Biofilms have been implicated in virtually every human infection and are particularly recalcitrant to antibiotic compounds and can persist despite sustained host defences. Biofilm formation is an important component of pulmonary colonisation and infection due to P. aeruginosa both in acute lung infections such as VAP and in chronic lung infections such as CF. Understanding bacterial physiology and the mechanisms by which P. aeruginosa protects itself from lethal concentrations of antibiotics is fundamental to designing interventions against resistant strains. This study compared the biofilm-forming ability of P. aeruginosa newly colonising the airway of MV patients and patients with longstanding CF during periods of chronic stability and acute pulmonary exacerbation and its associated effect on antibiotic susceptibility.
Biofilm-forming ability was greatest among P. aeruginosa
biofilms from newly colonised MV patients. Enhanced biofilm formation by isolates from ETAs suggests that these strains may have originated form P. aeruginosa
biofilms formed on the inner lumen of the ETT. Biofilm formation on the ETT is facilitated by multiple factors, including the polyvinylchloride plastic surface of the ETT, the lack of host defence mechanisms in the inner lumen and the accumulation of tracheobronchial secretions. Biofilms can then be dislodged from the ETT during routine suctioning and disseminate into the lower respiratory tract, contributing to the development of VAP. Whilst biofilm formation of P. aeruginosa
isolates colonising the ETT was high among the MV patients, only 50% of patients went on to develop VAP. Adair et al. [7
] showed that while 100% of patients (20/20) with VAP had ETTs covered with biofilm, 30% of patients (6/20) without VAP also had ETTs colonised with biofilms. Thus, while ETT colonisation is an important contributor to VAP, it is only one of many notable risk factors, including the severity of the patient’s underlying disease, prior surgery and prior exposure to antibiotics [15
In contrast, biofilm formation was lowest among the CF isolates and was highly variable among sequential clonal isolates. There was no correlation between biofilm formation and pulmonary status (e.g. chronic infection versus acute pulmonary exacerbation). The variability in biofilm formation among P. aeruginosa
CF isolates with similar genotypic profiles is supported by others [17
]. However, biofilm formation in relation to pulmonary status in sequential clonal isolates in CF patients has never been investigated. Differences in host factors and antibiotic selection pressure may account for the variability in biofilm formation and pulmonary status in CF patients. Impaired mucociliary clearance, the thick mucus layer overlying lung epithelium, and the presence of a microaerophilic or anaerobic environment are all factors that encourage the formation of P. aeruginosa
biofilms in CF [18
]. The reason for the heterogeneity in biofilm formation between MV and CF patients is likely multifactorial. Biofilms formed on living tissue have been shown to be morphologically different from biofilms formed on abiotic surfaces such as ETTs. Other factors include differences in host defences, prior antibiotic exposure and the differential expression of bacterial virulence factors [19
Comparison of MICs and BICs demonstrated a substantial difference in antibiotic resistance among the MV isolates. The greatest increase in biofilm antibiotic resistance occurred with the β-lactam antibiotics, with the majority of isolates exhibiting BICs ≥ 256 µg/mL. In contrast, the differences between the BIC and MIC for CF isolates were not as pronounced. At subinhibitory imipenem concentrations, imipenem has been shown to induce ampC
production, increase biofilm volume and increase alginate production in P. aeruginosa
PA01 biofilms and to induce biofilm formation in MDR Acinetobacter baumannii
]. Receipt of subtherapeutic concentrations of imipenem in MV patients likely contributed to the increased biofilm formation and potentially increased ampC
expression. This increased ampC
expression, in turn, may explain the increased ceftazidime and TZP resistance observed in this study. In contrast, the majority of CF isolates were susceptible to imipenem and none of the patients received imipenem for treatment of their acute pulmonary exacerbations, leading to potentially less induction of biofilm formation and ampC
expression. Tobramycin has also been shown to induce biofilm formation at subinhibitory concentrations in PA01. However, biofilm induction occurred in only approximately one-half of CF clinical isolates in a recent study and was independent of tobramycin susceptibility [22
]. This may explain the variability in biofilm-forming ability and BIC observed among the CF isolates in this study. It was also observed that resistance evolution in biofilms occurred rapidly, whilst resistance evolution in the planktonic mode of lifestyle remained relatively unchanged over time.
Regression analysis of biofilm formation with antimicrobial resistance demonstrated statistically significant positive correlations for three of the five antimicrobials tested. This suggests that physiological aspects specific to biofilms, such as exopolysaccharide production, biofilm-specific efflux pump expression or β-lactamase production, played a role in increased biofilm antimicrobial resistance. Interestingly, a negative correlation existed between biofilm formation and TZP resistance. Subinhibitory concentrations of TZP have been shown to lead to a decrease in bacterial adherence and biofilm formation among P. aeruginosa
clinical isolates [23
]. In general, increased antimicrobial resistance and biofilm formation were associated, underlining the importance of this mode of lifestyle in clinical infections and when considering treatment options.
Because of the important role of biofilms in the pathophysiology of VAP and CF, strategies targeting the biofilm have been developed. These include selective decontamination of the ETT and use of antiseptic- or antimicrobial-impregnated ETTs for prevention of VAP. In the North American Silver-Coated Endotracheal Tube (NASCENT) study, Kollef et al. [24
] randomised 2003 patients expected to require MV for ≥24 h to undergo intubation with either a silver-coated ETT or a conventional uncoated ETT. Compared with the uncoated ETT group, the silver-coated ETT group had a lower incidence of microbiologically confirmed VAP [37/766 (4.8%) vs. 56/743 (7.5%); P
= 0.03] and a statistically significant delay in the onset of VAP. Other strategies studied include removal of established biofilms and mucus secretions in the ETT using mechanical devices such as the Mucus Shaver. The Mucus Shaver has been shown to be effective in removing biofilm from the inner lumen of the ETT in intubated sheep [25
]. This technique is promising but further studies will be needed to determine whether removal of the biofilm is effective in preventing VAP. Whilst silver-coated ETTs are currently recommended by some groups, the Mucus Shaver cannot be recommended at this time in the absence of clinical data [27
Inhaled antibiotics (e.g. tobramycin and colistin) have also been studied as a method to prevent biofilm formation in the ETT and as adjunctive therapy for the treatment of VAP [28
]. Compared with systemic administration of antibiotics, inhaled antibiotics can achieve higher pulmonary concentrations, with levels far in excess of planktonic MICs [34
]. Falagas et al. [36
] conducted a meta-analysis of the effect of antibiotics administered via the respiratory tract for the prevention of ICU pneumonia. The incidence of VAP was reduced but there was no overall effect on mortality. Three recently published studies compared adjunctive inhaled colistin or colistimethate plus intravenous (i.v.) antibiotics with i.v. antibiotics alone for the treatment of VAP [29
]. Using a matched retrospective case–control design, Kofteridis et al. [29
] found no difference in eradication of Gram-negative organisms, including P. aeruginosa
, in the treatment group compared with the control group [17/34 patients (50%) vs. 19/42 patients (45%); P
= 0.679]. There was a trend towards improved clinical cure with inhaled plus i.v. colistin, but no statistically significant differences in either all-cause mortality or VAP-related mortality. Using a retrospective cohort analysis, Korbila et al. [30
] found inhaled colistin to be independently associated with VAP cure but also no improvement in mortality. In a recent randomised controlled trial, Rattanaumpawan et al. [37
] found that microbiological eradication was significantly improved in the treatment group compared with the control group (60.9% vs. 38.2%, P
= 0.03), but again no difference in clinical outcome. Thus, inhaled antibiotics may have a favourable effect on microbiological outcome, but this technique has not yet been shown to translate into improved patient outcomes.
The most promising strategy is to dose antibiotics based on biofilm susceptibilities in lieu of planktonic susceptibilities. Keays et al. [38
] retrospectively reviewed 110 CF patients who received antibiotic treatment for acute pulmonary exacerbation. Patients who received antibiotic combinations that inhibited bacteria (based on BIC testing) had a decrease in sputum bacterial density and hospital length of stay. Clinical trials examining the effectiveness of antibiotic dosing based on biofilm susceptibilities in CF are currently ongoing [39
]. Whilst this strategy has not yet been studied for the treatment of VAP, this is an area of potential benefit that will require further investigation.
This study is associated with certain limitations. Previous investigators have suggested that use of more mature biofilms may be more representative of in vivo conditions in CF and exhibit increased antibiotic resistance compared with biofilms formed after only 24 h [41
]. However, ETAs were collected from MV patients on a daily basis, thus the biofilm formed on the ETT of these patients would likely be more representative of young biofilm. Use of a 24-h biofilm model allowed direct comparisons between the CF and MV isolates to be made. Owing to the small number of MV and CF patients examined, additional studies examining biofilm formation and resistance in larger cohorts of patients will be needed.
These results indicate that the effect of antibiotics on biofilm formation is highly variable and unpredictable, with some antibiotics inducing biofilm formation while other antibiotics inhibit biofilm formation in these two patient populations. This unpredictability in biofilm formation and BIC make it increasingly difficult for clinicians to choose the most active antibiotic, particularly as biofilm susceptibility testing is not routinely performed as part of clinical care. In addition, the rapid induction and evolution of biofilm resistance in clonal sequential CF isolates, whilst planktonic antibiotic resistance remains stable, is worrisome. Biofilm formation and the associated development of antimicrobial resistance is a multifactorial process dependent on the presence or absence of prosthetic material, host immune status and disease state. These are factors that will require further investigation in order to determine the optimal antibiotic therapy to successfully treat biofilm-mediated infections.