**3.1 Surgical site infections: State of the art**

296 Gel Electrophoresis – Advanced Techniques

one rRNA operon, 62% display some degree of sequence divergence between 16S rRNA loci in a same genome (Case et al., 2007). In PCR-TTGE, the intra-genomic 16S rDNA heterogeneity can lead to multiple bands for a single OTU and then to an overestimation of OTU diversity. This pitfall inherent to the 16S rRNA gene marker will be avoided by band sequencing. However, as a pre-requisite of diversity analysis by PCR-TTGE, the major known species expected in a particular ecosystem should be individually studied by TTGE in order to explore heterogeneity in 16S rRNA gene copies (Roudière et al, 2007; Michon et

Alternative markers can also be proposed such as *rpoB* (Case et al., 2007) but universal *rpoB* PCR primers allowing the exploration of the whole bacterial diversity can not be designed

Several authors remarked that culture-independent methods regularly fail to identify species obtained using culture-dependent methods (Jany & Barbier, 2008). By contrast, culture-dependent methods have yielded information on the structure of microbial populations but they are limited by the in vitro growth capacity of most bacteria in a community. Such a discrepancy is not observed for all the communities studied. For instance, culture dependant and independent approaches of the premature neonate gut microbiome give globally congruent results (Roudière et al., 2009). However, it is accepted that culture-independent methods remain the only approach for monitoring the rapid dynamics of microbial communities. Nevertheless, the two types of methods reveal different images of the same community and combining culture-dependent and culture-independent methods may be worthwhile to obtain a more accurate view of the structure of the microbial

In spite of the limited growth capacity of most bacteria, culture-dependent methods remain the sole approach available for monitoring sub-populations selected on the basis of phenotypic traits such as dependence to metabolites or resistance to antimicrobial agents. In this context, PCR-TTGE can be used after culture in specific conditions in order to describe the diversity of cultivable population. The colonies growing in diverse conditions can be bulked and further analyzed by PCR-TTGE as described before. This culture- and geneticbased mixed approach is particularly suitable to describe dynamics of populations according to their level of resistance to antimicrobial drugs in natural environments

Culture-independent approaches have previously shown their interest in cardiology to detected new or atypical infectious agents (Marchandin et al., 2009; Daïen et al., 2010). Considering the interest and limitations of 16S rRNA PCR-TTGE, we will proposed a protocol for describing the diversity and following the dynamics of the bacterial community that colonize surgical wound of the patient during hospitalization for cardiac surgery. We will show how 16S rRNA PCR-TTGE is particularly suited to the low bacterial diversity encountered in aseptic surgical settings, where antibiotic prophylaxis and cutaneous

In addition, one example of the use of 16S PCR-TTGE in a culture-dependent analysis will be detailed. This approach associates determination of Minimal Inhibitory Concentration (MIC) at the community level and determination of the diversity by 16S PCR-TTGE in the

(personal data) and the databases remain poor in *rpo* sequences.

antisepsis effectively reduce the bacterial load of patients.

resistant sub-population at each concentration of antimicrobial agent.

al., 2011).

community (Case et al., 2007).

(Vanhove et al., 2011).

## **3.1.1 Epidemiology of surgical site infection**

Surgical site infections (SSIs) are among the most frequent healthcare associated infections (HAIs), along with urinary tract and pulmonary infections, and remain an unresolved problem for modern medicine, their occurrence having significant impact on patient morbidity, length of stay and cost of care. Data from longitudinal surveillance studies show SSI rates of 1 to 5% (Klevens et al., 2007; Astagneau et al., 2009; de Lissovoy et al., 2009), whereas higher rates are reported from interventional studies, where control groups can have up to 8.5% infection rates (Bode et al., 2010; Perl et al., 2002), owing to different population case mixes. Large-scale epidemiological studies have identified risk factors for SSI that can be grossly classified as related to patient condition, surgical procedure and environment. The American National Nosocomial Infection Surveillance system (NNIS) developed an easily calculated risk index that combines the patient-related risk assessment of the American Society of Anesthesiologists (ASA) score, and 2 surgical procedure-related factors: type of surgery as defined by pre- or per-operative microbial contamination (Altemeier classification, from I – clean surgery- to IV – septic surgery), and duration of operation exceeding 75th percentile for a given procedure. For all categories of surgery, there is a linear increase in the incidence of SSI when the NNIS risk index increases (Coello et al., 2005). Beside these surveillance-derived risk assessment scores, prospective controlled studies identified many more factors associated with a higher risk of SSI. Most important are poorly controlled diabetes mellitus, malignant diseases, smoking, advanced age, per operative hypothermia, emergency surgery (Coello et al., 2005). Specific risk factors have been identified for cardiac surgery: obesity, pre operative myocardial infarction, chronic obstructive broncho-pulmonary disease, duration of extra corporeal circulation, early post operative bleeding, combined valve and coronary bypass procedures… (Filsoufi et al., 2009).

In the mid 1980s, 30 to 35% of nosocomial (i.e. hospital-acquired) infections were deemed evitable (Haley et al., 1985). A recent analysis of infection control interventional studies estimates that as many as 26 to 54% of SSIs could be avoided by comprehensive implementation of evidence-based prevention strategies, foremost of which are pre operative cutaneous antisepsis, no pre operative shaving of surgical site, timely antibiotic prophylaxis and strict per operative glycemic control (Umsheid et al., 2011).

Still, even when all known preventive measures are implemented, even in low-risk (i.e. clean, non urgent) surgery for low risk patients, SSI can occur. This seemingly irreducible rate of "inevitable" infections raises the problem of how surgical site infections develop. Success of preventive measures based on antisepsis and optimization of patient status compounds the hypothesis that infection results from disequilibrium between host defense mechanisms and microbial infectiveness. In a 10-to-1 inequity between human cells and colonizing microbes, the balance of power in ensured by integrity of skin and mucous membranes, both obviously disrupted by surgery (Wenzel, 2010). However, the intimate mechanisms of infection are not known, starting with the origin of germs involved.

Temporal Temperature Gel Electrophoresis to Survey

sub-cutaneous tissue and skin were sampled again.

**3.2.1 Patients and samples** 

65°C, and 2 min at 72°C.

**3.2.3 TTGE migration** 

Pathogenic Bacterial Communities: The Case of Surgical Site Infections 299

Forty cotton swabs were collected from 5 patients who underwent CABG surgery at the Service of Thoracic and Cardiovascular Surgery of the Montpellier University Hospital (France). For each patient, swabs were sampled during intervention at the surgical site, superficially and deeply. The first sample was taken on the skin after cutaneous antisepsis just before incision and the second one in sub-cutaneous tissue once incision made. Thirdly, sternum edges were sampled after sawing. The fourth sample was mediastinal tissue after positioning of sternal retractors. At the end of the operation, mediastinum, sternum edges,

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

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

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

**3.2 Culture-independent approach: Use of TTGE to survey the dynamics of bacterial** 

**communities involved in surgical wound colonization and infection** 

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

electrophoresis in a 1.5 % agarose gel before TTGE migration.

#### **3.1.2 Are surgical site infections related to endogenous or exogenous bacteria?**

SSIs are 4.5 times more frequent in patients with nasal carriage of *Staphylococcus aureus* compared to non-carriers. In carrier patients who developed SSI, *S. aureus* isolated from infection site was identical to the one isolated from anterior nares in 84% of cases (Perl et al., 2002). The case for an endogenous bacterial origin is indirectly made by the 60% reduction rate in *S. aureus* SSIs obtained by thorough pre operative decontamination (Bode et al., 2010). Nasal carriage of *S. aureus* was found to be associated with a higher rate of SSIs in orthopedic surgery in a French multicentric study (Berthelot et al., 2010). However, in this study, only 27% of *S. aureus* infections were molecularly linked to an endogenous strain. This can be due to insufficient sensitivity of carriage detection, or to the fact that infections don't necessarily have an endogenous origin.

Indeed, bacteria involved in SSIs can also originate from an exogenous source in the per operative environment. Several studies report cases of cross contamination of surgical sites with bacteria molecularly linked to health care professionals or other patients (Perl et al., 2002). Contamination of surgical instruments and devices increases over time inside the operating room (Dalstrom et al., 2008). Under different air treatment devices, air contamination can vary from 8 to 34% (Knobben et al., 2006). In the air above the surgical site, *S. aureus* and coagulase negative staphylococci can be found, mostly molecularly linked to nasal and pharyngeal carriage by operating team personnel (Edminston et al., 2005).

## **3.1.3 From contamination to infection**

The origin of germs notwithstanding, what induces ordinarily commensal germs to become pathogenic is mostly unexplained. Indeed, in spite of the frequent presence of germs around and in the surgical site (up to 4.1% deep tissue samples found positive in clean orthopedic surgery (Byrne et al., 2007)), only very few infections actually develop.

Antibiotic prophylaxis certainly thwarts the development of most SSIs, at least insofar as bacterial load remains low and antibiotic spectrum and pharmacokinetics are adapted to the germs and tissues involved (Classen et al., 1992). However, the occurrence of SSIs in spite of adequate prophylaxis leads to consider the mechanism of infection from a bacterial point of view. Infections are often the consequences of disequilibrium in the relationships inside the microbiota or between the microbiota and the host. SSIs are typically infections related to microbiota disequilibrium, their major etiologic agents being members of the skin microbiota such as *S. aureus,* coagulase-negative Staphylococci and *Propionibacterium acnes.* The mechanism of selection of these bacteria from the skin community remains unknown and could be highlighted by dynamic survey of the microbial re colonization by a suitable method. Exploring the microbiota disequilibrium and its dynamics should provide insights into the mechanisms involved in SWI infections.

We hereafter present the use of TTGE in culture-independent and culture-based approaches to study the dynamics of bacterial communities involved in SSIs. The study was undertaken in the setting of clean cardiothoracic surgery, because SSI rates remain unacceptably high in coronary artery bypass grafts (CABG), in spite of ongoing efforts to minimize patient- and procedure-related risk factors (Filsoufi et al., 2009).

#### **3.2 Culture-independent approach: Use of TTGE to survey the dynamics of bacterial communities involved in surgical wound colonization and infection**

#### **3.2.1 Patients and samples**

298 Gel Electrophoresis – Advanced Techniques

SSIs are 4.5 times more frequent in patients with nasal carriage of *Staphylococcus aureus* compared to non-carriers. In carrier patients who developed SSI, *S. aureus* isolated from infection site was identical to the one isolated from anterior nares in 84% of cases (Perl et al., 2002). The case for an endogenous bacterial origin is indirectly made by the 60% reduction rate in *S. aureus* SSIs obtained by thorough pre operative decontamination (Bode et al., 2010). Nasal carriage of *S. aureus* was found to be associated with a higher rate of SSIs in orthopedic surgery in a French multicentric study (Berthelot et al., 2010). However, in this study, only 27% of *S. aureus* infections were molecularly linked to an endogenous strain. This can be due to insufficient sensitivity of carriage detection, or to the fact that infections

Indeed, bacteria involved in SSIs can also originate from an exogenous source in the per operative environment. Several studies report cases of cross contamination of surgical sites with bacteria molecularly linked to health care professionals or other patients (Perl et al., 2002). Contamination of surgical instruments and devices increases over time inside the operating room (Dalstrom et al., 2008). Under different air treatment devices, air contamination can vary from 8 to 34% (Knobben et al., 2006). In the air above the surgical site, *S. aureus* and coagulase negative staphylococci can be found, mostly molecularly linked to nasal and pharyngeal carriage by operating team personnel (Edminston et al.,

The origin of germs notwithstanding, what induces ordinarily commensal germs to become pathogenic is mostly unexplained. Indeed, in spite of the frequent presence of germs around and in the surgical site (up to 4.1% deep tissue samples found positive in clean orthopedic

Antibiotic prophylaxis certainly thwarts the development of most SSIs, at least insofar as bacterial load remains low and antibiotic spectrum and pharmacokinetics are adapted to the germs and tissues involved (Classen et al., 1992). However, the occurrence of SSIs in spite of adequate prophylaxis leads to consider the mechanism of infection from a bacterial point of view. Infections are often the consequences of disequilibrium in the relationships inside the microbiota or between the microbiota and the host. SSIs are typically infections related to microbiota disequilibrium, their major etiologic agents being members of the skin microbiota such as *S. aureus,* coagulase-negative Staphylococci and *Propionibacterium acnes.* The mechanism of selection of these bacteria from the skin community remains unknown and could be highlighted by dynamic survey of the microbial re colonization by a suitable method. Exploring the microbiota disequilibrium and its dynamics should provide insights

We hereafter present the use of TTGE in culture-independent and culture-based approaches to study the dynamics of bacterial communities involved in SSIs. The study was undertaken in the setting of clean cardiothoracic surgery, because SSI rates remain unacceptably high in coronary artery bypass grafts (CABG), in spite of ongoing efforts to minimize patient- and

surgery (Byrne et al., 2007)), only very few infections actually develop.

**3.1.2 Are surgical site infections related to endogenous or exogenous bacteria?** 

don't necessarily have an endogenous origin.

**3.1.3 From contamination to infection** 

into the mechanisms involved in SWI infections.

procedure-related risk factors (Filsoufi et al., 2009).

2005).

Forty cotton swabs were collected from 5 patients who underwent CABG surgery at the Service of Thoracic and Cardiovascular Surgery of the Montpellier University Hospital (France). For each patient, swabs were sampled during intervention at the surgical site, superficially and deeply. The first sample was taken on the skin after cutaneous antisepsis just before incision and the second one in sub-cutaneous tissue once incision made. Thirdly, sternum edges were sampled after sawing. The fourth sample was mediastinal tissue after positioning of sternal retractors. At the end of the operation, mediastinum, sternum edges, sub-cutaneous tissue and skin were sampled again.
