ESCMID∗ guideline for the diagnosis and treatment of biofilm infections 2014

Open ArchivePublished:January 14, 2015DOI:https://doi.org/10.1016/j.cmi.2014.10.024

      Abstract

      Biofilms cause chronic infections in tissues or by developing on the surfaces of medical devices. Biofilm infections persist despite both antibiotic therapy and the innate and adaptive defence mechanisms of the patient. Biofilm infections are characterized by persisting and progressive pathology due primarily to the inflammatory response surrounding the biofilm. For this reason, many biofilm infections may be difficult to diagnose and treat efficiently. It is the purpose of the guideline to bring the current knowledge of biofilm diagnosis and therapy to the attention of clinical microbiologists and infectious disease specialists. Selected hallmark biofilm infections in tissues (e.g. cystic fibrosis with chronic lung infection, patients with chronic wound infections) or associated with devices (e.g. orthopaedic alloplastic devices, endotracheal tubes, intravenous catheters, indwelling urinary catheters, tissue fillers) are the main focus of the guideline, but experience gained from the biofilm infections included in the guideline may inspire similar work in other biofilm infections. The clinical and laboratory parameters for diagnosing biofilm infections are outlined based on the patient’s history, signs and symptoms, microscopic findings, culture-based or culture-independent diagnostic techniques and specific immune responses to identify microorganisms known to cause biofilm infections. First, recommendations are given for the collection of appropriate clinical samples, for reliable methods to specifically detect biofilms, for the evaluation of antibody responses to biofilms, for antibiotic susceptibility testing and for improvement of laboratory reports of biofilm findings in the clinical microbiology laboratory. Second, recommendations are given for the prevention and treatment of biofilm infections and for monitoring treatment effectiveness. Finally, suggestions for future research are given to improve diagnosis and treatment of biofilm infections.

      Keywords

      Introduction

      Bacteria and fungi occur as individual, free-floating (planktonic) cells or clustered together in aggregates of cells (biofilms). A microbial biofilm is ‘a structured consortium of microbial cells surrounded by a self-produced polymer matrix’ [
      • Hall-Stoodley L.
      • Stoodley P.
      • Kathju S.
      • Høiby N.
      • Moser C.
      • Costerton J.W.
      • et al.
      Towards diagnostic guidelines for biofilm-associated infections.
      ] In addition to microorganisms, components from the host, such as fibrin, platelets or immunoglobulins, may be integrated into the biofilm matrix. Both bacteria and fungi can cause biofilm infections and biofilms may consist of one microorganism or be polymicrobial [
      • Hall-Stoodley L.
      • Stoodley P.
      • Kathju S.
      • Høiby N.
      • Moser C.
      • Costerton J.W.
      • et al.
      Towards diagnostic guidelines for biofilm-associated infections.
      ,
      • Ramage G.
      • Robertson S.N.
      • Williams C.
      Strength in numbers: antifungal strategies against fungal biofilms.
      ,
      • Lebeaux D.
      • Chauhan A.
      • Rendueles O.
      • Beloin C.
      From in vitro to in vivo models of bacterial biofilm-related infections.
      ,
      • Bjarnsholt T.
      • Ciofu O.
      • Molin S.
      • Givskov M.
      • Høiby N.
      Applying insights from biofilm biology to drug development—can a new approach be developed?.
      ]. Some biofilms adhere to natural or artificial surfaces in the host (including devices), while others may consist of aggregates associated with but not directly adherent to the surface (Fig. 1) [
      • Lebeaux D.
      • Chauhan A.
      • Rendueles O.
      • Beloin C.
      From in vitro to in vivo models of bacterial biofilm-related infections.
      ,
      • Bjarnsholt T.
      • Ciofu O.
      • Molin S.
      • Givskov M.
      • Høiby N.
      Applying insights from biofilm biology to drug development—can a new approach be developed?.
      ]. Importantly, biofilms are characterized by physiological and biochemical gradients (nutrients, oxygen, growth rate, genetics) from the surface to the deeper layers of the aggregated structure. Biofilm-growing microorganisms express different specific properties compared with planktonically (= non-aggregated) growing microorganisms [
      • Bjarnsholt T.
      • Ciofu O.
      • Molin S.
      • Givskov M.
      • Høiby N.
      Applying insights from biofilm biology to drug development—can a new approach be developed?.
      ]. The observation of aggregated microbes surrounded by a self-produced matrix adhering to surfaces or located in tissues or secretions is as old as microbiology since both Leeuwenhoek and Pasteur described the phenomenon [
      • Høiby N.
      A personal history of research on microbial biofilms and biofilm infections.
      ]. In environmental and technical microbiology, biofilms were already shown, 80–90 years ago, to be important for biofouling on submerged surfaces, e.g. ships. The concept of biofilm infections and their importance in medicine is, however, less than 40 years old and started by Høiby’s observations of heaps of Pseudomonas aeruginosa cells in sputum and lung tissue from chronically infected cystic fibrosis (CF) patients and Jendresen’s observations of acquired dental pellicles [
      • Høiby N.
      A personal history of research on microbial biofilms and biofilm infections.
      ]. The term biofilm was introduced into medicine in 1985 by Costerton [
      • Høiby N.
      A personal history of research on microbial biofilms and biofilm infections.
      ]. During the following decades it became obvious, that biofilm infections are widespread in medicine and odontology, and their importance is now generally accepted [
      • Høiby N.
      A personal history of research on microbial biofilms and biofilm infections.
      ].
      Figure thumbnail gr1
      FIG. 1Typical biofilm infections (3) (reproduced with permission).
      Biofilms typically cause chronic infections, which means that the infections persist despite apparently adequate antibiotic therapy and the host’s innate and adaptive defence mechanisms. Chronic infections are characterized by persistent and progressing pathology, mainly due to the inflammatory response surrounding the biofilm [
      • Høiby N.
      • Bjarnsholt T.
      • Givskov M.
      • Molin S.
      • Ciofu O.
      Antibiotic resistance of bacterial biofilms.
      ]. This also means that persisting local inflammation is the only common feature of various biofilm infections, whereas other signs and symptoms depend on the impairment—if any—of the function of the organ/foreign body infected by a microbial biofilm [
      • Høiby N.
      A personal history of research on microbial biofilms and biofilm infections.
      ,
      • Høiby N.
      • Bjarnsholt T.
      • Givskov M.
      • Molin S.
      • Ciofu O.
      Antibiotic resistance of bacterial biofilms.
      ]. The inflammatory cells dominating the persisting biofilm infection may be polymorphonuclear neutrophil leucocytes or mononuclear cells depending on whether the predominating immune response to the microbes of the biofilm is T helper type 2 (antibodies) or T helper type 1 (cell-mediated) polarized [
      • Høiby N.
      A personal history of research on microbial biofilms and biofilm infections.
      ,
      • Bjarnsholt T.
      • Jensen P.O.
      • Fiandaca M.J.
      • Pedersen J.
      • Hansen C.R.
      • Andersen C.B.
      • et al.
      Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
      ]. Many biofilm infections are difficult to diagnose and treat and currently no guidelines exist to help clinicians and clinical microbiologists with these infections.
      Most microorganisms in the environment grow as biofilms in order to be protected against the hostile environment [
      • Stoodley P.
      • Sauer K.
      • Davies D.G.
      • Costerton J.W.
      Biofilms as complex differentiated communities.
      ]. Traditionally, clinical microbiology laboratories have focused on culturing and testing planktonically (= non-aggregated) growing microorganisms and have reported the susceptibility to various antibiotics and antiseptics under planktonic growth conditions. Microorganisms in the biofilm mode of growth, however, cause chronic infections in tissues and on the surface of medical devices. The clinical implications of microorganisms growing as biofilms are that they may be more difficult to recover from clinical samples, and that they are physiologically much more resistant to the effects of antibiotics and disinfectants [
      • Høiby N.
      • Bjarnsholt T.
      • Givskov M.
      • Molin S.
      • Ciofu O.
      Antibiotic resistance of bacterial biofilms.
      ]. Moreover, antibiotic therapy based on susceptibility testing of planktonic microorganisms may be associated with treatment failure or recurrence of the infection. It is the purpose of the guideline to bring the current knowledge of biofilm diagnosis and therapeutic practice to the attention of clinical microbiologists and infectious disease specialists.
      The guideline addresses the diagnosis and treatment of biofilm infections. Such infections can be classified into those where biofilms are found in the tissue/mucus and not associated with foreign bodies and those where biofilms are adherent to foreign bodies [
      • Lebeaux D.
      • Chauhan A.
      • Rendueles O.
      • Beloin C.
      From in vitro to in vivo models of bacterial biofilm-related infections.
      ] (Fig. 1). The first group includes CF patients with chronic lung infections (FIG. 2, FIG. 3) and patients with chronic wound infections (Fig. 4), where the biofilms are found in the tissue and also in sputum of patients with CF [
      • Bjarnsholt T.
      • Jensen P.O.
      • Fiandaca M.J.
      • Pedersen J.
      • Hansen C.R.
      • Andersen C.B.
      • et al.
      Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
      ,
      • Kirketerp-Møller K.
      • Jensen P.O.
      • Fazli M.
      • Madsen K.G.
      • Pedersen J.
      • Moser C.
      • et al.
      Distribution, organization, and ecology of bacteria in chronic wounds.
      ,
      • Bjarnsholt T.
      • Alhede M.
      • Alhede M.
      • Eickhardt-Sorensen S.R.
      • Moser C.
      • Kuhl M.
      • et al.
      The in vivo biofilm.
      ]. The second group includes infections related to biofilms colonizing devices implanted inside the body or forming a connection between an inner or outer surface of the body where a normal microbial flora is present and a sterile anatomical compartment inside the body (FIG. 1, FIG. 5, FIG. 6, FIG. 7, FIG. 8, FIG. 9; Table 1). This group includes infections associated with orthopaedic alloplastic devices [
      • Gristina A.G.
      • Costerton J.W.
      Bacterial adherence to biomaterials and tissue. The significance of its role in clinical sepsis.
      ], endotracheal tubes [
      • Adair C.G.
      • Gorman S.P.
      • Feron B.M.
      • Byers L.M.
      • Jones D.S.
      • Goldsmith C.E.
      • et al.
      Implications of endotracheal tube biofilm for ventilator-associated pneumonia.
      ,
      • Friedland D.R.
      • Rothschild M.A.
      • Delgado M.
      • Isenberg H.
      • Holzman I.
      Bacterial colonization of endotracheal tubes in intubated neonates.
      ,
      • Gil-Perotin S.
      • Ramirez P.
      • Marti V.
      • Sahuquillo J.M.
      • Gonzalez E.
      • Calleja I.
      • et al.
      Implications of endotracheal tube biofilm in ventilator-associated pneumonia response: a state of concept.
      ,
      • Inglis T.J.
      • Lim T.M.
      • Ng M.L.
      • Tang E.K.
      • Hui K.P.
      Structural features of tracheal tube biofilm formed during prolonged mechanical ventilation.
      ], intravenous catheters [
      • Mermel L.A.
      • Allon M.
      • Bouza E.
      • Craven D.E.
      • Flynn P.
      • O'Grady N.P.
      • et al.
      Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America.
      ], indwelling urinary catheters or urethral stents [
      • Conway L.J.
      • Larson E.L.
      Guidelines to prevent catheter-associated urinary tract infection: 1980 to 2010.
      ,
      • Hooton T.M.
      • Bradley S.F.
      • Cardenas D.D.
      • Colgan R.
      • Geerlings S.E.
      • Rice J.C.
      • et al.
      Diagnosis, prevention, and treatment of catheter-associated urinary tract infection in adults: 2009 International Clinical Practice Guidelines from the Infectious Diseases Society of America.
      ] and tissue fillers [
      • Christensen L.
      • Breiting V.
      • Bjarnsholt T.
      • Eickhardt S.
      • Hogdall E.
      • Janssen M.
      • et al.
      Bacterial infection as a likely cause of adverse reactions to polyacrylamide hydrogel fillers in cosmetic surgery.
      ,
      • Bjarnsholt T.
      • Tolker-Nielsen T.
      • Givskov M.
      • Janssen M.
      • Chrsitensen L.
      Detection of bacteria by FISH in culture-negative soft tissue filler lesions.
      ] including, but not restricted to, breast implants [
      • Rieger U.M.
      • Mesina J.
      • Kalbermatten D.F.
      • Haug M.
      • Frey H.P.
      • Pico R.
      • et al.
      Bacterial biofilms and capsular contracture in patients with breast implants.
      ]. Many other biofilm infections are the subject of existing guidelines (e.g. endocarditis, otitis media, chronic sinusitis, biliary stents, shunts, contact lenses, voice prostheses, dialysis catheters, dental, intrauterine devices, artificial hearts, prosthetic valves). However, the present guideline will focus on the biofilm infections outlined above. The target professionals are clinical microbiologists and infectious disease specialists involved in diagnosis and treatment of biofilm infections.
      Figure thumbnail gr2
      FIG. 2Biofilm causing tissue infection. Biofilms of P. aeruginosa from sputum of cystic fibrosis patients. Gram-staining (a-e), PNA-FISH staining with a P. aeruginosa specific probe (f, g). The bacteria and the matrix are visible. A diversity of shapes of the biofilms are seen, a: with surrounding polymorphonuclear leukocytes (arrows), b & c: with a few leukocytes within the alginate matrix, d: with channel-like holes (arrow), e: with liberated planktonic bakteria (arrow). Magnification x 1000
      [
      • Bjarnsholt T.
      • Jensen P.O.
      • Fiandaca M.J.
      • Pedersen J.
      • Hansen C.R.
      • Andersen C.B.
      • et al.
      Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
      ]
      (reproduced with permission).
      Figure thumbnail gr3
      FIG. 3P. aeruginosa from sputum of a cystic fibrosis patient. Mucoid (large) and nonmucoid (small) colonies. The mucoid variant over-produces alginate which is the matrix in the P. aeruginosa biofilm in the respiratory tract of cystic fibrosis patients. Mucoid colonies are only found in patients with chronic biofilm infection and alginate from mucoid colonies is therefore a biofilm-specific antigen.
      Figure thumbnail gr4
      FIG. 4Biofilm causing tissue infection. Biofilm of S. aureus in a chronic wound. The bacteria were identified by a specific PNA-FISH probe (green stain) and the host cells visualised by DAPI (blue stain). Magnification x 1000
      [
      • Kirketerp-Møller K.
      • Jensen P.O.
      • Fazli M.
      • Madsen K.G.
      • Pedersen J.
      • Moser C.
      • et al.
      Distribution, organization, and ecology of bacteria in chronic wounds.
      ]
      (reproduced with persission).
      Figure thumbnail gr5
      FIG. 5Biofilm causing device-related infection and as the focus for systemic infection. A biofilm is located on a pancreatic-biliary stent from a 57 years old man. The biofilm was the focus of recurrent sepsis with E. coli (July 21-2003) where he was treated with adequate (planktonic susceptibility testing) antibiotics for 5 days and with the same E. coli strain and K. oxytoca (August 12, 2003) where he succumbed in spite of antibiotic treatment. Autopsy on August 14, 2003 showed growth from the biofilm of E. coli, K. oxytoca (same Pulsed Field Gel Electrophoresis (PFGE) type as isolated from the blood previously) and E. faecium which was not isolated from the blood. The figure with red arrows shows the stent held above a 9 cm petri dish with blood agar (upper left), the stent which has been opened (right), Methylene- and Gram-stained smears (magnification x 1000) and results of culture and susceptibility testing and PFGE. (111)(Reproduced with permission).
      Figure thumbnail gr6
      FIG. 6Biofilm causing device-related infection. PNA-FISH micrograph of catheter biofilm. Material scraped from a urinary catheter was hybridized with a universal bacterial probe (UUBmix) and an Enterobacteriaceae probe. Single cells and clusters of enterobacteria are visible in the biofilm. The large green background indicates unspecific binding of the EUBmix probe to the biofilm. E. faecalis and E. coli were isolated from the catheter tip by culture (82)(reproduced with permission).
      Figure thumbnail gr7
      FIG. 7Biofilm causing device-related infection. Three-dimensional confocal laser scanning microscopy of a biopsy from a grade 3 reaction following gel injection into the cheek 2 years previously. A PNA-FISH universal bacterial probe was used to visualize aggregates of bacteria (small red micropheres; arrows). The large blue dots represent DAPI nuclear counterstain of the predominating mononuclear cell types in these lessions. S. epidermidis had been identified by 16S rRNA gene sequencing. Magnification x 1000 (19)(reproduced with permission).
      Figure thumbnail gr8
      FIG. 8Microscopy studies of the endotracheal tube of a pig invasively mechanically ventilated for 72 hours, following oropharyngeal challenge with Pseudomonas aeruginosa (Li Bassi et al. Anesthesiology, accepted, 2013). a: Light microscopy of biofilm and respiratory secretions retrieved from the inner surface of the endotracheal tube (magnification x1000, oil immersion objective lens). Biofilm/secretions were spread on a glass slide, and stained with Congo Red and Crystal Violet for light microscopic studies
      [
      • Berra L.
      • De M.L.
      • Yu Z.X.
      • Laquerriere P.
      • Baccarelli A.
      • Kolobow T.
      Endotracheal tubes coated with antiseptics decrease bacterial colonization of the ventilator circuits, lungs, and endotracheal tube.
      ]
      . The black arrow indicates an aggregate of rod-shaped bacteria; microorganisms stain as purple, and the biofilm exopolysaccharide stains as pink. b: Confocal laser scanning micrograph of the internal surface of the endotracheal tube (magnification x250). The sample was stained with BacLight Live/Dead (Invitrogen, Barcelona, Spain). The white arrow depicts the endotracheal tube outer surface. A fully mature biofilm adherent to the endotracheal tube is shown and rod-shaped bacteria are embedded within the biofilm matrix. c: Scanning electron micrograph frontal-view of the internal surface of the endotracheal tube (magnification x1500) (Fernández-Barat et al. Crit Care Med 2012; 40: 2385-95)( Berra et al. Anesthesiology 2004; 100: 1446-56). Note presence of stage IV biofilm, characterized by multiple rodshaped bacteria embedded within an extracellular polymeric substance, as depicted by the white arrow. (Micrographs kindly provided by Laia Fernandez-Barat and Eli Aguilera Xiol, Hospital Clinic-CIBERES, Barcelona, Spain.)
      Figure thumbnail gr9
      FIG. 9Field emission scanning electron micrograph of a polymicrobial biofilm developed in the lumen of a Foley catheter removed from a patient affected by a catheter-associated urinary tract infection. The species identified by culture methods were Acinetobacter baumannii, Enterococcus faecalis and Escherichia coli (G. Donelli).
      TABLE 1Natural and pathogenic biofilms on human tissue and foreign bodies
      Organ/anatomic compartment A with normal flora (microbiome)Connection [may be via a foreign body]Organ/anatomic compartment B without normal flora (microbiome)
      Skin->Blood, peritoneum
      Pharynx->Bronchi, lungs
      Duodenum->Bile tract, pancreas
      Urethra->Bladder
      Vagina->Uterus
      Air in operating room, skin flora
      a Most frequently coagulase negative staphylococci and Propionibacteria, which exist as biofilms on detached epidermal cells.
      ->Alloplastic material, neurosurgical shunt
      No symptoms->Pathology
      Reproduced from ref.
      • Hall-Stoodley L.
      • Stoodley P.
      • Kathju S.
      • Høiby N.
      • Moser C.
      • Costerton J.W.
      • et al.
      Towards diagnostic guidelines for biofilm-associated infections.
      with permission.
      a Most frequently coagulase negative staphylococci and Propionibacteria, which exist as biofilms on detached epidermal cells.
      The overall burden of biofilm infections is significant [
      • Wolcott R.D.
      • Rhoads D.D.
      • Bennett M.E.
      • Wolcott B.M.
      • Gogokhia L.
      • Costerton J.W.
      • et al.
      Chronic wounds and the medical biofilm paradigm.
      ]. For example, among the >60 000 CF patients in developed western countries, nearly 80% will develop a chronic biofilm lung (and paranasal sinus) infection. For patients with chronic wound infections (1–2% of western populations) more than 60% have been shown to involve biofilms. For all patients with orthopaedic alloplastic device, 0.5–2% will develop an infection within the first two postoperative years [
      • Stoodley P.
      • Kathju S.
      • Hu F.Z.
      • Erdos G.
      • Levenson J.E.
      • Mehta N.
      • et al.
      Molecular and imaging techniques for bacterial biofilms in joint arthroplasty infections.
      ,
      • Johannsson B.
      • Taylor J.
      • Clark C.R.
      • Shamsuddin H.
      • Beekmann S.E.
      • Polgreen P.
      Treatment approaches to prosthetic joint infections: results of an Emerging Infections Network survey.
      ,
      • Cataldo M.A.
      • Petrosillo N.
      • Cipriani M.
      • Cauda R.
      • Tacconelli E.
      Prosthetic joint infection: recent developments in diagnosis and management.
      ]. Additionally, among patients with intravenous catheters, 5 per 1000 catheter days in the intensive care unit will develop catheter-related bloodstream infections (CR-BSI). The development of biofilm on the surface of endotracheal tubes (ETT) is related to the development of ventilator-associated pneumonia (VAP), which occurs in 9–27% of all intubated patients [
      Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia.
      ]. The rates for patients with indwelling urinary catheters are even higher because more than 50% of inserted catheters become colonized within the first 10–14 days of insertion [
      • Stickler D.J.
      Bacterial biofilms in patients with indwelling urinary catheters.
      ,
      • Hola V.
      • Ruzicka F.
      The formation of poly-microbial biofilms on urinary catheters.
      ] and for patients with tissue fillers where 1–2% experience adverse events of which almost all arise from biofilm infections. Postmastectomy tissue-expander infections occur in 2–24% of patients [
      • Viola G.M.
      • Raad II,
      • Rolston K.V.
      Breast tissue expander-related infections: perioperative antimicrobial regimens.
      ].
      For many of the above infections—with the possible exception of CF—there are controversies and uncertainties concerning both the diagnosis of biofilm infections and treatment or prophylactic approaches using antibiotics. These issues have been discussed in depth in all six American Society for Microbiology and three European Society for Clinical Microbiology and Infectious Diseases (ESCMID) conferences on biofilm infections from 1996 to 2013. These conferences and discussions about biofilm infections, as well as numerous textbooks, emphasize that in clinical settings the diagnosis and treatment of biofilm infections is inconsistent. This calls attention to the need for the development of evidence-based guidelines.
      For some of the biofilm infections mentioned above, e.g. CF [
      • Döring G.
      • Conway S.P.
      • Heijerman H.G.
      • Hodson M.E.
      • Høiby N.
      • Smyth A.
      • et al.
      Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus.
      ,
      • Döring G.
      • Høiby N.
      Early intervention and prevention of lung disease in cystic fibrosis: a European consensus.
      ,
      • Kerem E.
      • Conway S.
      • Elborn S.
      • Heijerman H.
      Standards of care for patients with cystic fibrosis: a European consensus.
      ,
      • Heijerman H.
      • Westerman E.
      • Conway S.
      • Touw D.
      • Döring G.
      Inhaled medication and inhalation devices for lung disease in patients with cystic fibrosis: a European consensus.
      ,
      • Döring G.
      • Flume P.
      • Heijerman H.
      • Elborn J.S.
      Treatment of lung infection in patients with cystic fibrosis: current and future strategies.
      ], intravenous catheters [
      • Mermel L.A.
      • Allon M.
      • Bouza E.
      • Craven D.E.
      • Flynn P.
      • O'Grady N.P.
      • et al.
      Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America.
      ], indwelling urinary catheters or urethral stents [
      • Conway L.J.
      • Larson E.L.
      Guidelines to prevent catheter-associated urinary tract infection: 1980 to 2010.
      ,
      • Hooton T.M.
      • Bradley S.F.
      • Cardenas D.D.
      • Colgan R.
      • Geerlings S.E.
      • Rice J.C.
      • et al.
      Diagnosis, prevention, and treatment of catheter-associated urinary tract infection in adults: 2009 International Clinical Practice Guidelines from the Infectious Diseases Society of America.
      ,
      • Siddiq D.M.
      • Darouiche R.O.
      New strategies to prevent catheter-associated urinary tract infections.
      ,
      • Bonkat G.
      • Rieken M.
      • Rentsch C.A.
      • Wyler S.
      • Feike A.
      • Schafer J.
      • et al.
      Improved detection of microbial ureteral stent colonisation by sonication.
      ] and periprosthetic joint infections [
      • Osmon D.R.
      • Berbari E.F.
      • Berendt A.R.
      • Lew D.
      • Zimmerli W.
      • Steckelberg J.M.
      • et al.
      Diagnosis and management of prosthetic joint infection: clinical practice guidelines by the Infectious Diseases Society of America.
      ,
      • Zimmerli W.
      • Trampuz A.
      • Ochsner P.E.
      Prosthetic-joint infections.
      ], there is sufficient scientific evidence of good quality to allow development of a guideline document. For others such as chronic wounds [
      • Kirketerp-Møller K.
      • Jensen P.O.
      • Fazli M.
      • Madsen K.G.
      • Pedersen J.
      • Moser C.
      • et al.
      Distribution, organization, and ecology of bacteria in chronic wounds.
      ,
      • James G.A.
      • Swogger E.
      • Wolcott R.
      • Pulcini E.D.
      • Secor P.
      • Sestrich J.
      • et al.
      Biofilms in chronic wounds.
      ,
      • Bjarnsholt T.
      • Kirketerp-Møller K.
      • Jensen P.O.
      • Madsen K.G.
      • Phipps R.
      • Krogfelt K.
      • et al.
      Why chronic wounds will not heal: a novel hypothesis.
      ] and tissue fillers [
      • Christensen L.
      • Breiting V.
      • Bjarnsholt T.
      • Eickhardt S.
      • Hogdall E.
      • Janssen M.
      • et al.
      Bacterial infection as a likely cause of adverse reactions to polyacrylamide hydrogel fillers in cosmetic surgery.
      ], there is good quality evidence for diagnostic approaches but probably not yet for therapy. As for VAP associated with ETT biofilm, in all available studies [
      • Adair C.G.
      • Gorman S.P.
      • Feron B.M.
      • Byers L.M.
      • Jones D.S.
      • Goldsmith C.E.
      • et al.
      Implications of endotracheal tube biofilm for ventilator-associated pneumonia.
      ,
      • Gil-Perotin S.
      • Ramirez P.
      • Marti V.
      • Sahuquillo J.M.
      • Gonzalez E.
      • Calleja I.
      • et al.
      Implications of endotracheal tube biofilm in ventilator-associated pneumonia response: a state of concept.
      ,
      • Inglis T.J.
      • Millar M.R.
      • Jones J.G.
      • Robinson D.A.
      Tracheal tube biofilm as a source of bacterial colonization of the lung.
      ,
      • Sottile F.D.
      • Marrie T.J.
      • Prough D.S.
      • Hobgood C.D.
      • Gower D.J.
      • Webb L.X.
      • et al.
      Nosocomial pulmonary infection: possible etiologic significance of bacterial adhesion to endotracheal tubes.
      ], the ETT biofilm has been evaluated following extubation. Hence, no reliable methods are currently available to detect ETT biofilm while the patient remains on invasive mechanical ventilation. Conversely, a few preventive and therapeutic strategies to reduce ETT biofilm formation and VAP have been tested in clinical settings [
      • Berra L.
      • Coppadoro A.
      • Bittner E.A.
      • Kolobow T.
      • Laquerriere P.
      • Pohlmann J.R.
      • et al.
      A clinical assessment of the Mucus Shaver: a device to keep the endotracheal tube free from secretions.
      ,
      • Kollef M.H.
      • Afessa B.
      • Anzueto A.
      • Veremakis C.
      • Kerr K.M.
      • Margolis B.D.
      • et al.
      Silver-coated endotracheal tubes and incidence of ventilator-associated pneumonia: the NASCENT randomized trial.
      ,
      • Biel M.A.
      • Sievert C.
      • Usacheva M.
      • Teichert M.
      • Wedell E.
      • Loebel N.
      • et al.
      Reduction of endotracheal tube biofilms using antimicrobial photodynamic therapy.
      ,
      • Liu W.
      • Zuo Z.
      • Ma R.
      • Zhang X.
      Effect of mechanical cleaning of endotracheal tubes with sterile urethral catheters to reduce biofilm formation in ventilator patients.
      ].
      The expert panel has completed a systematic review of the literature within the specific areas outlined above and prepared this document based on the systematic reviews including key questions concerning diagnosis, prophylaxis and treatment of biofilm infections and has evaluated the strength of the recommendations and quality of evidence (TABLE 2, TABLE 3).
      TABLE 2Definition of strength and quality of recommendations
      • Ullmann A.J.
      • Cornely O.A.
      • Donnelly J.P.
      • Akova M.
      • Arendrup M.C.
      • Arikan-Akdagli S.
      • et al.
      ESCMID* guideline for the diagnosis and management of Candida diseases 2012: developing European guidelines in clinical microbiology and infectious diseases.
      Table thumbnail fx1
      TABLE 3General features of clinical and laboratory indications for diagnosis of biofilm infections
      a Specific features depend on possible impairment of the function of the organ/foreign body involved.
      • Clinical signs of infection e.g. the classical but frequently low-grade inflammatory reactions tumor, rubor, dolor, loss of function and sometimes low-grade fever
      • Medical history of biofilm-predisposing condition (e.g. implanted medical device, cystic fibrosis)
      • Persisting infection lasting >7 days (this is unspecific, and other reasons are frequent such as resistance to the antibiotics used)
      • Failure of antibiotic treatment and recurrence of the infection (particularly if evidence is provided that the same organism is responsible on multiple time points)—typing of the pathogen
      • Documented evidence/history of antibiotic failure
      • Evidence of systemic signs and symptoms of infection that resolve with antibiotic therapy, only to recur after therapy has ceased.
      • Microbiological diagnostics:
      • Microscopic evidence from fluid/tissue samples obtained from the focus of the suspected infection
        • Microscopy revealing the presence of microbial aggregates and biofilm structure (smear or fluid sample, but ideally from tissue sample if possible)
        • Microscopy revealing evidence of microbial aggregates co-localized with inflammatory cells
        • Microbiological evidence of aggregated microorganisms consistent with infectious aetiology
      • Positive culture/non-culture-based techniques (PCR) of fluid or tissue sample
        • Culture-based identification of microbial pathogens (MALDI-TOF)
        • Presence of mucoid colonies or small colony variants of P. aeruginosa in culture positive samples—which may indicate antibiotic recalcitrance)
        • PCR, quantitative PCR or multiplex PCR positive results for pathogen associated with infection (e.g. Staphylococcus aureus with implant, Pseudomonas aeruginosa with cystic fibrosis)
        • fluorescence in situ hybridization positive results for known pathogen showing aggregated microoganisms
        • Non-culture-based identification of microbial pathogen (pyrosequencing, next-generation sequencing).
      • Specific immune response to identified microorganism—(e.g. P. aeruginosa antigens in cystic fibrosis patients) if the biofilm infection has been present for more than 2 weeks.
      Modified from ref.
      • Hall-Stoodley L.
      • Stoodley P.
      • Kathju S.
      • Høiby N.
      • Moser C.
      • Costerton J.W.
      • et al.
      Towards diagnostic guidelines for biofilm-associated infections.
      .
      a Specific features depend on possible impairment of the function of the organ/foreign body involved.

      Diagnosis of Biofilm Infections

      Generally: Biofilms are small in vivo, in tissues 4–200 μm, on foreign bodies 5–1200 μm (Table 4) [
      • Bjarnsholt T.
      • Alhede M.
      • Alhede M.
      • Eickhardt-Sorensen S.R.
      • Moser C.
      • Kuhl M.
      • et al.
      The in vivo biofilm.
      ]. Therefore, the search for biofilms in clinical samples may be difficult and time consuming, and may result in false-negative results if the samples are not representative of the focus of the biofilm infection. Biofilms can be released from artificial surfaces by sonication of fluids containing the sample [
      • Zhai Z.
      • Li H.
      • Qin A.
      • Liu G.
      • Liu X.
      • Wu C.
      • et al.
      Sonication fluid of prosthetic components for the diagnosis of infection after total joint arthroplasty: a meta-analysis.
      ].
      TABLE 4Biofilm size in chronic infections in otherwise sterile parts of the human body
      a The biofilm aggregate size was estimated by measuring the longest diameter or length directly on the micrograph images in the source articles. (adapted from ref.
      • Bjarnsholt T.
      • Alhede M.
      • Alhede M.
      • Eickhardt-Sorensen S.R.
      • Moser C.
      • Kuhl M.
      • et al.
      The in vivo biofilm.
      ).
      (See also Table 3)
      Biofilm demonstrated inVisualization methodApproximate diameterReference
      Lung infections (CF)Light microscopy∼4–8 μm
      • Høiby N.
      Pseudomonas aeruginosa infection in cystic fibrosis. Diagnostic and prognostic significance of Pseudomonas aeruginosa precipitins determined by means of crossed immunoelectrophoresis. A survey.
      Light microscopy∼5–100 μm
      • Baltimore R.S.
      • Christie C.D.
      • Smith G.J.
      Immunohistopathologic localization of Pseudomonas aeruginosa in lungs from patients with cystic fibrosis. Implications for the pathogenesis of progressive lung deterioration.
      FISH∼5–100 μm
      • Bjarnsholt T.
      • Jensen P.O.
      • Fiandaca M.J.
      • Pedersen J.
      • Hansen C.R.
      • Andersen C.B.
      • et al.
      Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
      FISH∼5–50 μm
      • Lefmann M.
      • Schweickert B.
      • Buchholz P.
      • Gobel U.B.
      • Ulrichs T.
      • Seiler P.
      • et al.
      Evaluation of peptide nucleic acid-fluorescence in situ hybridization for identification of clinically relevant mycobacteria in clinical specimens and tissue sections.
      Chronic woundsFISH∼5–200 μm
      • Bjarnsholt T.
      • Kirketerp-Møller K.
      • Jensen P.O.
      • Madsen K.G.
      • Phipps R.
      • Krogfelt K.
      • et al.
      Why chronic wounds will not heal: a novel hypothesis.
      FISH∼5–100 μm
      • Kirketerp-Møller K.
      • Jensen P.O.
      • Fazli M.
      • Madsen K.G.
      • Pedersen J.
      • Moser C.
      • et al.
      Distribution, organization, and ecology of bacteria in chronic wounds.
      Light and electron microscopy∼35–55 μm
      • James G.A.
      • Swogger E.
      • Wolcott R.
      • Pulcini E.D.
      • Secor P.
      • Sestrich J.
      • et al.
      Biofilms in chronic wounds.
      Soft tissue fillersFISH∼5–25 μm
      • Bjarnsholt T.
      • Tolker-Nielsen T.
      • Givskov M.
      • Janssen M.
      • Chrsitensen L.
      Detection of bacteria by FISH in culture-negative soft tissue filler lesions.
      Otitis mediaFISH∼15–25 μm
      • Hall-Stoodley L.
      • Hu F.Z.
      • Gieseke A.
      • Nistico L.
      • Nguyen D.
      • Hayes J.
      • et al.
      Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media.
      FISH∼10–80 μm
      • Nistico L.
      • Kreft R.
      • Gieseke A.
      • Coticchia J.M.
      • Burrows A.
      • Khampang P.
      • et al.
      Adenoid reservoir for pathogenic biofilm bacteria.
      FISH∼4–40 μm
      • Homøe P.
      • Bjarnsholt T.
      • Wessman M.
      • Sørensen H.C.
      • Johansen H.K.
      Morphological evidence of biofilm formation in Greenlanders with chronic suppurative otitis media.
      Implant-associatedElectron microscopy∼500 μm
      • Marrie T.J.
      • Nelligan J.
      • Costerton J.W.
      A scanning and transmission electron microscopic study of an infected endocardial pacemaker lead.
      FISH∼50 μm
      • Waar K.
      • Degener J.E.
      • van Luyn M.J.
      • Harmsen H.J.
      Fluorescent in situ hybridization with specific DNA probes offers adequate detection of Enterococcus faecalis and Enterococcus faecium in clinical samples.
      Electron microscopy∼5–15 μm
      • Costerton J.W.
      • Post J.C.
      • Ehrlich G.D.
      • Hu F.Z.
      • Kreft R.
      • Nistico L.
      • et al.
      New methods for the detection of orthopedic and other biofilm infections.
      FISH∼5–30 μm
      • Veeh R.H.
      • Shirtliff M.E.
      • Petik J.R.
      • Flood J.A.
      • Davis C.C.
      • Seymour J.L.
      • et al.
      Detection of Staphylococcus aureus biofilm on tampons and menses components.
      Catheter- and shunt-associatedElectron microscopy∼5–1000 μm
      • Marrie T.J.
      • Noble M.A.
      • Costerton J.W.
      Examination of the morphology of bacteria adhering to peritoneal dialysis catheters by scanning and transmission electron microscopy.
      Electron microscopy∼20–500 μm
      • Marrie T.J.
      • Costerton J.W.
      Scanning and transmission electron microscopy of in situ bacterial colonization of intravenous and intraarterial catheters.
      Fluorescence microscopy∼20–1200 μm
      • Stoodley P.
      • Braxton Jr., E.E.
      • Nistico L.
      • Hall-Stoodley L.
      • Johnson S.
      • Quigley M.
      • et al.
      Direct demonstration of Staphylococcus biofilm in an external ventricular drain in a patient with a history of recurrent ventriculoperitoneal shunt failure.
      FISH and electron microscopy>1000 μm
      • Parsa K.
      • Schaudinn C.
      • Gorur A.
      • Sedghizadeh P.P.
      • Johnson T.
      • Tse D.T.
      • et al.
      Demonstration of bacterial biofilms in culture-negative silicone stent and jones tube.
      Chronic osteomyelitisElectron microscopy∼25 μm
      • Gristina A.G.
      • Oga M.
      • Webb L.X.
      • Hobgood C.D.
      Adherent bacterial colonization in the pathogenesis of osteomyelitis.
      Electron microscopy∼25 μm
      • Marrie T.J.
      • Costerton J.W.
      Mode of growth of bacterial pathogens in chronic polymicrobial human osteomyelitis.
      Light and electron microscopy∼5–50 μm
      • Sedghizadeh P.P.
      • Kumar S.K.
      • Gorur A.
      • Schaudinn C.
      • Shuler C.F.
      • Costerton J.W.
      Microbial biofilms in osteomyelitis of the jaw and osteonecrosis of the jaw secondary to bisphosphonate therapy.
      Chronic rhinosinusitisElectron microscopy∼5–30 μm
      • Cryer J.
      • Schipor I.
      • Perloff J.R.
      • Palmer J.N.
      Evidence of bacterial biofilms in human chronic sinusitis.
      Fluorescence microscopy∼5–20 μm
      • Li H.
      • Wang D.
      • Sun X.
      • Hu L.
      • Yu H.
      • Wang J.
      Relationship between bacterial biofilm and clinical features of patients with chronic rhinosinusitis.
      Contact lensesElectron microscopy∼50–100 μm
      • Stapleton F.
      • Dart J.
      Pseudomonas keratitis associated with biofilm formation on a disposable soft contact lens.
      Abbreviations: CF, cystic fibrosis; FISH, fluorescence in situ hybridization.
      a The biofilm aggregate size was estimated by measuring the longest diameter or length directly on the micrograph images in the source articles. (adapted from ref.
      • Bjarnsholt T.
      • Alhede M.
      • Alhede M.
      • Eickhardt-Sorensen S.R.
      • Moser C.
      • Kuhl M.
      • et al.
      The in vivo biofilm.
      ).

       Q1-1 Which type of samples should be sent to the clinical microbiological laboratory to detect biofilm infections?

       CF with chronic lung infections

      A major problem in this type of infection is contamination of lower respiratory secretions with the normal oropharyngeal flora, particularly since members of the normal flora (e.g. Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, Moraxella catarrhalis) are common lung pathogens in CF [
      • Goddard A.F.
      • Staudinger B.J.
      • Dowd S.E.
      • Joshi-Datar A.
      • Wolcott R.D.
      • Aitken M.L.
      • et al.
      Direct sampling of cystic fibrosis lungs indicates that DNA-based analyses of upper-airway specimens can misrepresent lung microbiota.
      ]. Representative material originating from lower airways (sputum, protected samples through bronchoalveolar lavage, endolaryngeal suction (from small children), induced sputum) should be sent to the clinical microbiological laboratory (CML) [
      • Döring G.
      • Conway S.P.
      • Heijerman H.G.
      • Hodson M.E.
      • Høiby N.
      • Smyth A.
      • et al.
      Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus.
      ,
      • Döring G.
      • Høiby N.
      Early intervention and prevention of lung disease in cystic fibrosis: a European consensus.
      ,
      • Kerem E.
      • Conway S.
      • Elborn S.
      • Heijerman H.
      Standards of care for patients with cystic fibrosis: a European consensus.
      ,
      • Heijerman H.
      • Westerman E.
      • Conway S.
      • Touw D.
      • Döring G.
      Inhaled medication and inhalation devices for lung disease in patients with cystic fibrosis: a European consensus.
      ,
      • Döring G.
      • Flume P.
      • Heijerman H.
      • Elborn J.S.
      Treatment of lung infection in patients with cystic fibrosis: current and future strategies.
      ] (AII).

       Patients with chronic wound infection

      Biopsy tissues are considered the most reliable samples to reveal biofilm in wounds. The use of swabs to collect biofilm samples from the wound surface is considered an inadequate method (DII), due to contamination from the skin flora, the strong adherence of biofilm to the host epithelium and the growth of anaerobes in the deep tissues. If a moderate to severe soft tissue infection is suspected and a wound is present, a soft tissue sample from the base of the debrided wound should be examined. If this cannot be obtained, a superficial swab may provide useful information on the choice of antibiotic therapy [
      • Percival S.L.
      • Hill K.E.
      • Williams D.W.
      • Hooper S.J.
      • Thomas D.W.
      • Costerton J.W.
      A review of the scientific evidence for biofilms in wounds.
      ,
      • Lipsky B.A.
      • Berendt A.R.
      • Cornia P.B.
      • Pile J.C.
      • Peters E.J.
      • Armstrong D.G.
      • et al.
      Executive summary: 2012 Infectious Diseases Society of America clinical practice guideline for the diagnosis and treatment of diabetic foot infections.
      ] (AII).

       Patients with infections related to an orthopaedic alloplastic device (e.g. an artificial joint)

      If an orthopaedic implant-associated infection is suspected, synovial fluid should be sampled for cell counts and microbiological work-up. If the results suggest or confirm infection, debridement surgery is indicated (AIII). Intraoperative sampling includes biopsies from representative peri-implant tissue and removal of the device/prosthesis or modular parts of it (e.g. inlay, screws). This foreign material should be submitted for sonication to liberate the biofilm before being cultured in the CML [
      • Trampuz A.
      • Piper K.E.
      • Jacobson M.J.
      • Hanssen A.D.
      • Unni K.K.
      • Osmon D.R.
      • et al.
      Sonication of removed hip and knee prostheses for diagnosis of infection.
      ] (BII). The Clinical Practice Guidelines on Diagnosis and Management of Prosthetic Joint Infection by the Infectious Diseases Society of America [
      • Osmon D.R.
      • Berbari E.F.
      • Berendt A.R.
      • Lew D.
      • Zimmerli W.
      • Steckelberg J.M.
      • et al.
      Diagnosis and management of prosthetic joint infection: clinical practice guidelines by the Infectious Diseases Society of America.
      ] recommend sampling of three to six biopsies. The presence of at least two positive culture specimens with an identical microorganism confirms periprosthetic joint infection [
      • Catelas I.
      • Wimmer M.A.
      • Utzschneider S.
      Polyethylene and metal wear particles: characteristics and biological effects.
      ,
      • Zadaka A.
      • Gioe T.
      • Gertner E.
      Acute crystal-induced arthritis following arthroplasty.
      ,
      • Parvizi J.
      • Suh D.H.
      • Jafari S.M.
      • Mullan A.
      • Purtill J.J.
      Aseptic loosening of total hip arthroplasty: infection always should be ruled out.
      ,
      • Parvizi J.
      • Jacovides C.
      • Zmistowski B.
      • Jung K.A.
      Definition of periprosthetic joint infection: is there a consensus?.
      ,
      • Parvizi J.
      • Zmistowski B.
      • Berbari E.F.
      • Bauer T.W.
      • Springer B.D.
      • Della Valle C.J.
      • et al.
      New definition for periprosthetic joint infection.
      ]. The biopsies should be as large as possible (up to 1 cm3) and labelled and paired with the culture specimens to differentiate contamination from infection [
      • Portillo M.E.
      • Salvado M.
      • Sorli L.
      • Alier A.
      • Martinez S.
      • Trampuz A.
      • et al.
      Multiplex PCR of sonication fluid accurately differentiates between prosthetic joint infection and aseptic failure.
      ,
      • Vandercam B.
      • Jeumont S.
      • Cornu O.
      • Yombi J.C.
      • Lecouvet F.
      • Lefevre P.
      • et al.
      Amplification-based DNA analysis in the diagnosis of prosthetic joint infection.
      ,
      • Corvec S.
      • Portillo M.E.
      • Pasticci B.M.
      • Borens O.
      • Trampuz A.
      Epidemiology and new developments in the diagnosis of prosthetic joint infection.
      ,
      • Zimmerli W.
      • Moser C.
      Pathogenesis and treatment concepts of orthopaedic biofilm infections.
      ] (AIII).

       Patients with endotracheal tube biofilm, VAP

      There is a lack of validated methods to monitor biofilm formation within the ETT and to distinctly recognize its role in the development of VAP. First, in mechanically ventilated patients who develop VAP, biofilm within the ETT can be either the primary source of the infection or merely a concomitant colonized site. Second, respiratory secretions often accrue within the ETT; as a result, biofilm and respiratory secretions constitute a complex mixture that is difficult to characterize. The presence of sessile pathogens adherent to the ETT surface may be overestimated, because of the vast concentration of planktonic pathogens colonizing the airway secretions. Hence laboratory and clinical studies [
      • Gristina A.G.
      • Costerton J.W.
      Bacterial adherence to biomaterials and tissue. The significance of its role in clinical sepsis.
      ,
      • Gil-Perotin S.
      • Ramirez P.
      • Marti V.
      • Sahuquillo J.M.
      • Gonzalez E.
      • Calleja I.
      • et al.
      Implications of endotracheal tube biofilm in ventilator-associated pneumonia response: a state of concept.
      ] have found matching pathogens in both the ETT biofilm and secretions accrued within the airways/ETT in 56–70% of patients with VAP. A clinical guideline on the diagnosis of VAP [
      Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia.
      ] recommends that samples of lower respiratory tract secretions should be obtained and cultured from all patients with clinical suspicion of VAP, before change of antibiotic therapy, to identify the aetiology of VAP (AIII). Respiratory secretions can be obtained through endotracheal aspirate, bronchoalveolar lavage, or protected specimen brush depending on the situation and possibilities. In addition to these sampling procedures, in patients with clinical suspicion of ETT biofilm VAP, mucus from within the ETT can be aspirated and cultured to identify ETT pathogens in ETT biofilms that may have caused VAP (BII). This is not necessary for the diagnosis of the aetiology of VAP but may lead to a decision to replace the ETT. Devices serving to remove secretions and biofilm from the inner lumen of ETT, i.e. the Mucus Shaver (described in Q2-2) [
      • Berra L.
      • Coppadoro A.
      • Bittner E.A.
      • Kolobow T.
      • Laquerriere P.
      • Pohlmann J.R.
      • et al.
      A clinical assessment of the Mucus Shaver: a device to keep the endotracheal tube free from secretions.
      ,
      • Kolobow T.
      • Berra L.
      • Li B.G.
      • Curto F.
      Novel system for complete removal of secretions within the endotracheal tube: the Mucus Shaver.
      ,
      • Mietto C.
      • Foley K.
      • Salerno L.
      • Oleksak J.
      • Pinciroli R.
      • Goverman J.
      • et al.
      Removal of endotracheal tube debris obstruction by a clearing secretion device.
      ], can be employed to dislodge biofilm and identify the aetiology of ETT biofilm VAP (BIII). Mucus and biofilm retrieved from the inner surface of the ETT can be processed in the CML for microscopic examination of the presence of bacterial aggregates and biofilm on the ETT luminal wall (Fig. 8) [
      • Berra L.
      • De M.L.
      • Yu Z.X.
      • Laquerriere P.
      • Baccarelli A.
      • Kolobow T.
      Endotracheal tubes coated with antiseptics decrease bacterial colonization of the ventilator circuits, lungs, and endotracheal tube.
      ,
      • Wilson A.
      • Gray D.
      • Karakiozis J.
      • Thomas J.
      Advanced endotracheal tube biofilm stage, not duration of intubation, is related to pneumonia.
      ] (BIII). After extubation, the ETT inner surface can also be processed in the CML for microscopic examination of the presence of biofilms [
      • Berra L.
      • Coppadoro A.
      • Bittner E.A.
      • Kolobow T.
      • Laquerriere P.
      • Pohlmann J.R.
      • et al.
      A clinical assessment of the Mucus Shaver: a device to keep the endotracheal tube free from secretions.
      ,
      • Wilson A.
      • Gray D.
      • Karakiozis J.
      • Thomas J.
      Advanced endotracheal tube biofilm stage, not duration of intubation, is related to pneumonia.
      ,
      • Fernandez-Barat L.
      • Li B.G.
      • Ferrer M.
      • Bosch A.
      • Calvo M.
      • Vila J.
      • et al.
      Direct analysis of bacterial viability in endotracheal tube biofilm from a pig model of methicillin-resistant Staphylococcus aureus pneumonia following antimicrobial therapy.
      ,
      • Fernandez-Barat L.
      • Ferrer M.
      • Sierra J.M.
      • Soy D.
      • Guerrero L.
      • Vila J.
      • et al.
      Linezolid limits burden of methicillin-resistant Staphylococcus aureus in biofilm of tracheal tubes.
      ] (Fig. 8) (BIII).

       Patients with vascular catheters

      If the catheter is removed, the catheter tip (3 to 4 cm distal) should be sent to the CML for quantitative or semi-quantitative culture [
      • Mermel L.A.
      • Allon M.
      • Bouza E.
      • Craven D.E.
      • Flynn P.
      • O'Grady N.P.
      • et al.
      Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America.
      ,
      • Brun-Buisson C.
      • Abrouk F.
      • Legrand P.
      • Huet Y.
      • Larabi S.
      • Rapin M.
      Diagnosis of central venous catheter-related sepsis. Critical level of quantitative tip cultures.
      ,
      • Maki D.G.
      • Weise C.E.
      • Sarafin H.W.
      A semiquantitative culture method for identifying intravenous-catheter-related infection.
      ] (AII). In cases of totally implantable venous access port-related infection (e.g. Port-a-Cath®), also send the reservoir and/or the port to the CML [
      • Douard M.C.
      • Arlet G.
      • Longuet P.
      • Troje C.
      • Rouveau M.
      • Ponscarme D.
      • et al.
      Diagnosis of venous access port-related infections.
      ,
      • Longuet P.
      • Douard M.C.
      • Arlet G.
      • Molina J.M.
      • Benoit C.
      • Leport C.
      Venous access port-related bacteremia in patients with acquired immunodeficiency syndrome or cancer: the reservoir as a diagnostic and therapeutic tool.
      ] (AII). In cases of signs of local infection (tunnel or port-pocket infection), send aseptically removed material surrounding the port or tunnelled catheter, such as purulent fluid or necrotic skin, ideally during the surgical procedure, to reduce the risk of false-positive results [
      • Lebeaux D.
      • Fernandez-Hidalgo N.
      • Chauhan A.
      • Lee S.
      • Ghigo J.M.
      • Almirante B.
      • et al.
      Management of infections related to totally implantable venous-access ports: challenges and perspectives.
      ,
      • Whitman E.D.
      • Boatman A.M.
      Comparison of diagnostic specimens and methods to evaluate infected venous access ports.
      ]. Superficial swabs of skin and catheter hubs are associated with a high negative predictive value for short-term as well as long-term catheters [
      • Bouza E.
      • Alvarado N.
      • Alcala L.
      • Perez M.J.
      • Rincon C.
      • Munoz P.
      A randomized and prospective study of 3 procedures for the diagnosis of catheter-related bloodstream infection without catheter withdrawal.
      ,
      • Guembe M.
      • Martin-Rabadan P.
      • Echenagusia A.
      • Camunez F.
      • Rodriguez-Rosales G.
      • Simo G.
      • et al.
      Value of superficial cultures for prediction of catheter-related bloodstream infection in long-term catheters: a prospective study.
      ] (BII).
      If catheter-related infection is suspected and the catheter is still in situ, send paired blood cultures from the vascular catheter and peripheral blood taken simultaneously. Two types of blood cultures can be used: qualitative blood cultures with data of the time to positivity (TTP) [
      • Bouza E.
      • Alvarado N.
      • Alcala L.
      • Perez M.J.
      • Rincon C.
      • Munoz P.
      A randomized and prospective study of 3 procedures for the diagnosis of catheter-related bloodstream infection without catheter withdrawal.
      ,
      • Blot F.
      • Nitenberg G.
      • Chachaty E.
      • Raynard B.
      • Germann N.
      • Antoun S.
      • et al.
      Diagnosis of catheter-related bacteraemia: a prospective comparison of the time to positivity of hub-blood versus peripheral-blood cultures.
      ,
      • Raad I.
      • Hanna H.A.
      • Alakech B.
      • Chatzinikolaou I.
      • Johnson M.M.
      • Tarrand J.
      Differential time to positivity: a useful method for diagnosing catheter-related bloodstream infections.
      ,
      • Ruhnke M.
      • Rickerts V.
      • Cornely O.A.
      • Buchheidt D.
      • Glockner A.
      • Heinz W.
      • et al.
      Diagnosis and therapy of Candida infections: joint recommendations of the German Speaking Mycological Society and the Paul-Ehrlich-Society for Chemotherapy.
      ] (AII) and quantitative blood cultures [
      • Bouza E.
      • Alvarado N.
      • Alcala L.
      • Perez M.J.
      • Rincon C.
      • Munoz P.
      A randomized and prospective study of 3 procedures for the diagnosis of catheter-related bloodstream infection without catheter withdrawal.
      ,
      • Chatzinikolaou I.
      • Hanna H.
      • Hachem R.
      • Alakech B.
      • Tarrand J.
      • Raad I.
      Differential quantitative blood cultures for the diagnosis of catheter-related bloodstream infections associated with short- and long-term catheters: a prospective study.
      ] (AII). These two methods are considered equal, but TTP is the most convenient in the routine CML because the TTP is recorded by modern blood culture systems.

       Patients with indwelling urinary catheters or urethral stents

      The simplest way to reveal catheter-associated urinary tract infections is to collect urine samples from the catheter (CI), although this method can lead to false-negative results in biofilm-positive microorganisms in 50–64% [
      • Bonkat G.
      • Rieken M.
      • Rentsch C.A.
      • Wyler S.
      • Feike A.
      • Schafer J.
      • et al.
      Improved detection of microbial ureteral stent colonisation by sonication.
      ,
      • Hachem R.
      • Reitzel R.
      • Borne A.
      • Jiang Y.
      • Tinkey P.
      • Uthamanthil R.
      • et al.
      Novel antiseptic urinary catheters for prevention of urinary tract infections: correlation of in vivo and in vitro test results.
      ,
      • Hola V.
      • Ruzicka F.
      • Horka M.
      Microbial diversity in biofilm infections of the urinary tract with the use of sonication techniques.
      ].
      Removed catheters can also be analysed in the CML by microscopy and by cultivation or culture-independent techniques, preferably after sonication to investigate microbial diversity. This approach ensures the detection of biofilm infection because of isolation of only the adherent bacteria [
      • Bonkat G.
      • Rieken M.
      • Rentsch C.A.
      • Wyler S.
      • Feike A.
      • Schafer J.
      • et al.
      Improved detection of microbial ureteral stent colonisation by sonication.
      ,
      • Hola V.
      • Ruzicka F.
      • Horka M.
      Microbial diversity in biofilm infections of the urinary tract with the use of sonication techniques.
      ,
      • Morgan S.D.
      • Rigby D.
      • Stickler D.J.
      A study of the structure of the crystalline bacterial biofilms that can encrust and block silver Foley catheters.
      ,
      • Donelli G.
      • Vuotto C.
      Biofilm-based infections in long-term care facilities.
      ,
      • Choe H.S.
      • Son S.W.
      • Choi H.A.
      • Kim H.J.
      • Ahn S.G.
      • Bang J.H.
      • et al.
      Analysis of the distribution of bacteria within urinary catheter biofilms using four different molecular techniques.
      ,
      • Frank D.N.
      • Wilson S.S.
      • St Amand A.L.
      • Pace N.R.
      Culture-independent microbiological analysis of foley urinary catheter biofilms.
      ,
      • Xu Y.
      • Moser C.
      • Al-Soud W.A.
      • Sørensen S.
      • Høiby N.
      • Nielsen P.H.
      • et al.
      Culture-dependent and -independent investigations of microbial diversity on urinary catheters.
      ] (AII).

       Patients with infections connected to tissue fillers e.g. breast implants

      Removed material and contiguous material (BIII).

       Q1-2 Which methods should be used in the CML to detect biofilms in the samples?

      Detection of biofilms in the samples requires that microscopy should show evidence of an infective process, such as the presence of leucocytes, and that the microorganisms present are demonstrated to be microbial aggregates embedded in an apparently self-produced matrix distinct from the surrounding tissue or secretion. Microscopic analysis can be done using routine light microscopy and routine staining methods including Gram stain, which stains both tissue or mucus and the inflammatory cells, bacteria and the biofilm matrix [
      • Bjarnsholt T.
      • Jensen P.O.
      • Fiandaca M.J.
      • Pedersen J.
      • Hansen C.R.
      • Andersen C.B.
      • et al.
      Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
      ] (AII). Techniques such as confocal laser scanning microscopy and scanning electron microscopy are the most appropriate to reveal biofilms in biopsies but they are not available for routine diagnostic work in CML [
      • Malic S.
      • Hill K.E.
      • Hayes A.
      • Percival S.L.
      • Thomas D.W.
      • Williams D.W.
      Detection and identification of specific bacteria in wound biofilms using peptide nucleic acid fluorescent in situ hybridization (PNA FISH).
      ] (BIII).
      Specific microscopic identification of the biofilm microorganisms in samples (biopsies or swabs) can be done by means of species-specific fluorescence in situ hybridization probes and fluorescence microscopy [
      • Bjarnsholt T.
      • Jensen P.O.
      • Fiandaca M.J.
      • Pedersen J.
      • Hansen C.R.
      • Andersen C.B.
      • et al.
      Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
      ] (AII), whereas conventional culture methods or culture-independent methods based on PCR techniques (16S rRNA gene amplification, denaturant gradient gel electrophoresis, bacterial tag-encoded FLX amplicon pyrosequencing) [
      • Cataldo M.A.
      • Petrosillo N.
      • Cipriani M.
      • Cauda R.
      • Tacconelli E.
      Prosthetic joint infection: recent developments in diagnosis and management.
      ,
      • Corvec S.
      • Portillo M.E.
      • Pasticci B.M.
      • Borens O.
      • Trampuz A.
      Epidemiology and new developments in the diagnosis of prosthetic joint infection.
      ] cannot discriminate between planktonic and biofilm-growing bacteria [
      • Donelli G.
      • Vuotto C.
      Biofilm-based infections in long-term care facilities.
      ,
      • Choe H.S.
      • Son S.W.
      • Choi H.A.
      • Kim H.J.
      • Ahn S.G.
      • Bang J.H.
      • et al.
      Analysis of the distribution of bacteria within urinary catheter biofilms using four different molecular techniques.
      ,
      • Frank D.N.
      • Wilson S.S.
      • St Amand A.L.
      • Pace N.R.
      Culture-independent microbiological analysis of foley urinary catheter biofilms.
      ,
      • Xu Y.
      • Moser C.
      • Al-Soud W.A.
      • Sørensen S.
      • Høiby N.
      • Nielsen P.H.
      • et al.
      Culture-dependent and -independent investigations of microbial diversity on urinary catheters.
      ,
      • Malic S.
      • Hill K.E.
      • Hayes A.
      • Percival S.L.
      • Thomas D.W.
      • Williams D.W.
      Detection and identification of specific bacteria in wound biofilms using peptide nucleic acid fluorescent in situ hybridization (PNA FISH).
      ].
      Furthermore, in clinical samples planktonic microorganisms are released from biofilms and microorganisms may therefore be found in both forms. Release of microorganisms from biofilms may be achieved by imprint (and visualized by Gram stain), rolling (the Maki method), and semi-quantitative reporting of the number of colony-forming units (CFU) obtained, or by scraping, whirly-mixing, vortexing and/or sonication (Brun–Buisson method) [
      • Portillo M.E.
      • Salvado M.
      • Sorli L.
      • Alier A.
      • Martinez S.
      • Trampuz A.
      • et al.
      Multiplex PCR of sonication fluid accurately differentiates between prosthetic joint infection and aseptic failure.
      ,
      • Hola V.
      • Ruzicka F.
      • Horka M.
      Microbial diversity in biofilm infections of the urinary tract with the use of sonication techniques.
      ,
      • Kobayashi H.
      • Oethinger M.
      • Tuohy M.J.
      • Procop G.W.
      • Bauer T.W.
      Improved detection of biofilm-formative bacteria by vortexing and sonication: a pilot study.
      ,
      • Bjerkan G.
      • Witso E.
      • Bergh K.
      Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro.
      ,
      • Kobayashi N.
      • Bauer T.W.
      • Tuohy M.J.
      • Fujishiro T.
      • Procop G.W.
      Brief ultrasonication improves detection of biofilm-formative bacteria around a metal implant.
      ]. Crushing or tissue homogenization may be necessary if the biofilm is situated on the surface of a foreign body or in a bone or a cardiac valve (AII). Some microorganisms in biofilms may be viable but non-culturable [
      • Penterman J.
      • Nguyen D.
      • Anderson E.
      • Staudinger B.J.
      • Greenberg E.P.
      • Lam J.S.
      • et al.
      Rapid evolution of culture-impaired bacteria during adaptation to biofilm growth.
      ] when using routine media. In that case culture-independent methods should be used [
      • Pasquaroli S.
      • Zandri G.
      • Vignaroli C.
      • Vuotto C.
      • Donelli G.
      • Biavasco F.
      Antibiotic pressure can induce the viable but non-culturable state in Staphylococcus aureus growing in biofilms.
      ,
      • Zandri G.
      • Pasquaroli S.
      • Vignaroli C.
      • Talevi S.
      • Manso E.
      • Donelli G.
      • et al.
      Detection of viable but non-culturable staphylococci in biofilms from central venous catheters negative on standard microbiological assays.
      ] (BII). In areas of the body where a normal flora (microbiome) is present (in the mouth, pharynx, gut, skin) (Table 1), special precautions—such as washing or irrigating the area—should be taken to minimize contamination from the normal flora. Since culture or culture-independent techniques (e.g. PCR) cannot distinguish between biofilm-growing and planktonic microorganisms, the identification of biofilms relies on microscopic detection of microbial aggregates located in a matrix. It should be noted that low or non-pathogenic members of the normal flora may be important biofilm producers (e.g. Staphylococcus epidermidis biofilms on intravenous lines or on orthopaedic alloplastic material). Ideally the results of microscopy and culture should be quantitative or semi-quantitative and organisms should be identified to the species level to help discriminate between contaminants from the normal flora and infecting microorganisms, regardless of whether they have previously been associated with biofilm infections.

       Practice points

      In the case of tubes, urinary tract catheters and vascular catheters, (intravenous lines) biofilms may be located on both outer and inner surfaces.

       For diagnosis of catheter-related infection

      Microscopy of Gram-stained imprints from, for example, tips can be used as rapid detection of biofilm infections [
      • Mermel L.A.
      • Allon M.
      • Bouza E.
      • Craven D.E.
      • Flynn P.
      • O'Grady N.P.
      • et al.
      Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America.
      ] (AII).
      The culture of the catheter tip can be made with a quantitative or a semi-quantitative method. For quantitative culture, the tip is sonicated or vortexed in a 1-mL solution with a significant threshold of ≥103 CFU/mL to distinguish between colonization and contamination (Brun–Buisson method) (AII). For the semi-quantitative method, the catheter tip is rolled on an agar plate (Roll-plate method, also called the Maki method). The threshold is ≥15 CFU [
      • Maki D.G.
      • Weise C.E.
      • Sarafin H.W.
      A semiquantitative culture method for identifying intravenous-catheter-related infection.
      ] (AII). A meta-analysis published in 1997 seemed to favour the quantitative method [
      • Siegman-Igra Y.
      • Anglim A.M.
      • Shapiro D.E.
      • Adal K.A.
      • Strain B.A.
      • Farr B.M.
      Diagnosis of vascular catheter-related bloodstream infection: a meta-analysis.
      ] but a more recent paper stated that they could be considered equal for the diagnosis of tunnelled catheter-related infections [
      • Slobbe L.
      • El B.A.
      • Boersma E.
      • Rijnders B.J.
      Comparison of the roll plate method to the sonication method to diagnose catheter colonization and bacteremia in patients with long-term tunnelled catheters: a randomized prospective study.
      ]. Current Infectious Diseases Society of America guidelines consider that both methods can be used [
      • Mermel L.A.
      • Allon M.
      • Bouza E.
      • Craven D.E.
      • Flynn P.
      • O'Grady N.P.
      • et al.
      Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America.
      ] and we came to the same conclusion (AII).

       For the diagnosis of catheter-related bloodstream infections

      Paired blood cultures from a catheter and a peripheral vein with one of the two following differential criteria indicate biofilm infection: 1) TTP: ≥2 h earlier growth in the bottle with blood drawn from central venous catheter (CVC) [
      • Bouza E.
      • Alvarado N.
      • Alcala L.
      • Perez M.J.
      • Rincon C.
      • Munoz P.
      A randomized and prospective study of 3 procedures for the diagnosis of catheter-related bloodstream infection without catheter withdrawal.
      ,
      • Blot F.
      • Nitenberg G.
      • Chachaty E.
      • Raynard B.
      • Germann N.
      • Antoun S.
      • et al.
      Diagnosis of catheter-related bacteraemia: a prospective comparison of the time to positivity of hub-blood versus peripheral-blood cultures.
      ,
      • Raad I.
      • Hanna H.A.
      • Alakech B.
      • Chatzinikolaou I.
      • Johnson M.M.
      • Tarrand J.
      Differential time to positivity: a useful method for diagnosing catheter-related bloodstream infections.
      ] (AII); 2) quantitative blood culture with various thresholds published so far [
      • Bouza E.
      • Alvarado N.
      • Alcala L.
      • Perez M.J.
      • Rincon C.
      • Munoz P.
      A randomized and prospective study of 3 procedures for the diagnosis of catheter-related bloodstream infection without catheter withdrawal.
      ,
      • Chatzinikolaou I.
      • Hanna H.
      • Hachem R.
      • Alakech B.
      • Tarrand J.
      • Raad I.
      Differential quantitative blood cultures for the diagnosis of catheter-related bloodstream infections associated with short- and long-term catheters: a prospective study.
      ] (AII). Infectious Diseases Society of America guidelines propose the following threshold: a three-fold greater colony count in the blood drawn from CVC than in blood drawn from peripheral vein (AII) [
      • Mermel L.A.
      • Allon M.
      • Bouza E.
      • Craven D.E.
      • Flynn P.
      • O'Grady N.P.
      • et al.
      Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America.
      ].

       In case of totally implantable venous access port-related infection (e.g. Port-a-Cath®)

      Peripheral blood culture associated with a positive culture of the catheter tip (in situ located in the bloodstream) or the subcutaneous septum (to be penetrated by needles for injection of drugs into the catheter) [
      • Douard M.C.
      • Arlet G.
      • Longuet P.
      • Troje C.
      • Rouveau M.
      • Ponscarme D.
      • et al.
      Diagnosis of venous access port-related infections.
      ,
      • Longuet P.
      • Douard M.C.
      • Arlet G.
      • Molina J.M.
      • Benoit C.
      • Leport C.
      Venous access port-related bacteremia in patients with acquired immunodeficiency syndrome or cancer: the reservoir as a diagnostic and therapeutic tool.
      ] (AII). There is no consensus regarding methods to culture the port reservoir or septum. The following methods have been proposed: an adapted Brun–Buisson method of the septum after removal, the sampling of any macroscopic debris or clots present after septum removal or the swabbing of the internal surface of the port [
      • Whitman E.D.
      • Boatman A.M.
      Comparison of diagnostic specimens and methods to evaluate infected venous access ports.
      ,
      • Guembe M.
      • Martin-Rabadan P.
      • Echenagusia A.
      • Camunez F.
      • Rodriguez-Rosales G.
      • Simo G.
      • et al.
      Value of superficial cultures for prediction of catheter-related bloodstream infection in long-term catheters: a prospective study.
      ] (BIII). Some authors also propose the injection of sterile saline inside the port to recover fluid, which is subsequently cultured [
      • Lebeaux D.
      • Larroque B.
      • Gellen-Dautremer J.
      • Leflon-Guibout V.
      • Dreyer C.
      • Bialek S.
      • et al.
      Clinical outcome after a totally implantable venous access port-related infection in cancer patients: a prospective study and review of the literature.
      ]. No data are available to say which method is more accurate. Recently, a paper suggested that the best strategy was to combine catheter tip culture, port sonication fluid and internal surface biofilm cultures [
      • Bouza E.
      • Rojas L.
      • Guembe M.
      • Marin M.
      • Anaya F.
      • Luno J.
      • et al.
      Predictive value of superficial cultures to anticipate tunneled hemodialysis catheter-related bloodstream infection.
      ].

       Candida biofilms

      Traditional techniques require device removal followed by culture or microscopy of a catheter segment. Catheter-sparing diagnostic tests include paired quantitative blood cultures, differential time to positivity of paired blood culture, catheter-drawn quantitative blood cultures, acridine orange leucocyte cytospin with or without Gram stain [
      • Raad I.
      • Hanna H.
      • Maki D.
      Intravascular catheter-related infections: advances in diagnosis, prevention, and management.
      ]. These in situ techniques avoid catheter removal strictly for diagnostic purposes [
      • Mermel L.A.
      • Allon M.
      • Bouza E.
      • Craven D.E.
      • Flynn P.
      • O'Grady N.P.
      • et al.
      Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America.
      ,
      • Safdar N.
      • Fine J.P.
      • Maki D.G.
      Meta-analysis: methods for diagnosing intravascular device-related bloodstream infection.
      ]. Diagnosis of Candida biofilm (Fig. 10) using microbiological techniques poses major obstacles. Compared with many bacterial pathogens, blood cultures are not consistently positive for Candida, even in the case of systemic disease and previous antifungal therapy may also significantly impact the sensitivity of microbiological techniques, [
      • Bouza E.
      • Alcala L.
      • Munoz P.
      • Martin-Rabadan P.
      • Guembe M.
      • Rodriguez-Creixems M.
      Can microbiologists help to assess catheter involvement in candidaemic patients before removal?.
      ]. Furthermore, paired blood cultures have been poorly studied for fungal pathogens and are less accurate than for bacterial infection [
      • Bouza E.
      • Alcala L.
      • Munoz P.
      • Martin-Rabadan P.
      • Guembe M.
      • Rodriguez-Creixems M.
      Can microbiologists help to assess catheter involvement in candidaemic patients before removal?.
      ]. Sonication-vortexing recovers significantly more biofilm Candida CFU than brushing [
      • Bouza E.
      • Alcala L.
      • Munoz P.
      • Martin-Rabadan P.
      • Guembe M.
      • Rodriguez-Creixems M.
      Can microbiologists help to assess catheter involvement in candidaemic patients before removal?.
      ,
      • Percival S.L.
      • Kite P.
      • Eastwood K.
      • Murga R.
      • Carr J.
      • Arduino M.J.
      • et al.
      Tetrasodium EDTA as a novel central venous catheter lock solution against biofilm.
      ]. TTP of Candida species in culture bottles of peripheral blood may be a useful tool in the evaluation of patients with candidaemia who have an indwelling CVC, since TTP was shorter for definite catheter-related candidaemia (17.3 ± 2 h) than for candidaemia from other sources (38.2 ± 3 h). A TTP cut-off of 30 h was 100% sensitive and 51.4% specific for catheter-related candidaemia. So TTP in peripheral blood may be a sensitive, although non-specific, marker for catheter-related candidaemia and TTP of more than 30 h may help to exclude an intravascular catheter as the possible source of candidaemia (CIII) [
      • Ben-Ami R.
      • Weinberger M.
      • Orni-Wasserlauff R.
      • Schwartz D.
      • Itzhaki A.
      • Lazarovitch T.
      • et al.
      Time to blood culture positivity as a marker for catheter-related candidemia.
      ,
      • Ullmann A.J.
      • Akova M.
      • Herbrecht R.
      • Viscoli C.
      • Arendrup M.C.
      • Arikan-Akdagli S.
      • et al.
      ESCMID* guideline for the diagnosis and management of Candida diseases 2012: adults with haematological malignancies and after haematopoietic stem cell transplantation (HCT).
      ]. The number of positive peripheral blood cultures also seems to be a promising diagnostic tool to diagnose catheter-related candidaemia without directly removing the catheter [
      • Bouza E.
      • Alcala L.
      • Munoz P.
      • Martin-Rabadan P.
      • Guembe M.
      • Rodriguez-Creixems M.
      Can microbiologists help to assess catheter involvement in candidaemic patients before removal?.
      ].
      Figure thumbnail gr10
      FIG. 10Confocal laser scanning microscopy of a green-fluorescent-protein-tagged Candida albicans biofilm. Magnification x1000 (C. Imbert).

       In case of biofilm urinary tract infections in patients with indwelling urinary catheters or stents

      Freshly obtained urine from the bladder should be processed as in non-catheterized patients bearing in mind that this procedure gives more than 50% false-negative results in these patients. Examination of removed catheters or stents is necessary for detection of biofilm infection; however, the routine examination of removed urinary catheters or stents is not recommended.

       Biofilm-specific microbial phenotypes

      These have rarely been described with the exception of mucoid (and maybe small colony variant) P. aeruginosa in CF (Fig. 3) [
      • Bjarnsholt T.
      • Jensen P.O.
      • Fiandaca M.J.
      • Pedersen J.
      • Hansen C.R.
      • Andersen C.B.
      • et al.
      Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
      ,
      • Pressler T.
      • Karpati F.
      • Granstrom M.
      • Knudsen P.K.
      • Lindblad A.
      • Hjelte L.
      • et al.
      Diagnostic significance of measurements of specific IgG antibodies to Pseudomonas aeruginosa by three different serological methods.
      ]. This mucoid, slimy component is the polysaccharide alginate; however, no validated commercial methods to detect alginate in samples (e.g. sputum) are currently available. We recommend that growth of mucoid P. aeruginosa from clinical samples indicates the presence of biofilm infection and the mucoid phenotype should therefore be reported to the clinicians [
      • Bjarnsholt T.
      • Jensen P.O.
      • Fiandaca M.J.
      • Pedersen J.
      • Hansen C.R.
      • Andersen C.B.
      • et al.
      Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
      ,
      • Pressler T.
      • Karpati F.
      • Granstrom M.
      • Knudsen P.K.
      • Lindblad A.
      • Hjelte L.
      • et al.
      Diagnostic significance of measurements of specific IgG antibodies to Pseudomonas aeruginosa by three different serological methods.
      ] (AI).

       Q1-3 Are measurements of antibodies or inflammatory markers of any value to detect biofilm infections?

      Significantly elevated levels of IgG antibodies against crude or purified antigens from P. aeruginosa antigens measured by various methods including ELISA, these elevated levels are diagnostic for P. aeruginosa biofilm infections in CF patients and such tests are commercially available and have been validated (Table 5) [
      • Pressler T.
      • Karpati F.
      • Granstrom M.
      • Knudsen P.K.
      • Lindblad A.
      • Hjelte L.
      • et al.
      Diagnostic significance of measurements of specific IgG antibodies to Pseudomonas aeruginosa by three different serological methods.
      ] and are recommended for detection of P. aeruginosa biofilm infection in CF patients (AI). Likewise, significantly elevated antibodies against antigens from other bacteria causing biofilm infections in CF patients (e.g. Burkholderia cepacia complex, Achromobacter xylosoxidans, Stenotrophomonas maltophilia) have been reported, but no commercial tests are available. Some of the P. aeruginosa antibody tests have also been used in non-CF patients with chronic P. aeruginosa infections caused by mucoid phenotypes that also show significantly elevated antibody responses. IgM antibody response against biofilm-specific polysaccharide antigen in S. aureus and S. epidermidis alloplastic-related infections has also been reported [
      • Artini M.
      • Romano C.
      • Manzoli L.
      • Scoarughi G.L.
      • Papa R.
      • Meani E.
      • et al.
      Staphylococcal IgM enzyme-linked immunosorbent assay for diagnosis of periprosthetic joint infections.
      ,
      • Selan L.
      • Passariello C.
      • Rizzo L.
      • Varesi P.
      • Speziale F.
      • Renzini G.
      • et al.
      Diagnosis of vascular graft infections with antibodies against staphylococcal slime antigens.
      ] and can be recommended (BII). An elevated IgG and especially secretory IgA antibody response [
      • Aanaes K.
      • Johansen H.K.
      • Poulsen S.S.
      • Pressler T.
      • Buchwald C.
      • Høiby N.
      Secretory IgA as a diagnostic tool for Pseudomonas aeruginosa respiratory colonization.
      ] with simultaneous negative cultures may encourage a search for hidden foci (e.g. paranasal sinuses). This is done by means of further samplings by more invasive techniques (e.g. bronchoalveolar lavage or nasal irrigation) and use of culture-independent techniques (e.g. 16S rRNA gene PCR) to detect microorganisms suppressed/killed by ongoing antibiotic therapy [
      • Rudkjøbing V.B.
      • Thomsen T.R.
      • Alhede M.
      • Kragh K.N.
      • Nielsen P.H.
      • Johansen U.R.
      • et al.
      True microbiota involved in chronic lung infection of cystic fibrosis patients found by culturing and 16S rRNA gene analysis.
      ] and this is recommended (AII). There are however, no widely available antibody measurement methods or inflammatory markers specific for biofilm infections.
      TABLE 5Diagnostic use of three different anti-Pseudomonas antibody methods to detect chronic Pseudomonas aeruginosa biofilm infection in Scandinavian cystic fibrosis patients
      • Pressler T.
      • Karpati F.
      • Granstrom M.
      • Knudsen P.K.
      • Lindblad A.
      • Hjelte L.
      • et al.
      Diagnostic significance of measurements of specific IgG antibodies to Pseudomonas aeruginosa by three different serological methods.
      Crossed immune-electrophoresis (95% CI)Pseudomonas-CF-IgG ELISA (95% CI)Exotoxin A ELISA (95% CI)
      Specificity89% (86–92)83% (78–87)89% (85–92)
      Sensitivity96% (93–98)97% (94–99)93% (89–96)
      Positive predictive value87% (82–90)80% (75–84)86% (81–90)
      Negative predictive value97% (95–99)98% (95–99)95% (92–97)
      Positive predictive value after patients with other Gram-negative infections were excluded93% (89–95)85% (80–89)88% (84–92)
      Negative predictive value after patients with other Gram-negative infections were excluded97% (95–99)97% (94–99)95% (92–97)

       Inflammatory markers

      The non-specific inflammatory markers (C-reactive protein, procalcitonin, erythrocyte sedimentation rate, white blood cells), or various cytokines cannot distinguish between infections caused by planktonic cells and biofilm infections (DIII) [
      • Schuetz P.
      • Christ-Crain M.
      • Thomann R.
      • Falconnier C.
      • Wolbers M.
      • Widmer I.
      • et al.
      Effect of procalcitonin-based guidelines vs standard guidelines on antibiotic use in lower respiratory tract infections: the ProHOSP randomized controlled trial.
      ,
      • Worthington T.
      • Dunlop D.
      • Casey A.
      • Lambert R.
      • Luscombe J.
      • Elliott T.
      Serum procalcitonin, interleukin-6, soluble intercellular adhesin molecule-1 and IgG to short-chain exocellular lipoteichoic acid as predictors of infection in total joint prosthesis revision.
      ].

       Q1-4 How should the CML report to the clinician that biofilms have been detected?

      Since an antibiotic treatment regimen for acute infections may fail in the case of biofilm infections and since foreign bodies associated with biofilm infections may have to be removed/replaced or the infection suppressed by antimicrobial treatment, it is important that the CML reports include whether microbial biofilms have been detected in patients’ samples. It is important to note that identification of the presence of biofilms may be time consuming and needs to take into account the clinical condition of the patient so any clinician suspecting an infection due to a biofilm should contact the CML to ensure that appropriate diagnostic methods are employed. If a microbial biofilm is detected by microscopy it may be reported using descriptive terms e.g. ‘Microscopy shows Gram-negative rods in biofilm-like structures’ (FIG. 2, FIG. 4, FIG. 5, FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10) and the species should be reported if specific fluorescence in situ hybridization probes have been used (Fig. 2) (AIII). In other cases where microbial biofilm infection is suspected (e.g. culture positive or PCR positive only following sonication or after scraping, rolling etc.) the findings may be reported as ‘Growth of/PCR-detected microorganisms possibly from a biofilm infection’. Additionally the CML may offer assistance to the clinician regarding treatment regimens for biofilm infections e.g. ‘Please contact CML or infectious diseases specialist for consultancy concerning treatment of the biofilm infection’.

       Q1-5 Should routine antibiotic susceptibility testing employing planktonic bacteria from a biofilm infection be reported to the clinician? If yes—should the CML add an explanation about the interpretation of the results? How should such an explanation be written?

      Routine antibiotic susceptibility testing—from disc diffusion to microdilution-based automatic methods—are performed with planktonically growing bacteria or Candida spp. and the breakpoints, e.g. Susceptible, Intermediate and Resistant (S-I-R) based on pharmacokinetics of the various antibiotics and the typical minimum inhibitory concentrations (MIC) of the relevant microorganisms are given (EUCAST). Biofilm-growing microorganisms are significantly more tolerant to antibiotics [
      • Høiby N.
      • Bjarnsholt T.
      • Givskov M.
      • Molin S.
      • Ciofu O.
      Antibiotic resistance of bacterial biofilms.
      ] and corresponding breakpoints have not been established [
      • Döring G.
      • Flume P.
      • Heijerman H.
      • Elborn J.S.
      Treatment of lung infection in patients with cystic fibrosis: current and future strategies.
      ]. The S-I-R results can therefore not be used to predict therapeutic success in the case of biofilm infections and offer no guide to clinicians for treating such infections. However, biofilm infections are often foci for systemically spreading infections, e.g. bloodstream infections originating from biofilms in intravenous lines or in urinary catheters or stents [
      • Kizilbash Q.F.
      • Petersen N.J.
      • Chen G.J.
      • Naik A.D.
      • Trautner B.W.
      Bacteremia and mortality with urinary catheter-associated bacteriuria.
      ,
      • Høiby N.
      • Johansen H.K.
      • Ciofu O.
      • Jensen P.O.
      • Bjarnsholt T.
      • Givskov M.
      Foreign body infections—biofilms and quorum sensing.
      ] (Fig. 5). Such systemic spread of biofilm infections can be treated successfully with antibiotics based on the results of routine antibiotic susceptibility testing, which should therefore be reported [
      • Rodriguez-Tudela J.L.
      • Arendrup M.C.
      • Cuenca-Estrella M.
      • Donnelly J.P.
      • Lass-Florl C.
      EUCAST breakpoints for antifungals.
      ] (AIII). In that case, routine antibiotic susceptibility testing can efficiently predict treatment success or failure of the systemic infection (AII). Clinicians should be informed by the CML that recurrence of the infection from the biofilm focus may occur if the focus cannot be removed or suppressed by antimicrobial treatment. This may be reported as, for example, ‘Please contact CML or infectious diseases specialist for consultancy concerning treatment’ (AIII).

       Q1-6 Are there any routine in vitro antibiotic susceptibility tests that can reliably predict therapeutic success?

      Biofilm susceptibility tests have been designed and include the Calgary device, where biofilms are grown on pegs protruding from the lid of a microtitre plate which are subsequently exposed to various concentrations of antibiotics to establish their ‘biofilm eradication concentrations’. However, such testing has not yet resulted in reliable prediction of therapeutic success [
      • Döring G.
      • Flume P.
      • Heijerman H.
      • Elborn J.S.
      Treatment of lung infection in patients with cystic fibrosis: current and future strategies.
      ,
      • Moskowitz S.M.
      • Emerson J.C.
      • McNamara S.
      • Shell R.D.
      • Orenstein D.M.
      • Rosenbluth D.
      • et al.
      Randomized trial of biofilm testing to select antibiotics for cystic fibrosis airway infection.
      ,
      • Moskowitz S.M.
      • Foster J.M.
      • Emerson J.
      • Burns J.L.
      Clinically feasible biofilm susceptibility assay for isolates of Pseudomonas aeruginosa from patients with cystic fibrosis.
      ,
      • Waters V.
      • Ratjen F.
      Standard versus biofilm antimicrobial susceptibility testing to guide antibiotic therapy in cystic fibrosis.
      ] (DII).

       Q1-7 Which research is urgently needed to improve diagnosis of biofilm infections?

      • Algorithms for biofilm diagnosis and for handling the samples in the CML—why, when and how to demonstrate microbial biofilms.
      • Improved non-invasive methods (e.g. image-based diagnostic methods) for detecting biofilms in vivo in patients.
      • Host-specific markers that indicate a risk of biofilm infections.
      • How many samples should be taken and for how long a period.
      • How should samples be analysed for the detection of biofilm-growing microorganisms that may be viable-but-non-culturable in routine media.
      • Which bacterial species play an active role in biofilm infections since culture-independent molecular methods may identify a greater microbial diversity than previously demonstrated by culture. To what extent do detected species come from DNA released from non-viable bacteria?
      • Will enzymatic (e.g. DNases or other compounds) pretreatment of microbial biofilms improve diagnostic and therapeutic success?
      • Characterization of biofilm-specific antigens and detection of specific antibodies against biofilm-specific antigens.
      • Standardized biofilm susceptibility testing and establishment of biofilm-specific breakpoints for systemic and topically administered antibiotics.
      • Do some mycobacteria cause biofilm infection in patients?
      • Better animal models for relevant chronic biofilm infections [
        • Chauhan A.
        • Lebeaux D.
        • Decante B.
        • Kriegel I.
        • Escande M.C.
        • Ghigo J.M.
        • et al.
        A rat model of central venous catheter to study establishment of long-term bacterial biofilm and related acute and chronic infections.
        ].
      • Assess the clinical impact of 16S rRNA gene detection in intravenous catheters [
        • Bouza E.
        • Alvarado N.
        • Alcala L.
        • Perez M.J.
        • Rincon C.
        • Munoz P.
        A randomized and prospective study of 3 procedures for the diagnosis of catheter-related bloodstream infection without catheter withdrawal.
        ,
        • Warwick S.
        • Wilks M.
        • Hennessy E.
        • Powell-Tuck J.
        • Small M.
        • Sharp J.
        • et al.
        Use of quantitative 16S ribosomal DNA detection for diagnosis of central vascular catheter-associated bacterial infection.
        ,
        • Larsen M.K.
        • Thomsen T.R.
        • Moser C.
        • Høiby N.
        • Nielsen P.H.
        Use of cultivation-dependent and -independent techniques to assess contamination of central venous catheters: a pilot study.
        ]
      • Is there any evidence that ‘good bacteria’ reduce the occurrence of biofilm infection (prevention of biofilm infection by e.g. probiotics)?
      • Development of methods for the diagnosis of CR-BSI relying on the antigen-based detection of biofilm formation inside the catheter.
      • Do surveillance cultures and light-microscopy studies of accumulated mucus within the ETT have any benefits on early detection of ETT biofilm formation and incidence of VAP and therapeutic outcomes?

      Prevention and Treatment of Biofilm Infections

       Generally

      Antibiotics are used for:
      • 1)
        Prophylaxis i.e. to prevent infection in patients who are not yet infected or colonized in, for example, the lungs or bones/joints, if there is an unacceptable risk of development of an infection in such organs during a specific period or during a specific (e.g. surgical) procedure; that means that the antibiotic penetrates the site of a potential infection before the microorganisms (e.g. surgical wound).
      • 2)
        Pre-emptive treatment when colonization with a specific microorganism is detected on, for example, mucosal membranes and there is a known, unacceptable risk of development of severe clinical infection with that microorganism; that means that the antibiotic penetrates the site of a potential infection after the microorganisms, but before the establishment of an infection (e.g. pre-emptive therapy after open bone fracture to prevent biofilm infection on osteosynthetic material or pre-emptive eradication therapy of intermittent P. aeruginosa colonization in the lungs of CF patients to prevent biofilm infection).
      • 3)
        Empiric treatment is based on the clinical diagnosis of infection without knowledge of the microorganism but covering the most probable microorganisms in a specific clinical situation (e.g. staphylococci and E. coli in case of CR-BSI).
      • 4)
        Definitive treatment is based on clinical diagnosis and known culture and susceptibility testing results.
      These definitions are used in the following section.

       Q2-1 Can systemic antibiotics (oral, intravenous) be used to prevent some (which?) biofilm infections? Can the risk period be defined?

      Yes for some infections, not for other infections.

       Short-term (surgery prophylaxis, some urinary catheters) and intermediate-term (endotracheal tubes, some CVCs, some urinary catheters)

       Patients with infections related to their orthopaedic alloplastic devices

      Surgery-related biofilm infections can be prevented (their incidence reduced) by administration of prophylactic perioperative antibiotics (http://www.sign.ac.uk/pdf/sign104.pdf) [
      • Song Z.
      • Borgwardt L.
      • Høiby N.
      • Wu H.
      • Sørensen T.S.
      • Borgwardt A.
      Prosthesis infections after orthopedic joint replacement: the possible role of bacterial biofilms.
      ] (AI).

       Patients with indwelling urinary catheters or urethral stents

      Short-course systemic antibiotic therapy can postpone biofilm infections for up to 1–2 weeks [
      • Petronella P.
      • Scorzelli M.
      • Fiore A.
      • Corbisiero M.C.
      • Agresti E.
      • Esposito S.
      • et al.
      Antibiotic prophylaxis in catheter-associated urinary infections.
      ,
      • Sayin K.S.
      • Aybek Z.
      • Tekin K.
      • Okke D.
      • Akalin S.
      • Altintas S.
      • et al.
      Is short course of antimicrobial therapy for asymptomatic bacteriuria before urologic surgical procedures sufficient?.
      ], however we do not recommend the use of antibiotic prophylaxis because of concern about superinfection by multiresistant strains [
      • Conway L.J.
      • Larson E.L.
      Guidelines to prevent catheter-associated urinary tract infection: 1980 to 2010.
      ,
      • Hooton T.M.
      • Bradley S.F.
      • Cardenas D.D.
      • Colgan R.
      • Geerlings S.E.
      • Rice J.C.
      • et al.
      Diagnosis, prevention, and treatment of catheter-associated urinary tract infection in adults: 2009 International Clinical Practice Guidelines from the Infectious Diseases Society of America.
      ] (DIII).
      No systemic antibiotics can be recommended for the prophylaxis of CR-BSI [
      • Karanlik H.
      • Kurul S.
      • Saip P.
      • Unal E.S.
      • Sen F.
      • Disci R.
      • et al.
      The role of antibiotic prophylaxis in totally implantable venous access device placement: results of a single-center prospective randomized trial.
      ,
      • Di C I.
      • Toro A.
      • Pulvirenti E.
      • Palermo F.
      • Scibilia G.
      • Cordio S.
      Could antibiotic prophylaxis be not necessary to implant totally implantable venous access devices? Randomized prospective study.
      ,
      • van de Wetering M.D.
      • van Woensel J.B.
      Prophylactic antibiotics for preventing early central venous catheter Gram positive infections in oncology patients.
      ] (DI).
      There is no evidence to support the use of systemic antimicrobial agents to prevent biofilm infections in the treatment of wound-associated infections [
      • Lipsky B.A.
      • Berendt A.R.
      • Cornia P.B.
      • Pile J.C.
      • Peters E.J.
      • Armstrong D.G.
      • et al.
      Executive summary: 2012 Infectious Diseases Society of America clinical practice guideline for the diagnosis and treatment of diabetic foot infections.
      ,
      • O'Meara S.
      • Cullum N.
      • Majid M.
      • Sheldon T.
      Systematic reviews of wound care management: (3) antimicrobial agents for chronic wounds; (4) diabetic foot ulceration.
      ,
      • Howell-Jones R.S.
      • Wilson M.J.
      • Hill K.E.
      • Howard A.J.
      • Price P.E.
      • Thomas D.W.
      A review of the microbiology, antibiotic usage and resistance in chronic skin wounds.
      ] (CIII).
      There is no regimen that can be recommended regarding tissue fillers at the present time [
      • Viola G.M.
      • Raad II,
      • Rolston K.V.
      Breast tissue expander-related infections: perioperative antimicrobial regimens.
      ].
      Patients with endotracheal tube biofilm VAP: Please see Q2-2.
      Patients with intravenous catheters: see Antimicrobial lock therapy.

       Chronic disposing situation (CF, chronic wound, late alloplastic infections, some CVCs (>1 year for, for example, parenteral feeding), some urinary catheters, implants/fillers (absorbable/material repairing defects temporarily)

      Chronic P. aeruginosa lung infection in CF cannot be prevented by prophylactic use of systemic antibiotics in patients before the onset of intermittent P. aeruginosa colonization [
      • Heijerman H.
      • Westerman E.
      • Conway S.
      • Touw D.
      • Döring G.
      Inhaled medication and inhalation devices for lung disease in patients with cystic fibrosis: a European consensus.
      ] (DI). Urinary tract infections in patients chronically carrying urinary catheters cannot be prevented by prophylactic use of systemic antibiotic. No information currently exists about the other conditions.

       Q2-2 Can topical use of antimicrobials or antimicrobials attached to the surface of foreign bodies (e.g. intravenous lines, urinary catheters, tracheal tubes, artificial joints, bone cements for orthopaedic surgery) be used to prevent some (which?) biofilm infections? Can the risk period be defined?

       Short term (surgery prophylaxis, some urinary catheters)

       Orthopaedic alloplastics

      There is good evidence to suggest that antibiotic-impregnated materials (frequently gentamicin but also tobramycin and vancomycin) reduce the incidence of prosthesis-associated biofilm infections [
      • Johannsson B.
      • Taylor J.
      • Clark C.R.
      • Shamsuddin H.
      • Beekmann S.E.
      • Polgreen P.
      Treatment approaches to prosthetic joint infections: results of an Emerging Infections Network survey.
      ,
      • Parvizi J.
      • Saleh K.J.
      • Ragland P.S.
      • Pour A.E.
      • Mont M.A.
      Efficacy of antibiotic-impregnated cement in total hip replacement.
      ,
      • Marschall J.
      • Lane M.A.
      • Beekmann S.E.
      • Polgreen P.M.
      • Babcock H.M.
      Current management of prosthetic joint infections in adults: results of an Emerging Infections Network survey.
      ] (AI).

       Short-term urinary catheters

      Coating with antimicrobials (e.g. nitrofural) can only postpone but not prevent biofilm infections [
      • Viola G.M.
      • Raad II,
      • Rolston K.V.
      Breast tissue expander-related infections: perioperative antimicrobial regimens.
      ] (AI). Several studies conclude that the postponing effect is sufficient to prevent biofilm infections in short-term catheterization especially with nitrofurazone [
      • Petronella P.
      • Scorzelli M.
      • Fiore A.
      • Corbisiero M.C.
      • Agresti E.
      • Esposito S.
      • et al.
      Antibiotic prophylaxis in catheter-associated urinary infections.
      ,
      • Gaonkar T.A.
      • Sampath L.A.
      • Modak S.M.
      Evaluation of the antimicrobial efficacy of urinary catheters impregnated with antiseptics in an in vitro urinary tract model.
      ] (AI).

       Intermediate or long-term (endotracheal tubes, some CVC, some urinary catheters). Endotracheal tube biofilm (VAP)

      A few studies [
      • Adair C.G.
      • Gorman S.P.
      • Feron B.M.
      • Byers L.M.
      • Jones D.S.
      • Goldsmith C.E.
      • et al.
      Implications of endotracheal tube biofilm for ventilator-associated pneumonia.
      ,
      • Gorman S.
      • Adair C.
      • O'Neill F.
      • Goldsmith C.
      • Webb H.
      Influence of selective decontamination of the digestive tract on microbial biofilm formation on endotracheal tubes from artificially ventilated patients.
      ] have assessed the prophylactic effects of selective digestive decontamination, which involves the oropharyngeal/gastric administration of topical non-absorbable antibiotics, on ETT biofilm formation. There is consistent evidence that selective digestive decontamination does not inhibit ETT biofilm formation, and we do not suggest its use as a prophylactic strategy (DI). Nebulized antibiotics achieve higher concentrations in the artificially ventilated airways (mean gentamicin concentration 1 h after nebulization of 80 mg was 790 μg/ml) than antibiotics administered by the parenteral route and in one clinical study [
      • Adair C.G.
      • Gorman S.P.
      • Byers L.M.
      • Jones D.S.
      • Feron B.
      • Crowe M.
      • et al.
      Eradication of endotracheal tube biofilm by nebulised gentamicin.
      ], the use of nebulized gentamicin (80 mg in 4 ml saline every 8 h) in the prevention of VAP has been tested and it was effective in preventing the formation of ETT biofilm by the most common causative pathogens of VAP (CII). There have also been attempts to reduce ETT biofilm formation by coating the ETT surface with antimicrobial agents [
      • Berra L.
      • De M.L.
      • Yu Z.X.
      • Laquerriere P.
      • Baccarelli A.
      • Kolobow T.
      Endotracheal tubes coated with antiseptics decrease bacterial colonization of the ventilator circuits, lungs, and endotracheal tube.
      ,
      • Berra L.
      • Kolobow T.
      • Laquerriere P.
      • Pitts B.
      • Bramati S.
      • Pohlmann J.
      • et al.
      Internally coated endotracheal tubes with silver sulfadiazine in polyurethane to prevent bacterial colonization: a clinical trial.
      ,
      • Olson M.E.
      • Harmon B.G.
      • Kollef M.H.
      Silver-coated endotracheal tubes associated with reduced bacterial burden in the lungs of mechanically ventilated dogs.
      ,
      • Rello J.
      • Kollef M.
      • Diaz E.
      • Sandiumenge A.
      • del C.Y.
      • Corbella X.
      • et al.
      Reduced burden of bacterial airway colonization with a novel silver-coated endotracheal tube in a randomized multiple-center feasibility study.
      ]. Among all the available antimicrobial compounds used for ETT coating, silver (dispersion of silver-sulphadiazine or micro-dispersed silver ions in a polymer) has been the main focus in laboratory and clinical investigations [
      • Kollef M.H.
      • Afessa B.
      • Anzueto A.
      • Veremakis C.
      • Kerr K.M.
      • Margolis B.D.
      • et al.
      Silver-coated endotracheal tubes and incidence of ventilator-associated pneumonia: the NASCENT randomized trial.
      ,
      • Berra L.
      • Kolobow T.
      • Laquerriere P.
      • Pitts B.
      • Bramati S.
      • Pohlmann J.
      • et al.
      Internally coated endotracheal tubes with silver sulfadiazine in polyurethane to prevent bacterial colonization: a clinical trial.
      ]. It has been shown that silver-coated ETTs could exert antimicrobial effects within the proximal airways and hinder ETT biofilm formation. In addition, the silver-coated ETT exerted its maximal effect during the first 10 days of tracheal intubation [
      • Kollef M.H.
      • Afessa B.
      • Anzueto A.
      • Veremakis C.
      • Kerr K.M.
      • Margolis B.D.
      • et al.
      Silver-coated endotracheal tubes and incidence of ventilator-associated pneumonia: the NASCENT randomized trial.
      ,
      • Rello J.
      • Kollef M.
      • Diaz E.
      • Sandiumenge A.
      • del C.Y.
      • Corbella X.
      • et al.
      Reduced burden of bacterial airway colonization with a novel silver-coated endotracheal tube in a randomized multiple-center feasibility study.
      ] and reduced mortality in patients with VAP [
      • Afessa B.
      • Shorr A.F.
      • Anzueto A.R.
      • Craven D.E.
      • Schinner R.
      • Kollef M.H.
      Association between a silver-coated endotracheal tube and reduced mortality in patients with ventilator-associated pneumonia.
      ,
      • Ye X.
      • Rupnow M.
      • Bastide P.
      • Lafuma A.
      • Ovington L.
      • Jarvis W.R.
      Economic impact of use of chlorhexidine-impregnated sponge dressing for prevention of central line-associated infections in the United States.
      ] and the cost of VAP [
      • Kolobow T.
      • Berra L.
      • Li B.G.
      • Curto F.
      Novel system for complete removal of secretions within the endotracheal tube: the Mucus Shaver.
      ]. We recommend the use of silver-coated ETT in patients, who are expected to be ventilated for longer periods of time (≥1 week) and with risks for VAP (BI). The Mucus Shaver (National Institutes of Health, Bethesda, MD, USA) [
      • Kolobow T.
      • Berra L.
      • Li B.G.
      • Curto F.
      Novel system for complete removal of secretions within the endotracheal tube: the Mucus Shaver.
      ] has been devised to keep the ETT lumen free of mucus and to mechanically remove biofilm. It comprises an inflatable balloon with two or more 1.0-mm wide, 0.5-mm thick silicone rubber ‘shaving rings’. The balloon is inflated with air inside the ETT lumen sufficiently to force the two shaving rings firmly against the wall of the ETT. Thereafter the Mucus Shaver is gently retrieved followed by resumption of ventilation. A clinical study [
      • Berra L.
      • Coppadoro A.
      • Bittner E.A.
      • Kolobow T.
      • Laquerriere P.
      • Pohlmann J.R.
      • et al.
      A clinical assessment of the Mucus Shaver: a device to keep the endotracheal tube free from secretions.
      ] in a small population of patients showed that the Mucus Shaver is efficient in ETT biofilm removal. In a recent study [
      • Liu W.
      • Zuo Z.
      • Ma R.
      • Zhang X.
      Effect of mechanical cleaning of endotracheal tubes with sterile urethral catheters to reduce biofilm formation in ventilator patients.
      ] in paediatric intubated patients, ETT biofilm was mechanically removed through the inflatable balloon of a urethral catheter. This strategy reduced ETT biofilm formation and incidence of clinically confirmed VAP. Based on the limited number of studies, we support the routine use of dedicated devices to mechanically remove ETT biofilm (BII).

       Patients with CVCs

      Use of chlorhexidine-impregnated sponges or dressings reduces the incidence of CR-BSI and is considered cost-effective [
      • Timsit J.F.
      • Mimoz O.
      • Mourvillier B.
      • Souweine B.
      • Garrouste-Org
      • Alfandari S.
      • et al.
      Randomized controlled trial of chlorhexidine dressing and highly adhesive dressing for preventing catheter-related infections in critically ill adults.
      ,
      • Timsit J.F.
      • Schwebel C.
      • Bouadma L.
      • Geffroy A.
      • Garrouste-Org
      • Pease S.
      • et al.
      Chlorhexidine-impregnated sponges and less frequent dressing changes for prevention of catheter-related infections in critically ill adults: a randomized controlled trial.
      ,
      • Loveday H.P.
      • Wilson J.A.
      • Pratt R.J.
      • Golsorkhi M.
      • Tingle A.
      • Bak A.
      • et al.
      epic3: national evidence-based guidelines for preventing healthcare-associated infections in NHS hospitals in England.
      ,
      • Darouiche R.O.
      • Raad II,
      • Heard S.O.
      • Thornby J.I.
      • Wenker O.C.
      • Gabrielli A.
      • et al.
      A comparison of two antimicrobial-impregnated central venous catheters. Catheter Study Group.
      ] (AI). Coated CVCs should be considered if the incidence of CR-BSI is still high after implementation of all preventive measures. In that case, minocycline/rifampin-coated catheters are more efficient than chlorhexidine/silver sulphadiazine-coated catheters [
      • Casey A.L.
      • Mermel L.A.
      • Nightingale P.
      • Elliott T.S.
      Antimicrobial central venous catheters in adults: a systematic review and meta-analysis.
      ,
      • Gilbert R.E.
      • Harden M.
      Effectiveness of impregnated central venous catheters for catheter related blood stream infection: a systematic review.
      ,
      • Desrousseaux C.
      • Sautou V.
      • Descamps S.
      • Traore O.
      Modification of the surfaces of medical devices to prevent microbial adhesion and biofilm formation.
      ,
      • Campos R.P.
      • do Nascimento M.M.
      • Chula D.C.
      • Riella M.C.
      Minocycline-EDTA lock solution prevents catheter-related bacteremia in hemodialysis.
      ] (AI).

       Preventive antibiotic lock technique

      Antibiotic lock technique (ALT) is done by application of a small volume of concentrated antimicrobials, which is allowed to dwell for 12–24 h inside the catheter lumen. The use of ALT as prophylaxis should be restricted to patients who have experienced multiple CR-BSI despite optimal aseptic techniques (BI). Minocycline-EDTA ALT is more efficient in reducing the rate of CR-BSIs in haemodialysis patients than heparin locks [
      • Ferreira Chacon J.M.
      • Hato de A.E.
      • de Lourdes S.R.
      • Lazzarin CO V.
      • Alves B.C.
      • Mello de Andrea M.L.
      • et al.
      Randomized study of minocycline and edetic acid as a locking solution for central line (port-a-cath) in children with cancer.
      ,
      • Solomon L.R.
      • Cheesbrough J.S.
      • Bhargava R.
      • Mitsides N.
      • Heap M.
      • Green G.
      • et al.
      Observational study of need for thrombolytic therapy and incidence of bacteremia using taurolidine-citrate-heparin, taurolidine-citrate and heparin catheter locks in patients treated with hemodialysis.
      ]. A mix of taurolidine/citrate/heparin gave a reduced rate of CR-BSI in haemodialysis patients compared with heparin [
      • Betjes M.G.
      • van A.M.
      Prevention of dialysis catheter-related sepsis with a citrate-taurolidine-containing lock solution.
      ,
      • Allon M.
      Prophylaxis against dialysis catheter-related bacteremia with a novel antimicrobial lock solution.
      ]. Use of taurolidine/citrate without heparin is associated with an increased rate of thrombosis of the catheter and thereby significantly shorter catheter patency in patients undergoing haemodialysis [
      • Simon A.
      • Ammann R.A.
      • Wiszniewsky G.
      • Bode U.
      • Fleischhack G.
      • Besuden M.M.
      Taurolidine-citrate lock solution (TauroLock) significantly reduces CVAD-associated grampositive infections in pediatric cancer patients.
      ]. In paediatric cancer patients, an early paper showed no significant reduction of total number of CR-BSI but a significant reduction of CR-BSI due to coagulase negative staphylococci with taurolidine/citrate compared with heparin [
      • Dumichen M.J.
      • Seeger K.
      • Lode H.N.
      • Kuhl J.S.
      • Ebell W.
      • Degenhardt P.
      • et al.
      Randomized controlled trial of taurolidine citrate versus heparin as catheter lock solution in paediatric patients with haematological malignancies.
      ], whereas a more recent paper showed a significant reduction of CR-BSI with taurolidine/citrate as compared with heparin if used from the time of insertion [
      • Bisseling T.M.
      • Willems M.C.
      • Versleijen M.W.
      • Hendriks J.C.
      • Vissers R.K.
      • Wanten G.J.
      Taurolidine lock is highly effective in preventing catheter-related bloodstream infections in patients on home parenteral nutrition: a heparin-controlled prospective trial.
      ] (BI). In high-risk patients receiving parenteral nutrition, taurolidine/citrate reduces the rate of CR-BSI when initiated after the first episode of CR-BSI compared with heparin [
      • Broom J.K.
      • Krishnasamy R.
      • Hawley C.M.
      • Playford E.G.
      • Johnson D.W.
      A randomised controlled trial of Heparin versus EthAnol Lock THerapY for the prevention of Catheter Associated infecTion in Haemodialysis patients—the HEALTHY-CATH trial.
      ] (BI).

       Use of ethanol as ALT

      No significant reduction for ethanol 70% compared with heparin in reduction of CR-BSI incidence in haemodialysis was observed [
      • Sanders J.
      • Pithie A.
      • Ganly P.
      • Surgenor L.
      • Wilson R.
      • Merriman E.
      • et al.
      A prospective double-blind randomized trial comparing intraluminal ethanol with heparinized saline for the prevention of catheter-associated bloodstream infection in immunosuppressed haematology patients.
      ]. Among immunosuppressed haematology patients receiving chemotherapy, 70% ethanol significantly reduced CR-BSI compared with heparin in a randomized study [
      • Wales P.W.
      • Kosar C.
      • Carricato M.
      • de S.N.
      • Lang K.
      • Avitzur Y.
      Ethanol lock therapy to reduce the incidence of catheter-related bloodstream infections in home parenteral nutrition patients with intestinal failure: preliminary experience.
      ]. In cases of high-risk patients using in-home parenteral nutrition (one previous CR-BSI), ethanol 70% was assessed in a before–after study with a significant reduction of CR-BSI and need for catheter replacement in children [
      • Slobbe L.
      • Doorduijn J.K.
      • Lugtenburg P.J.
      • El B.A.
      • Boersma E.
      • van Leeuwen W.B.
      • et al.
      Prevention of catheter-related bacteremia with a daily ethanol lock in patients with tunnelled catheters: a randomized, placebo-controlled trial.
      ]. However, in a randomized placebo-controlled trial in adult haematology patients, no significant decrease in the incidence of CR-BSI and more adverse effects were reported in the ethanol group [
      • Redelman C.V.
      • Maduakolam C.
      • Anderson G.G.
      Alcohol treatment enhances Staphylococcus aureus biofilm development.
      ]. Furthermore, it has been reported that ethanol could increase pre-formed S. aureus biofilm in vitro [
      • Oliveira C.
      • Nasr A.
      • Brindle M.
      • Wales P.W.
      Ethanol locks to prevent catheter-related bloodstream infections in parenteral nutrition: a meta-analysis.
      ]. A recent meta-analysis showed that ethanol was more effective than heparin lock to reduce the incidence of CR-BSI and catheter replacement in paediatric parenteral nutrition but may increase the risk of thrombosis [
      • Novikov A.
      • Lam M.Y.
      • Mermel L.A.
      • Casey A.L.
      • Elliott T.S.
      • Nightingale P.
      Impact of catheter antimicrobial coating on species-specific risk of catheter colonization: a meta-analysis.
      ]. In conclusion we recommend the use of ethanol lock (CII).

       Antibiotic/antiseptic ointments

      The use of antibiotic ointments that have limited antifungal activity may serve to increase colonization and/or infection due to Candida species [
      • Walraven C.J.
      • Lee S.A.
      Antifungal lock therapy.
      ].
      Regarding the prevention of fungal infections, the most promising ALT includes use of amphotericin B, ethanol or echinocandins [
      • Lai K.K.
      • Fontecchio S.A.
      Use of silver-hydrogel urinary catheters on the incidence of catheter-associated urinary tract infections in hospitalized patients.
      ]. (CII).

       Indwelling urinary catheters

      Coating with antimicrobials can postpone biofilm infections but not prevent them [
      • Conway L.J.
      • Larson E.L.
      Guidelines to prevent catheter-associated urinary tract infection: 1980 to 2010.
      ,
      • Hachem R.
      • Reitzel R.
      • Borne A.
      • Jiang Y.
      • Tinkey P.
      • Uthamanthil R.
      • et al.
      Novel antiseptic urinary catheters for prevention of urinary tract infections: correlation of in vivo and in vitro test results.
      ,
      • Johnson J.R.
      • Kuskowski M.A.
      • Wilt T.J.
      Systematic review: antimicrobial urinary catheters to prevent catheter-associated urinary tract infection in hospitalized patients.
      ,
      • Srinivasan A.
      • Karchmer T.
      • Richards A.
      • Song X.
      • Perl T.M.
      A prospective trial of a novel, silicone-based, silver-coated foley catheter for the prevention of nosocomial urinary tract infections.
      ] (CI).

       Chronic disposing situation (e.g. CF, chronic wound, late alloplastic infections, some CVC (>1 year for parenteral feeding, for example), some urinary catheters, implants/fillers (absorbable/material repairing defects temporarily))

      Chronic P. aeruginosa lung infection in CF cannot be prevented by prophylactic use of nebulized antibiotics in patients before the onset of intermittent P. aeruginosa colonization [
      • Heijerman H.
      • Westerman E.
      • Conway S.
      • Touw D.
      • Döring G.
      Inhaled medication and inhalation devices for lung disease in patients with cystic fibrosis: a European consensus.
      ] (DI). Coating of long-term CVC or coating of indwelling urinary catheters and urethral stents by antibiotics or silver alloy cannot prevent chronic biofilm infection. The coating showed no or minimal effect on biofilm formation in several multicentre randomized controlled trials and cannot be recommended for common use [
      • Pickard R.
      • Lam T.
      • Maclennan G.
      • Starr K.
      • Kilonzo M.
      • McPherson G.
      • et al.
      Antimicrobial catheters for reduction of symptomatic urinary tract infection in adults requiring short-term catheterisation in hospital: a multicentre randomised controlled trial.
      ,
      • Srinivasan A.
      • Karchmer T.
      • Richards A.
      • Song X.
      • Perl T.M.
      A prospective trial of a novel, silicone-based, silver-coated foley catheter for the prevention of nosocomial urinary tract infections.
      ] (DI).
      There is little information regarding tissue implants/fillers where local irrigation has been used [
      • Viola G.M.
      • Raad II,
      • Rolston K.V.
      Breast tissue expander-related infections: perioperative antimicrobial regimens.
      ] (CIII).

       Q2-3 Can some biofilm infections be prevented or early biofilm infections be eradicated by early antibiotic treatment of colonization (no symptoms or signs) with bacteria known to be able to cause biofilm infections in the type of patients in question?

       Chronic P. aeruginosa lung infection in CF

      The chronic infections can be prevented by pre-emptive systemic and/or nebulized antibiotic eradication therapy of intermittent P. aeruginosa colonization of the lungs [
      • Döring G.
      • Høiby N.
      Early intervention and prevention of lung disease in cystic fibrosis: a European consensus.
      ,
      • Döring G.
      • Flume P.
      • Heijerman H.
      • Elborn J.S.
      Treatment of lung infection in patients with cystic fibrosis: current and future strategies.
      ] (AI).

       Patients with intravenous catheters

      Yes, ALT in case of repeated positive blood culture with identical coagulase-negative staphylococci or other microbes from CVC but no clinical signs of infection, also called CVC colonization [
      • Mermel L.A.
      • Allon M.
      • Bouza E.
      • Craven D.E.
      • Flynn P.
      • O'Grady N.P.
      • et al.
      Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America.
      ,
      • Walraven C.J.
      • Lee S.A.
      Antifungal lock therapy.
      ] (CIII). There are no data that point to preference of specific antimicrobial agents.

       Patients with VAP)

      No data available.

       Patients with chronic wound infection

      No data available.

       Patients with infections related to their orthopaedic alloplastic devices

      No data available.

       Patients with indwelling urinary catheters and urethral stents

      No data available.

       Patients with infections connected to tissue fillers, e.g. breast implants

      No data available.

       Q2-4 Can some non-foreign-body-related biofilm infections be eradicated with antibiotics? Can some foreign-body (e.g. artificial joints) related biofilm infections be eradicated with antibiotics without removal of the foreign body? Can biofilm infections in critically important organs (e.g. lungs) or implants (e.g. aortic grafts) be eradicated by antibiotics? Or rather persistently suppressed by antibiotics? Are there any methods to evaluate if the treatment is successful and can be stopped?

      Generally such biofilm infections afflict patients who need from a few weeks to life-long suppressive antibiotic treatment, if eradication cannot be achieved. There is a risk of development of resistance due to conventional mechanisms (the risk is reduced by combination therapy at least initially when the number of microorganisms—and therefore risk of mutation—is higher) and of adverse events caused by antibiotic treatment. It is unknown which antibiotic concentration/dose is needed to suppress biofilm infection, whereas this is known in some diseases regarding treatment of spreading planktonic microbial infections originating from the biofilm focus and thereby achieving containment of the biofilm infection (e.g. CF with chronic P. aeruginosa lung infection which is a focal infection in localized areas of the lungs). Currently only clinical signs and symptoms and paraclinical tests (detection of the microorganisms by culture or culture-independent methods, imaging techniques, inflammatory parameters) are available for evaluation of the effect of antibiotic therapy. However, even if the surrogate parameters indicate a favourable response, the microorganisms in the biofilm may survive and give rise to relapse in the case of cessation of antibiotic therapy. Antibody levels may stabilize or decrease slowly in the case of successful therapy, but are not a reliable marker of successful treatment. Generally, there are no data concerning when or if the chronic suppressive antibiotic therapy can be stopped if the underlying condition is still present.

       CF with chronic lung infections

      Chronic P. aeruginosa lung infection can be eradicated in a few patients within the first 1–2 years after the onset of the infection [
      • Döring G.
      • Høiby N.
      Early intervention and prevention of lung disease in cystic fibrosis: a European consensus.
      ,
      • Döring G.
      • Flume P.
      • Heijerman H.
      • Elborn J.S.
      Treatment of lung infection in patients with cystic fibrosis: current and future strategies.
      ] (AII). This is not possible in most cases of chronic lung infection. In these patients, chronic suppressive antibiotic therapy is given as daily nebulized antibiotics combined with systemic antibiotics either regularly every 3 months or at acute exacerbations and the patients’ lung function can thereby be maintained for decades [
      • Döring G.
      • Conway S.P.
      • Heijerman H.G.
      • Hodson M.E.
      • Høiby N.
      • Smyth A.
      • et al.
      Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus.
      ,
      • Heijerman H.
      • Westerman E.
      • Conway S.
      • Touw D.
      • Döring G.
      Inhaled medication and inhalation devices for lung disease in patients with cystic fibrosis: a European consensus.
      ,
      • Döring G.
      • Flume P.
      • Heijerman H.
      • Elborn J.S.
      Treatment of lung infection in patients with cystic fibrosis: current and future strategies.
      ] (AI).

       Patients with infections related to their orthopaedic alloplastic devices (e.g. joint prosthesis)

      Some foreign-body related biofilm infections can be eradicated with antibiotics without removal of the foreign body, if duration of symptoms of infection is ≤3 weeks, implant is stable, there is an absence of sinus tract and microorganisms are susceptible to suitable biofilm-validated antibiotics followed by debridement and antibiotic combination therapy, which includes an agent with efficacy on biofilms. Rifampicins have this property against staphylococci and fluoroquinolones have this property against Gram-negative bacilli, but should be given as combination therapy with another antibiotic due to the risk of development of resistance [
      • Zhao X.
      • Drlica K.
      Restricting the selection of antibiotic-resistant mutant bacteria: measurement and potential use of the mutant selection window.
      ]. Antibiotic combination therapy has to be combined with initial debridement surgery and exchange of modular parts of the implant [
      • Osmon D.R.
      • Berbari E.F.
      • Berendt A.R.
      • Lew D.
      • Zimmerli W.
      • Steckelberg J.M.
      • et al.
      Diagnosis and management of prosthetic joint infection: clinical practice guidelines by the Infectious Diseases Society of America.
      ] and has proved to be more effective than monotherapy [
      • Vilchez F.
      • Martinez-Pastor J.C.
      • Garcia-Ramiro S.
      • Bori G.
      • Macule F.
      • Sierra J.
      • et al.
      Outcome and predictors of treatment failure in early post-surgical prosthetic joint infections due to Staphylococcus aureus treated with debridement.
      ,
      • Cataldo M.A.
      • Petrosillo N.
      • Cipriani M.
      • Cauda R.
      • Tacconelli E.
      Prosthetic joint infection: recent developments in diagnosis and management.
      ,
      • Marschall J.
      • Lane M.A.
      • Beekmann S.E.
      • Polgreen P.M.
      • Babcock H.M.
      Current management of prosthetic joint infections in adults: results of an Emerging Infections Network survey.
      ,
      • Parvizi J.
      • Gehrke T.
      • Chen A.F.
      Proceedings of the International Consensus on Periprosthetic Joint Infection.
      ]. This strategy is recommended (AII).

       Patients with intravenous catheters

      Antimicrobial lock therapy is recommended in case of uncomplicated CR-BSI caused by coagulase-negative staphylococci, Enterobacteriaceae and possibly P. aeruginosa [
      • Funalleras G.
      • Fernandez-Hidalgo N.
      • Borrego A.
      • Almirante B.
      • Planes A.M.
      • Rodriguez D.
      • et al.
      Effectiveness of antibiotic-lock therapy for long-term catheter-related bacteremia due to Gram-negative bacilli: a prospective observational study.
      ,
      • Fernandez-Hidalgo N.
      • Almirante B.
      • Calleja R.
      • Ruiz I.
      • Planes A.M.
      • Rodriguez D.
      • et al.
      Antibiotic-lock therapy for long-term intravascular catheter-related bacteraemia: results of an open, non-comparative study.
      ,
      • Rijnders B.J.
      • Van W.E.
      • Vandecasteele S.J.
      • Stas M.
      • Peetermans W.E.
      Treatment of long-term intravascular catheter-related bacteraemia with antibiotic lock: randomized, placebo-controlled trial.
      ,
      • Fortun J.
      • Grill F.
      • Martin-Davila P.
      • Blazquez J.
      • Tato M.
      • Sanchez-Corral J.
      • et al.
      Treatment of long-term intravascular catheter-related bacteraemia with antibiotic-lock therapy.
      ,
      • del Pozo J.L.
      • Garcia C.M.
      • Hernaez S.
      • Martinez A.
      • Serrera A.
      • Aguinaga A.
      • et al.
      Effectiveness of teicoplanin versus vancomycin lock therapy in the treatment of port-related coagulase-negative staphylococci bacteraemia: a prospective case-series analysis.
      ] (AII). CR-BSI caused by S. aureus should lead to catheter removal because of a high risk of haematogenous complications, endocarditis and a low rate of treatment success in case of ALT [
      • Rijnders B.J.
      • Van W.E.
      • Vandecasteele S.J.
      • Stas M.
      • Peetermans W.E.
      Treatment of long-term intravascular catheter-related bacteraemia with antibiotic lock: randomized, placebo-controlled trial.
      ,
      • del Pozo J.L.
      • Garcia C.M.
      • Hernaez S.
      • Martinez A.
      • Serrera A.
      • Aguinaga A.
      • et al.
      Effectiveness of teicoplanin versus vancomycin lock therapy in the treatment of port-related coagulase-negative staphylococci bacteraemia: a prospective case-series analysis.
      ,
      • Fowler Jr., V.G.
      • Sanders L.L.
      • Sexton D.J.
      • Kong L.
      • Marr K.A.
      • Gopal A.K.
      • et al.
      Outcome of Staphylococcus aureus bacteremia according to compliance with recommendations of infectious diseases specialists: experience with 244 patients.
      ,
      • Fowler Jr., V.G.
      • Justice A.
      • Moore C.
      • Benjamin Jr., D.K.
      • Woods C.W.
      • Campbell S.
      • et al.
      Risk factors for hematogenous complications of intravascular catheter-associated Staphylococcus aureus bacteremia.
      ]. CR-BSI caused by Candida spp. should also lead to catheter removal, because non-removal is associated with an increased mortality and a more prolonged candidaemia [
      • Mermel L.A.
      • Allon M.
      • Bouza E.
      • Craven D.E.
      • Flynn P.
      • O'Grady N.P.
      • et al.
      Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America.
      ,
      • Ullmann A.J.
      • Akova M.
      • Herbrecht R.
      • Viscoli C.
      • Arendrup M.C.
      • Arikan-Akdagli S.
      • et al.
      ESCMID* guideline for the diagnosis and management of Candida diseases 2012: adults with haematological malignancies and after haematopoietic stem cell transplantation (HCT).
      ,
      • Lai K.K.
      • Fontecchio S.A.
      Use of silver-hydrogel urinary catheters on the incidence of catheter-associated urinary tract infections in hospitalized patients.
      ,
      • Segarra-Newnham M.
      • Martin-Cooper E.M.
      Antibiotic lock technique: a review of the literature.
      ,
      • Andes D.R.
      • Safdar N.
      • Baddley J.W.
      • Playford G.
      • Reboli A.C.
      • Rex J.H.
      • et al.
      Impact of treatment strategy on outcomes in patients with candidemia and other forms of invasive candidiasis: a patient-level quantitative review of randomized trials.
      ,
      • Nucci M.
      • Colombo A.L.
      • Silveira F.
      • Richtmann R.
      • Salomao R.
      • Branchini M.L.
      • et al.
      Risk factors for death in patients with candidemia.
      ,
      • Rex J.H.
      • Bennett J.E.
      • Sugar A.M.
      • Pappas P.G.
      • Serody J.
      • Edwards J.E.
      • et al.
      Intravascular catheter exchange and duration of candidemia. NIAID Mycoses Study Group and the Candidemia Study Group.
      ,
      • Nucci M.
      • Anaissie E.
      • Betts R.F.
      • Dupont B.F.
      • Wu C.
      • Buell D.N.
      • et al.
      Early removal of central venous catheter in patients with candidemia does not improve outcome: analysis of 842 patients from 2 randomized clinical trials.
      ,
      • Pappas P.G.
      • Kauffman C.A.
      • Andes D.
      • Benjamin Jr., D.K.
      • Calandra T.F.
      • Edwards Jr., J.E.
      • et al.
      Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America.
      ,
      • Cornely O.A.
      • Bassetti M.
      • Calandra T.
      • Garbino J.
      • Kullberg B.J.
      • Lortholary O.
      • et al.
      ESCMID* guideline for the diagnosis and management of Candida diseases 2012: non-neutropenic adult patients.
      ,
      • Cateau E.
      • Berjeaud J.M.
      • Imbert C.
      Possible role of azole and echinocandin lock solutions in the control of Candida biofilms associated with silicone.
      ]. Even if catheter removal is recommended, many patients are not candidates for catheter replacement because of their general condition. The role of antifungal lock therapy against Candida is not well defined. Azoles have poor activity against Candida biofilms both in vitro and in vivo; lipid formulations of amphotericin B are more effective than amphotericin B deoxycholate, and echinocandins have excellent activity [
      • Walraven C.J.
      • Lee S.A.
      Antifungal lock therapy.
      ,
      • Cateau E.
      • Berjeaud J.M.
      • Imbert C.
      Possible role of azole and echinocandin lock solutions in the control of Candida biofilms associated with silicone.
      ,
      • Toulet D.
      • Debarre C.
      • Imbert C.
      Could liposomal amphotericin B (L-AMB) lock solutions be useful to inhibit Candida spp. biofilms on silicone biomaterials?.
      ]. If conservative treatment with ALT is chosen, clinical status and blood cultures should be monitored to detect treatment failure, and therefore leading to catheter removal. In cases of CR-BSI, systemic antibiotics should always be associated with ALT (AII).

       Patients with endotracheal tube biofilm and VAP

      Systemic antibiotic therapy is not effective in clearing the biofilm present on the ETT [
      Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia.
      ] (DII). In patients with VAP, the pulmonary infection is treatable when appropriate antibiotics guided by microbiological findings are promptly administered [
      Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia.
      ] (AI). The duration of therapy should be based upon the clinical response. In the majority of cases, a 7-day treatment period is appropriate. Nevertheless, in VAP caused by P. aeruginosa or methicillin-resistant S. aureus, a longer treatment up to 14–21 days is preferable. It is recommended to reassess the clinical response to therapy after 48–72 h and therapy should be promptly changed in the case of lack of clinical improvement [
      Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia.
      ] (AI).

       Patients with indwelling urinary tract catheters or urethral stents

      Antibiotic therapy is not sufficient to clear the biofilm present on the stent or catheter, but only decreases the number of microorganisms in the urine and results in suppression of the symptoms [
      • Anaissie E.J.
      • Rex J.H.
      • Uzun O.
      • Vartivarian S.
      Predictors of adverse outcome in cancer patients with candidemia.
      ]. If the catheter is not removed or replaced, relapse will probably occur after the end of treatment, therefore antibiotic treatment without removal of stent or catheter cannot be recommended (AIII).

       Patients with chronic wound infection

      There is no reported evidence to support the use of systemic antimicrobial agents in the treatment of chronic wound-associated infections and their healing, even if this therapeutic approach is quite common in clinical practice [
      • Lipsky B.A.
      • Berendt A.R.
      • Cornia P.B.
      • Pile J.C.
      • Peters E.J.
      • Armstrong D.G.
      • et al.
      Executive summary: 2012 Infectious Diseases Society of America clinical practice guideline for the diagnosis and treatment of diabetic foot infections.
      ,
      • O'Meara S.
      • Cullum N.
      • Majid M.
      • Sheldon T.
      Systematic reviews of wound care management: (3) antimicrobial agents for chronic wounds; (4) diabetic foot ulceration.
      ,
      • Howell-Jones R.S.
      • Wilson M.J.
      • Hill K.E.
      • Howard A.J.
      • Price P.E.
      • Thomas D.W.
      A review of the microbiology, antibiotic usage and resistance in chronic skin wounds.
      ] (CIII).

       Patients with infections connected to tissue fillers e.g. breast implants

      No data available.

       Q2-5 What is the optimal antibiotic strategy for treatment of established biofilm infections?

       Cystic fibrosis

      Chronic suppressive therapy with nebulized antibiotics and systemic antibiotics either regularly every 3 months, or at acute exacerbations. Systemically administered antibiotics yield concentrations in the respiratory compartment of the lungs, which are adequate for planktonic bacteria, although insufficient for biofilm-growing bacteria, whereas the concentrations reached in the conductive compartment of the lungs (bronchi, sputum) are low. Topically administered antibiotics by inhalation, on the other hand, provide very high concentrations in the conductive compartment of the lungs, but low concentrations in the respiratory compartment. Combinations of systemic and topical antibiotics are therefore frequently used to reach both the respiratory and the conductive compartments of the lungs [
      • Döring G.
      • Conway S.P.
      • Heijerman H.G.
      • Hodson M.E.
      • Høiby N.
      • Smyth A.
      • et al.
      Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus.
      ,
      • Döring G.
      • Høiby N.
      Early intervention and prevention of lung disease in cystic fibrosis: a European consensus.
      ,
      • Heijerman H.
      • Westerman E.
      • Conway S.
      • Touw D.
      • Döring G.
      Inhaled medication and inhalation devices for lung disease in patients with cystic fibrosis: a European consensus.
      ,
      • Döring G.
      • Flume P.
      • Heijerman H.
      • Elborn J.S.
      Treatment of lung infection in patients with cystic fibrosis: current and future strategies.
      ,
      • Bensel T.
      • Stotz M.
      • Borneff-Lipp M.
      • Wollschlager B.
      • Wienke A.
      • Taccetti G.
      • et al.
      Lactate in cystic fibrosis sputum.
      ] (AI). Pharmacokinetics/pharmacodynamics of antibiotics against biofilm-growing microorganisms has only been carried out in animal studies and only with a few antibiotics, and the results indicate that β-lactam antibiotics show time-dependent killing and ciprofloxacin, colistin and tobramycin show concentration- or dose-dependent killing of biofilm-growing P. aeruginosa cells similar to planktonic growing cells. However, the concentrations of antibiotic needed were, in all cases, much higher even in the case of antibiotics with time-dependent killing [
      • Hengzhuang W.
      • Wu H.
      • Ciofu O.
      • Song Z.
      • Høiby N.
      Pharmacokinetics/pharmacodynamics of colistin and imipenem on mucoid and nonmucoid Pseudomonas aeruginosa biofilms.
      ,
      • Hengzhuang W.
      • Wu H.
      • Ciofu O.
      • Song Z.
      • Høiby N.
      In vivo pharmacokinetics/pharmacod