Corynebacterium*, *Propionibacterium* and *Micrococcaceae* in the phylum *Actinobacteria*. Gram-negative bacteria mainly belonged to *Proteobacteria* but also to *Flavobacteria.* This result confirmed that the nested-PCR TTGE approach gave a good representation of the skin microbiome even after antisepsis when the bacterial load is very low.

We have showed for the first time that the skin microbiota contaminated the surgical wound at all steps of the surgery in quantities that can be detected by molecular methods. The PCR-TTGE appeared as a suitable method to follow-up the microbiology of a clean wound such as a surgical wound. The approach could also give insights about the activity of antibiotics, e.g. agents used for antibioprophylaxis, at the community level and not only on isolated strains as performed currently.

In order to find relationships between the dynamics of microbiome and the clinical evolution of patients, a cohort of 120 patients will be studied for their surgical wound microbiota and their clinical outcome. Such clinical studies are necessary to understand to physio-pathological mechanism of wound re-colonization and SSI. PCR-TTGE appeared as easy and cost-effective enough to provide microbiological arguments at the community level in large clinical studies. Other very efficient methods such as pyrosequencing are until now not convenient for clinical studies on large cohorts of patients.

More generally the study presented here underlines that is now time for microbiome studies to exit from research labs and to take place on the benchtop of the medical microbiologist.

Current medical microbiology still rests on the shoulders of Leeuwenhoek, Koch and Pasteur. Although every microbiologist knows that a bacterium does not live alone but in complex communities, routine practice still consists in watching a small subset of isolated microbes under microscope and on Petri dishes. It is now clear that the human microbiome has profound effects on health and disease. Ambitious project are in progress such as the international Human Microbiome Consortium and the Human Microbiome Project. Highthroughput sequencing and metagenomics allow us to explore new fields of the

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relationships between human and bacteria, considering these relationships at the community level. Facing the huge volume of data generated by metagenomics, microbiologists and even more so physicians are perplexed. For instance, Grice et al. admit that "…hairy, moist underarms lie a short distance from smooth dry forearms, but these two niches are likely as ecologically dissimilar as rainforests are to deserts" (Grice et al., 2009). A conceptual revolution is occurring while in medical microbiology practice, skin is still shown as a sheet covered by *S. epidermidis*.

How can we deal with this new world we discover within and around ourselves with our PCR and sequencers? To change our understanding of health and disease, it is now time to develop ways of seeing patterns and interpret them among the staggering biodiversity of microbes. Extending microbial ecology to healthy and diseased microbiota as previously done for mouth (Bik et al., 2010), stomach (Bik et al., 2006), gut (Yang et al., 2009) and skin (Grice et al., 2009) could elucidate the physio-pathology of diseases. For instance, description of the baseline skin microbiome is a step toward testing the therapeutic potential of manipulating the microbiome in skin disorders (Grice et al., 2009). Studies on psoriasis (Gao et al., 2008) describe selective microbial shifts associated with diseases and suggest that therapies might require not only inhibiting the growth of pathogenic bacteria, but also promoting the growth of mutualistic bacteria. Moreover, antibiotic exposure and hygienic practices such as antisepsis modify the skin microbiome. Methods that help understand naturally occurring mutualistic microbial communities will provide insights into the conditions that favor the emergence of antibiotic-resistant organisms.

It is now urgent to confront 'microbiomology' data to clinical data in order to define new indicators for re-evaluating the risk of disease and for adapting therapeutics. Before the generalization of high-throughput metagenomics on the microbiologist's benchtop, alternative methods such as PCR-TTGE or other methods based on denaturant electrophoresis should be considered. Although they are less exhaustive and powerful than pyrosequencing, these approaches are easy to handle in all laboratories and are costeffective. They can be used to study large cohorts of patients, thus deriving clinical benefits from the conceptual revolution brought by contemporary researches on human microbial ecology.
