This clinical observational study was carried out at Skåne University Hospital in Lund, Sweden. The study protocol was reviewed and approved by the Regional Ethical Review Board, Lund, Sweden (protocol 2013/583). Informed consent, including permission to collect and publish anonymous data, was obtained from all patients or their relatives. The manuscript was prepared according to the STROBE guidelines for observational studies [27].
Patients and materials
We included patients 18 years and older who were admitted to our intensive care unit (ICU) and were expected to require invasive mechanical ventilation for at least 24 hours. Patients were allowed to participate only once and were included during six separate time periods from February 2014 to April 2017. Depending on the period, patients were intubated on clinical indications with one of the three different types of ETTs tested in our study. Each type of ETT was used in two of the six periods. The use of the different ETTs according to study period rather than by randomization was done for logistic reasons. Patients in study periods one and four received an uncoated PVC ETT (Oral/Nasal Endotracheal Tube, Mallinckrodt™, Medtronic, Dublin, Ireland ), which is standard in our hospital; patients in periods two and six received an SC ETT (Siliconized PVC, Oral/Nasal Soft Seal® Cuffed Tracheal Tube, Portex™, Smith’s Medical, Kista, Sweden); and patients in periods three and five received an NbMC PVC ETT, coated with a thin noble metal alloy coating consisting of gold, silver, and palladium (Bactiguard Infection Protection Endotracheal Tube, Bactiguard®, Tullinge, Sweden). Each of the six study periods was planned to last for about three months and the goal was to include a minimum of 15 patients in each period. Bundles of maneuvers (e.g. placing patients in a semi-recumbent position) to prevent VAP are standard at our facility. With the exception of use of different ETT materials during the study periods, all patients received standard intensive care according to diagnosis and the clinical decisions of the responsible physicians.
Microbiological procedures
Surveillance cultures (i.e., oropharyngeal swabs and endotracheal aspirates), were collected on days 1, 2, 3, 5, 7, 14 and 21, and thereafter once a week for all included patients. On the day of extubation, surveillance cultures were also collected if not previously scheduled.
All oropharyngeal swabs and endotracheal aspirates were processed in the same manner at the Department of Clinical Microbiology by use of standardized, extended microbiological procedures [28]. Samples were inoculated on three different selective plates, one differentiating and one non-selective agar plate as follows: (1) agar plate with 5% horse blood (LabM, Heywood, Lancashire, UK) supplemented with 10 mg/L colistin and 15 mg/L nalidixic acid; (2) agar plate with 5% horse blood supplemented with 2 mg/L gentamicin and 25 mg/L nalidixic acid for Gram-positive cocci including S. pneumoniae; (3) Hematin agar plate (OxoidTM, Thermo Science, Basingstoke, UK) supplemented with 300 mg/L bacitracin for fastidious Gram-negative rods including Haemophilus influenzae (selective); (4) Uriselect 4 agar (Bio-Rad Laboratories, Copenhagen, Denmark) supplemented with 10 mg/L vancomycin for non-fastidious Gram-negative rods (differentiating); and (5) Hematin agar with a colistin disk (non-selective). All plates were manufactured in-house, and they were inspected for growth after 16 and 40 hours of aerobic, anaerobic, or CO2 incubation at 35 - 37 °C. Identification of bacterial species was performed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MALDI Biotyper Microbial Identification system, Bruker, Boston, MA, USA). Differentiation of Candida spp was based on colony appearance on CHROM Candida agar (CHROMagar, Hägersten, Sweden) after 48 hours of incubation at 35 °C.
Patient data
Age, sex, body-mass-index (BMI), days with invasive ventilation, antibiotic and/or proton pump inhibitor (PPI) use during mechanical ventilation, Simplified Acute Physiology Score 3 (SAPS 3), and main diagnosis on ICU admission were documented for all patients. Data on the occurrence of VAP, together with data on microorganisms isolated in surveillance cultures and ETT biofilm were collected for all patients.
Processing of the ETT
Patients were extubated at the discretion of the treating physician or in the case of a patient’s death. After extubation, the ETTs were collected, avoiding contamination from other than oropharyngeal flora, and rinsed (inside and outside) with 1 L of sterile saline to eliminate excess mucus. Thereafter, the distal tip of the ETT was divided into four pieces for both scanning electron microscopy (SEM) (2 pieces) and microbial cultures (2 pieces). Finally, the ETT tip was cut in a cross-sectional manner 1.5 cm above the distal tip. Pieces of the ETT for microbial cultures were sonicated in phosphate-buffered saline (PBS) with 47 kHz for 1.5 minutes to dislodge biofilm microbes. The solution was then homogenized by vortex mixing and subsequently cultured using the same procedures as applied for the oropharyngeal swabs and endotracheal aspirates. A pilot study (not published) comparing different methods for processing of the ETTs indicated that the method outlined above was optimal for removing the biofilm and for dislodging the biofilms’ microbes before culturing. For SEM, pieces of an ETT were fixed in a solution of 4% formaldehyde diluted with PBS at room temperature for 30 minutes, dehydrated with crescent ethanol concentrations, air-dried overnight, and sputter-coated with 15 nm of Au/Pd thin film (Gatan PECS Mod 682, Gatan, Inc., Pleasanton, CA, USA) to prevent charging during SEM analysis.
Scanning electron microscopy and grading of the biofilm
The inner and outer surfaces of the ETTs were examined by SEM (Zeiss Supra 40VP, Carl Zeiss Microscopy GmbH, Jena, Germany). The analysis was performed using a secondary electron detector set at a working distance of 8–10 mm and electron acceleration of 3.68–4.05 kV. Low magnification (100x to 1,000x) was used to rank biofilm coverage as follows: 0, no biofilm; 1, scarce coverage of < 10%; 2, clusters with 10–70% coverage; 3, confluent film with > 70% coverage. High magnification (10,000x to 50,000x) was used to evaluate biofilm density (0, no biofilm; 1, low/very porous; 2 medium; 3, high/compact) and level of thickness (0, no biofilm; 1, thin 0.1–1.0 µm; 2, medium 1.1–7 µm; 3, thick > 7 µm). Film thickness was estimated as the difference in focus distance between the outer surface of the biofilm and the ETT surface. When measuring the thickness, the mean was calculated based on multiple representative points in the sample. The final grade of the biofilm was then calculated by adding the scores from coverage, density, and thickness together to give a score of 0 to 9. A high/advanced biofilm grade was defined as a score of ≥ 7. The grading system is summarized in Table 1. Grading of the biofilm was performed by a researcher at RISE who was blinded to all patient information including type of ETT analyzed.
Surveillance cultures and ETT tip cultures with growth of at least two species of bacteria usually found in the oral cavity were classified as having normal flora (negative cultures). Species not normally found in the oral cavity (e.g., pathogens, gut flora, or overgrowth of normal oral flora) were classified as abnormal flora (positive cultures). All culture results were reviewed by a microbiologist to ensure correct classification. The diagnosis of VAP was made by two independent physicians evaluating the cases based on clinical and radiological examinations. VAP was defined by the following: 1) new or progressive lobar infiltrate > 48 hours after intubation; 2) two or more of the minor criteria fever, leukocytosis/leukopenia, and/or purulent respiratory secretions; 3) microbiological confirmation in endotracheal aspirate [29]. Colonization with common VAP pathogens included cultures with Enterococcus faecium, E. faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumani, Pseudomonas aeruginosa, Streptococcus pneumoniae, Haemophilus influenzae, and other Enterobacteriaceae species.
VAP relapse was defined as previously reported[19]. Microbial persistence was defined as persistence of the causative agent of VAP in at least two surveillance cultures despite 48 hours of appropriate antibiotic therapy [30].
Statistical analysis
Results were expressed as median (interquartile range [q1-q3]) for continuous variables and numbers (percentages) for dichotomous variables. A p value of < 0.05 was considered significant, and all statistical tests were two-tailed. Groupwise comparisons were conducted using Fisher’s exact test for categorical variables and the Mann-Whitney U-test for continuous variables. Multivariable logistic regression analysis was applied to identify independent factors associated with the formation of high-grade biofilm on the ETT (score ≥ 7 in the scoring system described above). This cut-off was chosen based on data from an earlier study [18]. The Hosmer‐Lemeshow test was used to determine goodness of fit. Univariable logistic regression analyses were conducted to evaluate patients’ characteristics that could be associated with the development of VAP. Multivariable regression analysis of VAP was not performed due to too few cases of the condition (n = 12). Clinical studies comparing biofilm formation on ETTs using the scoring system applied in this study are lacking. Based on previous laboratory models using other scoring systems to grade biofilm formation [31,32] and given a power of 0.8 and an alfa level of 0.05, a sample size of 27 was needed in each group. We carried out all analyses with SPSS 26 (SPSS Inc, Chicago, IL, USA).