Clermont Université

Auvergne

Thèse D'exercice Pour Le Diplome D'état De Docteur

Présentée

Par Claudel

Catinot Noubam

Tchatat Directrice

Claire Madame

MCU Aumeran

Monsieur Ousmane Traore

Monsieur Frédéric Robin

Monsieur Patrick Merle

EN MÉDECINE SPECIALITE : BIOLOGIE MEDICALE

Evaluation de l'efficacité de deux détergents-désinfectants et d'un

détergent sur un biofilm de Klebsiella pneumoniae constitué de manière statique au sein de tubes de Tygon.

Use of endoscope

Bedside cleaning

Checking endoscope functions and leaktesting

Manual cleaning

Intermediate rinsing

High-level disinfection

Rinsing

Drying

Storage of endoscopes

The transmission of infections through contaminated endoscopes is a significant public health problem. Notable risk factors for the transmission of pathogens in endoscopes include the non-compliance with reprocessing guidelines, their complex design and damage of their inner surface, and the contamination of Endoscope Washer Disinfectors (EWD) [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF].

According to the Spaulding classification, most flexible endoscopes, such as gastrointestinal or bronchial, are classified as semi-critical devices because they are in contact with the intact mucous membranes and do not usually penetrate sterile tissue [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF]. Their reprocessing requires cleaning and high-level disinfection [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF] Flexible endoscopes are expensive instruments, designed to be reused, but that remain fragile because of their complexity. If some bronchoscopes can be sterilized by steam, most of flexible endoscopes are thermo-sensitive. To obtain a satisfactory quality of reprocessing, it is necessary to ensure that the endoscope is completely immersible and irrigable, especially for its critical components (valves and pistons; crutch; handles; internal channels) [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF][START_REF]DGOS/PF2/DGS/VSS1/2016/220 du 4 juillet 2016 relative à relative au traitement des endoscopes souples thermosensibles à canaux au sein des lieux de soins -Légifrance[END_REF][START_REF]3/2018/195 du 2 août 2018 relative à l'actualisation du traitement des endoscopes souples thermosensibles à canaux de type duodénoscope au sein des structures de soins -Légifrance[END_REF].

The aim of flexible endoscopes reprocessing is to prevent infectious risks for each patient undergoing endoscopy. Recommendations have been based on microbiological studies, literature review or expert opinions but clinical trials about endoscope decontamination are rare because of the reticence to expose patients to a risk of infection [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF].

Endoscope reprocessing is divided into several steps (Figure 1). Immediately after use, all channels are rinsed and flushed with standard care water. A leak-testing is performed, and the different functions of the endoscope checked. Then a manual cleaning including the brushing of channels and followed by an intermediate rinse with standard care water is performed. The next step is the high-level disinfection performed either manually or in an Endoscope Washer Disinfector (EWD). The minimum efficiency level of high-level disinfection is bactericidal, A thorough manual cleaning of endoscope with a detergent is an essential step in the endoscope reprocessing. It allows the removal of remaining organic soils that may interfere with the effectiveness of subsequent reprocessing steps and promote biofilm formation [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF][START_REF]DGOS/PF2/DGS/VSS1/2016/220 du 4 juillet 2016 relative à relative au traitement des endoscopes souples thermosensibles à canaux au sein des lieux de soins -Légifrance[END_REF][START_REF]3/2018/195 du 2 août 2018 relative à l'actualisation du traitement des endoscopes souples thermosensibles à canaux de type duodénoscope au sein des structures de soins -Légifrance[END_REF].

It should be done as soon as possible to avoid drying of organic soils. It includes the complete immersion of the endoscope in a detergent solution followed by the cleaning of all external surfaces, valve ports, channel openings, and distal ends and the brushing of all accessible channels until there is no visible soils. The cleaning is performed with disposable devices such as soft cloths, sponges, and/or brushes designed for this purpose. After the brushing, all channels are rinsed with standard care water to remove the organic residues (blood, tissue, stool, etc.). A difference between the French and European guidelines is that a double manual cleaning is recommended in France (Table 1) [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF][START_REF]DGOS/PF2/DGS/VSS1/2016/220 du 4 juillet 2016 relative à relative au traitement des endoscopes souples thermosensibles à canaux au sein des lieux de soins -Légifrance[END_REF][START_REF]3/2018/195 du 2 août 2018 relative à l'actualisation du traitement des endoscopes souples thermosensibles à canaux de type duodénoscope au sein des structures de soins -Légifrance[END_REF].

Many publications insist on the importance of following each step of the reprocessing and especially the manual cleaning to remove organic residues and biofilm [START_REF] Da | Evaluation of the ability of different detergents and disinfectants to remove and kill organisms in traditional biofilm[END_REF][START_REF] Martiny | The importance of cleaning for the overall results of processing endoscopes[END_REF]. Their removal is important because it prevents their adhesion during the high level-disinfection step [START_REF] Kampf | Surface fixation of dried blood by glutaraldehyde and peracetic acid[END_REF]. Some authors showed the importance of the detergent associated to brushing of endoscope channels to decrease bacterial burden and organic soil. The effect of detergents varied depending on the microbial species tested or whether the detergent was enzymatic or not [START_REF] Alfa | Simulated-Use Polytetrafluorethylene Biofilm Model: Repeated Rounds of Complete Reprocessing Lead to Accumulation of Organic Debris and Viable Bacteria[END_REF][START_REF] Ribeiro | The polytetrafluoroethylene (PTFE) channel model of cyclic-buildup biofilm and traditional biofilm: The impact of friction, and detergent on cleaning and subsequent high-level disinfection[END_REF]. It was underlined that products used during manual cleaning should be able to detach the biofilm to improve next steps of reprocessing [START_REF] Marion | Using an efficient biofilm detaching agent: an essential step for the improvement of endoscope reprocessing protocols[END_REF].

During the manual cleaning step of endoscopes, at a minimum, the product used must be a detergent. Its effectiveness and compatibility with the disinfectant and the endoscope are assured by the product manufacturer, who also provides its conditions of use (temperature and contact time). The product used can be only detergent (either neutral or with an enzymatic and/or alkaline booster) or a detergent associated to a disinfectant containing antimicrobial active substances [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF]. Detergents are usually surfactants that remove organic residues and microorganisms from surfaces and dissolve them [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF].

Neutral pH detergents are widely used because of their excellent compatibility with medical devices. They are available with or without enzymatic boosters that contain one or more kind of enzymes, such as protease, amylase, or lipase. Protease breaks down proteins into smaller, more soluble subunits, amylase catalyses the breakdown of starch, and lipase breaks down debris containing fats. Detergents with an alkaline booster contain alkaline chemicals that form a mild alkaline cleaner. These alkaline substances remove the dirt and help dissolve it in the cleaning solution. Strongly alkaline cleaners in the pH range > 11 are not recommended for cleaning flexible endoscopes due to possible incompatibility with the materials of certain parts of the endoscope. Some detergents are associated with alkaline and enzymatic booster that combine their different properties [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF].

Detergents associated to a disinfectant, as known as detergent-disinfectants, contain antimicrobial active substances commonly used as, for example, amine compounds, peracetic acid and its salts, and quaternary ammonium compounds [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF]. The use of pH-optimized peracetic acid is currently under discussion due to its potential binding of proteins to surfaces [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF]. The use of aldehydes should be avoided to prevent the fixation of organic residues (3). The standard NF EN 14885 describes the European Standards required for products with a microbicidal activity. Their minimum efficiency includes a bactericidal and fungicidal activity and activity against enveloped viruses [START_REF]Afnor EDITIONS[END_REF] Despite a reprocessing performed according to the guidelines, residual soil, and microorganisms could persist in endoscopes [START_REF] Neves | Effectiveness of current disinfection procedures against biofilm on contaminated GI endoscopes[END_REF][START_REF] Ofstead | Persistent contamination on colonoscopes and gastroscopes detected by biologic cultures and rapid indicators despite reprocessing performed in accordance with guidelines[END_REF][START_REF] Aumeran | Assessment on experimental bacterial biofilms and in clinical practice of the efficacy of sampling solutions for microbiological testing of endoscopes[END_REF]. The main identified causes of failure in endoscopes reprocessing [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF][START_REF] Kovaleva | Transmission of infection by flexible gastrointestinal endoscopy and bronchoscopy[END_REF] [START_REF] Rutala | Reprocessing endoscopes: United States perspective[END_REF].

In addition, the high microbial bioburden found in endoscopes after an endoscopy could explain too why the result of reprocessing is unsatisfactory [START_REF] Chu | Natural bioburden levels detected on flexible gastrointestinal endoscopes after clinical use and manual cleaning[END_REF][START_REF] Chu | The microbial flora of the gastrointestinal tract and the cleaning of flexible endoscopes[END_REF]. Alfa et al. counted 10 6 -10 7 colony-forming units (CFU) per bronchoscope or duodenoscope and 10 8 -10 9 CFU per colonoscope after using [START_REF] Alfa | Worst-case soiling levels for patient-used flexible endoscopes before and after cleaning[END_REF]. In used colonoscopes, this count was of 7.1 to 10.3 log10 CFU per device in the suction channel, and of 4.2 to 6.2 log10 CFU per device on outer surface. It has been shown that a proper manual cleaning step could reduce the number of bacterial cells by 2 to 5 log10 CFU per device [START_REF] Chu | Natural bioburden levels detected on flexible gastrointestinal endoscopes after clinical use and manual cleaning[END_REF][START_REF] Chu | The microbial flora of the gastrointestinal tract and the cleaning of flexible endoscopes[END_REF].

Biofilm is defined as a sessile microbial community characterized by cells that are irreversibly attached to a substratum or interface or to each other, are embedded in a matrix of extracellular polymeric substances that they have produced and exhibit an altered phenotype with respect to growth rate and gene transcription [START_REF] Donlan | Biofilms: survival mechanisms of clinically relevant microorganisms[END_REF]. The biofilm provides metabolic advantages. It is constituted of several microbial species which implement a metabolic cooperation [START_REF] Christensen | Metabolic commensalism and competition in a two-species microbial consortium[END_REF]. Its architecture facilitates the supply of oxygen and nutrients to the bacteria [START_REF] Donlan | Biofilms: survival mechanisms of clinically relevant microorganisms[END_REF]. The extracellular matrix protects bacteria from environmental stresses such as ultraviolet Endoscopies, performed in an environment mixing organic and artificial materials with complex devices, are subject to multiple types and levels of contamination favouring biofilm formation (16,28) (figure 2). The main cause of biofilm development is due to a defect in the reprocessing, which leads to the persistence of organic residues and microorganisms [START_REF] Roberts | The role of biofilms in reprocessing medical devices[END_REF]. It has been shown that a residual moisture, especially over a long period, allows surviving bacteria to form a biofilm [START_REF] Roberts | The role of biofilms in reprocessing medical devices[END_REF][START_REF] Otter | Surface-attached cells, biofilms and biocide susceptibility: implications for hospital cleaning and disinfection[END_REF]. Biofilm can develop within endoscopes and EWDs even when reprocessing guidelines are followed and particularly in the case of damaged channels [START_REF] Kovaleva | Transmission of infection by flexible gastrointestinal endoscopy and bronchoscopy[END_REF]. It has also been shown that microbial survival and persistence of organic residues could be the result of repeated endoscope cleaning/disinfection cycles [START_REF] Kovaleva | Transmission of infection by flexible gastrointestinal endoscopy and bronchoscopy[END_REF][START_REF] Alfa | Modeling microbial survival in buildup biofilm for complex medical devices[END_REF]. Finally, contamination of the water system may lead to persistent colonisation of disinfection systems, such as EWD, and result in secondary contamination of the disinfected endoscopes particularly during final rinsing [START_REF] Mackay | Water, water everywhere nor any a sterile drop to rinse your endoscope[END_REF]. extended-spectrum beta-lactamase. The epidemic strain was isolated from a duodenoscope where its development was favoured by deficiencies in manual cleaning and drying before the storage of the device [START_REF] Aumeran | Multidrugresistant Klebsiella pneumoniae outbreak after endoscopic retrograde cholangiopancreatography[END_REF]. This strain belonged to sequence type 14 and to the virulent capsular serotype K2 but was relatively non-virulent. It contained a conjugative plasmid carrying three resistance genes (blaCTX-M-15, blaOXA-1 and aac(6')-Ib-cr) and harboured few virulence genes (uge, wabG, kfu and mrkD). The ability to transfer its plasmid was notable, especially in biofilm conditions. The strain was highly resistant to cefotaxime (Minimum Inhibitory Concentration or MIC 516 mg/L) and the presence of this antibiotic at sub-MIC concentrations enhanced biofilm formation. The isolate was susceptible to ofloxacin (MIC 2 mg/L), but the bactericidal effect of this antibiotic was lesser in biofilm older than 24 hours [START_REF] Hennequin | Antibiotic resistance and plasmid transfer capacity in biofilm formed with a CTX-M-15-producing Klebsiella pneumoniae isolate[END_REF]. These characteristics may have given the strain a selective advantage for successful survival in a hospital environment.

The sequence of its genome is disponible on the MaGe (Magnifying Genomes) platform under the reference Klebsiella pneumoniae SB6610.

To our knowledge, very few studies have assessed both the efficiency of detergent and of detergent-disinfectants used during the manual cleaning step of endoscopes against a multiresistant Klebisella pneumoniae strain known to produce an important biofilm. The objective of this study was to evaluate the efficiency of two detergent-disinfectants and one enzymatic detergent on the Klebsiella pneumoniae SB6610 strain biofilm that was statically build in material of endoscopes channel known as Tygon tube.

Table 2: Description of the two detergent-disinfectants and of the enzymatic detergent evaluated.

I. Bacterial Strain

The strain used was a CTX-M-15 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (Klebsiella pneumoniae SB6610) responsible for an outbreak which affected 16

patients after an ERCP [START_REF] Aumeran | Multidrugresistant Klebsiella pneumoniae outbreak after endoscopic retrograde cholangiopancreatography[END_REF].

In accordance with standard NF EN 12353, aliquots of the strain, stored at -80°C in Luria-Bertani medium containing 20% glycerol, were used to grow a stock culture on tryptone soy agar (TSA) (Biomérieux, Lyon, France) that was kept for two months at +4°C.

The 10 8 CFU/mL bacterial suspension (optical density of 0.180 at 620 nm), used to produce the biofilm, was prepared from the second or third subculture on TSA of the stock culture. For the biofilm formation, the 10 8 CFU/mL suspension of K. pneumoniae SB6610 was diluted to obtain a 10 7 CFU/mL suspension.

II. Media and solutions

The nutrient medium required for bacterial adhesion and biofilm formation was Tryptone soy broth (TSB, CM0129, Oxoid, Basingstoke, England).

The recovery solution used to recover the biofilm and make all dilutions was the BioMérieux 2).

III. CTX-M-15 Klebsiella pneumoniae SB6610 Biofilm formation

The ability of the strain to form biofilm was tested on microplate with a modified crystal violet method [START_REF] O'toole | Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development[END_REF].

The biofilm was formed in a 12 cm flexible Tygon tube (Tygon® R3603, inned diameter 3 mm, outer diameter 5 mm, Saint-Gobain Performance, Charny-Orée-de-Puisaye, France) similar to tubing used for the inner channels of flexible endoscopes.

The 12 cm sterile Tygon tube was aseptically connected to sterile single-use 3-way stopcocks at both ends. The 24-hours biofilm was formed by inoculation of the Tygon tube with 1 mL of the 10 7 CFU/mL K. pneumoniae SB6610 suspension. The tube was sealed in a sterile bag and immersed in a water bath at 30°C with stirring.

After biofilm formation, under laminar flow hood, the outside of the Tygon tube was decontaminated with 95°C Ethanol and the bacterial suspension was removed by gentle air blowing.

For each trial, two 12 cm Tygon tubes were used, one for control of biofilm biomass and one to test the action of detergent-disinfectant or enzymatic detergent. For each tested product, a biological triplicate was performed. The tubes were further divided into 3-cm sections. The use of 3-cm sections made it possible to recover enough biomass to be analysed in each portion and to perform enough replicates on the same biofilm.

IV. Detergent-disinfectants and enzymatic detergent assays.

The action of the two detergent-disinfectants and the enzymatic detergent was evaluated on a 24-hours biofilm formed in the 12-cm Tygon tube. The chemical solution was injected with a syringe into the 12-cm Tygon tube, a visual check of the absence of bubbles was made, and the 3-way stopcocks at both ends were closed. The chemical agent was removed by gentle air blowing after 10 minutes contact for the two detergent-disinfectants, either at 20°C or at 35°C, and after 1 hour contact at 45°C for the enzymatic detergent.

Detergent-disinfectants and detergent supernatants were collected in a sterile tube for further analysis.

Recovery and numeration of viable bacteria from the biofilm

We used a mechanical technique to recover the biofilm based on scraping, vortexing, and ultrasonication, as previously described [START_REF] Aumeran | Assessment on experimental bacterial biofilms and in clinical practice of the efficacy of sampling solutions for microbiological testing of endoscopes[END_REF]. The 12-cm Tygon tube was cut under laminar flow hood into 3 portions of 3 cm.

Numeration of viable bacteria in supernatants

The viable bacteria were counted in supernatant of detergent and detergent-disinfectants.

After exposure to the biofilm, the supernatant of detergent-disinfectants and detergent were recovered by gentle air blowing in a sterile tube. The supernatant was then diluted up to 10 -7 and 0.5 mL of each dilution was plated on TSA, and incubated for 24 hours at 37°C.

Expression of results.

We expressed the results in CFU/cm² or in Log10 CFU/cm² of the Tygon tube with a detection threshold of 7 CFU/cm² or 0.82 Log10 CFU/cm 2 .

For each trial, the initial biomass of biofilm in the Tygon tube was determined by the average of the bacterial count (Log10 CFU/cm 2 ) in three 3-cm portions of tube.

For assays assessing the action of detergent-disinfectants or enzymatic detergent, the logarithmic ratio was calculated for each 3-cm tube section by calculating the ratio of the bacterial count (Log10 CFU/cm 2 ) of each tube section treated (by detergent-disinfectants or enzymatic detergent) to the average bacterial count (Log10 CFU/cm 2 ) of all control samples from each trial. The logarithmic reduction was calculated for each 3-cm tube section by calculating the differential between the average bacterial count (Log10 CFU/cm2) of all control samples from each trial to the bacterial count (Log10 CFU/cm2) of each tube section treated.

The comparison of the bacterial counts, the means of the logarithmic reduction and the means of the logarithmic ratios was carried out the Kruskall-Walis test and Wilcoxon -Mann Whitney test.

I. Homogeneity of the biofilm and reproducibility of assays

The reproducibility of 24-hours static biofilm formation in Tygon tube was assessed between assays. Fifteen reproducibility assays were performed and used as control. 3-cm sections were cut from each 12-cm Tygon tubes. The mean biofilm bacterial counts corresponding to the different trials were from 6.90  0.32 to 7.90  0.15 Log10 CFU/cm 2 (Figure 3). The standard deviations associated to the mean bacterial counts were low indicating the homogeneity of biofilm formation within the Tygon tube. Also, the comparisons between the bacterial counts of each assay did not show a significant difference (p=0.11) which underlines the reproducibility of the method (Figure 3).

II. Evaluation of the action of the two detergent-disinfectants and the multienzymatic detergent on a biofilm of CTX-M-15 producing Klebsiella pneumoniae SB6610

The action of the two detergent-disinfectants and the enzymatic detergent has been assessed on a 24-hours biofilm of CTX-M-15 producing Klebsiella pneumoniae SB6610. The detergentdisinfectants were used at 20°C and 35°C during 10 mn and the enzymatic detergent was used at 45°C during 60 mn as recommended by manufacturer. Each assay was performed in triplicate.

The count of viable bacteria after exposure to detergent-disinfectants was under the detection threshold of 0.82 Log10 CFU/cm 2 (Table 3A and3B). The mean logarithmic reduction was between 6.32 and 6.71 Log10 CFU/cm 2 for the two detergent-disinfectants, which corresponds to a mean logarithmic ratio of 0.11. The difference of bacterial counts between the control group and the detergent-disinfectant group was significative (p=0.0002) in all assays (Figure 4). There was no difference between the exposure temperatures.

Bacterial growth was more important with the enzymatic detergent with a mean of 7.10

Log10 CFU/cm 2 (Table 3C). The mean logarithmic reduction was of 0.42 Log10 CFU/cm 2 which corresponds to a mean logarithmic ratio of 0.94. The difference of bacterial counts between the control group and the detergent group was not significative (p=0. [START_REF] Chu | The microbial flora of the gastrointestinal tract and the cleaning of flexible endoscopes[END_REF]) in all assays (Figure 4)

III. Evaluation of biocide effect.

No bacterial growth was found in the supernatants of the two detergent-disinfectants with results under the detection threshold of a mean under the of 0.82 Log10 CFU/cm 2 . On the other hand, viable bacteria were found in the supernatant of the enzymatic detergent with a mean of 6.6 Log10 CFU/cm 2 . Thus, a more important biocidal effect was observed in the detergentdisinfectants than in the enzymatic detergents. Δ: The logarithmic reduction was calculated for each 3-cm tube section by calculating the difference between the average bacterial count (Log10 CFU/cm 2 ) of all control samples from each trial and the bacterial count (Log10 CFU/cm 2 ) of each tube section treated (by detergentdisinfectants or enzymatic detergent). Comparisons between groups were carried out the Kruskall-Walis test with a significance level set at 0.05.

p-value of mean logarithmic reductions: EnziQure ® at 45°C versus control p = 0,19; DDN9 ® at 20°C versus control p = 0,0002; DDN9 ® at 35° versus control p = 0,0002; Aniozyme X3 ® at 20°C versus control p = 0,0002; Aniozyme X3 ® at 35°C versus control p = 0,0002.

We conducted a study about the efficiency of one enzymatic detergent, and one enzymatic and one non-enzymatic detergent-disinfectant on a biofilm of a multi-resistant Klebsiella pneumoniae strain. These three products are commonly used during the manual cleaning step of endoscopes reprocessing. The 24-hours biofilm was statically constituted in Tygon tubes that are the material of inner channels of endoscopes. Biofilm formation was homogeneous in Tygon tubes between assays. The effectiveness of products was evaluated by logarithmic reduction of the bacterial load after exposure.

We observed that the two detergent-disinfectants removed the biofilm from Tygon tubes (logarithmic reduction higher than 6 Log10 CFU/cm 2 ) and that no bacterial growth has been found in detergent-disinfectants supernatants. No difference of efficiency has been observed

between the enzymatic and the non-enzymatic detergent-disinfectant regardless of temperature use. Enzymatic detergent was less effective in removing biofilm (logarithmic reduction lower than 1 Log10 CFU/cm 2 ). A bacterial growth was observed in the enzymatic detergent supernatant. A preliminary work comparing the effectiveness between water and an enzymatic detergent showed no bacterial logarithmic reduction after exposure (data not shown), and this is why we wanted to explore both the action of detergents and detergent-disinfectants used during the manual cleaning step of endoscopes reprocessing.

Several works regarding effects of chemical products used during the manual cleaning phase has been reported in the literature but very few compare at the same time both detergentdisinfectants and detergents, with or without enzymatic agents. They are mainly carried out on biofilm, soil, or a combination of the two.

The addition of enzymes in detergent products remains a controversial subject. Their interest is to be found in the improvement of the digestion of organic residues, which would provide a significant advantage during the cleaning phase, particularly in the presence of a biofilm [START_REF] Stiefel | Enzymes Enhance Biofilm Removal Efficiency of Cleaners[END_REF][START_REF] Beilenhoff | ESGE-ESGENA guideline: Cleaning and disinfection in gastrointestinal endoscopy[END_REF]. Removal of biofilm and soil has been significantly improved with the compositions containing enzymes [START_REF] Alfa | Simulated-Use Polytetrafluorethylene Biofilm Model: Repeated Rounds of Complete Reprocessing Lead to Accumulation of Organic Debris and Viable Bacteria[END_REF][START_REF] Stiefel | Enzymes Enhance Biofilm Removal Efficiency of Cleaners[END_REF][START_REF] Gonzalez | Onsite comparison of an enzymatic detergent and a non-enzymatic detergent-disinfectant for routine manual cleaning of flexible endoscopes[END_REF]. Other studies showed a better efficiency of the non-enzymatic detergent [START_REF] Fang | A study of the efficacy of bacterial biofilm cleanout for gastrointestinal endoscopes[END_REF][START_REF] Vickery | Removal of biofilm from endoscopes: evaluation of detergent efficiency[END_REF][START_REF] Vickery | The effect of multiple cycles of contamination, detergent washing, and disinfection on the development of biofilm in endoscope tubing[END_REF]. These differences could be explained by the nature of the biofilm more or less associated to soil and by the enzymatic composition of detergents. It was shown that a biofilm of Enterococcus faecalis was more easily eliminated by an enzymatic formulation compared to a biofilm of Pseudomonas aeruginosa [START_REF] Ribeiro | The polytetrafluoroethylene (PTFE) channel model of cyclic-buildup biofilm and traditional biofilm: The impact of friction, and detergent on cleaning and subsequent high-level disinfection[END_REF]. The effectiveness of enzymatic detergents on soil could vary with: (i) the nature of their enzymatic activity (such as protease, amylase and lipase) that affect different components of soil; (ii) their number, because more than two enzymes seems improve the effectiveness; (iii) their conditioning (encapsulated or not) that could affect their stability [START_REF] Stiefel | Enzymes Enhance Biofilm Removal Efficiency of Cleaners[END_REF][START_REF] Loukili | Effectiveness of detergent-disinfecting agents on Escherichia coli 54127 biofilm[END_REF]. A better efficacy of enzymatic products after five repeated rounds of reprocessing suggested that "linker layer" of the biofilm left after using an enzymatic cleaner was less stable than that left after using a non-enzymatic cleaner [START_REF] Alfa | Simulated-Use Polytetrafluorethylene Biofilm Model: Repeated Rounds of Complete Reprocessing Lead to Accumulation of Organic Debris and Viable Bacteria[END_REF][START_REF] Verran | The relationship between substratum surface roughness and microbiological and organic soiling: A review[END_REF].

Few works evaluated the action of enzymatic and non-enzymatic detergent-disinfectants.

Loukili et al. evaluated fifteen detergent-disinfectants on a biofilm of Escherichia coli constituted only on glass. Five of these products were enzymatic versions of other products tested. Three non-enzymatic detergent-disinfectants showed low efficacy, six showed moderate efficacy, and six showed high efficacy. The addition of enzymes improved the efficacy of only three of five products [START_REF] Loukili | Effectiveness of detergent-disinfecting agents on Escherichia coli 54127 biofilm[END_REF]. It is known that the support of biofilm can have an impact on the activity of the biocide tested [START_REF] Verran | The relationship between substratum surface roughness and microbiological and organic soiling: A review[END_REF].

One study showed a superiority of enzymatic detergents over non-enzymatic detergentdisinfectants in the phase of manual cleaning of endoscopes [START_REF] Gonzalez | Onsite comparison of an enzymatic detergent and a non-enzymatic detergent-disinfectant for routine manual cleaning of flexible endoscopes[END_REF].

Our results are different because we found a better efficiency of detergent-disinfectants than of enzymatic detergents. There was no difference between enzymatic and non-enzymatic detergent-disinfectants. Our study suggests the impact of the kind of products and of their biocide effect on biofilm during the manual cleaning phase. It has been shown it they also have an impact on the global quality of endoscopes reprocessing [START_REF] Rahman | Duodenoscope-Associated Infections: Update on an Emerging Problem[END_REF]. The manual cleaning is critical to reduce the high bacterial load associated with organic soil early on endoscopes reprocessing because it prevents desiccation of organic residues, biofilm development and facilitates the high-level disinfection [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF][START_REF] Chu | Natural bioburden levels detected on flexible gastrointestinal endoscopes after clinical use and manual cleaning[END_REF]. No recommendations exist to reduce organic residues after cleaning, but it has been shown that an appropriate cleaning should reduce number of microorganisms and organic debris by 4 logs or 99.99% [START_REF] Chu | Natural bioburden levels detected on flexible gastrointestinal endoscopes after clinical use and manual cleaning[END_REF]. The cleaning phase require at least a product with detergent properties, but we showed that it could be interesting to associate a disinfectant activity.

The bacterial strain was a Klebsiella pneumoniae producing a CTX-M-15 extendedspectrum beta-lactamase that was implicated in an outbreak associated with endoscopic retrograde cholangiopancreatography (ERCP) in our center [START_REF] Aumeran | Multidrugresistant Klebsiella pneumoniae outbreak after endoscopic retrograde cholangiopancreatography[END_REF]. This strain had good biofilm producing properties, was able to survive in the hospital environment and possessed virulence factors as well as a high transfer rate antibiotic resistance plasmid [START_REF] Hennequin | Antibiotic resistance and plasmid transfer capacity in biofilm formed with a CTX-M-15-producing Klebsiella pneumoniae isolate[END_REF]. Many reports have related endoscopy to nosocomial infections [START_REF] Rahman | Duodenoscope-Associated Infections: Update on an Emerging Problem[END_REF][START_REF] Kovaleva | Transmission of infection by flexible gastrointestinal endoscopy and bronchoscopy[END_REF]. Pseudomonas aeruginosa is currently the most common organism isolated from contaminated endoscopes but reports of duodenoscopy associated outbreaks caused by multidrug-resistant Enterobacteriaceae have surfaced recently [START_REF] Rahman | Duodenoscope-Associated Infections: Update on an Emerging Problem[END_REF][START_REF] Kovaleva | Transmission of infection by flexible gastrointestinal endoscopy and bronchoscopy[END_REF]. In several reports, the bacterial persistence in endoscopes was related to biofilm formation [START_REF] Rahman | Duodenoscope-Associated Infections: Update on an Emerging Problem[END_REF][START_REF] Kovaleva | Transmission of infection by flexible gastrointestinal endoscopy and bronchoscopy[END_REF][START_REF] Aumeran | Multidrugresistant Klebsiella pneumoniae outbreak after endoscopic retrograde cholangiopancreatography[END_REF]. These events, occurred despite a reprocessing more or less adequate, lead to revisit existing guidelines and offer supplementary recommendations for duodenoscope reprocessing [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF][START_REF]DGOS/PF2/DGS/VSS1/2016/220 du 4 juillet 2016 relative à relative au traitement des endoscopes souples thermosensibles à canaux au sein des lieux de soins -Légifrance[END_REF][START_REF]3/2018/195 du 2 août 2018 relative à l'actualisation du traitement des endoscopes souples thermosensibles à canaux de type duodénoscope au sein des structures de soins -Légifrance[END_REF]. This work has some limits. The first limit is the static condition of biofilm formation without the addition of organic soil that do not reflect the reality of biofilm formation in endoscopes [START_REF] Alfa | Simulated-Use Polytetrafluorethylene Biofilm Model: Repeated Rounds of Complete Reprocessing Lead to Accumulation of Organic Debris and Viable Bacteria[END_REF]. In endoscopes, biofilm formation is promoted by a stronger absorption of hydrophilic bacteria to a hydrophobic surface such as Tygon [START_REF] Vickery | Removal of biofilm from endoscopes: evaluation of detergent efficiency[END_REF], by the flows through endoscope channels during use that induces shear forces enhancing biofilm adhesion [START_REF] Denyer | Microbial biofilms: formation and control[END_REF] and by the presence of organic soil that protect bacteria from disinfection and desiccation and constitute a reserve of nutrients for bacteria [START_REF]The effect of interfering substances on the disinfection process: a mathematical model -Lambert[END_REF][START_REF] Lakkis | The Effect of Organic Soil on Hydrogen Peroxide Disinfection Efficacy During Extended Storage[END_REF]. We wanted to study only the biocide efficiency of products on a preformed biofilm but not their cleaning aspect. If we had added soil, it could have represented interfering substances that could disturb the interpretation of the results. To complete our study, the same product could be evaluated on a dynamic biofilm formation associated with organic soil. The second limit is the exposure of biofilm to the products without a mechanical cleaning (such as brushing) which means that only the chemical action has been evaluated. The manual cleaning phase is systematically associated with a brushing [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF][START_REF]DGOS/PF2/DGS/VSS1/2016/220 du 4 juillet 2016 relative à relative au traitement des endoscopes souples thermosensibles à canaux au sein des lieux de soins -Légifrance[END_REF][START_REF]3/2018/195 du 2 août 2018 relative à l'actualisation du traitement des endoscopes souples thermosensibles à canaux de type duodénoscope au sein des structures de soins -Légifrance[END_REF]. In our study, the absence of a mechanical cleaning could be a cause of the poor efficiency of enzymatic detergent. It has been shown that the mechanical cleaning is important for the success of the manual cleaning phase because it add a physical removal of biofilm to the chemical action [START_REF] Beilenhoff | Reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: Position Statement of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology Nurses and Associates (ESGENA) -Update 2018[END_REF][START_REF] Ribeiro | The polytetrafluoroethylene (PTFE) channel model of cyclic-buildup biofilm and traditional biofilm: The impact of friction, and detergent on cleaning and subsequent high-level disinfection[END_REF]. We nevertheless applied a flush to remove the detergent from the Tygon tube which can mimic a small mechanical shearing action. The third limit is the evaluation of biofilm only by bacterial culture and microbial count. We did not use imaging techniques such as scanning electron microscopy to visualise biofilm and confocal microscopy to observe viable but non-cultivable bacteria. The majority of studies have shown a good correlation between the bacterial Our study evaluated the effectiveness of two detergents-disinfectants and one detergent, usually used during the manual cleaning phase of endoscopes reprocessing, on the reduction of a multi-drug resistant Klebsiella pneumoniae CTX-M-15 biofilm. The 24-hours biofilm was statically constituted in Teflon tubes before its exposure to the products and the bacterial logarithmic reduction was calculated.

The biofilm production was homogeneous and comparable between the different assays. We showed an efficiency of the two detergent-disinfectants on the reduction of bacterial load because no growth has been found on Tygon after their exposure. Moreover, any viable bacteria were found in the supernatant of the both detergent-disinfectants, which underlines the effectiveness of their biocide effect. On the contrary, no effectiveness in decreasing the bacterial load has been observed after exposure to the enzymatic detergent on the Tygon and viable bacteria were found in the supernatant of the detergent.

Our results demonstrate the importance of the choice of the products used during the manual cleaning phase of endoscopes reprocessing. Guidelines recommend at least a product with detergent activity particularly to remove organic soil while combining brushing. Yet the bacterial load post-endoscopy can be high and embedded in a biofilm. So, our study shows that combining detergent and disinfectant activity can be interesting to decrease this bacterial load which facilitate the disinfection step of endoscope reprocessing. It indicates that the guidelines on manual cleaning of endoscopes should be strictly followed but that once constituted the biofilm in endoscope tubing will be very difficult to eradicate using only a detergent (even enzymatic) product.

Our findings are probably not generalizable due to the tested products and the specificities of the Klebsiella pneumoniae strain that been involved in an outbreak from contaminated Results: Biofilm production was homogeneous between the assays. The bacterial count was below the detection limit on Tygons after exposure to detergent-disinfectants and the log reduction was between 6.32 and 6.1 Log10 CFU/cm 2 . No viable bacteria were found in their supernatants. No impact of the exposure temperature was found on the detergent-disinfectant group. On the contrary, no decrease of the bacterial load was observed on the Tygon after exposure to the enzymatic detergent and viable bacteria were found in the supernatant.

Conclusions:

Our results demonstrate the importance of the choice of products used during the manual cleaning phase. They also show the interest of combining a detergent and disinfectant activity to decrease the bacterial load during the manual cleaning phase.

INTRODUCTION 13 Figure 1 :

 131 Figure 1: Steps of endoscope reprocessing.

Figure

  Figure adapted from Rahman MR, Perisetti A, Coman R, Bansal P, Chhabra R, Goyal H.

Figure 2

 2 Figure 2 Biofilm in scanning electron microscopy at different magnifications. Biofilm was developed in PTFE channels overnight and then subjected to full cleaning and

  The most common infections after endoscopy are endogenous infections involving the patients own flora. Exogenous infections are associated with contaminated endoscopes and could theoretically prevented. Failures in manual cleaning are known to be the cause of endoscope associated outbreaks[START_REF] Rahman | Duodenoscope-Associated Infections: Update on an Emerging Problem[END_REF]. Between 2008 and 2009, at the University Hospital Center (CHU) of Clermont-Ferrand, occurred an outbreak of nosocomial infections related to endoscopic retrograde cholangiopancreatography (ERCP). Over nine months, 16 patients presented either an infection (eight bacteraemia and four biliary tract infections) or a colonisation (four faecal carriages) with a Klebsiella pneumoniae producing a CTX-M-15

  DNP + Thiosulfate solution (Biomérieux, Lyon, France) containing Polysorbate 80, lecithin, histidine, and thiosulfate. This solution allows a dilution and a neutralisation of the main disinfectants. Three different chemical agents were used: one enzymatic detergent-disinfectant Aniozyme X3  (Anios, Lezennes, France); one non-enzymatic detergent-disinfectant DDN 9  (Franklab, Montigny le Bretonneux, France) and one multi-enzymatic detergent multi-enzymatic detergent PHAGO'CLEAN enziQure  (Phagogène, OneLife, Louvain-la-Neuve, Belgium). According to the manufacturer's recommendations the two detergents-disinfectants were used at two different temperatures (20°C and 35°C) during 10 minutes, and the enzymatic detergent was used à 45°C during 60 minutes (Table

Each 3 -

 3 cm portion was flushed with 10 mL of DNP + Thiosulfate solution (Biomérieux®, Marcy-L'Etoile, France) then immersed in this recovery solution before being divided half lengthwise and scraped with a scalpel. The 10-ml solution and the two halves of each Tygon tube portion were vortexed together two times (Vortex Genie, shake 8, Scientific Industries inc, Bohemia, USA) for 3 minutes, interspersed with ultrasonication (Branson 2210, Bransonic, Danbury, USA) for 5 minutes. The solution was then diluted up to 10 -7 and 0.5 mL of each dilution was plated on TSA, and incubated for 24 hours at 37°C.

Figure 3

 3 Figure 3 Bacterial recovery of 24-hours biofilm from 15 assays on Tygon tubes. For each

Figure 4 :

 4 Figure 4: Mean logarithmic reduction after of viable bacteria from 24-hours biofilm of CTX-

  Our results are only valid for the chemical products formulations used and the bacterial species tested because the Klebsiella pneumoniae SB6610 showed a selective advantage for successful survival in a hospital environment. Further research is needed on the physico-chemical mechanisms of biofilm adhesion and elimination particularly during the manual cleaning phase in endoscopes reprocessing. It could enable the optimisation of detergents and detergent-disinfectants composition.

  duodenoscopes and showed a selective advantage for successful survival in a hospital environment. A perspective of this work would be to expose different biofilm-producing strains embedded in organic soil at different detergent and detergent-disinfectants, whether enzymatic or not. CONCLUSION Nous avons évalué l'efficacité de deux détergents-désinfectants et d'un détergent, utilisés lors du nettoyage manuel des endoscopes, sur un biofilm de Klebsiella pneumoniae productrice d'une béta-lactamase à spectre étendu de type CTX-M-15. Le biofilm de 24 heures a été constitué de manière statique au sein de tubes de Tygon avant d'être exposé aux produits testés. Les résultats étaient exprimés par la réduction logarithmique de la biomasse bactérienne. La production du biofilm était homogène et comparable entre les essais. Aucune croissance bactérienne n'était observée sur le Tygon après exposition aux deux détergent-désinfectants montrant l'efficacité sur la réduction de la charge bactérienne. Aucune bactérie vivante n'était retrouvée dans le surnageant des détergent-désinfectants soulignant leur effet biocide. En revanche, la charge bactérienne ne diminuait pas après exposition au détergent enzymatique et des bactéries vivantes étaient retrouvées dans le surnageant du détergent. Nos résultats montrent l'importance du choix des produits utilisés lors de la phase de nettoyage manuel des endoscopes. Les recommandations préconisent l'utilisation d'un produit au minimum détergent pour éliminer les souillures organiques en lui associant un brossage mécanique. Post-endoscopie, la charge bactérienne présente dans l'endoscope est importante et potentiellement sous la forme d'un biofilm. Notre étude montre que combiner une activité détergente et désinfectante est intéressant pour diminuer la charge bactérienne afin de faciliter l'étape de désinfection des endoscopes. Cela souligne la nécessité de respecter les recommandations de nettoyage manuel des endoscopes mais aussi qu'éradiquer un biofilm constitué peut être difficile en utilisant uniquement un produit détergent (même enzymatique). Ces résultats ne sont probablement pas généralisables du fait des types de produits testés et des spécificités de cette souche de Klebsiella pneumoniae qui a été impliquée dans une épidémie d'infections nosocomiales liées à des duodenoscopes contaminés et a montré un avantage sélectif pour une survie en milieu hospitalier. Une des perspectives de ce travail serait d'évaluer l'action de différents produits détergents et détergents-désinfectants, enzymatiques ou non, sur différentes souches productrices de biofilm en association à des souillures organiques Contexte et objectifs de l'étude : Les endoscopes peuvent être des vecteurs de transmission d'agents pathogènes et être à l'origine d'épidémie d'infection nosocomiale. Ils sont soumis à un nettoyage et une désinfection de haut niveau (DHN) après chaque utilisation. Le nettoyage manuel est une étape importante de la DHN. Il permet d'éviter la formation de biofilm et doit être effectué avec des produits ayant une action détergente. L'objectif de cette étude était d'évaluer l'efficacité de deux détergents-désinfectants et d'un détergent enzymatique, utilisés lors du nettoyage manuel des endoscopes sur un biofilm de Klebsiella pneumoniae. Matériel et méthode : Des tests en Triplicat technique et biologique ont été effectués sur un biofilm de 24 heures constitué de manière statique au sein de tubes de Tygon. Après exposition du biofilm aux détergents-désinfectants à 20°C et 35°C, et au détergent enzymatique à 45°C, un compte des bactéries viables a été effectué sur le Tygon, et la réduction logarithmique ainsi que le ratio logarithmique de la biomasse bactérienne ont été calculés. Par la suite, une numération des bactéries viables dans le surnageant des produits a été effectuée. Résultats : La formation du biofilm était homogène entre les essais. Le compte bactérien des Tygons après exposition aux détergents-désinfectants était en dessous du seuil de détection, la réduction logarithmique était entre 6.32 et 6.1 Log10 UFC/cm 2 . Aucune bactérie viable n'était retrouvée dans leurs surnageants. La température d'exposition n'avait pas d'influence sur l'efficacité des détergents-désinfectants. Aucune diminution de la charge bactérienne n'était retrouvée après exposition au détergent enzymatique et des bactéries viables ont été retrouvées dans le surnageant.Conclusions : Nos résultats montrent l'importance du choix des produits utilisés lors de la phase de nettoyage manuelle et l'intérêt de combiner une activité détergente et désinfectante pour la diminution de la charge bactérienne.Background and objectives of the study: Endoscopes can be vectors for the transmission of pathogens and can be the cause of nosocomial infection outbreaks. Cleaning and High-level disinfection (HLD) of endoscopes are performed after each use. Manual cleaning is an important step in HLD. It prevents the formation of biofilm and should be performed with a product have at least a detergent property. The objective of this study was to evaluate the effectiveness of two detergent-disinfectants and an enzymatic detergent used during the manual cleaning phase of endoscope on a Klebsiella pneumoniae biofilm.Materials and methods: Three technical and biological replicates assays were performed on a 24-hour biofilm was statically constituted in Tygon tubes. A bacterial count was performed on the Tygon and the bacterial logarithmic reduction and logarithmic ratio were calculated after exposure detergent-disinfectants at 20°C and 35°C, and enzymatic detergent at 45°C to the biofilm. Subsequently, a count of viable bacteria in the supernatant of the products was performed.

Table 1 :

 1 Comparison of French and European guidelines about endoscopes reprocessing (2-4).

	Bedside cleaning	France	ESGE
	External surface: Wipe using a soft, disposable cloth/sponge	X	X
	Air/water channel: Aspiration / Insufflation
	Standard care water	X
	Sterile water		X
	Instrument/suction channel:
	Aspiration / insufflation with standard care water	X
	flushing with a detergent		X
	Transported to the reprocessing room.	X	X
	Manual cleaning Single cleaning (ESGE) and Double cleaning (France)	France	ESGE
	Leak test before immersion	X	X
	Detergent or detergent-disinfectant without Aldehyde	X	X
	1st cleaning in detergent or detergent-disinfectant solution:	X	X
	Cleaning (Brushing) of all external surfaces	X	X
	Disassembly and cleaning (Brushing) valve ports, channel openings, distal tips	X	X
	Flushing of all channels	X	X
	Brushing of all accessible channels	X	X
	Elimination of the detergent or detergent-disinfectant solution	X	X
	1st Rinse +

canal irrigation with standard care water X 2nd cleaning in new detergent or detergent-disinfectant solution: X

  fungicidal, mycobactericidal and virucidal. Although sporicidal activity is not required, the EN ISO 15 883-4 standard recommends that the disinfectant used in EWD be sporicidal for a prolonged contact time at a minimum concentration and temperature (5). The last steps are terminal rinsing with bacteriologically controlled water and drying. Drying can be done before the storage and/or in storage cabinets with a drying function (Table1) (2-4).

	Full immersion of the endoscope.	X
	Flushing of all channels	X
	Purge all channels	X
	Elimination of the detergent or detergent-disinfectant solution	X
	Intermediate rinsing	France	ESGE
	Rinsing of external surfaces and all channels with drinking water		X
	Channel irrigation and external rinsing with standard care water	X
	Purge water from air channels	X
	Cleaning and disinfection in endoscope washer-disinfector	France	ESGE
	Connection to the EWD according to the manufacturer's instruction	X	X
	Steps: Initial rinsing, cleaning, intermediate rinsing, High level-disinfection, final rinsing, drying	X	X
	Water quality:
	Bacteriologically controlled water at each step	X
	Drinking water for intermediate rinsing		X
	Sterile filtered water for final rinsing		X
	Drying after disinfection et before storage	France	ESGE
	Compressed air specially provided for drying channels and outer surfaces	X	X
	Storage of endoscopes	France	ESGE
	Storage cabinets with/without a drying function	X	X

Table 3 :

 3 Impact of detergent-disinfectants and detergent on 24-h biofilm of CTX-M-15

	producing Klebsiella pneumoniae SB6610
	SD, standard deviation; CFU, colony forming unit
	3A Exposure to non-enzymatic detergent-disinfectant DDN9 ®
			Bacterial count from each section of Teflon tube (log10 CFU/cm 2 )
		Exposure at 20°C during 10 mn	Exposure at 35°C during 10 mn
	Section	Assay Count Ratio Reduction Section	Assay	Count Ratio Reduction
	1	A2	0,82	0,11	6,65	1	A3	0,82	0,11 6,71
	2	A2	0,82	0,11	6,65	2	A3	0,82	0,11 6,71
	3	A2	0,82	0,11	6,65	3	A3	0,82	0,11 6,71
	4	B2	0,82	0,11	6,65	4	B3	0,82	0,11 6,71
	5	B2	0,82	0,11	6,65	5	B3	0,82	0,11 6,71
	6	B2	0,82	0,11	6,65	6	B3	0,82	0,11 6,71
	7	C2	0,82	0,11	6,65	7	C3	0,82	0,11 6,71
	8	C2	0,82	0,11	6,65	8	C3	0,82	0,11 6,71
	9	C2	0,82	0,11	6,65	9	C3	0,82	0,11 6,71
	Mean		0,82	0,11	6,65	Mean		0,82	0,11 6,71
	SD		0,00			SD		0,00
	Controls					Controls
	Mean	7,47				Mean	7,53
	SD	0,07				SD	0,09
	3B Exposure to enzymatic detergent-disinfectant Aniozyme X3 ®
			Bacterial count from each section of Teflon tube (log10 CFU/cm 2 )
		Exposure at 20°C during 10 mn	Exposure at 35°C during 10 mn
	Section	Assay Count Ratio Reduction Section	Assay	Count Ratio Reduction
	1	A4	0,82	0,11	6,32	1	A5	0,82	0,11	6,43
	2	A4	0,82	0,11	6,32	2	A5	0,82	0,11	6,43
	3	A4	0,82	0,11	6,32	3	A5	0,82	0,11	6,43
	4	B4	0,82	0,11	6,32	4	B5	0,82	0,11	6,43
	5	B4	0,82	0,11	6,32	5	B5	0,82	0,11	6,43
	6	B4	0,82	0,11	6,32	6	B5	0,82	0,11	6,43
	7	C4	0,82	0,11	6,32	7	C5	0,82	0,11	6,43
	8	C4	0,82	0,11	6,32	8	C5	0,82	0,11	6,43
	9	C4	0,82	0,11	6,32	9	C5	0,82	0,11	6,43
	mean		0,82	0,11	6,32	mean		0,82	0,11	6,43
	SD		0,00			SD		0,00
	Controls					Controls
	Mean	7,14				Mean	7,25
	SD	0,14				SD	0,10

Table 3 (

 3 Suite): Impact of detergent-disinfectants and detergent on 24-h biofilm of CTX-M-

	15 producing Klebsiella pneumoniae SB6610
	SD, standard deviation; CFU, colony forming unit
	3C Exposure to enzymatic detergent EnziQure ®
	Bacterial count from each section of Teflon tube (log10 CFU/cm 2 )
	Section	Assay	Count	Ratio	Reduction
	1	A1	7,19	0,96	0,33
	2	A1	7,23	0,96	0,29
	3	A1	7,30	0,97	0,22
	4	B1	7,26	0,97	0,26
	5	B1	7,07	0,94	0,45
	6	B1	6,78	0,90	0,74
	7	C1	7,33	0,97	0,19
	8	C1	7,18	0,96	0,34
	9	C1	6,55	0,87	0,97
	Mean		7,10	0,94	0,42
	SD		0,09
	Controls
	Mean	7,52
	SD	0,16

Remerciements

Aux membres de mon jury de thèse,

LIST OF ABREVIATIONS

 DHN : Désinfection de haut niveau.  EWD : Endoscope Washer Disinfectors.  HLD: High-level disinfection.

 ERCP: Endoscopic retrograde cholangiopancreatography

MATERIALS AND METHODS

RESULTS

DISCUSSION

quantification and the presence of biofilm on imaging, but Stiefel et al. showed a discrepancy for some non-enzymatic detergents that reduce the number of bacteria with poor biofilm removal [START_REF] Stiefel | Enzymes Enhance Biofilm Removal Efficiency of Cleaners[END_REF]. We used a technique of recovery and evaluation of biofilm that has been validated in the laboratory and was considered as robust and mastered by our team [START_REF] Aumeran | Assessment on experimental bacterial biofilms and in clinical practice of the efficacy of sampling solutions for microbiological testing of endoscopes[END_REF][START_REF] Cholley | Klebsiella pneumoniae survival and regrowth in endoscope channel biofilm exposed to glutaraldehyde and desiccation[END_REF] as shown by the homogeneity of our controls.

BIBLIOGRAPHY