Endotracheal tube biofilms in ventilator-associated ...



Implications and current control strategies for ventilator-associated pneumonia

Ching-Yee Loo1.2, Wing-Hin Lee1,2, Paul M. Young2,3, Rosalia Cavaliere4, Cynthia B. Whitchurch4, and Ramin Rohanizadeh1*

1Advanced Drug Delivery Group, Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia

2 Woolcook Institute of Medical Research and Discipline of Pharmacology, Sydney Medical School, University of Sydney, NSW 2006, Australia

3Discipline of Pharmacology, Sydney Medical School, University of Sydney, NSW 2006, Australia

4The ithree institute, University of Technology Sydney, Ultimo, NSW 2007, Australia.

*Corresponding author:

Dr Ramin Rohanizadeh

Faculty of Pharmacy (A15), University of Sydney, Sydney, New South Wales 2006, Australia. E-mail: ramin.rohanizadeh@sydney.edu.au

Summary

Ventilator-associated pneumonia (VAP) remains a major burden to the healthcare system and intubated patients in intensive care units (ICU). In fact, VAP is responsible for at least 50% of prescribed antibiotics to patients who need mechanical ventilation. One of the factors contributing to VAP pathogenesis is believed to be rapid colonization of biofilm-forming pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus on the surface of inserted endotracheal tubes. These biofilms serve as a protective environment for bacterial colonies and provide enhanced resistance towards many antibiotics. Several strategies have been adopted to reduce the occurrence of VAP such as control of cuff pressure, aspiration of subglotic secretion and use of endotracheal tubes with ultrathin cuff membranes, to eliminate or prevent biofilm formation. This review presents and discusses an overview of current strategies to inhibit the colonization and formation of biofilm on endotracheal tubes, including antibiotic treatment, surface modification and antimicrobial agent incorporation onto endotracheal tube materials.

Keywords: biofilm, Pseudomonas aeruginosa, Staphylococcus aureus, ventilator associated pneumonia, silver nanoparticle, surface modification

1. Introduction

Hospital-acquired pneumonia (HAP) is the second most common lung infection caused by microorganisms and pathogens, and is responsible for one quarter of infections in intensive care units (ICU) [1,2]. Ventilator-associated pneumonia (VAP) is defined as a nosocomial infection occurring in patients that rely on mechanical ventilators, via invasive methods (tracheostomy tube and endotracheal intubation). VAP accounts for more than 80% of HAP [2]. Statistics have shown that VAP affects up to 28% of intubated patients and the incidence rate escalates with time [1,3]. The probability of developing VAP is the highest during early intubation; with ~3% infection/day for the first 5 days of intubation decreasing to 2% per day from day 5 to 10 [1,4].

Numerous studies have highlighted the role of the endotracheal tubes in the pathogenesis of VAP [5,6]. In the case of critically ill patients, endotracheal tubes are used to provide ventilation to patients. However, these endotracheal tubes often impair normal mucocilliary clearance, leading to accumulation of tracheobronchial secretions whilst increasing the risk of pneumonia infection [7]. Secondly, the action of endotracheal tube insertion may cause injury and introduce exogenous and/or endogenous bacterial flora into the mucosa [8]. This provides an active reservoir for bacterial colonization, which can develop into multiple-antibiotic resistant biofilms [9]. A common hypothesis is that the biofilm arise from aspirated secretions, environmental contamination of “breathe-air” through ventilators and accumulation of secretions into tracheobronchial sections [9-11]. Irrespective of the contamination source, it is agreed that the biofilm harbors pathogenic microorganisms that cause systemic infections and significant mortality. Pseudomonas aeruginosa and Staphylococcus aureus are major causal pathogens of endotracheal tube associated infections with 41.7% and 36.7% VAP cases being attributed to them respectively [3]. Other microorganisms contributing to VAP are Streptococcus pneumoniae, Acinetobacter baumannii, Enterobacteriacea and other Gram-negative aerobic bacteria [2,3].

Many guidelines for the prevention of VAP have been published [12-16], which including a ventilator bundle launched in 2006 by US agencies and scientific groups [16]. In the following year, guidelines across European were reviewed and several new guidelines established [13,14]. Among them, the use of endotracheal tubes with subglottic secretion drainage (SSD) is recommended in all guidelines while tracheostomy is not endorsed [13-15]. Coating and impregnation of endotracheal tubes with silver fall in the gray area as these approaches are recommended by some guidelines while some are against them [13-15]. In 2010, Lorente and co-workers reviewed emerging strategies that had not been included in the previous guidelines [17]. These strategies include using endotracheal tubes with ultrathin cuff membranes and SSD, endotracheal tubes with low cuff pressure, device with balloons to remove biofilm, and saline instillation [17].

Significant research has been devoted to understand the underlying mechanism of biofilm formation and strategies utilised by these microorganisms to survive antimicrobial attack [18-21]. With these insights, approaches have been devised which could be generalized in two categories: 1) inhibition of bacterial colonization [22-28] and 2) eradication or detachment of formed biofilms [29-33]. As the functional biology of biofilms is broad and extensive literature is available, this review we will mainly focus on the emerging strategies for biofilm treatment in these two categories; with emphasis on the pre-clinical development phase.

2. Biofilm and the mechanism of formation

P. aeruginosa is the most studied biofilm forming bacterial species [34,35]. In simple terms, biofilms are sessile communities of bacteria, which attach to biotic or abiotic surfaces in aqueous environments. The cascade of events of biofilm formation are 1) deposition of a conditioning layer on a surface, 2) transport of planktonic cells to the surface via diffusive, convective or active flagella-driven transport, 3) initial surface contact followed by reversible adsorption/desorption of cells on the surface (initial colonization), 4) irreversible binding onto the surface, rapid propagation and microcolony formation, 5) matrix production and biofilm maturation; and 6) detachment (release) of pioneer cells [36]. This process is outlined in Figure 1.

The colonization of bacteria onto surfaces is universally regarded as the initiation of biofilm communities. At this stage, planktonic cells move along the surface via motility or Brownian motion. Once the cells are within close proximity of a surface, reversible colonization occurs when the net attractive forces outweighed the repulsive forces generated between the bacterial cell and contact surface [37]. This contact results in the formation of a monolayer of cells on the surface. At this stage, the bacterial cells are still susceptible to antibiotic treatment.

Based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) adhesion theory, the main interactions between cells and the surface are through van der Waals and Coulomb forces [38]. In aqueous conditions, counter ions generated as a result of surface charges are attracted to each surface to form electric double layers. As bacterial cells are negatively charged, repulsive electrostatic interactions between them are intensified in surfaces that having the same net charge as a result of an overlapping electric double layer [39,40]. Additionally, to ensure the bacteria remain irreversible anchored to the solid surface prior to or after attachment, exopolysaccharides and eDNA surface conditioning materials are secreted by pioneer cells to act as binding bridges [37]. Finally, bacterial cells can utilize their pili and flagella to pierce through the potential energy barriers resulting from repulsive forces owing to their small radii. A study by O’Toole and Kolter showed that the pili and flagella are necessary for biofilm formation in P. aeruginosa [41]

The next step of biofilm formation involves multiplication and aggregation of attached monolayer cells into microcolonies. At an appropriate time, these microcolonies differentiate into mature biofilms which are enveloped within an extrapolymeric substance (EPS) matrix [42]. EPS comprises polysaccharides (50 to 90% of the organic mass of biofilm mass), proteins, DNA and lipids [42-44]. EPS are highly hydrated and tenaciously bound to the surface. Water channels in EPS allow transport of essential nutrients and oxygen to cells embedded within the biofilm. In vitro, mature biofilms can grow up to 50 μm in thickness with tower-like and mushroom-shaped structures. Bacterial biofilms at this stage are extremely well-tolerant to antimicrobial agents, and various mechanisms are activated which include limiting biocide penetration, reduced growth rate and the presence of persister cells for enhanced survival rate. The final step in biofilm development involves the dispersion of biofilm in order to liberate and spread bacterial cells to other locations for initiation of new biofilms.

3. Approaches to prevent biofilm establishment and/or eradication of preformed biofilms

3.1 Antimicrobial agents

3.1.1 Antibiotics

The inappropriate use of antibiotics is a key reason contributing to the emergence of antibiotic resistance. To achieve optimal outcome in mechanically ventilated patients with biofilm infections the appropriate selection of existing antibiotics, duration of treatment, combination therapy, inhaled antibiotic formulation and development of novel antibiotics should be considered. The selection of appropriate antibiotics is often difficult mainly due to the lack of comparative randomized double-blinded studies demonstrating significant differences between each antibiotic treatment group [45]. Furthermore, some antibiotics are prone to developing resistance during therapy, which ultimately causes treatment failure. For example, as bacterial tolerance towards gentamicin during therapy is common, one attempt to replace gentamicin with amikacin instead was not successful because once amikacin treatment was discontinued and gentamicin was re-used, the resistance level returned [46]. Faced with these challenges, a general empiric antibiotic therapy regimen has been adopted over the years for suspected VAP cases caused by bacterial biofilm infections. Generally, upon suspicion of P. aeruginosa biofilm occurrence, combination therapies of anti-pseudomonal antibiotics are used. Commonly, a β-lactam (cephalosporin) is used in conjunction with a fluoroquinolone (ciprofloxacin or levofloxacin) or an aminoglycoside (amikacin or tobramycin) [47]. On the other hand, patients with suspected Gram-positive bacterial infections are often treated with vancomycin as an initial choice coupled with anti-methicillin resistant Staphyloccus aureus (MRSA) agents such as linezolid [47]. Recommended therapy for early infections with no risk factors of multidrug resistant microorganisms is mono-antibiotic therapy with fluoroquinolones, ertapenem, ceftriaxone or ampicillin [47,48].

In recent years, inhaled antibiotics have been evaluated as a method to prevent biofilm formation on endotracheal tubes and as a possible adjunctive therapy for VAP [49-53]. Inhalation therapy is associated with high local antibiotic concentrations within the lung, superior penetrability and much lower systemic toxicity [54]. In a study by Palmer and co-workers, the concentration of aerosolized antibiotic in sputum was 200-fold higher than that achieved through systemic administration [55]. To date, the antibiotics used for inhalation administration include gentamicin, colistin, tobramycin, polymyxin B, amikacin, ceftazidim and pentamidine [49,51-53,56-60]. Both aerosolized tobramycin and colistin have been specifically formulated for administration to mechanically ventilated patients [49-52,57]. Adair and co-workers compared the efficacy of nebulised gentamicin and parenterally administered cephalosporin to inhibit biofilm formation on endotracheal tubes [56]. Nebulised gentamicin demonstrated superior performance in terms of higher local concentration on the tube and controlling bacterial infection compared to parenteral administration. In addition, biofilm formation was evident on all endotracheal tubes for patients receiving parenterally administered antibiotic while only 40% of tubes dosed with nebulized gentamicin showed biofilm formation [56]. Czosnowski and co-workers demonstrated that inhaled formulation of antibiotic was successful in treating >70% of ventilator associated infections including infections by multidrug resistant pathogens [61]. In a recent Phase II clinical trial, 29 mechanically ventilated patients with VAP were randomized to either receive aerosolized amikacin or placebo every 12 h for 7–14 days [62]. Consistent with other findings, delivery of aerosolized amikacin sustained high concentrations in the lower respiratory tract with negligible adverse effects [62-64]. In another double-blind, placebo controlled trial with mechanical ventilated patients infected with Gram-negative pathogens, adjunctive therapy of inhaled amikacin (400 mg) twice per day in addition to systemic antibiotics helped to substantially reduce systemic antibiotics administration by 50% on day 7 [63]. Inhaled colistin has been developed as a response to multidrug resistance in P. aeruginosa and A. baumannii infections in seriously ill mechanically ventilated patients [49]. Most studies with inhaled colistin provided encouraging results, with at least 80% cure in patients [57]. Both ciprofloxacin and aztreonam are currently under evaluation as potential inhaled therapeutic agents. A Phase III clinical trial for inhaled ciprofloxacin is underway to investigate its effectiveness towards mechanically ventilated patients [45].

New antibiotics proposed for the treatment of biofilms include drugs such as tigecycline, ceftobiprole, and telavancin [31,65-68]. Tigecycline is a derivative of tetracycline with broad spectrum activities against Gram-negative, Gram-positive and anaerobic strains including MRSA [68]. In a comparison study between antibiotics on their actions against MRSA biofilm eradication, rifampin, daptomcyin and tigecycline were able to detach mature biofilms while linezolid, tobramcyin and levofloxacin were only effective against young biofilms [31]. Recently, tigecycline, a known bacteriostatic agent was coupled with gentamicin to treat biofilms of clinical MRSA isolates [68]. Both antibiotics demonstrated synergistic killing towards MRSA biofilms compared to either antibiotic alone [68]. Telavancin is a new lipoglycopeptide antibiotic which is similar structurally to vancomycin yet has been modified to incorporate a lipophilic side chain. With such similarity, televancin is expected to possess potent bactericidal activity towards MRSA. Recent evidence revealed that telavancin is more effective compared to vancomycin. The observed MIC for telavancin against MRSA ranged from 0.06 to 1.0 μg/mL while that of vancomycin was between 0.5 to 2.0 μg/mL [69,70]. With regard to anti-biofilm activity, telavancin was also consistently more potent than vancomycin [66,67]. For instance the minimal biofilm eradication concentration for telavancin was 0.125 to 2 μg/mL, while, for the same action, the required concentration for vancomycin exceeded 512 μg/mL [66].

Other approaches such as the use of electrical and ultrasound therapies to enhance antibiotic potency have been reported to treat biofilms. Ultrasound therapy is associated with aiding antibiotic transport through the biofilm or may behave as a stimulus to induce antibiotic release from coatings [33]. As demonstrated by Norris and co-workers, formation of P. aeruginosa biofilm was significantly impeded on ciprofloxacin-coated hydrogels when they were exposed to ultrasound therapy (43 kHz) [33]. Furthermore, by applying low levels direct currents (70 μA/cm2) in adjuvant with 1.5 μg/mL tobramycin, it was found that more than 90% of the subpopulation of persister cells in both planktonic and those embedded in biofilm could be efficiently killed [32]. As such, one could envisage designs of endotracheal tubes containing antibiotic reservoirs so that, upon infection with a biofilm, the release of antibiotic could be triggered with external high-intensity ultrasound [33]. Another interesting idea is to utilize photodynamic therapy to kill bacterial biofilms. The principle behind this strategy is based upon the generation of reactive oxygen species by photosensitizing drugs to cause oxidative damage to biofilms. This was evident in a study in which photosensitizer methylene blue eradicated biofilm in endotracheal tubes by >99.9% (p < 0.05) after a single treatment [71].

3.1.2. Silver-based compounds

For centuries elemental silver and silver salts have been known for their antimicrobial activities [72,73]. However, the advent of antibiotics has dramatically reduced the medical applications of silver. The history of silver as an antimicrobial agent in clinical settings dates back to 1844 where a German obstetrician used 1% silver nitrate solution to treat blindness in newborns caused by Postpartum infections [72]. The renewed interest in silver may be a consequence of the emergence of multiple antibiotic-resistances in bacteria. In principle, silver-based therapy is advantageous because a) it is effective against various multidrug resistant microorganisms; b) it causes simultaneous and multiple antibacterial actions on cells which reduces the chance of cells acquiring resistance and c) it has low systemic toxicity [72]. Different forms of silver based compounds are used as antimicrobial therapy, including silver nanoparticles (AgNPs), silver salts, dendrimer-silver complexes and polymer-silver nanocomposites [29,72-79]. Although the mechanism of silver toxicity is mainly believed to derive from the release of Ag+ ions, some reports have pointed out that additional toxicity routes exist with AgNPs.

Recent evidence has demonstrated that in addition to having potent antimicrobial effects, silver also exerts anti-biofilm activities [29,30,77,78,80-84]. Bjarnshlolt and co-workers demonstrated that the addition of 5–10 μg/mL silver sulfadiazine was effective at eradicating established mature P. aeruginosa biofilms while 1 μg/mL had no apparent effect [78]. In comparison, tobramycin had negligible effect even at high concentrations (100 μg ml–1). Two interesting findings were noted; a) the tolerance of P. aeruginosa was dependent on both drug dose and mode of cell growth. Planktonic P. aeruginosa were more susceptible towards silver sulfadiazine compared to biofilms where a 100-fold higher dose was required; b) the resistance mechanism of P. aeruginosa biofilms towards silver sulfadiazine was quorum sensing independent. In other words, the regulation of cell-cell signaling towards the development of increased antimicrobial resistance was not an effective protective mechanism against silver since this compound demonstrated no significant difference in eradicating both quorum sensing activated- and deficient- biofilm systems [78,84]. Chaudhari and co-workers demonstrated that quorum sensing signal of S. aureus biofilm communities was quenched in the presence of AgNPs alone but not in the presence of antibiotic [84]. The surface conditioning layers (e.g., polysaccharides) deposited by bacteria were thought to be neutralized by AgNPs which thus effectively hindered biofilm formation [84]. In another study, 100 nM biologically synthesized AgNPs used against P. aerugionsa and S. epidermis biofilms resulted in ~95% eradication of the biofilms [75]. The authors argued that the diffusion of AgNPs was not hindered by EPS matrix as these nanoparticles reach bacterial cells within the biofilm matrix through the existing water channels, thus were able to impart their antimicrobial activity [75]. Another type of bio-based AgNPs synthesized by leaf extract broth of Azadirhacta indica showed uniform spherical particles with average sizes of 50 to 60 nm [85]. These nanoparticles caused distinct biofilm formation retardation in S. aureus clinical isolates by interrupting the secretion of carbohydrates and proteins necessary to form the biofilm matrix [85].

The efficacy of AgNPs as anti-biofilm agents varies according to the nanoparticle size, coating and shape, particle diffusion into the biofilm and type of microorganism [29,30,77,80,82]. Spherical citrate-capped AgNPs (average mean size of 8 nm) synthesized via chemical reduction method with sodium borohydride as a reducing agent was used to establish the correlation between inactivation of planktonic cells and biofilm formation of P. aeruginosa [80]. Treatment using 10 μg ml–1 of AgNPs hindered more than 60% biofilm formation even though half of the planktonic cells population survived the treatment. Complete killing of planktonic cells and inhibition of biofilm formation was not achievable even at AgNPs concentrations higher than 90 μg ml–1. Although EPS was indeed produced by these AgNPs-treated cells, the cells had seemingly lost the ability to adhere firmly to solid surfaces. In addition, it is hypothesized that the presence of AgNPs caused a change in energy balance, which subsequently activated a stress response in the bacterial cells [74,80]. Crucial intracellular components such as DNA or ribosomes were found crystallized in the centre of bacterial cells upon exposure to AgNPs or silver ions [80,86]. Kora and Arunachalam reported that 45-nm sodium dodecyl sulfate (SDS)-capped AgNPs synthesized by means of UV photoreduction demonstrated superior activity against biofilm formation. Complete biofilm inhibition was observed at concentration of AgNPs as low as 1 μg ml–1 [77]. It was probable that these SDS-capped AgNPs exerted their toxicity towards P. aeruginosa through the generation of reactive oxygen species (ROS) radicals which in turn damaged cell membranes, increased cell permeability and caused leakage of intracellular contents [77].

It is interesting to consider the net surface charge differences of stabilizers (i.e. SDS, citrate or polyvinylpyrrlidone PVP) on the efficacy of silver particles (i.e. via retention of the silver ions or inhibition of local oxidation). The cell wall of Gram-negative bacteria are comprised of a lipopolysaccharide membrane and inner peptidoglycan layer in which the amino, carboxyl and phosphate groups on its cellular membrane provide the bacterium with a negative charge. Citrate is anionic in nature and PVP is cationic while SDS is a known amphiphilic molecule. Being amphiphilic, SDS-capped AgNPs can be incorporated easily into the phospholipid bilayer of the bacterial membrane, which increases the interactions between AgNPs and membrane proteins. In comparison however, there exists a certain amount of electrostatic repulsion between negatively charged citrate-capped AgNPs and P. aeruginosa cells, which limits the cell-AgNPs interactions. Furthermore, Kittler and co-workers showed that the dissolution rate of AgNPs stabilized using a negatively charged molecule (i.e. citrate) was lower than a positively charged molecule (i.e. PVP), probably owing to the role of citrate as a chemical barrier, preventing the release of Ag+ [76].

AgNPs are reported to be lethal to all microorganisms associated within biofilms [87], though a recent study by Martinez-Gutierrez and co-workers suggested that the anti-biofilm behavior of these nanoparticles was strain-dependent [30]. Gram-negative bacteria biofilms (P. aeruginosa, Acinetobacter baumanii) were more susceptible towards AgNPs compared to Gram-positive staphylococci biofilms [30]. An approximate1-log reduction was noted for Gram-negative strains compared to 20-nm AgNPs (69%) > 35-nm AgNPs (52%) [29]. The presence of charged organic matter also altered biofilm resistance to both AgNPs and dissolved silver ions [88]. Stable non-agglomerated AgNPs was found to exert greater toxicity to P. fluorescens biofilms compared to both highly agglomerated particles and dissolved ions. Dissolved silver ions were non-toxic to bacterial cells due to the complexation of cationic Ag+ with anionic biomacromolecules present on the surface of biofilm matrix, thus reducing the number of available ionized silver for bactericidal action [88]. The authors suggested that the transport of AgNPs aggregates was hindered by EPS, limiting AgNPs delivery directly to adherent cells. In comparison, non-aggregated AgNPs could diffuse into the biofilm matrix easily and exert their toxicity either via direct nanoparticle-cell interactions or through slow AgNP dissolution. In this way, stable AgNPs remained toxic to bacterial biofilms and inactivation of ionic compounds (Ag+) with surface EPS is minimised [88].

3.2 Surface modification and incorporation of antimicrobial agents

In general, the three main factors that influence bacterial adhesion onto solid surfaces are i) microenvironment (pH, ionic strength, nutrient availability); ii) type and characteristics of the bacteria; and iii) physicochemical and morphological properties of the surface (hydrophobicity, surface roughness, energy) [22,28,89-93]. Surface modification of biomaterials (i.e. endotracheal tubes) to hinder bacterial colonization is an effective biofilm control strategy (Table 1).

As discussed, mature biofilms are extremely recalcitrant. Therefore, many believe that delaying or inhibiting bacteria attachment in the first place is a logical strategy. [22,24-26,28]. To date, strategies for surface modification of medical materials include i) physical modification to alter the surface topography of materials without addition of antimicrobial agents; ii) physical deposition using plasma, ion beam or corona discharge; iii) covalent binding of antimicrobial agents onto surfaces; iv) coating with a low energy polymer; and v) surface oxidation.

Lopez-Lopez and co-workers evaluated the kinetics of adherence of S. aureus, S. epidermis and P. aeruginosa on medical plastics made from different materials such as PVC, Teflon®, siliconised latex, polyurethane and Vialon® [93]. The authors found that material type was an important parameter for the rate of bacterial colonization whereby both polyurethane and Vialon® were the most hostile surface for staphylococci attachment while E. coli and P. aeruginosa tended to adhere the least to Teflon® [93]. Since then, many studies have revealed various factors are involved in the irreversible attachment of cells to a surface. Of particular significance in cell attachment is the surface properties including surface hydrophobicity, surface energy, porosity and chemical composition [90-92,94].

3.2.1 Hydrophilic vs. hydrophobic

A hydrophobic surface, as possessed by medical polymers, is often thought to be favored for bacterial colonization. However, conclusive remarks on this topic are difficult, as many conflicting data on the role of hydrophobicity on bacterial adhesion have been reported. This is likely to be predominately due to the classification of hydrophobicity, surface chemistry and nano-macroscopic structure. Many reports have demonstrated reduced bacterial adhesion to hydrophobic surfaces, however this is often linked to an increase in contact angle due to nanoscopic roughness (i.e. the lotus leaf affect). Conversely other studies have shown reduced adhesion to hydrophilic surfaces likely to be attributed to surface chemistry. This aspect is an important consideration when reviewing the literature.

To increase the surface hydrophilicity of PVC endotracheal tubes, Balazs and co-workers employed a chemical surface modification technique via oxygen glow discharge to introduce oxygenated functional groups on the tube [94]. Oxygen-plasma treated PVC surfaces became significantly more hydrophilic, as demonstrated by a decrease in water contact angle from 80° to 8–20°. Meanwhile, these treated surfaces showed large defects in the order of 15–30 μm, which could be a direct consequence of incorporating oxygenated functional groups. The authors found that the treated hydrophobic PVC tubes showed a 70% reduction in adhesion of four different strains of P. aeruginosa [94]. In another study, the adhesion of eighteen clinical isolates of P. aeruginosa on oxygen-plasma treated PVC endotracheal tubes was reported [92]. As expected, a more hydrophilic surface was better at hindering P. aeruginosa adhesion by as much as 70%. It is noteworthy to mention that the bacteria adhesion behavior differed significantly between strains. For example, four clinical strains isolated from endotracheal aspiration of ICU patients adhered at least 600% better to a surface than the model P. aeruginosa PAO1 laboratory strain, which indicates that future interventional strategies aimed to reduce bacterial adhesion should be studied on various P. aeruginosa strains rather than relying on a single laboratory strain [92]. Sousa and co-workers showed that the affinity of S. epidermis towards acrylic-based material (less hydrophobic) is lower than silicone (more hydrophobic) [90].

In contrast, Tang and co-workers reported that in vitro bacterial adhesion was reduced 42–89% with a more hydrophobic surface. [91]. However, it is likely that this may be reflective of the surface roughness rather than chemistry. The susceptibilities of smooth and nano-rough PVC endotracheal tubes to bacterial adhesion were evaluated in a recent study [28]. For this, commercially available endotracheal tubes (Sheridan® 6.0 mm ID, uncuffed) were subjected to lipase digestion to create a nano-rough texture. Using various validation techniques to determine the textural preferences of P. aeruginosa, it was concluded that nano-rough PVC endotracheal tubes reduced colonization by about 40% compared to smooth surfaces [28].

3.2.2. Modification of endotracheal tube surface topography at a nano-scale

Surface roughness at the nano-scale is suggested to minimize contact between the bacterial cell wall and the surface, thereby reducing the electrostatic interactions for initial cell attachment [28]. Furthermore, coating of a sugar metabolite (i.e. fructose) onto nano-rough PVC endotracheal tube surfaces was shown to reduce both planktonic growth and biofilm formation on treated nano-rough surfaces [26]. Sugar metabolites such as fructose have also been used in tandem with aminoglycosides to treat persister cells of both Gram-positive and Gram-negative microorganisms. Fructose is believed to act as stimulator to switch dormant persister cells into actively growing cells that are more susceptible to antibiotics [95]. Interestingly, serum was found to actively inhibit the formation of P. aeruginosa biofilm on medical devices, possibly owing to hindrance of interaction between microorganisms and the surface or by inhibiting directed bacterial twitching motility [96].

Inspired by the non-wetting and self-cleaning behavior of the lotus leaf, a recent study attempted to replicate the characteristics of lotus leafs to achieve an anti-bacterial anti-adhesion surface [22]. In particular, the treatment of PVC plastics using solvent (tetrahydrofuran, THF) and anti-solvent (ethanol or methanol) induced morphological changes in surfaces of endotracheal materials with notable increase in surface hydrophobicities [22]. The authors showed that ethanol-treated PVC surfaces were super-hydrophobic containing submicron-textured structures; resulting in significant delay of P. aeruginosa attachment without using antibiotics. Untreated smooth control PVC surface was colonized with P. aeruginosa as early as 6 h and initiation of biofilm maturation was evident at 24 h. On the other hand, bacterial cells were only visible on treated super-hydrophobic PVC surface at 24 h [22].

Artificial adaptation of shark’s skin has led to design novel biomimetic surfaces composed of pillars or spikes with varying heights, diameter and space separation [25]. These imprinted micro-patterns of sub-micron pillars are believed to control microorganism attachment [24,25,97,98] without altering mechanical properties and compatibility of the bulk material. Chung and co-workers proved that these patterned surfaces were resistant to S. aureus biofilm formation up to 21 days. Meanwhile, mature biofilm was initiated on smooth surface at day 7 [97]. A similar approach was employed to investigate the effects of micro-patterning texture of medical plastics on staphylococci adhesion and biofilm formation [24]. Using a soft-lithography technique, polyurethane biomaterial surface was imprinted with ordered and uniformed pillars having an available (accessible) surface contact area of ~25% compared to smooth and non-textured surface [24]. Initial adhesion of S. aureus was reduced to 90% thus subsequent biofilm development was markedly impaired [24]. As these pillars had an average height of 700 nm and the largest pillar space separation was of sub-bacterial dimension, these effectively restricted the contact of S. aureus to the material surface, leaving the top of the pillars the only point cells could interact with the surface [24]. A novel biomimetic submicron-patterned surface in an endotracheal tube-like polymer was engineered recently to evaluate the effect of patterned topography on S. aureus biofilm formation [25]. The use of these micro-patterned textures resulted in 89% inhibition when biofilms were grown for 4 days in standard culture containing mucin [25].

3.3.3 Coating an impregnation of endotracheal tubes

Coating of antimicrobial agents using chemical modification techniques has also been a subject of intense interest lately. Silver-impregnated coating on endotracheal tubes is probably the most studied with extensive investigations at clinical levels [99-104]. Other antimicrobial agents currently under investigation in vitro (or pre-clinical) settings include other silver-based compounds, zinc oxide nanoparticles, thiocyanates, bronopol, benzalkonium chloride, chlorohexidine, triclosan and hexetidine [23,27,105,106]. The immobilization of zinc oxide (ZnO) nanoparticles onto PVC endotracheal tubes was found to retain the bacteriostatic nature of ZnO as the ratio of live to dead S. aureus cells compared PVC alone [27]. The antibacterial activity was attributed to the release of bacteriostatic Zn2+ from nanoparticles, bacterial cell membranes binding to ZnO nanoparticles and formation of reactive oxygen species in cells [27]. On the other hand, thiocyanation of PVC surfaces, via covalent immobilization, imparted hydrophilicity and bactericidal characteristics to the treated surface. Although the bactericidal effect of immobilized thiocyanate was lower than that of free soluble thiocyanate, this did not affect the anti-adhesion effect of the treated surfaces, which was shown by lower adhesion of staphylococci in the thiocyanate treated PVC compared to non-treated PVC surfaces [105]. Physical deposition of triclosan and bronopol onto medical grade PVC using a plasma immersion ion implantation technique was applied to induce antibacterial properties on PVC surfaces. This approach is primarily based on the coatings of triclosan and bronopol onto surfaces pre-treated with oxygen plasma, followed by modification of coated molecules with argon plasma to improve their antibacterial characteristics [23]. From these results, both triclosan and bronopol were effective against S. aureus with ~80% cells killed after 10 days of culture but were unable to hinder the colonization of S. aureus [23]. On the other hand, triclosan-treated surfaces were more effective against E. coli compared to bronopol. The adherence of survived E. coli cells onto PVC surfaces was also significantly lower than S. aureus [23]. The deposition of antimicrobial agents onto medical grade PVC using a multistep physicochemical approach was achieved via surface discharge plasma and graft copolymerization to produce high-density structures and functionalization with antimicrobial agents such as bronopol, bezalkonium chloride and chlorohexidine [106]. Both bronopol and bezalkonium chloride were only effective against E. coli (up to 80% inhibition in adhesion) while the adhesion of S. aureus was not different compared to the control group. In comparison, the incorporation of chlorohexidine effectively blocked the attachment of both Gram-positive and Gram-negative bacteria [106].

To-date, coatings of endotracheal tubes with silver-based compounds have received the most significant attention. Early in vitro, in vivo and pre-clinical trials on the effects of silver-coated endotracheal tubes on bacterial colonization, and bacterial resistance have demonstrated promising results [99-103,107]. In 1999, Hartmann and co-workers reported the first investigation on silver coated endotracheal tubes. The authors used an in vitro oropharynx-larynx-lung model which was continuously exposed to P. aeruginosa and mechanically ventilated up to 50 h to closely mimic the actual clinical scenario [107]. This study found that non-coated control tubes were colonized to a greater extent compared to silver-coated tubes [107]. A randomized double-blinded controlled experiment using a mechanically ventilated dog model challenged with buccal administration of P. aeruginosa, demonstrated that silver-coated endotracheal tubes exerted sustained antimicrobial effect within the lung airways and hindered biofilm formation on the tubes [102]. Delayed onset of lung colonization of P. aeruginosa was observed for subjects receiving silver-coated tubes (1.8 ± 0.8 vs. 3.2 ± 0.8 days, p = 0.02) and the total count of bacterial burden in lung parenchyma was also significantly reduced (44.8 ± 0.8 vs. 5.4 ± 9 log CFU g–1, p = 0.01) compared to uncoated tubes [102]. Similarly, Berra and co-workers also performed a controlled, randomized study in 16 sheep, mechanically ventilated with either control uncoated endotracheal tubes or silver-sulfadiazine and chlorhexidine coated tubes to compare the rate of bacterial colonization on the tubes and ventilator circuits after 24 h [101]. All control tubes were severely colonized (up 108 CFU g–1) and demonstrated thick, densely packed and aggregated bacterial biofilms (193.3–405.6 μm). The trachea and lungs in five of eight control groups were infected with pathogenic bacteria. However, bacterial colonization within coated tubes and the entire ventilator circuits was hindered in seven of eight ventilated sheep. Biofilm formation on endotracheal tubes was also not evident and the lungs showed no traces of bacterial colonization [101]. Berra and co-workers evaluated the feasibility of silver sulfadiazine coated endotracheal tubes challenged with 104–106 CFU ml–1 P. aeruginosa every 24 h in in vitro as well as in animals [100]. In the in vitro setting, the coated tubes remained bacteria free up to 72 hours while non-coated endotracheal tubes were heavily colonized with bacteria (up to 3.2 x 109 CFU g–1) [100]. This was further supported in animal studies where thick mucoid biofilm layers were formed on the non-coated tube, ventilator tubing and lower respiratory tract (p ˂ 0.01). The common aerobic microorganisms found were α-hemolytic Streptococcus spp., K. pneumoniae, Moraxella spp., Pasteurella haemolytica, P. multocida, Pseudomonas aeruginosa and Staphylococcus aureus [100]. The mechanically ventilated sheep with silver sulfadiazine coated tubes meanwhile were associated with decreased bacterial burden on the tubes, ventilator circuit and respiratory tract. No local or systemic toxicity were observed [100]. Similar findings were also recorded in a series of investigations using in vitro and animal (rabbits) models, [103].

A prospective, randomized phase I–II clinical trial was carried out with forty-six patients undergoing cardiac surgery to primarily establish the interventions of silver sulfadiazine coating onto endotracheal tubes to reduce bacterial burden in patients receiving mechanical ventilation for 12–24 h [99]. The patients, above 18 of age, who require mechanical ventilation in anesthesia condition were randomized into two groups to receive either a silver sulfadiazine coated or a conventional standard endotracheal tube [99]. In another independent small clinical trial, the safety of silver-coated endotracheal tubes and associated bactericidal activity in the lung airways were reported [104]. For this 121 patients who needed mechanical ventilation for more than 24 h and did not have prior respiratory infections were recruited to randomly receive either silver-coated or non-coated endotracheal tubes [104]. In this study, Ag+ was dispersed in a polymer on both the inner and outer lumens and could migrate to the surface of endotracheal tube to provide sustained bactericidal effect [104]. In general, both studies demonstrated that the use of silver coated endotracheal tubes was associated with reduced bacterial colonization on the tube lumen and non-appearance of biofilm formation. Furthermore, these tubes were safely implemented, easy to manage and well-tolerated with no significant adverse reactions [99,104]. Furthermore, probably due to the small sample groups, the impact on VAP incidence was not sufficiently demonstrated [104].

In a large randomized North American silver-coated endotracheal tube (NASCENT) single-blind trial study, a total of 2003 patients expected to use ventilators for more than 24 h were recruited and randomized to receive either silver-coated or conventional endotracheal tubes [108]. Their findings could be summarized as follows: a) silver-coated endotracheal tubes reduced VAP incidence from 7.5% to 4.8% which corresponded to a relative risk reduction of 35.9% and an absolute risk reduction of 2.7%; b) silver-coated endotracheal tubes were not successful to achieve significant reduction in mortality rate, duration of intubation, duration of ICU stay or the severity of adverse effects compared to conventional tubes [108]. The same group further evaluated mortality in patients who developed VAP in the previous NASCENT trial [108] using retrospective cohort analysis [109]. The silver-coated tube was associated with reduced mortality with VAP (14%) compared to control (36%). However, no differences were observed for those who did not develop VAP [109]. In addition, the use of silver-coated endotracheal tubes as a preventative measure for VAP might result in potential hospital cost savings [110].

4. Conclusions

Numerous studies have aimed to investigate the role of endotracheal tubes as causative agent on VAP as it remains the most commonly acquired infections in intubated patients. It is generally accepted that rapid formation of multiple-drug-resistant bacterial biofilms is a main contributor to VAP. The insertion of endotracheal tubes into patients bypasses the body’s primary host defenses, in which the lung becomes a suitable site for bacterial colonization, proliferation and biofilm formations. The prevention of VAP or rather the inhibition of bacterial biofilms on endotracheal tubes is becoming a priority for hospitals. To date, many strategies have been attempted to eradicate or prevent biofilm contamination on endotracheal tubes, which include use or biocide, antibiotics and dispersal agents, ultrasound, chelation, enzymatic digestion or surface modification of surface. Although these methods are promising, many afford only temporary relief as microorganisms are quick to develop inherent resistance. The main question is, how much understanding do we have on the intricate systems of biofilm communities? As these bugs are constantly evolving, it is desirable to design therapeutic approaches that combine several modes of antimicrobial actions synergistically to achieve greater sensitivity of biofilms

5. Expert commentary

Ventilator associated pneumonia in ICU is a significant healthcare problem, affecting up to 28% of intubated patients. The intubation tube provides the first point of call for bacterial infection, since this invasive medical device is open to bacterial adhesion and biofilm formation. There is no standardized guidelines for preventing VAP in hospital and a number of recommendations exist including endotracheal tubes with low cuff pressure, devices with balloons to remove biofilms, and saline instillation and use of silver. At a research and development level, a number of stratergies are under development to tackle bacterial adhesion, biofilm formation and to target bacteria locally, with a view to reduce and treat VAP. One approach is to modify the surface chemistry or surface topology of the endotracheal tube to be more hydrophilic or contain nano-rough surfaces. While these may reduce bacterial adhesion, this is likely to only temporarily prevent adhesion, since bacteria express and lay down conditioning media that will eventually overcome this approach. A second approach is to incorporate antibiotics or antimicrobial compounds into/onto the tube. Approaches such as the incorporating silver nanoparticles may enhance bacterial killing however; again this approach is likely to be overcome with time. Ultimately, a multifaceted approach is likely to be the answer to preventing VAP; utilizing tubes that incorporate both modified surfaces and antibacterials.

6. Five-year view

Currently, the main focus of clinical trials has been to reduce and treat VAP via impregnation or coating of endotracheal tubes with silver. Research within the field has mainly focused on two areas, incorporation of antibacterials into/onto tubes or surface modification to hinder bacteria adhesion and thus biofilm formation. Over the next five years it is likely that we will see both these novel approaches reach a clinical setting. The most-likely successful approach, however is to take a multi-faceted approach, via modification of surface texture and incorporation of antibacterials into the surface structure. Importantly, however, the success of modified endotracheal tubes in reducing VAP, is going to come down to cost effectiveness. A successful clinical product is likely to be cheap to manufacture and not require extensive processing over normal manufacturing processes. Furthermore, incorporation of currently used antibacterials and modification of polymers already used in the clinic would ensure rapid translation and uptake in the clinic if proved effective in preventing VAP.

7. Key issues

• Endotracheal tubes in ICU are essential for ventilation but are a major source of infection and ventilator associated pneumonia (VAP).

• VAP affects up to 28% of intubated patients and the incidence rate escalates with time post ventilation.

• After bacterial adhesion to intubation tubes, biofilm formation can occur making it difficult to remove potential sources of VAP via conventional antibiotics.

• Guidelines for reducing VAP are not uniform and there is no cohesive clinical strategy for reducing VAP associated mortality.

• Reducing bacterial adhesion is a key strategy for reducing biofilm formation and VAP through surface modification of intubation tubes.

• Actively ‘targeting bacteria’ by incorporating antibacterial drugs or metal ions/nanoparticles into tubes is another strategy.

• Clinical trials have shown some success using silver coated endotracheal tubes.

• Developing improved endotracheal tubes that prevent VAP and reduce VA related mortality has the potential to improve healthcare outcomes and reduce ICU costs.

Figure captions

Figure 1: Schematic representative of the major stages in the development of P. aeruginosa biofilm

Figure 2: Proposed biofilm resistance mechanisms. (A) The presence of EPS provides physical barrier, which limits penetration and diffusion of antimicrobial agents. (B) Metabolic activities and growth rate of bacterial cells within biofilm matrix as a function of cell depth is determined by various factors such as nutrient and oxygen availability. The zone showing the lightest color represents cells with the highest growth rate and hence the most susceptibility to antimicrobial agents. Cells at the most-inner layers (zone with the darkest orange color) often survive in dormant state or live in anaerobic conditions. (C) Activation of stress regulator (RpoS) which could mediate the overexpression of antimicrobial agent-destroying enzymes. The activation of biofilm resistance genes such as efflux pumps effectively removes antimicrobial agents from ctyoplasms. (D) Survival of persister cells which lead to revolution into recurrent ‘superbug’ biofilm infections with extremely high resistance to antimicrobial agents.

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* of interest; **of considerable interest.

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