Patients admitted to intensive care units (ICUs) frequently require mechanical ventilation to support their respiratory function. Although this intervention is lifesaving, a significant risk of complications, most notably ventilator-associated pneumonia (VAP), remains. The prevalence of VAP is high in this patient population, making this group particularly relevant for investigation. Ventilator-associated pneumonia is a serious hospital-acquired infection of the lung parenchyma that develops in patients receiving mechanical ventilation. Ventilator-associated pneumonia is associated with increased patient morbidity, prolonged ICU stays, higher healthcare costs, and elevated mortality rates among critically ill patients.(1) The primary mechanism underlying VAP involves the aspiration of pathogenic microorganisms from the oropharynx into the lower respiratory tract.(2,3) This mechanism highlights the oral cavity as a significant reservoir for potential respiratory pathogens.

The oral microbiome in healthy individuals is a diverse and balanced ecosystem. However, the oral microbiome in critically ill patients receiving intubation is frequently disrupted, leading to dysbiosis.(4,5) Factors such as underlying systemic diseases, reduced salivary flow, impaired immune responses, and the presence of endotracheal tubes can profoundly alter the oral environment. These changes favor the proliferation of opportunistic pathogens, shifting the oral microbiota towards one dominated by potentially virulent microorganisms.(6,7,8) Periodontal disease, a chronic inflammatory condition, is also recognized as a potential source of respiratory pathogens, further emphasizing the link between oral health and systemic infections, particularly among vulnerable populations.(9,10)

Among the bacterial species most frequently implicated in VAP are Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, and Pseudomonas aeruginosa. S. aureus is a versatile pathogen, and its antibiotic resistance, particularly methicillin-resistant S. aureus (MRSA), poses a significant clinical challenge.(11) K. pneumoniae is known for its rapidly evolving antibiotic resistance mechanisms and its association with severe healthcare-associated infections.(12) S. pneumoniae is a common etiological agent of pneumonia, whereas P. aeruginosa is an opportunistic pathogen frequently encountered in hospital settings, known for its intrinsic resistance to many antibiotics and its ability to form biofilms.(13) The persistent presence of these bacteria, even in clinically stable patients receiving mechanical ventilation, signifies ongoing colonization and a predisposition to severe respiratory infections.

The importance of oral healthcare in preventing VAP has been widely acknowledged. Interventions such as regular tooth brushing and the use of antiseptic mouthwashes, including chlorhexidine, have been explored for their potential to reduce the bacterial load in the oral cavity and subsequently decrease the incidence of VAP.(14,15) Studies suggest that oral hygiene can reduce bacterial transport and colonization during intubation.(16,17,18,19) However, the optimal methods and consistent effectiveness of oral care in this patient population remain subjects of ongoing research and debate.(20) The unique challenges associated with delivering comprehensive oral care to patients receiving mechanical ventilation, coupled with potential adverse effects of certain agents and limitations in reaching all colonized sites,(21) necessitate a deeper understanding of bacterial dynamics in this critical population. Therefore, understanding the relationship between oral and tracheal bacterial populations is crucial for effective VAP prevention and management.

Despite increasing evidence linking oral dysbiosis to VAP, limited data are available regarding the simultaneous temporal dynamics of oral and tracheal colonization by specific respiratory pathogens in critically ill patients receiving mechanical ventilation. Understanding these microbial interactions may contribute to the development of more targeted preventive strategies.

To investigate the prevalence, co-occurrence, and temporal dynamics of S. aureus, K. pneumoniae, S. pneumoniae, and P. aeruginosa in oral and tracheal samples from critically ill patients receiving mechanical ventilation.

Of the 49 patients initially screened for intubation in the ICU of Hospital Regional de Uberaba (25 women and 24 men), 21 patients (11 men and 10 women) with a mean age of 70.7 years (standard deviation [SD]: 12.5) met the criteria for prospective analysis. The remaining 28 patients were excluded because of early extubation (within 24 h after intubation), death, clinical instability on the day of follow-up sample collection, or withdrawal of consent. Importantly, none of the 21 patients receiving mechanical ventilation who were included in the study exhibited clinical signs or symptoms of pneumonia or any other active infectious process at either the initial (S1, T1) or 48-h (S2, T2) collection time points.

Oral and tracheal samples from these 21 patients were analyzed using PCR to detect S. aureus, K. pneumoniae, S. pneumoniae, and P. aeruginosa, all of which are frequently implicated in VAP. Table 2 summarizes the detection frequencies of these microorganisms in the collected samples. Statistical analysis revealed no significant differences in the overall detection (presence or absence) of these bacteria when comparing collection time points or sample types (p>0.09, q<1.56). These findings indicate persistent detection of these pathogens in both oral and tracheal samples throughout the study period.

Despite the absence of clinical infection, more than 43% of the analyzed oral and tracheal samples tested positive for at least one of the target bacteria at both collection time points. Notably, most initial oral samples were positive. Although P. aeruginosa showed increased detection in oral samples from the first to the second collection, the remaining bacterial species generally exhibited decreased oral detection over the 48-h period.

Figure 1 illustrates the positive (gray) and negative (white) detection of bacteria in oral (S1/S2) and tracheal (T1/T2) samples from the 21 patients at both collection time points. Correlation analysis of the presence or absence of S. aureus between oral and tracheal samples revealed a significant positive correlation at both the initial collection (S1 versus T1; p=0.0028, r=0.61) and the 48-h collection (S2 versus T2; p=0.0021, r=0.63). Specifically, S. aureus was detected in both S1 and T1 samples from eight patients, whereas another eight patients showed no detection in either. Furthermore, a positive correlation for S. aureus was also observed between the initial and 48-h oral samples (S1 versus S2; p=0.01, r=0.52), with seven patients remaining positive and eight remaining negative at both time points. This longitudinal correlation was not observed between the initial and 48-h tracheal samples (p=0.59, r=0.14).

Figure 1

For K. pneumoniae, a significant positive correlation in detection was found only at the initial collection between oral and tracheal samples (S1 versus T1; p=0.009, r=0.55), with nine patients showing detection in both samples and seven showing no detection in either sample. No other significant correlations for K. pneumoniae were observed among S1, S2, T1, and T2 (p>0.11, r>0.05).

In contrast, no significant positive correlations were observed for the detection of P. aeruginosa and S. pneumoniae across most oral and tracheal samples or collection time points (S1, S2, T1, and T2; p>0.11, r>0.05). However, a significant positive correlation was identified for S. pneumoniae when comparing detection in the initial oral samples (S1) with detection in the 48-h tracheal samples (T2; p=0.03, r=0.47). Specifically, six patients showed no detection in either S1 or T2, whereas nine showed detection in both samples. No similar delayed correlation was observed for S. aureus (p=0.13, r=0.33), K. pneumoniae (p=0.16, r=0.31), or P. aeruginosa (p=0.13, r=0.33).

Figure 2 illustrates the mean fluorescence intensity, representing the relative bacterial load, for each detected bacterium in oral and tracheal samples. Quantitative analysis of relative bacterial load revealed no statistically significant differences for S. aureus when comparing S1 and S2 (p=0.10), T1 and T2 (p=0.63), S1 and T1 (p=0.16), S2 and T2 (p=0.59), or S1 and T2 (p=0.19). Similarly, S. pneumoniae showed no significant differences in relative bacterial load between S1 and S2 (p=0.42), T1 and T2 (p=0.70), S1 and T1 (p=0.12), S2 and T2 (p=0.06), or S1 and T2 (p=0.14). P aeruginosa also demonstrated no significant differences across these comparisons (p>0.36). In contrast, K. pneumoniae showed a statistically significant increase in relative bacterial load in tracheal samples between T1 and T2 (p=0.04). Furthermore, the relative bacterial load of K. pneumoniae was significantly higher in T2 samples than in S2 samples (p=0.01). No significant differences were found for K. pneumoniae between S1 and S2 (p=0.26), S1 and T1 (p=0.27), or S1 and T2 (p=0.72). These findings indicate a specific increase in K. pneumoniae relative bacterial load within tracheal samples during the 48-h study period.

Figure 2

This study investigated the presence, detection patterns, and relative bacterial load of key respiratory pathogens—S. aureus, K. pneumoniae, S. pneumoniae, and P. aeruginosa—in oral and tracheal samples from clinically stable patients receiving mechanical ventilation. The study population is particularly relevant because mechanical ventilation creates specific risk factors for VAP. A significant finding was that more than 43% of patients tested positive for at least one target bacterium at both the initial and 48-h sample collections despite the absence of clinical signs of infection. This high prevalence is consistent with previous research identifying the oral cavity as an important reservoir of opportunistic pathogens in critically ill hospitalized patients.(7,8) Such colonization represents a continuous risk for the development of VAP.(1) Initially, oral samples (S1) showed high detection rates for K. pneumoniae (62%), S. pneumoniae (67%), and S. aureus (48%). Although the detection rate of P. aeruginosa increased in oral samples from S1 to S2 (52% to 67%), the remaining bacterial species generally showed decreased oral detection during the 48-hour study period.

Despite these individual changes, statistical analysis revealed no significant differences in the overall detection (presence or absence) of these bacteria across collection time points or sample types (p>0.09, q<1.56). These findings indicate persistent colonization, suggesting that although the detection of individual bacterial species may fluctuate, both oral and tracheal sites remain colonized. The oral microbiota of patients receiving mechanical ventilation may undergo dysbiosis,(4,5) and the presence of these microorganisms within the oral biofilm has been well documented.(7,2) Correlation analyses evaluating bacterial presence or absence provided additional insights into the potential relationships between oral and tracheal colonization.

A significant positive correlation for S. aureus was observed between oral and tracheal samples at both the initial (S1 versus T1; p=0.0028, r=0.61) and 48-h (S2 versus T2; p=0.0021, r=0.63) collections. This association, with S. aureus detected in both oral and tracheal samples from eight patients at S1/T1 and absent from both samples in another eight patients, strongly supports the hypothesis that aspiration may contribute to the transfer of pathogens to the lower respiratory tract.(3,2) Furthermore, a positive correlation for S. aureus detection was found between the initial and 48-h oral samples (S1 versus S2; p=0.01, r=0.52), indicating relatively stable oral colonization over time. However, a similar association was not observed between the initial and 48-h tracheal samples (T1 versus T2; p=0.59, r=0.14), suggesting that tracheal colonization may be influenced by factors beyond persistent oral colonization.

For K. pneumoniae, a significant positive correlation was observed only between oral and tracheal samples collected at the initial time point (S1 versus T1; p=0.009, r=0.55). The absence of significant correlations across the remaining sample comparisons (p>0.11, r>0.05) suggests that the relationship between oral and tracheal colonization by K. pneumoniae may be more complex after the initial colonization. Conversely, P. aeruginosa and S. pneumoniae generally showed no significant positive correlations between oral and tracheal detection or across collection time points (p>0.11, r>0.05). However, a significant positive correlation was identified between S. pneumoniae detection in the initial oral samples (S1) and 48-h tracheal samples (T2; p=0.03, r=0.47). This delayed association suggests that oral colonization by S. pneumoniae may contribute to subsequent tracheal colonization in patients receiving mechanical ventilation.(9)

Oral biofilm can serve as a reservoir for these microorganisms, which may subsequently be aspirated into the lower respiratory tract.(7,2) Beyond the presence or absence of bacterial detection, quantitative analysis of relative bacterial load revealed distinct patterns. For S. aureus, S. pneumoniae, and P. aeruginosa, no statistically significant differences in relative bacterial load were observed across the evaluated comparisons (p>0.06). These findings suggest that the relative bacterial load of these pathogens remained stable during the 48-h study period in clinically stable patients. However, a significant finding emerged for K. pneumoniae. The relative bacterial load was significantly higher in 48-h tracheal samples (T2) than in both the initial tracheal samples (T1; p=0.04) and 48-h oral samples (S2; p=0.01). These findings suggest an increase in K. pneumoniae bacterial load within tracheal samples during mechanical ventilation, despite the absence of a corresponding increase in oral samples.

The identification of persistent patterns of oral and tracheal colonization reinforces the importance of integrating oral healthcare protocols into ICU management. Moreover, the observed increase in tracheal K. pneumoniae relative bacterial load suggests that standard oral hygiene measures alone may not fully prevent lower airway colonization in patients receiving mechanical ventilation. Although maintaining good oral hygiene is fundamental for reducing bacterial load and preventing VAP,(6,14,15,16,17,18,19) the present findings indicate that K. pneumoniae may exhibit distinct colonization dynamics within the lower respiratory tract.(12,13) This dynamic increase in tracheal relative bacterial load may justify closer microbiological surveillance and future investigation of targeted preventive strategies for K. pneumoniae-associated VAP, particularly considering the inconsistent effectiveness reported for some oral care interventions, including chlorhexidine mouthwash.(20) The potential use of topical antibiotics within the oral cavity to reduce bacterial migration into the lower respiratory tract also remains an area of ongoing investigation.(21)

This study has several limitations that should be acknowledged, including the relatively small sample size, the single-center design, and the short 48-h follow-up period. Additionally, molecular detection by qPCR does not distinguish viable from non-viable bacteria and does not allow assessment of antimicrobial resistance profiles. Therefore, larger longitudinal studies integrating microbiological culture and sequencing approaches are warranted.

In summary, this study demonstrated a high prevalence and dynamic colonization patterns of S. aureus, K. pneumoniae, S. pneumoniae, and P. aeruginosa in patients receiving mechanical ventilation, even in the absence of clinical signs of infection. Significant correlations between oral and tracheal detection of S. aureus support a potential association between oral colonization and lower airway microbial presence. Quantitative analysis further demonstrated a significant increase in the relative bacterial load of K. pneumoniae in tracheal samples during the study period, suggesting distinct colonization dynamics for this pathogen. These findings reinforce the relevance of oral health management in patients receiving mechanical ventilation and suggest that pathogen-specific microbial behavior should be considered when developing future preventive strategies for ventilator-associated pneumonia. Future strategies must integrate these insights to develop precise risk stratification tools and targeted prophylactic measures, ultimately enhancing patient safety and outcomes in critical care.