Endotracheal Tube Biofilm Research Articles

Microbial investigations in throat swab and tracheal aspirate specimens are beneficial to predict the corresponding endotracheal tube biofilm flora among intubated neonates with ventilator-associated pneumonia.

Ventilator-associated pneumonia (VAP) is a common nosocomial infection in neonatal intensive care units with high morbidity and mortality. Bacterial biofilm in the endotracheal tube (ET) provides a notable and persistent source of pathogens that may cause VAP, and thus is important for VAP detection. However, during intubation microbial investigations in ET, samples are unavailable due to the infeasibility of collecting ET samples during intubation of neonates. It is therefore of great importance to find alternative sources of samples that can help identify the ET biofilm flora. In the present study, the microbial signatures of throat swabs and tracheal aspirates were compared with ET biofilm samples from VAP neonates using 16S ribosomal RNA gene polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Sequences were assigned to phylogenetic species using BLAST. Microbial diversity and richness among the three types of specimens were compared based on their DGGE fingerprints, and taxonomic characteristics based on the BLAST results. The microbial richness and diversity of ET biofilms were similar to tracheal aspirate yet significantly different from throat swab samples (P<0.05). Compared with ET biofilms, the overall constituent ratio of microflora was significantly different in throat swab and tracheal aspirate samples (P<0.05). However tracheal aspirate samples were useful for predicting Staphylococcus sp. in ET biofilms with a sensitivity of 85.7% and a specificity of 83.3%. The sensitivity for the combination of tracheal aspirate and throat swab samples to detect Staphylococcus sp. in ET biofilms was 100%. The detection of Pseudomonas sp. in throat swabs assisted its identification in ET biofilms (sensitivity 33.3% and specificity 100%). The results of the present study suggest that microbial investigations in throat swab and tracheal aspirate samples are beneficial for identifying the ET biofilm flora. There may therefore be clinical applications of using substituent samples to identify pathogens in ET biofilms for VAP surveillance among intubated neonates.

Streptococcus sp. in neonatal endotracheal tube biofilms is associated with ventilator-associated pneumonia and enhanced biofilm formation of Pseudomonas aeruginosa PAO1

Ventilator-associated pneumonia (VAP) is a serious complication of mechanical ventilation leading to high morbidity and mortality among intubated neonates in neonatal intensive care units (NICUs). Endotracheal tube (ETT) biofilm flora were considered to be responsible for the occurrence of VAP as a reservoir of pathogens. However, regarding neonates with VAP, little is known about the complex microbial signatures in ETT biofilms. In the present study, a culture-independent approach based on next generation sequencing was performed as an initial survey to investigate the microbial communities in ETT biofilms of 49 intubated neonates with and without VAP. Our results revealed a far more complex microflora in ETT biofilms from intubated neonates compared to a previous culture-based study. The abundance of Streptococci in ETT biofilms was significantly related to the onset of VAP. By isolating Streptococci in ETT biofilms, we found that Streptococci enhanced biofilm formation of the common nosocomial pathogen Pseudomonas aeruginosa PAO1 and decreased IL-8 expression of airway epithelia cells exposed to the biofilm conditioned medium of PAO1. This study provides new insight into the pathogenesis of VAP among intubated neonates. More studies focusing on intubated neonates are warranted to develop strategies to address this important nosocomial disease in NICUs.

Effect of the endOclear® Device on Biofilm in Endotracheal Tubes.

Organisms trapped in biofilms cause more than 80% of medical infections. Significant investments are being made to develop methods of removing these biofilms. The endOclear® device is reported to remove biofilm from endotracheal tubes (ETTs) and to decrease pneumonia rates and ventilator time. This was an observational study performed at a university Level 1 trauma center intensive care unit. A series of 40 ETTs were collected at extubation, with half of the patients having been treated daily with the endOclear® device. Biofilms were quantified from a standardized point on the distal ETT. The patients' standard and biofilm cultures were reviewed. The mean hours of intubation for the control group was 135 and for the device group 138. This difference was not statistically significant (p = 0.91). Eleven patients in the device group were found to have pneumonia compared with six in the control group (p = 0.34). Ventilator data after device use showed a mean increase of 29.9 cc in tidal volume and a mean decrease in peak pressures of 0.39 cm H2O. Comparisons between biofilm stage or hours of intubation and a diagnosis of pneumonia found no correlation. Only nine of 40 ETTs had congruence between the microbiata of the biofilm and standard bronchoalveolar lavage (BAL) fluid, a divergence rate of 78%. Comparison of the endOclear® group and controls demonstrated a trend toward a higher pneumonia rate in the former. Additionally, the device achieved very small, clinically insignificant, changes in ventilator settings, and no difference was seen in the time on the ventilator. Comparisons between biofilm and standard BAL cultures continue to show the biofilm is more diverse than previously thought. In this study, no statistical significance was found between biofilm stage and the pneumonia rate. This study provides additional evidence that there is no correlation between biofilm stage and duration of intubation.

Community analysis of dental plaque and endotracheal tube biofilms from mechanically ventilated patients

PurposeMechanically ventilated patients are at risk for developing ventilator-associated pneumonia, and it has been reported that dental plaque provides a reservoir of respiratory pathogens that may aspirate to the lungs and endotracheal tube (ETT) biofilms. For the first time, metataxonomics was used to simultaneously characterize the microbiome of dental plaque, ETTs, and non-directed bronchial lavages (NBLs) in mechanically ventilated patients to determine similarities in respective microbial communities and therefore likely associations. Material and methodsBacterial 16S rRNA gene sequences from 34 samples of dental plaque, NBLs, and ETTs from 12 adult mechanically ventilated patients were analyzed. ResultsNo significant differences in the microbial communities of these samples were evident. Detected bacteria were primarily oral species (e.g., Fusobacterium nucleatum, Streptococcus salivarius, Prevotella melaninogenica) with respiratory pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcuspneumoniae, and Haemophilus influenzae) also in high abundance. ConclusionThe high similarity between the microbiomes of dental plaque, NBLs, and ETTs suggests that the oral cavity is indeed an important site involved in microbial aspiration to the lower airway and ETT. As such, maintenance of good oral hygiene is likely to be highly important in limiting aspiration of bacteria in this vulnerable patient group.

Molecular analysis of endotracheal tube biofilms and tracheal aspirates in the pediatric intensive care unit.

BackgroundVentilator-associated pneumonia (VAP) is a known complication of mechanically ventilated children in the pediatric intensive care unit (PICU). Endotracheal tube (ETT) biofilms are often implicated in the development of VAP by providing a conduit for pathogens to the lower respiratory tract.MethodsA prospective cohort study from April 2010–March 2011 of children 4 weeks to 18 years of age ventilated for greater than 72 hours to determine the microbiota of ETT biofilms and tracheal aspirates.ResultsThirty-three patients were included with a mean age of 6.1 years (SD ± 5.1 years) and average length of intubation of 8.8 days (SD ± 5.0 days). Bacterial communities from tracheal aspirates and the proximal and distal ends of ETTs were determined using 16S rRNA gene libraries. Statistical analysis utilized two-part statistics and the Wilcoxon signed rank sum test for comparison of bacterial communities. Sequencing revealed a predominance of oropharyngeal microbiota including Prevotella and Streptococcus spp. Pathogenic bacterial genera including Staphylococcus, Burkholderia, Moraxella, and Haemophilus were also represented. Bacterial load was greatest at the proximal aspect of the ETT. Duration of intubation did not significantly impact bacterial load. Morisita Horn analysis across sites showed similar communities in 24/33 (72%) of patients.ConclusionsETT biofilms and tracheal aspirates of intubated patients in the PICU primarily consisted of oropharyngeal microbiota, but had a significant representation of potentially pathogenic genera. While the majority of patients had similar microbiota when comparing their ETT biofilms and tracheal aspirates, a subset of patients showed a divergence between communities that requires further investigation.

Biofilms in ventilator-associated pneumonia.

Biofilms develop rapidly following endotracheal intubation and represent a persistent source of unnecessary pathogens in the critically ill patient. Overall, the imbalance in the lung microbiome caused by an endotracheal tube and its role in biofilm formation and in ventilator-associated pneumonia is still unclear. Although endotracheal tube-biofilm preventive measures are being tested, no outcome impact has ever been demonstrated, and therefore no approach has been clinically recommended. Nonetheless, an accurate description of the actual biofilm morphology in vivo could be useful to implement effective preventive measures. The combined use of in vitro biofilm models, in vivo animal models and clinical research is vitally important to the attainment of a comprehensive understanding of biofilms in ventilator-associated pneumonia in the near future.

Respiratory pathogen colonization of dental plaque, the lower airways, and endotracheal tube biofilms during mechanical ventilation

PurposeIn mechanically ventilated patients, the endotracheal tube is an essential interface between the patient and ventilator, but inadvertently, it also facilitates the development of ventilator-associated pneumonia (VAP) by subverting pulmonary host defenses. A number of investigations suggest that bacteria colonizing the oral cavity may be important in the etiology of VAP. The present study evaluated microbial changes that occurred in dental plaque and lower airways of 107 critically ill mechanically ventilated patients. Materials and MethodsDental plaque and lower airways fluid was collected during the course of mechanical ventilation, with additional samples of dental plaque obtained during the entirety of patients' hospital stay. ResultsA “microbial shift” occurred in dental plaque, with colonization by potential VAP pathogens, namely, Staphylococcus aureus and Pseudomonas aeruginosa in 35 patients. Post-extubation analyses revealed that 70% and 55% of patients whose dental plaque included S aureus and P aeruginosa, respectively, reverted back to having a predominantly normal oral microbiota. Respiratory pathogens were also isolated from the lower airways and within the endotracheal tube biofilms. ConclusionsTo the best of our knowledge, this is the largest study to date exploring oral microbial changes during both mechanical ventilation and after recovery from critical illness. Based on these findings, it was apparent that during mechanical ventilation, dental plaque represents a source of potential VAP pathogens.

Scanning Electron Microscopic Biofilm Grading and Ventilator Associated Pneumonia in Relation to Duration of Intubation

Background: Ventilator associated pneumonia (VAP) accounts for 7-70% of infections in intensive care units (ICUs) leading to increase in hospitalization, mortality and cost. Endotracheal tube (ETT) is a risk factor for developing VAP caused by biofilm producing organisms resistant to antimicrobial agents. This study aimed to determine guidelines for ETT extubation through detection of biofilm formation and development of VAP. Objectives: Our aim was to assess biofilm formation on the luminal surface of ETTs in intubated patients in ICU in relation to duration of intubation bacteriologically and morphologically using Scanning Electron Microscope (SEM). Antimicrobial resistance and VAP development were also studied. Methodology: Our work was conducted on 20 ETTs divided into two groups: Group (1); 8 patients with ETTs<5 days, and group (2); 12 patients with ETTs of 5-12 days. Culture of the interior of the ETTs, identification of bacterial species, antibiotic susceptibility and presence with staging of biofilm into 3 grades by SEM were performed. Results: A highly significant relationship was observed between the duration of intubation and biofilm grading (p value <0.001). Also, the duration of intubation and VAP development were significantly correlated (p<0.001). A highly significant relationship was observed between VAP development and biofilm grading by SEM (p<0.001). A moderate significant relationship was found between VAP development and prognosis of patients (p<0.02). It was also observed that prolonged intubation was associated with prevalence of polymicrobial colonization with the predominance of Gram negative bacilli. Furthermore, a moderate significant relationship was observed between antimicrobial resistance and both biofilm grading and duration of intubation (p<0.02). Conclusion: Biofilm formation and grading as well as bacterial colonization with multidrug-resistant (MDR) bacteria were time dependent in patients on mechanical ventilation in ICU which may enhance their morbidity and mortality rates. Also a role of ETT biofilm is emphasized in the pathogenesis and prognostic outcome of VAP in patients intubated for a prolonged period. Accordingly, we recommend that ETT should be exchanged every 4 days to avoid such morbidity and mortality consequences.

Effect of N-acetylcysteine inhalation on ventilator-associated pneumonia caused by biofilm in endotracheal tubes

To observe the formation of the biofilm in endotracheal tubes, the characteristics of etiology, drug resistance and effect on the biofilm and ventilator-associated pneumonia (VAP) of inhaled N-acetylcysteine (NAC). We selected 117 tracheally intubated and undergoing mechanical ventilation for ≥48 h in our hospital ICU from September 2010 to August 2012. All the cases were randomly divided into control group (60 cases) and study group (57 cases). The patients in the study group were treated with different doses of aerosolized NAC according to different ages, starting the first administration within 12 hours of mechanical ventilation, once every 8 hours, until stopping mechanical ventilation. Comparison was performed on the two groups in biofilm structure under the scanning electron microscopy, biofilm culture positive rate, VAP incidence, the etiology and drug resistance of the lower airway secretions and biofilms. (1) Electron microscopy showed that biofilm had formed in the endotracheal tube inner wall in early period of mechanical ventilation. With prolonged mechanical ventilation, biofilm structure improved. At the same time of mechanical ventilation, the thickness of biofilm in the study group decreased as compared with the control group. (2) Biofilm culture positive rate and incidence of ventilator-associated pneumonia decreased in the study group compared with in the control group (65%(37/57) vs. 80%(48/60), P<0.05; 11% (6/57)vs. 32%(19/60), P<0.01). (3) A large number of pathogenic bacteria colonized in the biofilm and gram-negative bacilli dominated. With prolonged mechanical ventilation, the cultured pathogens converged from the lower airway secretions and biofilm. With prolonged mechanical ventilation, biofilm structure was improved. Inhalation of NAC can inhibit biofilm formation and reduce the incidence of VAP.

Effect of nebulized eucalyptus on contamination of microbial plaque of endotracheal tube in ventilated patients.

Background:Formation of biofilm and bacterial colonization within the endotracheal tube (ETT) are significant sources of airway contamination and play a role in the development of ventilator-associated pneumonia (VAP). This study was conducted to examine the effect of nebulized eucalyptus (NE) on bacterial colonization of ETT biofilm.Materials and Methods:We performed a randomized clinical trial in three intensive care units (ICUs) of an educational hospital. Seventy intubated patients were selected and randomly divided into intervention (n = 35) and control (n = 35) groups. The intervention group received 4 ml (5%) of eucalyptus in 6 ml normal saline every 8 h. The placebo group received only 10 ml of normal saline in the same way. On extubation, the interior of the tube was immediately sampled using a sterile swab for standard microbiological analysis. Chi-square and Fisher's exact tests were used for statistical analysis in SPSS. P values less than 0.05 were considered statistically significant.Results:In both samples, Klebsiella pneumoniae and Acinetobacter baumannii were the most frequently isolated bacteria. In the control group, heavy colonization was greater than in the intervention group (P = 0.002). The frequency of isolation of K. pneumoniae in the intervention group was lower than in the control group (P < 0.001). However, there was no difference between the two groups in other isolated bacteria.Conclusions:NE can reduce microbial contamination of the endotracheal tube biofilm in ventilated patients. Moreover, K. pneumoniae was the most sensitive to NE.

Antifungal Susceptibility Patterns of Candida Species Recovered from Endotracheal Tube in an Intensive Care Unit.

Aims. Biofilms formed by Candida species which associated with drastically enhanced resistance against most antimicrobial agents. The aim of this study was to identify and determine the antifungal susceptibility pattern of Candida species isolated from endotracheal tubes from ICU patients. Methods. One hundred forty ICU patients with tracheal tubes who were intubated and mechanically ventilated were surveyed for endotracheal tube biofilms. Samples were processed for quantitative microbial culture. Yeast isolates were identified to the species level based on morphological characteristics and their identity was confirmed by PCR-RFLP. Antifungal susceptibility testing was determined according to CLSI document (M27-A3). Results. Ninety-five strains of Candida were obtained from endotracheal tubes of which C. albicans (n = 34; 35.7%) was the most frequently isolated species followed by other species which included C. glabrata (n = 24; 25.2%), C. parapsilosis (n = 16; 16.8%), C. tropicalis (n = 12; 12.6%), and C. krusei (n = 9; 9.4%). The resulting MIC90 for all Candida species were in increasing order as follows: caspofungin (0.5 μg/mL); amphotericin B (2 μg/mL); voriconazole (8.8 μg/mL); itraconazole (16 μg/mL); and fluconazole (64 μg/mL). Conclusion. Candida species recovered from endotracheal tube are the most susceptible to caspofungin.

Visualizing biofilm formation in endotracheal tubes using endoscopic three-dimensional optical coherence tomography.

Biofilm formation has been linked to ventilator-associated pneumonia, which is a prevalent infection in hospital intensive care units. Currently, there is no rapid diagnostic tool to assess the degree of biofilm formation or cellular biofilm composition. Optical coherence tomography (OCT) is a minimally invasive, nonionizing imaging modality that can be used to provide high-resolution cross-sectional images. Biofilm deposited in critical care patients’ endotracheal tubes was analyzed in vitro. This study demonstrates that OCT could potentially be used as a diagnostic tool to analyze and assess the degree of biofilm formation and extent of airway obstruction caused by biofilm in endotracheal tubes.

Endotracheal tube biofilm translocation in the lateral Trendelenburg position.

IntroductionLaboratory studies demonstrated that the lateral Trendelenburg position (LTP) is superior to the semirecumbent position (SRP) in the prevention of ventilator-associated pulmonary infections. We assessed whether the LTP could also prevent pulmonary colonization and infections caused by an endotracheal tube (ETT) biofilm.MethodsEighteen pigs were intubated with ETTs colonized by Pseudomonas aeruginosa biofilm. Pigs were positioned in LTP and randomized to be on mechanical ventilatin (MV) up to 24 hour, 48 hour, 48 hour with acute lung injury (ALI) by oleic acid and 72 hour. Bacteriologic and microscopy studies confirmed presence of biofilm within the ETT. Upon autopsy, samples from the proximal and distal airways were excised for P.aeruginosa quantification. Ventilator-associated tracheobronchitis (VAT) was confirmed by bronchial tissue culture ≥3 log colony forming units per gram (cfu/g). In pulmonary lobes with gross findings of pneumonia, ventilator-associated pneumonia (VAP) was confirmed by lung tissue culture ≥3 log cfu/g.ResultsP.aeruginosa colonized the internal lumen of 16 out of 18 ETTs (88.89%), and a mature biofilm was consistently present. P.aeruginosa colonization did not differ among groups, and was found in 23.6% of samples from the proximal airways, and in 7.1% from the distal bronchi (P = 0.001). Animals of the 24 hour group never developed respiratory infections, whereas 20%, 60% and 25% of the animals in group 48 hour, 48 hour-ALI and 72 hour developed P.aeruginosa VAT, respectively (P = 0.327). Nevertheless, VAP never developed.ConclusionsOur findings imply that during the course of invasive MV up to 72 hour, an ETT P.aeruginosa biofilm hastily colonizes the respiratory tract. Yet, the LTP compartmentalizes colonization and infection within the proximal airways and VAP never develops.

Impact of persistent endotracheal tube biofilm on ventilator-associated pneumonia clinical and microbiological response

Ventilator-associated pneumonia (VAP) is the most common infectious complication in critically ill patients. The formation of biofilm on endotracheal tube (ETT) surface plays an important role in pathogenesis of this infection, because it facilitates the growth and survival of microorganisms and difficults the action of antimicrobial therapy.

Could saline instillation displace bacteria from the endotracheal tube biofilm to lower airways during suctioning procedure?

A biofilm is found on the inner side of endotracheal tubes (ETT) in mechanically ventilated patients. Saline instillation inside the ETT during the suctioning procedure is very common. This procedure could displace bacteria to the lower airways, increasing the risk of ventilator-associated pneumonia (VAP).

The characteristics of bacterial biofilm formation in endotracheal tubes in ventilated patients and the relationship between the biofilm and ventilator-associated pneumonia

Objective To observe the formation of the biofilm in endotracheal tubes, the characteristics of etiology, drug resistance and relationship between the biofilm and ventilator-associated pneumonia(VAP). Methods A total of 60 cases of ventilated children patients whose mechanical ventilation time were ≥48 h in the ICU from September 2010 to September 2012, according to the mechanical ventilation time, all cases were divided into 2 to 6 d group, 7 to 14 d group and ≥15 d group.The incidence of VAP, biofilm structure under the electron microscope, etiology culture positive rate of the lower airway secretions and biofilm, etiological characteristics and drug resistance were prospectively studied. Results (1) A total of 19 cases occurred VAP in 60 cases of mechanical ventilation, the incidence of VAP was 31.7%.(2) Observed by electron microscope, biofilm had formed in the endotracheal tube inner wall in early period of mechanical ventilation.With prolonged mechanical ventilation, biofilm structure had improved, as well as VAP incidence rate from 9.1%(2 to 6 d group ) increased to 44.4%(7 to 14 d group) and 88.9%(≥15 d group). (3) A large number of pathogenic bacteria colonized in the biofilm.Gram-negative bacilli were dominate and drug resistance was high.(4) With prolonged mechanical ventilation, the cultured pathogens from the lower airway secretions and biofilm converged. Conclusion Biofilm could form in the endotracheal tube with mechanical ventilation patients, and is associated with the occurrence of VAP and refractory infections. Key words: Endotracheal tube; Biofilm; Ventilator-associated pneumonia; Pathogens; Drug resistance

A study of biofilm on endotracheal tubes in pediatric intensive care unit

Aim This study was performed to evaluate the existence of biofilm in the endotracheal tube (ETT) of mechanically ventilated children, and to study a microbial link between biofilm flora and bacteria causing ventilator-associated pneumonia (VAP). Patients and methods This was a prospective study conducted on 20 children with ETT who were evaluated for biofilm existence using scanning electron microscopy. Results A total of 20 children were enrolled in the study. Of them, 17 (85%) children showed biofilm formation on the luminal surface of ETT. A significant relationship was observed between duration of intubation and biofilm stage. Of the 17 positive cases with biofilm formation, colonization of the inner ETT surface occurred in 14 (70%) cases and 17 isolates were recovered. Five isolates were Gram positive, whereas the majority of isolates were Gram-negative bacilli. Seventeen patients developed pneumonia. All patients who developed biofilm also developed VAP. The occurrence of multidrug resistance among detected microorganisms was high. Conclusion We concluded that the density of ETT biofilm increased with increased duration of intubation. We also concluded that ETT colonization with biofilm-producing organisms increased the risk of developing VAP with highly resistant microbes.

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Pirrone, Massimiliano1; Pinciroli, Riccardo1; Mietto, Cristina1; Imber, David1; Chenelle, Christopher1; Kacmarek, Robert1; Berra, Lorenzo1 Author Information

Description and microbiology of endotracheal tube biofilm in mechanically ventilated subjects.

A biofilm is found on the inner side of endotracheal tubes (ETT) in mechanically ventilated patients, but its features and role in pneumonia remain unclear. This prospective, observational, monocentric study included critically ill ventilated subjects. Measurement of the ETT inner volume was first performed before extubation using the acoustic reflection method. After extubation, the biofilm was studied by means of optical and atomic force microscopy. Bacteriological analysis was then performed and compared with clinical documentation. Twenty-four subjects were included. Duration of intubation lasted from 2 to 79 d (mean ± SD: 11 ± 15 d). The mean percentage of ETT volume loss evaluated in situ (n = 21) was 7.1% and was not linked with the duration of intubation. Analyses with atomic force microscopy (n = 6) showed a full coverage of the inner part of the tube with biofilm, even after saline rinse. Its thickness ranged from 0.8 to 5 μm. Bacteriological cultures of the biofilm (n = 22) often showed the same bacteria as in tracheal secretions, especially for pathogenic organisms. Pseudomonas aeruginosa and Candida albicans were among the most frequent microorganisms. In subjects who had experienced a successfully treated episode of ventilator-associated pneumonia (n = 5), the responsible bacteria were still present in the biofilm. ETT biofilm is always present in intubated patients whatever the duration of intubation and appears quickly after intubation. Even after soft rinse, a small but measurable part of biofilm remains always present, and seems strongly adherent to the ETT lumen. It contains potentially pathogenic bacteria for the lung.

Microbial composition and antibiotic resistance of biofilms recovered from endotracheal tubes of mechanically ventilated patients.

In critically ill patients, breathing is impaired and mechanical ventilation, using an endotracheal tube (ET) connected to a ventilator, is necessary. Although mechanical ventilation is a life-saving procedure, it is not without risk. Because of several reasons, a biofilm often forms at the distal end of the ET and this biofilm is a persistent source of bacteria which can infect the lungs, causing ventilator-associated pneumonia (VAP). There is a link between the microbial flora of ET biofilms and the microorganisms involved in the onset of VAP. Culture dependent and independent techniques were already used to identify the microbial flora of ET biofilms and also, the antibiotic resistance of microorganisms obtained from ET biofilms was determined. The ESKAPE pathogens play a dominant role in the onset of VAP and these organisms were frequently identified in ET biofilms. Also, antibiotic resistant microorganisms were frequently present in ET biofilms. Members of the normal oral flora were also identified in ET biofilms but it is thought that these organisms initiate ET biofilm formation and are not directly involved in the development of VAP.