The biofilm is the most common form found in nature for many bacterial species. To increase their probability of survival in their environment, bacteria secrete a layer of extracellular polymeric substances (EPS) (1–3). The particular architecture of the biofilm effectively protects the bacteria from external environmental aggressions such as UV irradiation, antibiotics and disinfection. These bacterial species are more resistant than planktonic bacteria. (4–6). These characteristics make it difficult to remove the biofilm. Several methods have been reported for the analysis of biofilms.(3). If biofilm persists on surgical instruments or medical implants, living bacteria can lead to hospital-acquired infections, resulting in public health problems and increased hospital costs. (7, 8). For example, flexible endoscopes used in gastroenterology are ideal surfaces for biofilm growth. Many viable bacteria have been found on endoscopes despite the cleaning, disinfection and sterilization process in hospitals. (9, 10).
From clinical point of view, biofilm occurs in several situation. For instance biofilm growth occurs in the lungs of cystic fibrosis patients (11). The biofilm structure acts as a shield and protects the bacteria from the antimicrobials. In patients undergoing mechanical ventilation, the formation of biofilm on endotracheal tubes is an early and frequent event. Moreover, high-grade biofilm formation on an endotracheal tube is associated with the development of ventilator-associated pneumonia (12).
Regarding infections associated with biomaterials (BAI), the main source of contamination is the patient's skin. The bacterial flora of human skin consists mainly of Staphylococcus epidermidis and Staphylococcus aureus. When a medical device is implanted, contact with the skin is sufficient to contaminate the implant (13). Fragile patients with comorbidities are the most susceptible to nosocomial infections. All implants are at risk of being colonized by bacteria. Studies find 60%-70% of nosocomial infections caused by contaminated medical implants. (14). Contamination of the medical implant can lead to device malfunction, systemic infection by hematogenous spread of the bacterial agent, and even to tissue destruction resulting in severe disease and death. (15).
All medical implants are at risk of bacterial colonization and infection such as cardiac prostheses, orthopedic implants, silicone breast implants, dental implants, intravascular catheters, artificial pumps left ventricular assist devices, pacemakers, vascular prostheses, cerebrospinal fluid shunts, urinary catheters, voice prostheses, ocular prostheses, contact lenses and intrauterine contraceptive devices (16, 17).
Several challenges are encountered when attempting to treat infections related to biofilms covering medical implants. These include chronic infection, impaired wound healing and acquired antibiotic resistance. The biofilm grows and can lead to the dissemination of infectious emboli. (14, 15, 18). When an implant is placed, the human body identifies the implant as a foreign body. A physiological balance is established between the host (the human body) and the implant. This phenomenon, called biocompatibility, can be seriously compromised if bacteria adhere to the surface of the implant, which can lead to a form of rejection of the implant. (19). For example, infections related to orthopedic implants can result in osteomyelitis with destruction of the bone and surrounding soft tissue. Bone is a very poorly vascularized tissue, which makes treatment of these infections with antibiotics difficult and ineffective (20–23). Thus, treatment of infections in orthopedic devices requires a multi-step procedure. In the first stage, the infected implant is removed, the patient is treated for infection, and then a new device is implanted in the second stage when no further signs of infection are present. This multi-stage procedure results in high morbidity with bed rest, cardiovascular problems and difficulty walking.
Capsular contracture (CC) is the contraction of fibrotic scar tissue around the silicone breast implant. It is the most common complication of breast augmentation. It can lead to asymmetry, pain, and its treatment requires a surgical revision (24). Studies have reported incidence rates of CC ranging from 1.3 to 45% (25–28). The fibrotic tissue around the implants was analyzed by scanning electron microscopy confirming the presence of bacterial biofilm. The most common germ found in capsular contracture was Staphylococcus epidermidis (29). The severity of capsular contracture is assessed according to the Baker scale. It has been shown that the higher the Baker grade, the higher the number of bacteria in the human periprosthetic capsule (30) and in the porcine model (31). In 2011, the FDA alerted to a strong association between large cell anaplastic lymphoma (BIA-ALCL) and textured breast implants (32). This is a rare non-Hodgkin's T-cell or null lymphoma first described by Stein and colleagues (33). The clinical symptomatology of this pathology is common and misleading with the appearance of a late peri-implant seroma (the pathology occurs on average after 8 years of implant placement) containing malignant cells in one breast. Occasionally, a tumor mass attached to the capsule may be found. Lymph node involvement is found in 5 to 10% of patients. The pathophysiology is not yet elucidated, but a serious hypothesis focuses on infection by the biofilm, associated with a genetic predisposition of the patient. The chronic inflammation caused by the periprosthetic bacterial biofilm activates the immune response, which activates T lymphocytes and triggers polyclonal proliferation. This chronic inflammation can lead to monoclonal proliferation of T lymphocytes, which can lead to the development of ALCL (34). It was found that bacterial adhesion to silicone is significantly higher than to polyurethane or Teflon (35).
To avoid as much as possible this kind of complications, new materials limiting the adhesion of bacteria are currently being studied. The prevention of biofilm formation in medical implants can be controlled by following various novel emergent strategies like polymer coatings, antimicrobial coatings, nanostructured coatings, surface modifications, and biosurfactants. Non antibiotic based therapies are proposed such as enzyme-mediated approaches, phage therapy or immunotherapy, (36).
Nevertheless, it is essential to correctly quantify the biofilm on new biomaterials to determine their ability to avoid biofilm formation and to compare them with the current ones.
Biofilm analysis:
Several approaches have been developed to study biofilm (37), including bacterial counting, colorimetric methods with dyes (crystal violet, SYTO9 staining) and imaging methods such as optical microscopy, electron microscopy, fluorescence microscopy, and confocal microscopy. These methods provide different kind of information that seem sometimes incoherent. For instance a discrepancy between biofilm size and number of viable bacteria has been reported (38).
The adhesion of microorganisms to prosthetic surfaces reduces their detection (39). Therefore, to measure viable bacteria present in the biofilm, detaching efficiently the biofilm surrounding the implants is essential. The detachment procedure must effectively detach and separate individual cells to generate reliable colony forming units (CFU) values (40) while maintaining their cultivability (3, 37). Furthermore, most studies use scrapping, enzymatic or ultrasonic detachment procedures (41–43). Despite microbiology culture techniques’ play a key role in diagnosing these complex implant-related infections there is a universal lack of standardized and shared procedures for microbiological sampling and processing (39).
Biofilm removal methods:
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Sonication is ultrasonic energy applied to the biomaterial surface to disrupt adherent biofilm (41). There are two types of sonication: direct sonication via a tip coming into direct contact with the implant and indirect sonication with the implant placed in a water bath. In this study, the indirect sonication method was used.
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Enzymatic techniques attempt to break chemical bonds in the extracellular matrix of the biofilm to detach bacteria (43). Digest-EUR® is a mucolytic composed of dithiothreitol for rapid digestion and mucus fluidification (44).
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Mechanized bead mill process: the implants are placed in a sterile tube with 3ml of distilled water and 1 mm-diameter stainless steel beads before the agitation bead mill (6000 rpm) with the Ultra Turrax® Tube Drive disposal (45).
In this study, artificial biofilm was formed on four different medical implants of interest for our daily clinical and/or research practice. We investigated the best conventional technic to dislodge the biofilm on the implants and quantify the number of bacteria. The type of implants selected for the study included i.silicone implants, ii catheters and iii.endotracheal tube.
i.Silicone implants are widely used for breast augmentation and breast reconstruction. Bacterial biofilms have been implicated with breast implant complications, including capsular contracture (46–49), and breast implant-associated anaplastic large-cell lymphoma (BI-ALCL) (50).
ii.Catheter related infections are a major cause of morbidity and mortality worldwide. In the United States, 250,000 hospital-acquired bloodstream infections per year have been reported and 23,000 related to central venous catheter infection in 2009 (51). Another study conducted in the USA reported a mortality rate of 27% in catheter-associated bacteremia (all types) (52). A peripherally inserted central venous catheter (PICC-line) is an intravenous access that can be used for a prolonged period for chemotherapy regimens, extended antibiotic therapy, or total parenteral nutrition.
iii. Endotracheal tubes are used daily for ventilation during surgery under general anesthesia but also in intensive care units for invasive ventilation. High-grade biofilm formation on an endotracheal is associated with the development of ventilator-associated pneumonia (12, 53) We currently perform a study to compare the microbiote detected in biofilm that developed in vivo on endotracheal tubes and the bacteria responsible for pneumonia acquired under mechanical ventilation.