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

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Mouthwash preparation

For the 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 0.12% was prepared in the Pharmaceutical Unit of Songklanagarind Hospital.

Both types of mouthwash were straw-colored and filled in non-identical 10ml bottles.

Oral care

Prior to the oral hygiene intervention, the modified BOAS was recorded, which ranged from 5 to 20, with 5 to 10 representing normal to mild dysfunction, 11 to 15 representing moderate dysfunction, and 16 to 20 representing severe dysfunction . The first oropharyngeal aspiration (T0) in the lower gingivo-buccal sulcus to quantify the bacterial inoculum was performed after randomization. Thereafter, toothbrushing (Colgate) with toothpaste (Colgate) was performed by the bedside nurses. The mouthwash of the first group was performed with 10 mL of Moraceae solution with CHX every 8 h. The mouthwash 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 aspiration. Oral care with interventional mouthwashes was performed for 7 days or until patients were extubated. The third sample (T2) was collected on the morning of day 4 before the oral care procedure.

The samples were taken immediately after the mouthwash to avoid a decrease in the saliva concentration of the mouthwash and bacterial colonization11. Considering that the oral microflora transforms into VAP pathogen 48 to 72 h after hospitalization5.17, samples were also collected on day 4. The modified BOAS was recorded at 3-4 p.m. daily by the first author (PS). Patient satisfaction was surveyed at the end of the intervention in conscious patients.

Microbiological study

Saliva samples were transferred to sterile 1.5 ml microcentrifuge tubes and stored on ice for a few hours for transport to research laboratories. 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 on day 4 (T2), and assessed for Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae using real-time PCR.

Microbial evaluation by real-time PCR

Whole saliva samples were collected from individual subjects at baseline, immediately after mouthwash use, and 4 days after mouthwash use by spitting into sterile plastic utensils. DNA was extracted from saliva samples using a PureDirex Genomic DNA Isolation Kit (Bio-Helix Co., LTD., Keelung City, Taiwan) following the manufacturer’s protocol for saliva bacteria, and the Bacterial DNA was stored at -20 ºC until further use. The amounts of targeted bacteria in saliva at baseline (T0), immediately after mouthwash use (T1) and 4 days after mouthwash use (T2) were assessed by performing real-time PCR . Total bacterial DNA (5 μL) was added to a Sensi-FAST™ SYBR kit (Bioline Reagent Ltd., CA, USA). The primer sequences used were: total bacteria (5′-TCCTACGGGAGGCACGCAGT-3′ and 5′-GGACTACCAGGGTATCTAATCCTGTT-3′)18, A.baumannii (5′-CATTATCACGGTAATTAGTG-3′ and 5′-AGAGCACTGTGCACTTAAG-3′)19, K. pneumoniae (5′-AGAGTATTGGTTGACTGCAGGATTT-3′ and 5′- AAACATCAAGCCATATCCATTGG-3′)20, P. aeruginosa (5′-CCTGACCATCCGTCGCCACAAC-3′ and 5′-CGCAGCAGGATGCCGACGCC-3′)21and S. aureus (5′-GAAATCGATGGTGACAGTAAATAA-3′ and 5′-CTACGTTCATTTGCACCYGATAA-3′)22. 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 hybridization 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 sec. 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 analyzed in duplicate and the number of target bacteria in the saliva samples was quantified using a standard curve.

The standard curve of each target bacterium has already been reported23. 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 OD600nm= 2.0 using a UV/VIS spectrophotometer (Biochrom Ltd., Cambridge, UK). Bacterial cell suspensions were diluted twice, which were then divided into two aliquots. To determine the number of bacteria in colony forming units (CFU)/mL, the first aliquot was measured using the culture 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 the log CFU/mL against the corresponding Cq, showing a high correlation coefficient (R2> 0.99).

Evaluation criteria and data collection

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

Secondary endpoints included oral health using the modified BOAS, patient satisfaction, and VAP at admission. The evaluation of patient satisfaction was carried out according to the clinical evaluation reported by Sarvizadeh et al.24. Conscious patients were asked whether they experienced pain or discomfort during and after mouthwashing and were classified as tolerable and intolerable. The diagnosis of VAP was based on the criteria of the Centers for Disease Control and Prevention25.

Baseline data included age, gender, comorbidities, reason for ICU admission, SOFA score, antibiotic use, mechanical ventilation days, and length of ICU stay. For adverse events related to interventional rinse, we monitored for signs of mucositis, gingivitis, or anaphylaxis.

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

statistical analyzes

The primary outcome was a reduction in total CFU over time after mouthwashing in each group. An a priori effect size was difficult to determine due to the lack of sufficiently precise prior data on which the calculation was based. According to Julious SA report26 , the appropriate sample size for a pilot study is 12 participants per group. With a dropout rate of 20%, we planned to enroll a total of 30 patients. No interim analysis was planned.

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

The results of repeated measures such as the number of bacteria in the patients’ saliva and the modified BOAS at different time points were compared between the groups by the GEE with the population mean model. The structure of the working correlation matrix for the GEE analysis was guided by the lowest quasi-likelihood under the independence model criterion (QIC). Finally, group interaction over time was reported as the β coefficient and Wald’s 95% confidence interval (CI).

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