**3.2.2 Cell lysis, extraction of bacterial DNA and PCR amplification**

Bacterial genomic DNA was directly extracted from bulk cells present on cotton swabs using an enzymatic method (MasterPure Gram positive DNA purification kit, EPICENTRE Biotechnologies ®) according to the manufacturer's recommendations with modifications as described by (Roudière et al., 2009). This method has been previously described as efficient on a wide range of bacteria including Gram-positive bacteria (Roudière et al., 2009; Jacquot et al., 2011). A fragment about 1465 bp of the 16SrRNA gene was amplified using the primers 27f (5'-GTGCTGCAGAGAGTTTGATCCTGGCTCAG-3') and 1492r (5'- CACGGATCCTACGGGTACCTTGTTACGACTT-3'). The PCRs were carried out in 50 μL of reaction mixture containing 200 nM of each primer (Sigma Genosys), 200 nM each dNTP (Fermentas), 1U of *Taq* polymerase (Promega) in the appropriate reaction buffer, and 1 μL of crude DNA extract as the template. PCR conditions were 30 cycles of 1 min at 94°C, 1 min at 65°C, and 2 min at 72°C.

The 199-bp fragment (from position 338 to position 536, *Escherichia coli* numbering) overlapping the 16S rDNA V2-V3 variable region (Neefs et al., 1993; Sundquist et al., 2007) was amplified using the primers HDA1-GC (primer HDA1 with a fragment rich in GC – the 'GC clamp' – added to the 59 extremity) and HDA2 (Ogier et al., 2002). The reaction mixture (50 μl) consisted of 200 nM of each primer (Sigma Genosys), 200 mM each dNTP (Fermentas), 2.5 U FastStart Taq DNA polymerase (Roche, France) in the appropriate reaction buffer, with 1.8 mM MgCl2. One μL of DNA previously amplified was added to the reaction buffer and the thermal cycling was as follows: 95°C for 2 min; 35 cycles of 95°C for 1 min, 62°C for 30 s, 72°C for 1 min ; and 72°C for 7 min. PCR products were checked by electrophoresis in a 1.5 % agarose gel before TTGE migration.

### **3.2.3 TTGE migration**

TTGE migration was performed in the DCode Universal Mutation Detection System (Bio-Rad Laboratories). Gels were composed of 8 % (w/v) bisacrylamide (37.5: 1), 7 M urea, 40 ml N,N,N9,N9-tetramethylethylenediamine, and 0.1 % (w/v) ammonium persulfate, and were run in 16 Tris/acetate/EDTA buffer at pH 8.3. DNA was loaded on the gel with inhouse dye marker (50% sucrose, 0.1% bromophenol blue) using capillary tips. The electrophoresis conditions were 46 V for 16 h with an initial temperature of 63°C and a final temperature of 70°C corresponding to an increase of 0.4°C h-1. In order to obtain thin

Temporal Temperature Gel Electrophoresis to Survey

before suture; AD12, skin at the end of the procedure.

**3.2.6 PCR-TTGE profiles and diversity index** 

Pathogenic Bacterial Communities: The Case of Surgical Site Infections 301

sample AD6 In the corresponding TTGE profile, the faint bands seen in single PCR-TTGE profile were very faint in the nested PCR-TTGE profile while original and intense bands appeared in the latter profile. The reproducibility is also satisfactory (data not shown).

Fig. 1. Migration in agarose gel after single (A) and nested PCR (B) for samples from the patient AD. AD5, skin before incision; AD6, sub-cutaneous tissue after incision; AD7, sternum edges; AD8, mediastinum at the beginning of the operation; AD9, mediastinum at the end of intervention; AD10, edges of sternum before closing; AD11, sub-cutaneous tissue

Fig. 2. TTGE migration after single PCR (sp) and nested PCR (np) on samples from the patient AD. AD6 sub-cutaneous tissue after incision; AD9, mediastinum before suture; AD11, sub-cutaneous tissue before suture; AD12, skin at the end of the surgery.

About fifteen samples from the sternal region were recovered during hospital stay of each patient undergoing programmed CABG surgery. TTGE profiles were obtained for each sample after nested-PCR and showed clear bands easily separated from each other (data not shown). This result confirmed that 16S rRNA gene PCR-TTGE was a suitable method to survey the bacterial community from skin and wound tissues in cardiac surgery. By numbering the bands in each profile, crude diversities index (DI) were determined. Crude DI varied from 2 to 12 (mean value = 6). The bacterial communities presenting the lower mean value of crude DI (DI=4.2) were sampled on the skin just after preoperative antisepsis. Subcutaneous tissues sampled just before the closure of the wound at the end of the operation displayed the higher crude DI (DI=8.2). The dynamics of evolution of mean crude DI over the surgical operation showed that the bacterial diversity globally increased from the start to the end of the surgery,

discrete bands, a pre-migration for 15 min at 63°C and 20 V was done after loading. Premigration and migration were performed with additional magnetic shaking in the electrophoresis chamber. Gels were stained for 15 min with 0.5 mg ethidium bromide ml-1 in 1x TAE buffer, washed for 45 min in 1x TAE buffer, and photographed under UV illumination.

### **3.2.4 TTGE band sequencing**

Each TTGE band for further analysis was cut out of the gel with a disposable sterile scalpel to avoid contamination between bands. Gel slices were washed twice in molecular biology grade water and incubated overnight at 37 °C in 10 mM Tris buffer (pH 8.5) to allow DNA diffusion. Amplification of a single 16S rRNA gene V3 region copy was performed using 1 l band eluate and the primers HDA1 without a GC-clamp and HDA2. The PCR was carried out in 50 ml reaction mixture containing 200 nM of each primer, 200 mM each dNTP, 2.5 mM MgCl2 and 2.5 U Taq DNA polymerase (Promega) in the appropriate buffer. PCR conditions were 94 °C for 2 min; 35 cycles of 45 s at 95 °C, 30 s at 62 °C, 1min at 72 °C; and 10 min at 72 °C. PCR products were checked by electrophoresis in a 1.5 % agarose gel and sequenced on an ABI 3730xl sequencer (Cogenics). Each sequencing chromatograph was visually inspected and corrected. The sequences were analyzed by comparison with Genbank (http://www.ncbi.nlm.nih.gov/) and RDPII databases (http://rdp.cme.msu.edu/) using Basic Local Alignment Search Tool (BLAST) and Seqmatch programs, respectively and affiliated to an OTU as recommended (Drancourt et al., 2000; Stackebrandt & Goebel, 1994).

#### **3.2.5 Optimization of DNA amplification in samples with low load of template**

In most assays, the bacterial load is low because samples originated from quasi-sterile sites, i.e. the mediastinum during the surgical intervention. When applied on DNA samples extracted from a low number of bacteria (<103/tube) (Roudière et al., 2009), a single PCR produced a faint or no signal in agarose gel electrophoresis (data not shown). We showed that the signal was equivalent to the positive control for DNA extracted from skin, a site with a resident microbiota but faint for the samples taken from sub-cutaneous and mediastinal tissues, sites expected to be almost sterile (data not shown). To gain in sensitivity, we performed a nested-PCR approach with pre-amplification of the almost complete 16S rRNA gene, further used as template for a second PCR focused on the V2-V3 hypervariable regions of the gene. As expected, nested-PCR resulted in an improved signal in agarose gel for all samples including samples with low bacterial load (Figure 1A and 1B). However, nested-PCR could induce biases such as amplification of contaminant DNA, preferential amplification of a sub-population of template and heteroduplex formation, among others (Park & Crowley, 2010. Consequently, the enhanced quantity of amplified DNA in agarose gel had to be confirmed in TTGE in order to assess the quality of amplified DNA and qualitatively compare single and nested PCR. Figure 2 shows the TTGE profiles after single- and nested-PCR obtained for 4 samples. We observed that nested-PCR led to an enrichment of the profiles while all the bands detected by single PCR-TTGE were also observed in nested PCR-TTGE. The PCR-TTGE profiles presented in Figure 2 displayed neither obvious heteroduplex bands nor smears due to amplification of miscellaneous contaminant DNA. However, preferential amplification was observed particularly for

discrete bands, a pre-migration for 15 min at 63°C and 20 V was done after loading. Premigration and migration were performed with additional magnetic shaking in the electrophoresis chamber. Gels were stained for 15 min with 0.5 mg ethidium bromide ml-1 in 1x TAE buffer, washed for 45 min in 1x TAE buffer, and photographed under UV

Each TTGE band for further analysis was cut out of the gel with a disposable sterile scalpel to avoid contamination between bands. Gel slices were washed twice in molecular biology grade water and incubated overnight at 37 °C in 10 mM Tris buffer (pH 8.5) to allow DNA diffusion. Amplification of a single 16S rRNA gene V3 region copy was performed using 1 l band eluate and the primers HDA1 without a GC-clamp and HDA2. The PCR was carried out in 50 ml reaction mixture containing 200 nM of each primer, 200 mM each dNTP, 2.5 mM MgCl2 and 2.5 U Taq DNA polymerase (Promega) in the appropriate buffer. PCR conditions were 94 °C for 2 min; 35 cycles of 45 s at 95 °C, 30 s at 62 °C, 1min at 72 °C; and 10 min at 72 °C. PCR products were checked by electrophoresis in a 1.5 % agarose gel and sequenced on an ABI 3730xl sequencer (Cogenics). Each sequencing chromatograph was visually inspected and corrected. The sequences were analyzed by comparison with Genbank (http://www.ncbi.nlm.nih.gov/) and RDPII databases (http://rdp.cme.msu.edu/) using Basic Local Alignment Search Tool (BLAST) and Seqmatch programs, respectively and affiliated to an OTU as recommended (Drancourt et

**3.2.5 Optimization of DNA amplification in samples with low load of template** 

In most assays, the bacterial load is low because samples originated from quasi-sterile sites, i.e. the mediastinum during the surgical intervention. When applied on DNA samples extracted from a low number of bacteria (<103/tube) (Roudière et al., 2009), a single PCR produced a faint or no signal in agarose gel electrophoresis (data not shown). We showed that the signal was equivalent to the positive control for DNA extracted from skin, a site with a resident microbiota but faint for the samples taken from sub-cutaneous and mediastinal tissues, sites expected to be almost sterile (data not shown). To gain in sensitivity, we performed a nested-PCR approach with pre-amplification of the almost complete 16S rRNA gene, further used as template for a second PCR focused on the V2-V3 hypervariable regions of the gene. As expected, nested-PCR resulted in an improved signal in agarose gel for all samples including samples with low bacterial load (Figure 1A and 1B). However, nested-PCR could induce biases such as amplification of contaminant DNA, preferential amplification of a sub-population of template and heteroduplex formation, among others (Park & Crowley, 2010. Consequently, the enhanced quantity of amplified DNA in agarose gel had to be confirmed in TTGE in order to assess the quality of amplified DNA and qualitatively compare single and nested PCR. Figure 2 shows the TTGE profiles after single- and nested-PCR obtained for 4 samples. We observed that nested-PCR led to an enrichment of the profiles while all the bands detected by single PCR-TTGE were also observed in nested PCR-TTGE. The PCR-TTGE profiles presented in Figure 2 displayed neither obvious heteroduplex bands nor smears due to amplification of miscellaneous contaminant DNA. However, preferential amplification was observed particularly for

illumination.

**3.2.4 TTGE band sequencing** 

al., 2000; Stackebrandt & Goebel, 1994).

sample AD6 In the corresponding TTGE profile, the faint bands seen in single PCR-TTGE profile were very faint in the nested PCR-TTGE profile while original and intense bands appeared in the latter profile. The reproducibility is also satisfactory (data not shown).

Fig. 1. Migration in agarose gel after single (A) and nested PCR (B) for samples from the patient AD. AD5, skin before incision; AD6, sub-cutaneous tissue after incision; AD7, sternum edges; AD8, mediastinum at the beginning of the operation; AD9, mediastinum at the end of intervention; AD10, edges of sternum before closing; AD11, sub-cutaneous tissue before suture; AD12, skin at the end of the procedure.

Fig. 2. TTGE migration after single PCR (sp) and nested PCR (np) on samples from the patient AD. AD6 sub-cutaneous tissue after incision; AD9, mediastinum before suture; AD11, sub-cutaneous tissue before suture; AD12, skin at the end of the surgery.

#### **3.2.6 PCR-TTGE profiles and diversity index**

About fifteen samples from the sternal region were recovered during hospital stay of each patient undergoing programmed CABG surgery. TTGE profiles were obtained for each sample after nested-PCR and showed clear bands easily separated from each other (data not shown). This result confirmed that 16S rRNA gene PCR-TTGE was a suitable method to survey the bacterial community from skin and wound tissues in cardiac surgery. By numbering the bands in each profile, crude diversities index (DI) were determined. Crude DI varied from 2 to 12 (mean value = 6). The bacterial communities presenting the lower mean value of crude DI (DI=4.2) were sampled on the skin just after preoperative antisepsis. Subcutaneous tissues sampled just before the closure of the wound at the end of the operation displayed the higher crude DI (DI=8.2). The dynamics of evolution of mean crude DI over the surgical operation showed that the bacterial diversity globally increased from the start to the end of the surgery,

Temporal Temperature Gel Electrophoresis to Survey

Pathogenic Bacterial Communities: The Case of Surgical Site Infections 303

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

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

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

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

procedure.

performed as described in previous section.

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

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