Changes in Resistome After Dental Extraction and Amoxicillin.

Trial Design This is a prospective, longitudinal, observational, cohort study investigating young adults undergoing surgical extraction of a wisdom tooth with and without antibiotics post-operatively. The prescription of antibiotics will be based on clinical judgment based on case complexity, trauma and length of procedure. No randomization will be performed. Four observational time points will be undertaken for collecting 3 bacterial samples (tongue coating, saliva and stool sample at: baseline, 1 week, 1 month and 6 months.

To survey the extent and persistence of a range of bacterial resistance genes within the oral and gut microbiota of a cohort of systemically healthy young adults before and after the surgical extraction of third molars who receive and do not receive amoxicillin.

Hypothesis It is anticipated that a single course of amoxicillin (250 mg three times daily for five days) prescribed after a surgical tooth extraction, such as an impacted third molar, alters the short-term and long-term antibiotic resistance profile and causes a significant shift in the composition of the oral and gut resistome in a healthy cohort.

From analysis of the bacterial resistance, comparison from the baseline data over time will give insight on the impact of increased antibacterial resistance. Understanding of this should help pave the way for determining guidelines for the rational prescription of antibiotics for dental surgical clinical procedures and so form a basis for antibiotic stewardship as well as determine a potential risk to benefit awareness when prescribing antibiotics.

This baseline data can be followed-up in a longer timeframe to examine the persistence of resistance genes over time in a later study. It is expected a significant number of patients will be from student cohorts at HKU which will make later recall straightforward. Patients will be asked to keep a record log of antibiotic consumption after the initial study that may inform later research.

Subjects scheduled for surgical extractions who give a written consent to be a part of the study will be recruited. An experienced clinical operator with specialist maxillofacial surgery training (ML Co-A) will be responsible for performing clinical treatment. Prescription of antibiotics will be based on case complexity, difficulty of the operative surgical procedure and time taken. Therefore patients will not be randomized but based on obtaining 10 patients in each group. Three sets of samples (Saliva, tongue dorsum scrapings and stool samples) will be collected at the following four time points.

The subjects will be asked to refrain from brushing their teeth the night before sample collection is to be performed and similarly asked not to use dental mouthwashes. Apart from collection time points, subjects will be asked to maintain their 'normal', routine oral hygiene procedures for the duration of the study.

After collection of the baseline samples the surgical extraction of the third molar tooth will be performed. The subject will be given post extraction instructions and analgesics for pain relief for all the subjects and Amoxicillin for those requiring antibiotics. Compliance will be checked for by asking the patient and checking the tablet wrapper strips of amoxicillin at the recall appointments. The subject will be asked to refrain from using any mouth rinse during the six month study period (if they did not already do so) and a toothpaste without antibacterial content will be provided for use.

Tongue coating, saliva and stool samples for DNA extraction will be collected at all the specified time points as described below.

Baseline- antibiotic group On the day of surgery Baseline- no antibiotic On the day of surgery

1 week

1 week

1 month 6 months

Samples from the oral cavity will be taken in the morning at least two hours after meals. Subjects will be advised to collect the stool samples within 24 hours of the scheduled appointment.

A sterile micro brush will be used to collect the tongue coatings The tongue coating sample will be taken after stroking the surface of the tongue for three times from the back to the front side on one half of the anterior two-thirds of the tongue for each time point of sampling. After the sample collection, the tip of the micro brush will be cut and placed inside an Eppendorf vial. The samples will be stored at -800C until DNA extraction is performed.

For the saliva samples, the subject will be asked to spit out into a sterile container which will be immediately transferred into ice and then stored at - 800C. Collection of the stool samples will be performed based on the guidelines for stool sample collection given for the human microbiome project (16). DNA extraction will be performed from the samples using the DNA extraction kit provided by (Qiagen, USA).

Metagenomic shotgun sequencing For metagenomic sequencing extracted genomic DNA will be physically sheared to an average fragment size of 250 bp. The fragmented gDNA will be sequenced bi-directionally (100 bp each direction) on a lane of an Illumina HighSeq. Quality filtering will be performed with Trimmomatic (17), after which the paired and unpaired forward reads will be combined and reads with more than 10% ambiguous bases will be removed. Next, the reads will be screened for human sequences with Best Match Tagger v3.101 (18), after which human reads and duplicate reads will be removed.

Relative abundance of resistant genes The blaTEM primer used will be as described by Kim et al (22). Primers to analyze the presence of erm (G), erm (B) and erm (F) resistance genes will be as described by Jansonn et al (23). Primers for Tet (M) and Tet (W) genes will as described by Diaz-Torez et al (24). 16S universal primers will be used for the relative quantification as a standard.

12 The relative gene abundance will be assessed using real time PCR. Conventional PCR reactions will be used to generate gene-specific amplicons used as template DNA standards for qPCR. Amplicons will be purified using a gel/PCR extraction kit QIAquick PCR Purification Kit (Qiagen, USA) and quantified fluorometrically using a Quant-iTTM PicoGreen® dsDNA Assay Kit (Invitrogen). The number of DNA molecules will be calculated based on the size and mass of the amplicons. Tenfold serial dilutions of the amplicons will be prepared and used following the procedure by Yu et al. (25).

Selection and Withdrawal of Subjects Patients will be recruited from the waiting list from the Prince Philip Dental Hospital, The University of Hong Kong who have been clinically screened and wish to have their impacted wisdom teeth removed.

Sample size We would present the outcome in log 10 scale compared to day 0 in community DNA extracted from fecal samples. From the previous study (15), we anticipate the standard deviation as 1.25 units. By the sample size determination with the use of software G*Power version 3.1.9.2 (Franz Faul, Universitat Kiel, Germany), we would need at least 8 per group to have a power of 0.8 to detect the 1.25 unit difference at the 0.05 significance level. Therefore, in this research, we collect total sample size as 16 (8 per group) in the initial stage.

Treatment of Subjects This is a prospective, longitudinal, observational, cohort study investigating young adults undergoing surgical extraction of a wisdom tooth with and without antibiotics post-operatively. Antibiotics and analgesics will be given post- operatively.

Amoxicillin Intra orally, three times daily 250 mg for 5 days Analgesics - Arcoxia 120mg for three days and Paracetamol 500 mg QID PRN for 3 days or Paracetamol 1 g Plus 16 mg Codeine Phosphate. The prescription of antibiotics will be based on clinical judgment based on case complexity, trauma and length of procedure. No 14 randomization will be performed. Four observational time points will be undertaken for collecting 3 bacterial samples (tongue coating, mouth rinse and stool sample at: baseline, 1 week, 1 month and 6 months.

If the patients have to take additional antibiotics in the review period of six months, they will be asked to inform the investigator.

Compliance will be checked for by asking the patient and checking the tablet wrapper strips of amoxicillin at the recall appointments.

Assessment of Efficacy The study is not looking into the efficacy of a new drug. The treatment procedures being carried out are according to the conventional treatment protocol.

Assessment of Safety The study is not looking to the safety of a new drug. The treatment procedures being carried out are according to the conventional treatment protocol.