Nosocomial pneumonia represents approximately one quarter of all nosocomial infections and tracheal intubation increases the risk for infection by six to 20 times higher.1,2 Among patients under mechanic ventilation on intensive care unit (ICU), ventilator-associated pneumonia (VAP) represents the most frequent type of nosocomial infection, which extends the length of hospitalization, increase mortality and costs of ICU treatment.1 The clinical diagnosis of VAP has been based upon local and systemic signs and symptoms of infection in addition to radiological criteria.1,2 Furthermore, bacteriological diagnosis using protected specimen brush (PSB), bronchoalveolar lavage (BAL), and protected endotracheal aspirates have been fully standardized but lack specificity, as it relies upon the identification of microorganisms growing in tracheal secretion.1–4 Nevertheless, bacteria growing in biofilms attached to the endotracheal tube may also play a role in the pathogenesis of VAP.5,6 Indeed, the knowledge of biofilm formation by microorganisms in a series of medical devices and its association to difficult-to-treat infections has become a concerning in the nosocomial setting.7 Therefore, the VAP represents a clear example of multifactorial infection in which the biofilm may be implicated not only on the pathogenesis, but equally on the high prevalence of multiresistant bacteria.8–12 The endotracheal tube (ET) allows direct entry of microorganisms from the oropharynx environment into the lower respiratory tract, whereas the innate immune system components are ineffective.12–14 Thus, ET allows microorganisms presenting natural ability to form biofilm to attach on the tube surface through production of a matrix of polysaccharides and proteins that provides protection and consequent survival in the environment.10 According to previous publications, bacteria within the biofilm can infect the lungs by several ways: through detachment of biofilm portions, thus reaching the lungs and by aspiration into deeper airways of aerosolized planktonic pathogens detached from the biofilm.13,14

Recently, microbial diagnosis of implant-associated infections has been incremented by using vortexing and sonication technique applied to dislodge sessile microorganisms attached to a variety of medical implants, by disrupting the polymer matrix on the surface of the biofilm while maintaining the integrity of the pathogen.15–17 Trampuz et al., Inacio et al., and Vandecandelaere et al. were some of the authors whose studies applied sonication for identifying pathogens from biofilms attached to different human implants.15,17–20 Despite the lack of data, ET sonication may be an interesting tool to increase the yield of detection of sessile pathogens and its possible effect on the pathogenesis of VAP.

Therefore, we aimed to compare the yield of cultured microorganisms collected by ET centrifuged and non-centrifuged sonication fluid with microorganisms collected by conventional endotracheal aspiration of intubated pediatric ICU patients.

Microbiological data showing ET biofilm flora and upper airway colonization remains insufficient, even though it has been frequently recognized as potential risk factors for nosocomial pneumonia. Therefore, we aimed to provide information regarding the microbiota attached to ET inner surfaces biofilms by use of sonication and compared to conventional tracheal aspirates cultures, among colonized pediatric ICU patients. Sonication has been frequently applied for dislodging biofilm sessile pathogens, and therefore validated as an adds-on technique for microbial diagnosis of different human device-associated infection.15–17,19 Vandecandelaere et al. applied sonication on ET for biofilm detachment and compared with TA surveillance cultures among mechanical ventilated adult patients, and found correlation between antibiotic-resistant pathogens identified either on ET biofilms and tracheal secretion cultures.20 Gil-Perotin et al. analyzed the formation of ET biofilm and its implication to VAP by scraping the biofilm from the inner surface of ET followed by vortexing. Biofilm was found almost universally on ET and 50% of bacteria associated to VAP were also cultured from biofilms.11

To our knowledge, no previous studies have applied sonication and vortexing on ET for biofilm microbial analysis in the ICU pediatric population. Unlike others authors, we investigated microbial biofilm formation on ET mainly among subjects with no clinical diagnosis of VAP (with one exception), even though they were all on systemic antimicrobial therapy for varied reasons. We found that even on colonized mechanically ventilated pediatric patients, there were a high prevalence (96.7%) of ET biofilm formation after intubation. Previous authors have already acknowledged the association between upper respiratory tract colonization, microaspiration, and biofilm formation on the surface of ET as risk factor for nosocomial pneumonia.5,6,8,9,11 Another important aspect of our study was the special attention to investigate the microbial results of SF using a concentration step. Indeed, many authors have added a SF concentration step using samples centrifugation to identify higher number of microorganisms on the sample.15–19,24 At least for biofilms attached to ET inner surface, centrifugation of sonication fluid was not statistically superior to non-centrifuged SF to increase microbial diagnosis.

Along with other results,5,11 in this study TA and SF cultures yielded mostly Gram-negative bacilli, mainly P. aeruginosa and A. baumannii (40%). These pathogens have been highly associated with severe acute and chronic nosocomial respiratory infections as they are easily capable of expressing biofilm-forming and antibiotic-resistant genes.1,5–11,20,25,26 Previous studies analyzed the microorganisms present in dental plaque of ICU patients and showed that more isolates of P. aeruginosa were identified when compared to the general population.27–28 Besides, Pseudomonas spp., although not a common inhabitant of oral microflora is an important respiratory pathogen frequently associated with late-onset VAP.7,11,27 The remarkable ability of P. aeruginosa to adhere and form complexes biofilm structures in sputum, lung tissues, and ET and therefore producing difficult-to-treat chronic infections, such as cystic fibrosis and VAP, was the very beginning of studies connecting biofilm science with medicine.5,13,14 Broad-spectrum antimicrobial therapy was offered for all patients in our studied sample, which might have contributed to the high prevalence of A. baumannii isolated. Indeed, late-onset VAP have been frequently caused by multidrug-resistant A. baumannii, an environmental opportunistic biofilm-forming low-virulent pathogen capable of surviving to moist and dry conditions for a prolonged period, and associated to previous use of systemic empirical antibiotics.25,26,29,30 Of note, common oral microbiota such as Streptococcus spp., which presents a wide variety of species, including S. mutans, a biofilm promoter, were also frequently isolated.27 Coagulase-negative staphylococci, which have a well-known genetic predisposition to form biofilms, were also frequently detected on cultures. Our results seemed to be in accordance with a recent study that applied traditional culture methods and molecular techniques to assess the microbial diversity of ET biofilms, and eventually identified coagulase-negative staphylococci, mainly S. epidermidis as the most frequently encountered organism on cultures.18

In our study, the overall microbial culture results observed from each collection technique were concordant (83%), which suggests that culture of all three techniques (TA, SF, and CSF) may have isolated both sessile and planktonic cells. A study by Vandecandelaere et al. also suggests an agreement of results between surveillance cultures and ET biofilm cultures using sonication technique.20 However, in a situation of VAP, according to ESCMID guideline for the diagnosis and treatment of biofilm infections, it is difficult to determine if the biofilm within the ET can be either the primary source of infection or merely a concomitant colonized site.6 Also, respiratory secretions can accumulate in the ET leading to a vast concentration of planktonic pathogens in airway secretion, underestimating the presence of sessile pathogens.

Regarding the presence of discordant results among reintubated patients and discrepancy in antibiotics susceptibility, biological variation and change of microbial flora due to a longer stay in ICU might be the reason behind the discordance, once common in-hospital pathogens were found at the second moment, like Acinetobacter spp., P. aeruginosa and Candida albicans. We also hypothesize that planktonic forms are more susceptible to create drug-resistant strains and are easily detectable through aspirate cultures. Besides, the biofilm itself represents a resistance mechanism to antibiotics, and once the bacteria are dispersed they are detected and rapidly become susceptible to antibiotics. As no significant differences were found between the techniques assessed in this study, the tracheal aspirate could be an option for the identification of pathogens from the biofilm, in which was found a similar microbial sessile flora when compared to the isolates of planktonic pathogens. This study, however, had a small sample size, which limited the microbiological findings, and the strength of our statistical analysis. The absence of more patients diagnosed with VAP could also underestimate the findings by showing less pathogens, as different sensibility patterns in comparison to the results obtained. Although our study demonstrated 83.3% of concordance between techniques we do not recommend using sonication as a diagnostic test without further studies, which should be performed to better understand the relation between VAP pathogenesis and ET biofilms.

The authors declare no conflicts of interest.