Preoperative oral hygiene treatment reduces bacterial transport and colonization during intubation for orthopedic surgery

Abstract

Purpose: The process of infection by bacteria and viruses involves invasion, establishment, growth, and parasitization. Poor oral hygiene and dysbiosis are significant risk factors for pneumonia. The aim of this study was to evaluate bacterial transport into the trachea during intubation for orthopedic surgery and the impact of oral hygiene treatment.

Methods: The study cohort included 53 patients with fracture who underwent surgical procedures under general anesthesia and were divided into two groups: an oral hygiene treatment (OHT) group (n = 27) and a control group (n = 26). Before intubation, the OHT group underwent preoperative oral hygiene treatment. Microbiological culture was used for detection and counting of bacteria from the oropharynx, trachea, and tip of the endotracheal tube (ETT).

Results: Patients in the OHT group had a lower pathogen detection rate and lower degree of bacterial colonization in the oropharynx, trachea, and ETT tip.

Conclusion: Preoperative oral hygiene treatment is able to reduce bacterial transport and colonization during orthopedic surgery, thus providing an important adjunct to pre-anesthesia care.

Introduction

Although a prerequisite for mechanical ventilation (MV) in patients under general anesthesia, the presence of an endotracheal tube (ETT) interferes with the normal physiological mechanisms of respiratory function [1], especially the natural barrier function of the trachea, creating a direct channel for transfer of bacteria to the trachea from the oropharynx through the open glottis [2].

Pneumonia can be a postoperative complication of general anesthesia, and bacterial pneumonia is a common cause of mortality and morbidity [3]. Poor oral hygiene and dysbiosis are important risk factors for pneumonia [4] and the majority of pathogens have been confirmed to have colonized the oral cavity before pneumonia is diagnosed [5]. For patients with a tracheal cannula, the oral cavity may be an important reservoir of bacteria responsible for postoperative pneumonia [3], and thus if fewer oral bacteria are present, fewer will be transported to the lung via the ETT.

Previous research has demonstrated reductions in lung infection rates with oral hygiene treatment, including chlorhexidine [6], povidone-iodine [7], and even purified water [8], and oral care with chlorhexidine (CHX) is now included in the European care bundle for prevention of ventilator-associated pneumonia [9]. Nevertheless, most studies to date have focused on acute and critical patients in the intensive care unit [10], and limited data are available for larger series of patients who require surgery under general anesthesia, especially those with limited self-care ability. In such patients, reduction of daily oral cleaning routines can result in poor oral health, and the oral microorganisms present may differ from those in healthy adults [11].

Currently, there is evidence that tooth brushing and rinsing with CHX are beneficial for reduction of dental plaque and oropharyngeal colonization [12,13], but the effect on bacterial transport is unknown. Therefore, the main aim of this study was to evaluate the number of culturable bacteria transported during endotracheal intubation in patients undergoing surgery for fractures and the impact of preoperative oral hygiene treatment on microorganism transport via ETT.

Materials and Methods

A predicted sample size was calculated based on an unpublished preliminary experiment. The CFU count at the ETT tip was the outcome index. This suggested that the CFU count for the control group would be 60.33 ± 3.81, and that this was expected to decrease by 53.71 in the OHT group. Therefore, the calculated sample size was 9 participants in each group on the basis of PASS 15.0 (NCSS Medical LLC, Phoenix, AZ, USA) (Alpha = 0.05, Power = 0.9). Considering the possibility that 10-20% of the data would be missing and not wasting oral hygiene treatment supplies, the final sample size was determined as 28 participants in each group.

Study design

This study was designed and conducted as a pilot randomized, examiner-blind, controlled clinical trial. Eligible patients were those undergoing surgical procedures for fractures under endotracheal intubation and mechanical ventilation. The exclusion criteria included emergency surgery, patient age <18 or >75 years, presence of oral or periodontal disease, missing teeth and use of dentures, severe liver and kidney insufficiency, heart failure, or other serious diseases, known hypersensitivity to CHX, a confirmed diagnosis of pneumonia at the baseline, and immunosuppressive drug or antibiotic use within the previous three months. The eligible patients were enrolled after admission between June and November 2022 and divided randomly into two groups: an oral hygiene treatment (OHT) group and a control group.

Patients in the OHT group received oral hygiene treatment, including toothbrushing using the Bass technique [14] with fluoride toothpaste and a soft-bristled toothbrush for 3 min and gargling with 15 mL of 0.12% chlorhexidine (Enwei & Zhongxin Bio-Medical Co. Ltd., Nanjing, Jiangsu, PR China) for 1 min, 30 min before anesthesia, whereas the control received no intervention. All treatment procedures were performed by a trained operator who assigned participants to the interventions by grouping.

Specimen preparation

Microorganism specimens were collected from 4 areas per participant. After the intervention, microorganisms were sampled at specified sampling points using a disposable sterile swab, including the gingiva of the right upper lateral incisor, the midpoint of the upper jaw, and the root of the tongue, which are in the path of the ETT as it enters the trachea (Area-A). General anesthesia was then induced with sufentanil (0.4 µg/kg), propofol (2.0-3.0 mg/kg), penehyclidine (0.5 mg) and rocuronium (0.6 mg/kg). The anesthesiologist, who was wearing sterile gloves and unaware of the grouping, executed the intubation using a video laryngoscope. Microorganism specimens were collected after intubation (Area-B) and at the end of surgery (Area-C) 1 cm distal to the ETT by brushing with an electronic bronchoscope. Before emergence from general anesthesia, all participants were extubated gently using a laryngoscope to prevent the ETT tip from touching the oral cavity, and a laryngeal mask was used to guarantee MV. Then, 1 cm of the anterior ETT segment was removed with sterile scissors (Area-D). All intubation equipment, including the ETT (ID = 7.0), laryngoscope and electronic bronchoscope, was manipulated in a sterile manner (Fig. 1).

Each microorganism specimen was suspended in 1 mL of phosphate-buffered saline for 180 s, and 5 µL of the suspension was then inoculated onto blood, MacConkey, and chocolate agar plates (Zhengzhou AntuBiological Engineering Co. Ltd., Zhengzhou, Henan, PR China). To determine the total number of bacteria, the number of colony-forming units (CFUs) after a 48-h incubation at 37°C under 5-10% CO₂ was manually counted by an individual blinded to the grouping. The VITEK2 Compact system was used to identify pure cultures and dominant pathogens, facilitating rapid identification of bacteria by automatic determination of changes in metabolites in terms of color and light transmittance. Subsequently, the results were accepted only when the dominant strains were consistent with Gram staining results.

The study also recorded demographic and clinical data, including sex, age, body mass index (BMI), smoking, Brinkman index, tooth-brushing frequency at home and during hospitalization, and operation position, and the following parameters were recorded daily after surgery: temperature, leukocyte count, antibiotic agent type and indication, and chest radiography results.

Fig. 1 Specimen collection flow chart

Postoperative pneumonia

Postoperative pneumonia was diagnosed on the basis of the Clinical Pulmonary Infection Score (CPIS), a CPIS of at least 6 being very suggestive of pneumonia. Pneumonia was diagnosed from chest X-rays showing a new, persistent progressive infiltrate and an increase of suctioned secretions, and one or more of the following: fever exceeding 38.0°C for more than 4 h; blood leukocytosis (>10 × 10⁹/L); and an increase in the fraction of inspired oxygen (FIO₂) of 0.2 needed to maintain arterial oxygen saturation, sustained for more than 4 h.

Randomization and blinding

A randomization sequence was created using IBM SPSS Statistics 26.0 (IBM, Armonk, NY, USA), and participant allocation was performed by the operator based on these random numbers. The examiners and counters were unaware to the study allocation, but the participants and operator were aware of whether or not they have completed the treatment.

Statistical analysis

IBM SPSS Statistics 26.0 for Windows (IBM) was used for statistical analysis. Categorical variables were presented as percentages. Additionally, continuous variables with normal and non-normal distributions were presented as means (SDs) and medians (IQRs). Parametric (t-test) and non-parametric (Fisher’s exact test, Mann-Whitney U test) statistical analyses were employed for paired comparisons, and Spearman’s correlation was used to evaluate relationships among multiple specimens. Statistical significance was determined at P < 0.05.

Results

This study assessed bacterial transport into the airways during orthopedic surgery intubation and the effects of oral hygiene treatment on pathogen colonization and the incidence of postoperative pneumonia. Between June and November 2022, 56 randomized patients were recruited, but three patients were excluded because a long operation time (>10 h) prevented specimens being sent promptly for examination (OHT: n = 27; control: n = 26). The baseline characteristics of the two groups were comparable, including age, body weight, sex distribution, smoking, anesthesia duration, operation position, and tooth-brushing frequency. (Table 1)

To assess the ETT as a channel for entry of oral cavity bacteria into the trachea, bacteria sampled from the oral cavity, trachea, and ETT were cultured. Pathogenic bacteria were cultured from 28 (26.92%) of 104 specimens in the control group, and this proportion was significantly lower (4.63%) (P < 0.001) in the OHT group (Fig. 2). Bacterial identification showed that the main pathogens were Staphylococcus aureus (14, 13.46%), Klebsiella pneumoniae (4, 3.86%), and Pseudomonas aeruginosa (3, 2.88%) in the control group, whereas P. aeruginosa (2, 1.85%), Haemophilus parainfluenzae (2, 1.85%), and Staphylococcus pyogenes (1, 0.93%) were detected in the OHT group (Table 2).

Two patients in the control group (7.69%) were diagnosed as having postoperative pneumonia. The incidence of pneumonia did not differ significantly between the two groups (P = 0.236). However, the degree of bacterial colonization in the OHT group was lower than that in the control group at all collection sites (Table 3, Fig. 3).

Spearman’s correlation analysis showed that the number of CFUs in the oral cavity specimens (Area-A) was correlated positively with that for areas B-D (Spearman’s γ = 0.748, 0.610, and 0.759, respectively; P < 0.05). Additionally, the remaining three specimens were correlated positively. Older age was negatively correlated with brushing frequency at home (Spearman’s γ = −0.437; P < 0.05), and there was a positive correlation between the frequency of brushing at home and that during hospitalization (Spearman’s γ = 0.433; P < 0.05). Other factors, such as body weight, sex distribution, smoking, anesthesia duration, and operation position showed a low correlation (Table 4).

Finally, patients with different fracture sites were found to have different brushing frequencies during hospitalization. Those with upper limb fractures had a significantly higher brushing frequency than those with lower limb or spinal fractures (Table 5).

Table 1 Patient characteristics

	Control (n = 26)	OHT (n = 27)	P
Age, years	52.08 (12.51)	45.26 (13.18)	0.059b
Male/female	14/12	14/13	1.000a
BMI	24.20 (3.89)	24.59 (3.18)	0.688b
Smoker/non-smoker	15/11	16/11	1.000a
Brinkman index	0 (225)	0 (200)	0.743c
BF1, /day	1 (1)	2 (1)	0.065c
BF2, /day	1 (1)	0.5 (1)	0.721c
Anesthesia duration, min	191 (84)	207 (84)	0.468b
Operation position, supine/lateral/prone	14/0/12	18/1/8	0.325a

BMI: body mass index; BF₁: brushing frequency at home; BF₂: brushing frequency during hospitalization; n: number of participants. ^aDifferences were assessed using the Fisher exact test. ^bUnpaired Student’s t-test. ^cMann-Whitney U-test

 

Fig. 2 Proportions of pathogenic and non-pathogenic bacteria

Table 2 Characteristics of pathogens in specimens obtained during the perioperative period

	Control (n = 104)	OHT (n = 108)	P
Pathogenic/non-pathogenic, case	28/76	5/103	<0.001*
Pathogenic bacteria
Staphylococcus aureus, %	14 (13.46)	0 (0)	<0.001*
Pseudomonas aeruginosa, %	3 (2.88)	2 (1.85)	0.679
Haemophilus parainfluenzae, %	2 (1.92)	2 (1.85)	1.000
Enterobacter cloacae, %	2 (1.92)	0 (0)	0.498
Staphylococcus pyogenes, %	1 (0.96)	1 (0.93)	1.000
Klebsiella pneumoniae, %	4 (3.86)	0 (0)	0.056
Streptococcus pneumoniae, %	2 (1.92)	0 (0)	0.239

n: the number of specimens (microorganism specimens were collected from 4 areas per participant.). *Differences were assessed using Fisher’s exact test.

 

Table 3 Effects of oral hygiene treatment on bacterial transport (CFUs/mL [10³])

	Control (n = 26)	OHT (n = 27)	P
Area-A	52.50 (125.00)	6.00 (4.00)	<0.001*
Area-B	6.25 (57.75)	0.00 (2.00)	<0.001*
Area-C	5.00 (19.50)	0.00 (2.00)	0.003*
Area-D	72.50 (59.50)	6.80 (5.00)	<0.001*

CFU: colony-forming unit; n: number of participants. *Mann-Whitney U-test

 

Fig. 3 Effects of oral hygiene treatment on bacteria transport

Table 4 Correlation analysis

	Age	BF1	BF2	Area-A	Area-B	Area-C	Area-D
Age	1	DP	NS	NS	NS	NS	NS
BF1	−0.437*	1	DP	DP	DP	DP	DP
BF2	NS	0.433*	1	NS	NS	NS	NS
Area-A	NS	−0.336*	NS	1	DP	DP	DP
Arae-B	NS	−0.407*	NS	0.748*	1	DP	DP
Area-C	NS	−0.330*	NS	0.610*	0.624*	1	DP
Area-D	NS	−0.393*	NS	0.759*	0.692*	0.622*	1

BF₁: brushing frequency at home; BF₂: brushing frequency during hospitalization; NS: non-significant correlation; DP: the data have been presented in other space of the table. *P < 0.05

 

Table 5 Tooth brushing frequency of patients

BF1, /day	2.0 (1.0)
Upper limb fractures	2.0 (1.0)	P = 0.092b
Vertebral fractures	1.0 (1.0)
Lower limb fractures	2.0 (1.0)
BF2, /day	1.0 (1.0)
Upper limb fractures	1.0 (1.5)	P = 0.012b
Vertebral fractures	0.5 (1.0)
Lower limb fractures	0.0 (1.0)

BF₁: brushing frequency at home; BF₂: brushing frequency during hospitalization. ^bKruskal-Wallis test

Discussion

This study aimed to evaluate the levels of bacterial transport caused by ETT and the impact of preoperative oral hygiene treatment. It was found that the number of bacterial colonies cultured from oral cavity samples was lower after preoperative oral hygiene treatment, being consistent with previous findings [15]. More interestingly, oral hygiene treatment reduced bacterial colonization of the trachea and ETT, as reported previously [7]. However, the development of pneumonia did not differ between the groups, contradicting earlier results obtained in the ICU [16,17]. These findings support the contention that preoperative oral hygiene treatment can reduce bacterial transport and colonization during intubation, but does not affect the development of postoperative pneumonia in these participants.

The oral cavity, the upper respiratory tract and the ETT route may contain >500 bacterial species, including pneumonia-associated pathogens such as Streptococcus pneumoniae, H. parainfluenzae, and S. aureus [18]. Previous studies have suggested a possible etiological role of the oral cavity in the pathogenesis of pneumonia, particularly dental plaque, the dorsum of the tongue, and foreign bodies, which may serve as the main source of potentially pathogenic microorganisms in patients with poor oral hygiene [12]. This study revealed a pathogen detection rate of 26.92% in the control group, constituting a potential risk of infection by pathogenic microorganisms. A systematic review [19] of tooth-brushing in patients with pneumonia showed significantly reduced rates with a low risk of bias. Considering all trials, the trend was toward lower pneumonia rates, suggesting the importance of brushing to maintain oral health as a means of preventing pneumonia, particularly in unhealthy individuals. Mechanical oral hygiene [19] and oral disinfection [20] are required for optimal oral health, but this may be challenging for patients with fractures. Poor oral hygiene and lack of mechanical elimination can lead to increased bacterial proliferation and dental plaque accumulation, thus posing a higher risk of pneumonia [21,22].

For patients with fractures requiring surgery, ETT after general anesthesia necessitates mouth-opening, and this can lead to changes in the oral environment [4]. For example, the inflated cuff blocks normal mucociliary flow, and the ETT acts as a channel through which bacteria can move toward the lower airways, as shown in previous studies [1]. Moreover, a biofilm layer covers the ETT surface over time, and the bacteria in this biofilm may detach and colonize the lower airways [23]. Because endotracheal intubation is an invasive procedure, it likely introduces bacteria into the subglottic area mechanically [1]. Generally, in this study, bacterial colonization of the oral cavity was highly correlated with lower airway colonization after intubation (Spearman’s γ = 0.748, 0.610, and 0.759, P < 0.05), indicating that the ETT immediately introduced oral bacteria into the subglottic region.

Infection is the process by which bacteria invade, establish, grow, and parasitize. If these microorganisms have pathogenic properties, the host may develop an illness. Therefore, reduction of bacterial colonization in the oral cavity is important, particularly in relation to dental plaque [15]. Currently, oral hygiene treatment with the antiseptic CHX, povidone-iodine, or purified water is used to reduce the rates of bacterial colonization and respiratory tract infection [5,8], and this was confirmed in the present study. After oral hygiene treatment, oral bacterial colonization was significantly reduced, followed by a decrease of bacterial load in the subglottic area and ETT. However, use of a single oral hygiene treatment before endotracheal intubation did not prevent pneumonia to any significant degree (OHT: 0%; control: 7.69%, P = 0.236), which may have been attributable to the generally low incidence of pneumonia in the study cohort. Because routine antibiotic use after surgery has become standard in China, no conclusions about any differences in pneumonia prevention can be reached. However, the present findings confirmed a reduction in the number of pathogens during intubation after oral hygiene treatment, including the rates of pathogenic bacterial colonization and detection. More research on oral hygiene treatment will be necessary to evaluate its potential for preventing pneumonia in high-risk populations, especially elderly and immunocompromised patients. Furthermore, although most hospitals in China implement oral care protocols for all patients, particularly in the ICU, limited data are available for patients undergoing general anesthesia, and no oral care guidelines exist for patients at risk of pneumonia, as recommended by anesthesiologists or anesthesia nurses. This study focused specifically on bacterial invasion rather than the effect of a single preoperative oral hygiene treatment on the incidence of pneumonia; hence the goal was to provide an important supplement to pre-anesthesia care to reduce risk factors for pneumonia development.

In addition, patients with lower limb or spinal fractures are more likely to neglect tooth- brushing during hospitalization. This may be because such patients are more likely to be bedridden than those with upper limb injuries, thus reducing their adherence to oral hygiene. Both medical workers and families of patients should provide more help to patients, particularly with regard to personal hygiene. Osteoporosis in elderly patients may also increase the risk of load-bearing fractures, which can cause patients to be bedridden, and thus age is an independent risk factor for development of pneumonia [24]. Hence, more attention should be given to elderly patients with lower limb or spinal fractures, and the role of oral hygiene treatment in prevention of pneumonia in these patients requires further evaluation.

The present results showed that the incidence of pneumonia did not differ significantly between the two groups, and the results should be interpreted cautiously. Preoperative oral hygiene treatment clearly reduced the sources of bacteria during intubation, and future studies should include a larger number of participants, particularly the elderly or immunocompromised, to investigate this issue. CHX has a broad range of activity against Gram-positive microorganisms and can be used for oropharyngeal decontamination, although its activity against Gram-negative microorganisms may be lower [25]. In the OHT group, P. aeruginosa and H. parainfluenzae were still detectable in the samples. The addition of other hygiene measures should also be investigated and more attention should be focused on establishing standardized treatment protocols. In comparison to the gold standard 16S rRNA assessment, the VITEK2 Compact system employs bacterial metabolic products to facilitate precise identification of bacteria, making it a widely applicable and convenient method for bacterial identification in hospital settings. In future studies, both methods will need to be compared.

In conclusion, preoperative oral hygiene treatment, including mechanical and chemical oral cleansing, was found to have no significant effect on the incidence of pneumonia in the study patients, although it was effective for reducing microorganism transport and colonization, which are key factors involved in bacterial infection. Therefore, as a convenient and low-cost treatment, preoperative oral hygiene should be promoted for more patients undergoing general anesthesia, particularly those with limited self-care ability.

Abbreviations

CFUs: colony-forming units; CHX: chlorhexidine; CPIS: clinical pulmonary infection score; ETT: endotracheal tube; MV: mechanical ventilation; OHT: oral hygiene treatment VAP: ventilator-associated pneumonia

Ethical Statements

This study was approved by the Medical Ethics Committee of The Second Affiliated Hospital of Zunyi Medical University (KYLL-2022-042) on June 22, 2022 and registered before patient enrollment (ChiCTR2200061196, www.chictr.org.cn/index.aspx, Yu Shuai, 15/06/2022). All methods on human subjects were conducted in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) and all procedures were carried after obtaining informed written consent from the subjects. The participants were recruited between June and November 2022, and all provided written informed consent.

Conflicts of Interest

The authors have no conflicts of interest to declare.

Funding

This work was funded by the Guizhou High-Level Innovative Talent Training Program “Thousand” Level Talents Program and the Collaborative Innovation Center of the Chinese Ministry of Education (2020-39).

Author Contributions

YS: conceptualization, investigation, visualization, writing - original draft. XW: investigation, writing - original draft. SC and TH: data curation, investigation. ZW: writing - review & editing. YZ: conceptualization, writing - review & editing, supervision.

ORCID iD

¹⁾YS: [e-mail], https://orcid.org/0000-0002-3540-457X

¹⁾XW: [e-mail], NA

²⁾SC: [e-mail], NA

²⁾TH: [e-mail], NA

¹⁾ZW: [e-mail], NA

¹⁾YZ*: [e-mail], NA

Acknowledgments

Assistance with the study: The authors are grateful to all the laboratory staff for their excellent collaboration.

Data Availability Statements

Data and materials are available upon reasonable request. (Email: [e-mail])

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