**3.2.7 Operational Taxonomic Units diversity**

Each band with specific migration distance was cut from the gel and the eluted DNA was reamplified, sequenced and compared to genetic databases for OTU affiliation. For the five patients presented here, we detected 16 different OTUs (Table 1). Four major OTU, *Staphylococcus* sp., *Flavobacteriaceae*, *Comamonadaceae* and *Propionibacterium* sp., were detected in all patients. *Acinetobacter* sp. and *Corynebacterium* sp. are also frequently detected. In contrast, OTUs such as *Bosea* sp. and *Bacillus* sp. were specific of one single patient. Most major OTUs were present in all patient at all step of the surgery operation and in all sampling sites. *Flavobacteriaceae* and *Comamonadaceae* were atypical Gram-negative bacilli recently described in the skin metagenome (Grice et al., 2009). These atypical Gram-negative bacilli were particularly represented in subcutaneous and in deep tissues (Figure 4). Band identification by sequencing allowed refining the crude DI by the elimination of multiple bands corresponding to a single OTU, as observed for bacteria that displayed 16S rRNA genes heterogeneity. In some cases, the DI dramatically decreased as shown in Figure 3.

#### **3.3 Culture-dependent approach: Use of PCR-TTGE to assess the impact of antimicrobial prophylaxis on the whole skin microbiome**

#### **3.3.1 Methods**

Sternal skin microbiome was sampled with cotton swabs, before hospitalization, in four patients requiring CABG surgery. This sample reflects the normal resident skin microbiome of the patients. Sternal skin samples were streaked on 5% blood and nutrient agar plates supplemented with different concentrations of cefamandole (0, 0.125, 0.25, 0.5, 2, 4, 8 and 32 g/mL) and incubated for 24h at 37°C in anaerobiosis and aerobiosis. The resulting colonies

from 4.2 to 7.6. The increase of crude DI was also observed at each site of sampling: 4.2 to 7.6 for skin, 7.4 to 8.2 for subcutaneous tissues, 4.4 to 5.2 for sternum banks and 5.4 to 6.8 for

Fig. 3. Representative evolution of crude and optimized DI during the course of the surgical

Each band with specific migration distance was cut from the gel and the eluted DNA was reamplified, sequenced and compared to genetic databases for OTU affiliation. For the five patients presented here, we detected 16 different OTUs (Table 1). Four major OTU, *Staphylococcus* sp., *Flavobacteriaceae*, *Comamonadaceae* and *Propionibacterium* sp., were detected in all patients. *Acinetobacter* sp. and *Corynebacterium* sp. are also frequently detected. In contrast, OTUs such as *Bosea* sp. and *Bacillus* sp. were specific of one single patient. Most major OTUs were present in all patient at all step of the surgery operation and in all sampling sites. *Flavobacteriaceae* and *Comamonadaceae* were atypical Gram-negative bacilli recently described in the skin metagenome (Grice et al., 2009). These atypical Gram-negative bacilli were particularly represented in subcutaneous and in deep tissues (Figure 4). Band identification by sequencing allowed refining the crude DI by the elimination of multiple bands corresponding to a single OTU, as observed for bacteria that displayed 16S rRNA genes heterogeneity. In some cases, the DI dramatically decreased as shown in Figure 3.

**3.3 Culture-dependent approach: Use of PCR-TTGE to assess the impact of** 

Sternal skin microbiome was sampled with cotton swabs, before hospitalization, in four patients requiring CABG surgery. This sample reflects the normal resident skin microbiome of the patients. Sternal skin samples were streaked on 5% blood and nutrient agar plates supplemented with different concentrations of cefamandole (0, 0.125, 0.25, 0.5, 2, 4, 8 and 32 g/mL) and incubated for 24h at 37°C in anaerobiosis and aerobiosis. The resulting colonies

**antimicrobial prophylaxis on the whole skin microbiome** 

mediastin. Figure 3 showed a representative DI dynamics for a patient.

operation of the patient AD.

**3.3.1 Methods** 

**3.2.7 Operational Taxonomic Units diversity** 


Table 1. Repartition of the OTUs detected in the skin microbiota of five patients.

Fig. 4. Number of patients carrying the major OTUs according to the step of the surgery procedure.

were counted and the whole bacterial cultures were then harvested for DNA extraction. DNA extraction, single PCR HDA, TTGE migration and band affiliation to an OTU were performed as described in previous section.

#### **3.3.2 Results: Impact of antimicrobial prophylaxis on skin microbiota**

Most samples displayed cultivable microbiota susceptible to cefamandole, i.e. no culture was observed on agar plates with 8mg/L, which was the cefamandole critical concentration used to define susceptibility versus resistance. At lower concentrations (from 0.125 mg/L to 4 mg/L), we observed a decrease of colonies in number and in diversity, comparatively to

Temporal Temperature Gel Electrophoresis to Survey

**medical microbiologist?** 

strains as performed currently.

very low.

Pathogenic Bacterial Communities: The Case of Surgical Site Infections 305

concentrations in the surgical wound (0.125mg/L to 4mg/L), TTGE profiles showed a modification of the microbiota with loss of bands affiliated to the genus *Staphylococcus* and appearance of other bands affiliated to the genera *Corynebacterium* and *Micrococcus* or to *Bacillus cereus* group. These bands corresponding to OTUs with a decreased susceptibility to cefamandole revealed minor OTUs that were undetected in the total cultivable microbiota, i.e. on plates without antibiotics. For instance in the patient CB (Figure 5), a band corresponding to *Bacillus* sp. was observed from 0,5 à 4mg/L cefamandole and a band

corresponding to *Corynebacterium* sp. was observed in at least 8mg/L cefamandole.

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

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

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

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

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

now not convenient for clinical studies on large cohorts of patients.

the control plate. The mean colony counts went from 80 CFU/plate without antibiotics to 30 CFU/plate with 0.125 mg/L cefamandole and to 6 CFU/plate with 1mg/L cefamandole. All the colonies were bulked and analyzed by 16S rRNA gene PCR-TTGE. Profiles comparison allowed to determine the concentration of antimicrobial agents that inhibited 25%, 50%, 75% and 90% of the microbial diversity and to identify the resistant bacteria to the species level. These data will be related to dynamics of wound re-colonization after surgery.

Fig. 5. TTGE gel illustrating the culture-dependent approach on skin sample of the patient CB3. The gel shows differences in banding patterns according to the concentration of cefamandole and the culture atmosphere. Lanes 1 and 8, culture without cefamandole; Lanes 2 and 9, cefamandole 0.125mg/L; Lanes 3 and 10, cefamandole 0.25mg/L; Lanes 4 and 11, cefamandole 0.5mg/L; Lanes 5 and 12, cefamandole 1 mg/L; Lanes 6 and 13, cefamandole 2mg/L; Lanes 7 and 14, cefamandole 4mg/L; Lane 15, cefamandole 8mg/L. Bands corresponding to OTUs are shown with arrow: a for *Staphylococcus* sp., b for *Bacillus*

sp., c for *Corynebacterium* sp. and d for *Propionibacterium* sp.

The TTGE profiles from colonies harvested on control plates without antibiotics showed the predominant cultivable population. For the four patients studied herein, the corresponding bacteria belonged to the genera *Staphylococcus* (4 to 6 species) and *Propionibacterium*. For two patients, the cultivable skin microbiota was susceptible to cefamandole because no colonies were observed on plates with more than 8 mg/L, which was the cefamandole critical concentration used to define susceptibility versus resistance. For the two other patients, colonies were observed for 8 mg/L plates incubated in aerobiosis and even for 32 mg/L plates incubated in anaerobiosis. Resistant bacteria belonged to the genera *Bacillus* and *Corynebacterium*. For lower concentrations, which could correspond to the tissue

the control plate. The mean colony counts went from 80 CFU/plate without antibiotics to 30 CFU/plate with 0.125 mg/L cefamandole and to 6 CFU/plate with 1mg/L cefamandole. All the colonies were bulked and analyzed by 16S rRNA gene PCR-TTGE. Profiles comparison allowed to determine the concentration of antimicrobial agents that inhibited 25%, 50%, 75% and 90% of the microbial diversity and to identify the resistant bacteria to the species level.

Fig. 5. TTGE gel illustrating the culture-dependent approach on skin sample of the patient CB3. The gel shows differences in banding patterns according to the concentration of cefamandole and the culture atmosphere. Lanes 1 and 8, culture without cefamandole; Lanes 2 and 9, cefamandole 0.125mg/L; Lanes 3 and 10, cefamandole 0.25mg/L; Lanes 4 and 11, cefamandole 0.5mg/L; Lanes 5 and 12, cefamandole 1 mg/L; Lanes 6 and 13, cefamandole 2mg/L; Lanes 7 and 14, cefamandole 4mg/L; Lane 15, cefamandole 8mg/L. Bands corresponding to OTUs are shown with arrow: a for *Staphylococcus* sp., b for *Bacillus*

The TTGE profiles from colonies harvested on control plates without antibiotics showed the predominant cultivable population. For the four patients studied herein, the corresponding bacteria belonged to the genera *Staphylococcus* (4 to 6 species) and *Propionibacterium*. For two patients, the cultivable skin microbiota was susceptible to cefamandole because no colonies were observed on plates with more than 8 mg/L, which was the cefamandole critical concentration used to define susceptibility versus resistance. For the two other patients, colonies were observed for 8 mg/L plates incubated in aerobiosis and even for 32 mg/L plates incubated in anaerobiosis. Resistant bacteria belonged to the genera *Bacillus* and *Corynebacterium*. For lower concentrations, which could correspond to the tissue

sp., c for *Corynebacterium* sp. and d for *Propionibacterium* sp.

These data will be related to dynamics of wound re-colonization after surgery.

concentrations in the surgical wound (0.125mg/L to 4mg/L), TTGE profiles showed a modification of the microbiota with loss of bands affiliated to the genus *Staphylococcus* and appearance of other bands affiliated to the genera *Corynebacterium* and *Micrococcus* or to *Bacillus cereus* group. These bands corresponding to OTUs with a decreased susceptibility to cefamandole revealed minor OTUs that were undetected in the total cultivable microbiota, i.e. on plates without antibiotics. For instance in the patient CB (Figure 5), a band corresponding to *Bacillus* sp. was observed from 0,5 à 4mg/L cefamandole and a band corresponding to *Corynebacterium* sp. was observed in at least 8mg/L cefamandole.
