Efficacy of Moraceae with chlorhexidine mouthwash on the microbial flora of critically ill intubated patients: a randomized controlled pilot study

Mouthwash preparation

For Moraceae mouthwash preparation, we used 0.02% Artocarpus lakoocha extract, 0.0005% CHX, and cremophor RH40 in the mouthwash base, which was prepared in the standard laboratory of the Faculty of Dentistry, Prince of Songkla University (Patent no. 9519). CHX mouthwash of 0.12% was prepared in the pharmaceutical unit of Songklanagarind hospital.

The two types of mouthwashes were straw-yellow colored and filled in non-identical, 10 mL bottles.

Oral care

Before oral hygiene intervention, The Modified BOAS was recorded, which ranged between 5 and 20, with 5–10 representing normal to mild dysfunction, 11–15 representing moderate dysfunction, and 16–20 representing severe dysfunction. The first oropharyngeal suction (T0) in the lower gingivobuccal sulcus to quantify bacterial inoculum was performed after randomization. Subsequently, tooth brushing (Colgate) with toothpaste (Colgate) was performed by bedside nurses. The mouth washing of the first group was performed with 10 mL of Moraceae with CHX solution every 8 h. The mouth washing of the second group was performed with 0.12% CHX solution alone every 8 h. After mouth washing, the second sample (T1) was collected by oropharyngeal suction. Oral care with intervention mouthwashes was performed for 7 days or until the patients were extubated. The third sample (T2) was collected on the morning of day 4 before the oral care procedure.

The samples were collected immediately after mouth washing to avoid a decrease in the mouthwash salivary concentration and bacterial colonization¹¹. Considering that the oral microflora changes to VAP pathogen at 48 to 72 h after hospitalization^5,17the samples were also collected on day 4. The Modified BOAS was recorded at 3–4 pm daily by the first author (PS). Patient satisfaction was surveyed at the end of the intervention in conscious patients.

Microbiological study

The saliva samples were transferred to 1.5 mL sterile microcentrifuge tubes and stored on ice for a few hours for transportation to the research laboratories. The saliva samples were immediately stored at − 80 °C until used for real-time PCR. Saliva samples were collected at baseline (T0), after mouthwash (T1), and day 4 (T2), and evaluated for Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae using real-time PCR.

Microbial evaluation using real-time PCR

The whole salivary samples were collected from individual subjects at baseline, immediately after using mouthwash, and 4 days after using mouthwash by having them spit into sterile plasticware. DNA was extracted from the salivary samples using a PureDirex Genomic DNA Isolation kit (Bio-Helix Co. ., LTD., Keelung City, Taiwan) following the manufacturer’s protocol for salivary bacteria, and bacterial DNA was stored at − 20 ºC until further use. The quantities of targeted bacteria in the saliva at baseline (T0), immediately after using mouthwash ( T1), and 4 days after using mouthwash (T2) were evaluated by performing real-time PCR. Total bacterial DNA (5 μL) was added to a Sensi-FAST™SYBR kit (Bioline Reagent Ltd., California, USA). The sequences of primers used were as follows: total bacteria (5′-TCCTACGGGAGGCAGCAGT-3′ and 5′-GGACTACCAGGGTATCTAATCCTGTT-3′)¹⁸, A. baumannii (5′-CATTATCACGGTAATTAGTG-3′ and 5′-AGAGCACTGTGCACTTAAG-3′)¹⁹, K. pneumoniae (5′-AGAGATTGGTTGACTGCAGGATTT-3′ and 5′-AAACATCAAGCCATATCCATTGG-3′)²⁰, P. aeruginosa (5′-CCTGACCATCCGTCGCCACAAC-3′ and 5′-CGCAGCAGGATGCCGACGCC-3′)^{twenty one}and S. aureus (5′-GAAATCGATGGTGACAGTAATAA-3′ and 5′- CTACGTCATTTGCACCYGATAA-3′)^{twenty two}. The PCR cycles consisted of an initial step of 10 min at 95 °C followed by 2 min at 50 °C and 40 cycles at 95 °C for 20 s with different annealing temperatures, including 60 °C for total bacteria and P. aeruginosa59 °C for K. pneumoniae57 °C for S. aureusand 52 °C for A. baumannii for 20 s. The polymerization temperature was 72 °C for 25 s. Amplification, detection, and data analysis were performed using the CFX96™ Real time system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each sample was run in duplicate and the number of targeted bacteria in the saliva samples was quantified using a standard curve.

The standard curve of each target bacteria has been previously reported^{twenty three}. The bacterial pellets of the target bacteria were collected after centrifugation at 3000 g for 5 min, washed twice with phosphate-buffered saline (pH 7.0), and adjusted to OD_600nm= 2.0 using a UV/VIS spectrophotometer (Biochrom Ltd., Cambridge, UK). The bacterial cell suspensions were two-fold diluted, which were then divided into two aliquots. To determine the bacterial number as colony-forming units (CFU)/ mL, the first aliquot was measured using the cultivation method. The second aliquot was used for DNA extraction, as mentioned above, to determine the quantification cycle (Cq) of real-time PCR using the CFX96 Touch™ Real-Time PCR detection system ( Bio-Rad Laboratories). A linear standard curve was plotted for each bacterial species from log CFU/mL against the corresponding Cq, showing a high correlation coefficient (R²> 0.99).

Study endpoints and data collection

The primary endpoint of the study was a reduction in oral bacterial total counts as indicated by CFU after mouthwash exposure. We used the following common pathogens: A. baumannii, K. pneumoniae, P. aeruginosaand S. aureus.

The secondary endpoints included oral health using the Modified BOAS, patients’ satisfaction, and VAP during admission. Patients’ satisfaction evaluation was performed according to the clinical assessment reported by Sarvizadeh et al.^{twenty four}. The conscious patients were asked whether they experienced pain or discomfort during and after mouth washing and graded as tolerable and intolerable. VAP diagnosis was based on the Centers for Disease Control and Prevention criteria²⁵.

Baseline data included age, gender, comorbidities, the reason for ICU admission, SOFA score, antibiotic use, mechanical ventilator days, and length of ICU stay. For adverse events related to intervention mouthwash, we monitor the signs of mucositis, gingivitis, or anaphylaxis .

All patients were followed up until discharge or death, whichever occurred first.

Statistical analysis

The primary outcome was a reduction in total CFU over time after mouth washing in each group. An a priori effect size was difficult to determine because of the lack of appropriately precise previous data on which the calculation was based. According to the report by Julious SA²⁶ the appropriate sample size for a pilot study is 12 participants per group. With a 20% dropout rate consideration, we planned to enroll a total of 30 patients. There was no planned interim analysis.

The study was analyzed on an intention-to-treat basis. No imputation was performed. The Shapiro–Wilk test was used to assess the normal distribution of continuous variables. Continuous data are expressed as the mean and standard deviation or median and interquartile range, depending on data distribution. Numbers and percentages were used to describe the categorical variables. The differences in patient characteristics and outcomes between the two groups were compared using the Wilcoxon-rank sum test, Fisher’s exact test, and χ²test, as appropriate.

Repeated-measures outcomes such as bacterial counts from patients’ saliva and Modified BOAS at different times were compared between the groups by the GEE with the population-average model. The working correlation matrix structure for GEE analysis was guided by the lowest quasi-likelihood under the independence model criterion (QIC). Finally, the interaction of the group with time was reported as the β-coefficient and 95% Wald confidence interval (CI).

A P A value of < 0.05 was considered statistically significant for all comparisons. All statistical analyses were performed using STATA version 16 (StataCorp, College Station, TX, USA) and SigmaPlot (Systat Software, San Jose, CA).