Impact of tooth brushing on oral bacteriota and health care-associated infections among ventilated COVID-19 patients: an intervention study

Study design and participants

Recruitment for this prospective cohort study took place in the University Hospital in Krakow, Poland. Patients were offered the opportunity to participate in the study and asked for the signed consent form on admission to the hospital. During hospitalization, 56 of them qualified for intubation and were hospitalized in the temporary intensive care unit (ICU) for COVID-19 patients between 1st September and 31st January 2022.

The inclusion criteria were as follows:

1.

SARS-CoV-2 infection confirmed by real-time reverse transcriptase-polymerase chain reaction (RT‒PCR) assay of nasal and pharyngeal swabs upon hospital admission

2.

Signed consent to participate in the study

3.

Patients admitted to the ICU

4.

Intubation due to COVID-19-related pneumonia and acute respiratory distress syndrome (ARDS) within 36 h preceding study procedures

All medical procedures in the ICU were performed according to the local standards. To prevent leakage of fluids below the laryngeal/tracheal cuff, VBM medical manometers 21 [21] were used. The cuff pressure was measured by the nursing staff 3 times/day, with a normal range between 40 and 60 mmHg.

Demographic and clinical data were gathered from the hospital electronic medical records, including but not limited to age, sex, date of COVID-19 diagnosis, admission to the hospital and ICU, date of intubation, selected comorbidities, pre- and postintubation treatment, including systemic steroids and antibiotics, days of antibiotic treatment (DOT) before and after intubation (the number of Days a patient receives an antibiotic). Selected baseline and maximal laboratory results were also extracted.

Oral health assessment

On admission to the ICU, the patients’ oral health was assessed utilizing the modified Beck Oral Assessment Score (BOAS), consisting of 5 subscales: assessment of lips, mucosa and gingiva, tongue, teeth, and saliva. A higher score reflects dysfunction or tissue injury. BOAS scores range from 5 (no oral dysfunction) to 20 (severe dysfunction). A score greater than 5 is abnormal [22, 23] (Additional file 1: Appendix A1).

Oral cavity sampling methods

Samples were taken two times, first within 36 h of intubation (baseline) and again after 7 days of intubation (follow-up).

In each sampling, four oral habitats were sampled: buccal mucosa, tongue, buccal dental surface and gingival pocket. Every sample was taken by a trained dentist.

Sampling the posterior part of the dorsum of the tongue and buccal mucosa, we used ESwab™. ESwab combines a COPAN-invented flocked swab with 1 mL of liquid amine in a plastic, screw cap tube. Dental plaque was collected from the buccal dental surface side using Tooth Cleanic KerrHawe—KWX-OP-SZ-011, and after collection, the brush was placed in 1 mL of Liquid Amies in a plastic screw cap tube. Three pieces of PerioPaper Strips, designed to absorb or carry 0–1.2 µl of fluid, were used to collect gingival cerficular fluid (GCF) samples. The strips were placed in the gingival pocket for 30–45 s until its surface soaked up. To minimize the risk of preanalytical errors during sample collection, sterile gauze was used to remove excess saliva from the mucosae and dry the dental surfaces, preventing salivary contamination of GCF.

Intervention

Patients were divided into 2 groups depending on the oral care procedure:

1.

Standard oral procedure (SP, cleaning and moisturizing of oral cavity, suction of excess fluid)

2.

Extended oral procedure with tooth brushing (EP, cleaning and moisturizing of the oral cavity, tooth brushing, suction of excess fluid)

The procedures were performed twice daily in each patient. Detailed information on these procedures is included in Additional file 1: Appendix A2. The allocation protocol was based on the patients’ location in the ward. Each room was assigned one type of procedure (standard—even number or extended—uneven number of rooms) that was permanent during the study period. The efficacy of two different procedures for oral care in an ICU setting was assessed.

Microbiological cultures

After the collection of patient samples, they were delivered as soon as possible to the microbiological laboratory for the purpose of microorganism isolation. Banking and identification of bacterial strains from HAIs of all hospitalized patients (samples were collected throughout the duration of the whole hospitalization, not only during the intervention) in the temporary units for COVID-10 patients in the UH were performed in the Department of Microbiology, UH, Krakow. Various diagnostic samples were collected, cultured and assessed for the microbial etiology of infections. In this group, 11 K. pneumoniae strains from HAIs of patients under surveillance (with SP or EP) were used for further analysis: 5 from PNU, 3 from BSI and 3 from UTI cases. One strain from an oral sample of patient 6 was not stored. Strains from oral samples and HAIs were stored in the Department of Microbiology, UH, Krakow, at − 70 °C using Microbank® (Biomaxima, Lublin). Dilutions of samples (from − 1 to − 6) were inoculated on the following media: McConkey (Graso, Biotech), Columbia (Lab-Agar, Biomaxima), Scheadler (Scheadler-Agra, Biomaxima), Bile Esculine Azide (Lab-Agar, Biomaxima), and MRS Agar (Oxoid). Simultaneously, swab samples were inoculated with the qualitative culture method using the same media as in the dilution method. Media were aerobically incubated at 37 °C for 24 h (McConkey, Columbia, Bile Esculine Azide) or anaerobically at 37 °C for 48 h (M.R. S and Scheadler, GENbag Atmosphere Generators, BioMérieux, France). Next, the colonies were counted and phenotypically reported, and the results are presented as CFU/mL (colony forming unit). The microorganisms were identified using MALDI TOF MS mass spectrometry (Vitek MS Home bioMérieux).

Laboratory-confirmed health care-associated infections

The bacterial health care-associated infection (HAI) cases were analyzed retrospectively using definitions from the Healthcare-Associated Infections Surveillance Network (HAI-Net) [24], including bloodstream infections (BSI), pneumonias (PNU), urinary tract infections (UTI) and others. The cases and samples were obtained via passive surveillance defined as identifying and reporting of HAIs by nontrained personnel. In 2020–2022, no active, prospective surveillance and registration of HAIs was carried out in the hospital; therefore, there was no other source of information on HAIs that had not been microbiologically confirmed. LOS was defined as the sum of the days of stay (patient days, pds), including the day of admission and the day of discharge.

Microbiological samples were routinely acquired when there was suspicion of infection by taking blood, bronchoalveolar lavage (BAL) or urine samples. Only laboratory-confirmed cases based on culture growth qualified for the analysis; with multiple samples from the same infection case, only the first isolate from each patient was selected for microbiological analysis, with subsequent patient and HAI cultures excluded.

Multiple logistic regression models were built to identify predictors of HAI of A. baumannii, Enterococcus faecalis and Klebsiella. pneumoniae etiology (3 etiology-specific models) that included the type of oral care procedure applied, identification of A. baumannii/E. faecalis/K. pneumoniae in the baseline oral sampling, baseline BOAS score category and pre-ICU antibiotic use.

Pulsed-field gel electrophoresis (PFGE)

All isolates of Klebsiella pneumoniae were analyzed using the standardized PFGE protocol developed at the Centers for Disease Control and Prevention by the PulseNet program (version for Escherichia coli). Data from previous studies showed that K. pneumoniae strains isolated in Polish hospitals (mainly from ICUs for adults and neonates and pediatric units) often belong to one clone. For this reason, a PFGE study was performed for this species to check for clonal spreading of strains coming from oral bacteriota samples and HAI cases. Genomic DNA was prepared in situ in agarose blocks and was subsequently digested with restriction enzymes: XbaI (25 U per block, Thermo Scientific. The digested products were separated on a CHEF III PFGE system (Bio-Rad, Warsaw, Poland) in 0.5 × Tris-borate-EDTA buffer at 14 °C at 6 V for 22 h with a starting pulse of 2 s and a final pulse of 35 s. GelCompar (Applied Maths, Kortrijk, Belgium) was used for cluster analysis with the unweighted pair group method with an arithmetic mean and the Dice coefficient. Similarity must be > 90% for the pattern to be considered to belong to the same pulsotype. The results of the PFGE analysis are presented for each patient with a number that they were assigned on recruitment (1–56). The term “case” refers to HAI diagnoses.

Ethics statement

The study and its protocol were approved by the Jagiellonian University Bioethics Committee, decision number 1072.6120.333.2020; Dec 7, 2020, and 1072.6120.353.2020. Dec 16, 2020. Written informed consent was obtained from each subject prior to participation.

Statistical analysis

The PS Imago Pro ver. 7.0 was used for all statistical analyses. The normality of the continuous variable distribution was assessed using the Shapiro‒Wilk test. Differences between groups were analyzed with Student’s t test or nonparametric test (Mann–Whitney U test) when appropriate. Paired data were analyzed using the Wilcoxon test. Continuous variables are presented as the arithmetic mean (x̄) ± standard deviation (SD) or as the median with interquartile range (IQR) when the data were not normally distributed. The distribution of categorical variables was described as counts and percentages. Statistical testing was completed to compare categorical variables using an independent sample chi-squared test or Fisher's exact test when appropriate and dependent samples with McNemar’s test. We built a multivariate logistic regression model including an arbitrarily chosen number of predictors. We assured that the multicollinearity assumption was fulfilled. Negelkerke’s index was used as the coefficient of determination, R2. We measured the strength association by the odds ratio (OR) and 95% confidence intervals (CI). Statistical inference was set at p < 0.05.

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