Antimicrobial Effect of a Single Dose of Amoxicillin on the Oral Microbiota Cecilia Larsson Wexell, DDS, PhD;*† Henrik Ryberg, PhD;‡§ Wivi-Anne Sjöberg Andersson, DDS;¶ Susanne Blomqvist, BSc;** Pieter Colin, PhD;††‡‡ Jan Van Bocxlaer, PhD;†† Gunnar Dahlén, DDS PhD**

ABSTRACT Purpose: Amoxicillin is commonly used in oral surgery for antimicrobial prophylaxis against surgical-site infection and bacteremia because of its effect on oral streptococci. The aim of this study was to determine whether amoxicillin reaches the break-point concentrations in saliva and has any effect on the salivary microbiota, colonizing bacteria on mucosal membranes and on the gingival crevice after a single dose of amoxicillin. Material and Methods: Twenty subjects received 2 g of amoxicillin, per os. The facultative and strictly anaerobic microflora, as well as the streptococcal microflora specifically, were followed from baseline and after 1, 4, and 24 hours. Samples were taken for microbial analysis from saliva, the dorsum of the tongue, and the gingival crevice, and were inoculated and cultured. Plasma samples and saliva samples were analyzed for amoxicillin concentrations (free and protein bound) using liquid chromatography and mass-spectrometry. Results: Amoxicillin was detected in concentrations over the break-point (>2 μg/mL) of amoxicillin in plasma after 1 and 4 hours but not after 24 hours. The dose had a significant effect on the streptococci in the gingival crevice. Conclusion: A single dose given as prophylaxis to prevent a surgical-site infection results in a significant reducing effect on the oral streptococcal microflora in the gingival crevice and may have an impact on bacteria spreading into tissues and the bacteremia of streptococci. KEY WORDS: amoxicillin, LC-MS/MS, oral microbiology, oral surgery, plasma, saliva

*Senior consultant oral and maxillofacial surgeon, Department of Oral and Maxillofacial Surgery, Södra Älvsborg Hospital, Borås, Sweden; †Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; ‡Senior Chemist, Centre for Bone and Arthritis Research, Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; §Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden; ¶Consultant oral medicine and special care dentistry, Wivi-Anne Sjöberg-Andersson, Clinic of Oral Medicine and Special Care Dentistry, Östra Sjukhuset, Gothenburg, Sweden; **professor, Department of Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; ††Post-Doctoral researcher, Laboratory of Medical Biochemistry and Clinical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Belgium; ‡‡Associate Professor, Department of Anesthesiology, University Medical Center Groningen, University of Groningen, The Netherlands

INTRODUCTION Antimicrobial resistance is a severe global threat, and it is crucial to prevent the overprescription and overuse of antibiotics.1 In general, antibiotic treatment and prophylaxis are considered to be given unnecessarily in noncompromised patients within dentistry.2 After surgery, during the postoperative follow-up period, the prevention of postoperative complications and the risk of compromising surgical efforts may drive the decision to administer prophylactic antibiotics to healthy patients in connection with oral and dentoalveolar surgery. Kreutzer and colleagues made a systematic review of publications between 2000 and 2013 and eight reports of 532 fulfilled their requirements for further analyses when searching for the best evidence for or against prophylactic antibiotics.3 Evidence exists of the beneficial use of prophylactic antibiotics for surgical tooth extractions in healthy patients with a reduction in the risk of infection, pain, and dry socket. However,

Corresponding Author: Dr. Cecilia Larsson Wexell, Department of Oral and Maxillofacial Surgery, Södra Älvsborg Hospital, SE-501 82 Borås, Sweden; e-mail: [email protected] Conflict of Interest: The authors declare no conflict of interest. © 2015 Wiley Periodicals, Inc. DOI 10.1111/cid.12357

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prophylaxis can cause side effects that may do more harm than good with respect to antibiotic resistance.4 For dental implant treatment, it is reported that antibiotic administration, perioperatively, significantly reduces the rate of osseointegration failure during the early time period,5–7 but the incidence of postoperative infections is not affected.6,7 Different antimicrobial prophylactic protocols may be used when operating on patients with mild or severe systemic diseases, as well as on patients with ongoing infections and malignancies. Age, obesity, diabetes, altered immune response, chronic corticosteroid use, and treatment with antiresorptives and concomitant drugs may influence the protocol. Hoefert and Eufinger8 demonstrated that long-term preoperative antibiotic treatment could lead to complete healing in 70% to 87% of cases, after surgery, in contrast to 35% to 53% with a short-term regimen for patients suffering from osteonecrotic jaw as a side effect of antiresorptive treatment. In patients with cancer, the stage of malignancy may affect the risk of infection postoperatively. A clean-contaminated intervention is defined as a surgical procedure performed under controlled circumstances in a healthy but colonized body surface area such as the oral cavity. The oral surfaces are colonized by microorganisms belonging to the resident (“normal”) microbiota, and they are most likely to be responsible for most infections following clean-contaminated procedures.9 Antimicrobial prophylaxis (AMP) or antibiotic prophylaxis (AP) refers to the short administration of an antimicrobial agent orally or intravenously. A single dose is usually administered 30 to 60 minutes before the surgical incision is made through the mucosa. The clinical effect on oral streptococci was compared between a 3-day therapy and a 7-day course of orally administered amoxicillin after the extraction of an infected tooth.10 The oral microbiota was collected after 0, 9, and 30 days, and the results showed that the two courses had a similar clinical effect and a similar reducing effect on oral streptococci susceptible to amoxicillin. The microbial contamination of a surgical site precedes infection, and it has been demonstrated that the contamination of a surgical site with >105 microorganisms per gram of tissue markedly increases the risk of surgical-site infection.11 Microorganisms from the mucosa usually occur in the surgical wound during surgical procedures in the oral cavity. Postoperative infections after oropharyngeal surgery are usually poly-

microbial and involve both aerobic and anaerobic species, predominantly various streptococci. AMP or AP is believed to reduce the amount of bacteria at the surgical site and to prevent potential harmful bacteria penetrating and causing bacteremia. The extent to which a single dose of amoxicillin reaches the surgical site and influence the bacteria at that location is not known. The aim of the present study was to investigate the extent to which oral microbiota in general and oral streptococci specifically are influenced at the different oral sites such as saliva, mucosa (tongue) and the gingival crevice 1, 4, and 24 hours after administration per os of a 2 g single dose of amoxicillin. We also determined the amoxicillin concentrations in saliva and plasma at the same time points during the experimental period. MATERIALS AND METHODS Subjects and Experimental Design Twenty healthy volunteers (age range 25–70 years) with healthy oral conditions were identified and enrolled in the study. The study was performed at two different departments at two different occasions. In the first occasion, blood was also sampled. Ten subjects (eight women and two men) were recruited from among the staff at the Clinic of Oral Medicine and Special Care Dentistry, whereas 10 subjects (seven women and three men) were recruited from among the staff at the Department of Oral Microbiology. The study was approved by the Regional Ethical Review Board, Gothenburg (Dnr 57611), and all volunteers gave their informed consent to participate. The trial is registered under EudraCT 2014–003234-16. The use of alcohol and nicotine (smoking or snuff) was not permitted from the day before the study started until the end of the study in order to get the group as homogenous as possible. An allergy to penicillin, ongoing infection (any part of the body), or treatment with antibiotics during the last 6 weeks were regarded as exclusion criteria. In the first group of 10 subjects, venous blood and saliva were sampled for quantification of the amoxicillin concentration. For all 20 subjects, samples from saliva, the gingival crevice, and tongue were collected for microbiological analysis at baseline and 1, 4, and 24 hours after supervised administration of 2 g of amoxicillin (Amimox®, Meda AB, Solna, Sweden).

Antimicrobial Effect of Amoxicillin on Oral Streptococci

Biochemical Analyses of Amoxicillin and Assay Method Blood Sampling. Venous blood samples were collected by direct vein puncture from the median cubital vein into tubes (6 and 9 mL) with and without ethylenediaminetetraacetic acid (EDTA) (vacuette K2 EDTA and vacuette Z Serum Clot Activator) on each occasion (0, 1, 4, and 24 hours). The samples were centrifuged at 2,800g for 10 minutes (Hettich Rotina, Hettich Laboratory Technology, Tuttlingen, Germany) within 2 hours and immediately stored at −70°C (Thermo Scientific, AB Ninolab, Upplands Väsby, Sweden). Saliva Sampling. An amount of 1 to 2 mL of nonstimulated saliva was collected in a 5 mL tube and immediately stored in a −70°C freezer (Thermo Scientific, AB Ninolab) at baseline and 1, 4, and 24 hours post administration of 2 g amoxicillin. Reagents. Amoxicillin was purchased from SigmaAldrich (St. Louis, MO, USA) and 2H4-Amoxicillin was purchased from Toronto Research Chemicals (Ontario, Canada). High-performance liquid chromatography (HPLC)-grade water was prepared using a commercial Millipore Synergy 185system (Millipore, Billerica, MA, USA). All other reagents were of analytical grade. The Oasis MCX 96-well μ-elution plate came from Waters (Milford, MA, USA) and the RED dialysis 48-well device came from ThermoFisher Scientific (Waltham, MA, USA). Instrumentation. The Liquid chromatography–mass spectrometry (LC/MS) set-up consisted of an Acquity UPLC system (Waters, Milford, MA, USA), equipped with an Acquity HSS T3 column (50 mm × 2.1 mm, 1.7 mm particle size) and an Acquity BEH C18 guard column (5 mm × 2.1 mm, 1.7 mm particle size), all from Waters (Milford, MA, USA), and a Waters Quattro Ultima triple quadrupole system (Micromass Waters, Manchester, UK) equipped with an electrospray source (orthogonal Z-spray) operated in negative ionization (ESI-) mode. The column was kept at 40°C, and the injection volume was 1 μL. The mobile phases consisted of a 1 mM CH3COOH/CH3COONH4 buffer and acetonitrile was used as an organic solvent. Components were eluted using gradient elution at a flow rate of 0.6 mL/ minute. From 0 to 1 minute, the mobile phase contained 5% of acetonitrile, whereas from 1 to 2 minute, the

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amount of acetonitrile in the mobile phase was increased linearly to 25%. The MS instrument was operated with a capillary voltage of 3.5 kV, source block temperature of 150°C and cone voltage of 10 V. Nitrogen, 400°C, at 750 L/h was used as the desolvation gas and argon was used as the collision gas. The dwell time and interscan delays were set at 50 ms and 20 ms. Peak areas were integrated using MassLynx 4.1 software (Micromass Waters, Manchester, UK). Solid Phase Extraction Procedure. Frozen samples were allowed to thaw at room temperature and prior to analysis, the samples were centrifuged at 800 × g for 10 minutes at room temperature to precipitate any solid particles that were present. Oasis MCX μ-elution 96-well plates were conditioned and equilibrated using 200 μL of methanol and 200 μL of water respectively. The 50 μL aliquots of plasma samples and subsequently 50 μL of internal standard solution were added to an empty 96-well plate. Prior to homogenization by repeated aspiration with a multichannel pipette, 400 μL of a 2.5% (v/v) H3PO4 solution was added to the wells. Of this mixture, 200 μL was loaded onto the Oasis μ-elution plate. Two hundred microliters of a 2% (v/v) formic acid solution was used to wash the Oasis plate prior to elution with 30 μL of a 5% (v/v) NH4OH solution in acetonitrile/methanol (60:40). Finally, eluates were diluted with 90 μL of a 1 M CH3COOH/CH3COONH4 buffer. After capping the 96-well plate with a polypropylene cap mat, sample plates were placed in the autosampler at 5°C until injection. To determine the unbound amoxicillin concentration in plasma and saliva, 4-hours equilibrium dialysis, at 37°C, using the RED® device, was performed. The dialysis buffer used was a 52 mM HEPES buffer (supplemented with NaCl to guarantee isotonicity) at pH 7.4. Afterwards, dialysis samples were quantified against a calibration curve constructed in HEPES buffer. The unbound fraction was calculated as the concentration in the dialysate over the concentration in the matrix compartment of the RED® device. Mass Spectrometry. For amoxicillin multiple reaction monitoring (MRM) transition, 364.2 - > 222.6 was used as a quantifier and MRM transition 364.2 - > 319.9 was used as a qualifier. The collision energy was 6 eV. For the internal standard 2H4-amoxicillin, MRM transition, 368.3 - > 227.2 was used as a quantifier and the collision

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energy used was 8 eV. The calibration curve consisted of the following points; 0.43, 0.80, 2.13, 3.19, 6.38, 12.76, 25.52, and 51.05 μg/mL. The imprecision of the method is 8.2% intra-day Coefficient of variation (CV) and 2.5% inter-day CV for the 0.57 μg/mL QC level, 8.1% intra-day CV and 9.7% inter-day CV for the 5.74 μg/mL QC level and 9.2% intra-day CV and 5.4% inter-day CV for the 40.14 μg/mL QC level.

tative and anaerobic bacteria. Amoxicillin-tolerant bacteria were isolated and identified using Gramstaining, microscopic morphology, selective media, and API-Zym (Les Balmes de Grottes, Montalieu, France). The susceptibility (minimal inhibitory concentration (MIC) ) of amoxicillin was estimated for the isolates using an E-test (AB Biodisk, Solna, Sweden).

Microbiological Analyses

All analyses were assessed at individual level. Microbiological data were transformed into logarithms of the number of CFUs. A repeated measures analysis of variance (ANOVA) test was used to identify differences in the number of bacteria at four different time points (before as well as 1, 4, and 24 hours after the administration of 2 g per os of amoxicillin). In the event of the ANOVA providing statistically significant results at p < .05, post hoc tests were performed to make pairwise comparisons between bacteria and the different time points. The Bonferroni–Dunn test was used. According to the Bonferroni correction, the levels of significance of the test were 0.0125 (i.e., 0.05 divided by 4, the number of time points according to the study design). The PASW Statistics 21.0 statistical software package (SPSS Inc, Chicago, IL, USA) was used.

Microbiological Sampling. Three different samples were taken from each individual at each time point: 1) Unstimulated saliva was collected. One milliliter was transferred to a transport medium, VMGA II S.12 2) One sample was taken from the dorsum of the tongue by scraping firmly over an area of 1 cm2 of the surface with a cotton pellet. The yield was transferred to transport medium VMGAIII.12 3) The third sample was taken with paper points from the gingival crevice at the buccal site of teeth 36 and 46 and transferred to transport medium VMGAIII. Microbiological Processing. The samples were kept in room temperature and processed within 24 hours. They were serially diluted 10-fold in VMGA I.13 From each dilution, 0.1 mL was inoculated on the following media: one blood agar plate (BBL, 4.3%, containing 0.3% Bacto Agar (Difco Laboratories, Detroit, MI, USA), 5% defibrinated horse blood, 0.5% hemolyzed human red blood cells, and 5×10−5 mg/L menadione (Becton, Dickinson and Company, Sparks, MD, USA) incubated aerobically in 10% CO2 for 2 to 3 days and one Brucella blood agar (BBL) incubated anaerobically with the hydrogen combustion method for 5 to 7 days. Furthermore, one Mitis-Salivarius agar (Difco, Becton, Dickinson and Company) for the selective culture of streptococci was incubated in 10% CO2 for 2 to 3 days. In addition, one blood agar and one Brucella blood agar plate containing 10 μg/mL of amoxicillin (SigmaAldrich, Sweden AB, Stockholm, Sweden), corresponding to the mean concentration achieved in serum after one dose of 2 g amoxicillin, was incubated in parallel with the other plates. After incubation, the total colony-forming units (CFUs) were counted on plates containing 30 to 300 colonies and the total log counts of CFU were calculated for 1) facultative bacteria, 2) streptococci, 3) anaerobically cultured bacteria and 4) amoxicillin-tolerant facul-

Statistics

RESULTS Samples from all volunteers were collected at the four time points. The volunteers took their normal medicines, and the medications used by the participants were considered not interfering with amoxicillin. No adverse events were registered during the study. Biochemical Analyses There was a relatively large variation in the amoxicillin concentrations among the subjects. The mean plasma concentrations were 8.26 μg/mL (0.446–18.1) and 6.86 μg/mL (3.45–12.5) (median values 7.64 and 6.24) at 1 and 4 hours postadministration, respectively (Table 1). The unbound fraction was 70 1 8.5% for the 1-hour time point and 77 1 4.2% for the 4-hour time point (mean 1 standard error of the mean). At 24 hours postadministration, the amoxicillin concentrations were below the lower limit of quantification of our assay. Microbiological Analyses Table 2 shows the total number of facultative anaerobic bacteria, streptococci, anaerobically cultured bacteria,

Antimicrobial Effect of Amoxicillin on Oral Streptococci

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TABLE 1 Concentrations of Total Plasma Amoxicillin (μg/mL) and Free Plasma Amoxicillin (Ratio to Total Plasma) Patient

1 2 3 4 5 6 7 8 9 10

Baseline Levels

1 Hour Postadministration

4 Hours Post Administration

24 Hours Post Administration

0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

0.446/0 18.1/0.854 14.2/0.832 9.70/0.96 5.98/0.866 3.93/0.705 8.21/0.642 4.82/0.675 10.1/0.634 7.08/0.825

12.5/0.712 4.64/0.788 10.0/0.744 6.85/0.855 3.89/0.716 5.82/0.688 10.0/0.911 3.45/1.026 6.67/0.75 4.74/0.543

0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

Levels of total and free amoxicillin in plasma before and after the administration of 2 g of amoxicillin. The amount of amoxicillin in saliva was below the level of detection (0.43 μg/mL).

and amoxicillin-resistant anaerobes in samples from the tongue, saliva, and gingival crevice at baseline and 1, 4, and 24 hours after a single dose of 2 g of amoxicillin. All samples were predominated by the streptococci, which constituted the absolute majority of the facultative anaerobic bacteria and anaerobically cultured bacteria (Table 2). Tongue and saliva samples showed a similar microbiological pattern from baseline to 24 hours – with a marginal and nonsignificant decrease in facultative

anaerobic bacteria and streptococci, as well as anaerobically cultured bacteria, with the lowest counts noted after 4 hours. The reduction in percent of baseline was calculated to be between 20% to 35% after 24 hours. It should be noted that the total viable count on the aerobic plate exceeded the counts on the anaerobic plate and the number of strict anaerobes could not be calculated by a simple subtraction. By making a selective calculation based on colony morphology and excluding the

TABLE 2 Mean (1SD) Total Log Counts (CFU) of Various Bacterial Groups in Samples from the Tongue, Saliva, and Gingival Crevice at Baseline and 1, 4, and 24 Hours after a Single Dose of Amoxicillin2 in 20 Healthy Adult Subjects

Sampling Site

Tongue

Saliva

Crevicular fluid

*Significant difference.

Sampling Time

Total Log Counts of Aerobically Grown Bacteria Mean 1 SD

Total Log Counts of Streptococci Mean 1 SD

Total Log Counts of Amoxicillin-Tolerant (10 μg/mL) Anaerobic Bacteria Mean 1 SD

Total Log Counts of Anaerobically Grown Bacteria Mean 1 SD

Baseline 1 hours 4 hours 24 hours Baseline 1 hour 4 hours 24 hours Baseline 1 hour 4 hours 24 hours

5.96 1 0.45 5.67 1 0.47 5.23 1 0.90 5.59 1 0.43 6.06 1 0.50 6.06 1 0.62 5.67 1 0.67 5.89 1 0.56 5.24 1 0.67 5.04 1 0.96 4.95 1 0.85 4.49 1 0.80

5.61 1 0.62 5.57 1 0.46 5.13 1 0.63 5.66 1 0.70 5.89 1 0.64 5.91 1 0.56 5.32 1 0.78 5.58 1 0.83 5.04 1 0.94 5.00 1 0.79 4.53 1 1.06 4.19 1 1.00*

2.90 1 1.74 3.05 1 1.20 2.70 1 1.82 3.27 1 1.78 3.69 1 1.54 3.39 1 1.80 3.54 1 1.46 3.90 1 2.08 1.45 1 1.68 2.02 1 1.64 1.21 1 1.56 2.22 1 1.43

5.92 1 0.39 5.76 1 0.36 5.33 1 0.66 5.71 1 0.56 6.00 1 0.82 5.93 1 0.81 5.73 1 0.65 5.84 1 0.85 5.41 1 0.58 5.40 1 0.65 4.73 1 1.24 4.31 1 1.15*

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streptococci, an increase of >100% of strict anaerobes was noted during the experimental period. Amoxicillintolerant bacteria did not display any significant change in total log counts from baseline during 24 hours in saliva and on the tongue, although an average increase of around 200% was noticed for tongue and saliva samples. The gingival crevice samples showed a small yet not significant decrease during 24 hours for the total number of facultative anaerobic bacteria (Table 2), but a greater and statistically significant decrease was recorded for the total number of anaerobically cultured bacteria (p = .03), as well as for streptococci (p = .004), between baseline and 24 hours and between 1 hour and 24 hours (p = .002). The average percentage reduction between baseline and 24 hours was 71% for total facultative bacteria and 87% for streptococci. All individuals displayed the presence of a few (56 μg/mL). Large numbers of B. longum were found in five subjects and Capnocytophaga spp. in one subject, and this change may be of biological significance in these individuals, although both Bifidobacterium spp. and Capnocytophaga spp. are not considered pathogenic in oral infections.9 It is noteworthy, however, that, even after a single dose, antibiotics can select less susceptible strains to become more predominant in the gingival crevice microbiota. It is possible to speculate that this change in microbial homeostasis is biologically significant when amoxicillin is used for prophylaxis. Numerous studies have been conducted to investigate the effect of various systemic antibiotic regimens in

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the treatment of periodontitis, used solely or as an adjunct to mechanical debridement in order to reduce (“eliminate”) putative periodontal pathogens and prevent disease from further progression. The benefit of antibiotics has been controversial, and comparisons are extremely difficult to make because of numerous and variable treatment protocols. Few studies have investigated the immediate effect (within hours) on the gingival crevice microbiota and, to our knowledge, no study has determined the effect of a single dose. It is therefore important to conclude that there is a significant reduction in bacterial counts in the gingival crevice following a single dose of amoxicillin that lasts over 24 hours. Another important finding from this study is the very clear satellite phenomenon found in some subject samples. This illustrates that, in the complex microbial community, such as the subgingival microbiota, as well as the one on the dorsum of the tongue and saliva, resistant microorganisms also make it possible for more susceptible strains to survive in the neighborhood of the resistant strains. This is one of the mechanisms that make polymicrobial biofilms difficult therapeutic targets.23 Biofilms in general display an increased tolerance to antimicrobials due to the presence of extracellular matrix, poor penetration, increased level of mutations and quorum-sensing regulated mechanisms, and a substantial proportion of dormant cells.24 When it comes to the target bacteria for antibiotics in the oral cavity, regardless of whether they come through the salivary glands or through the gingival crevice, the influence appears to be limited to the oral microbiota that are present in biofilms on all oral surfaces. On the other hand, the effect on the streptococcal microflora that was noted when it was exposed to concentrations obtained in plasma, such as in the gingival exudate, indicates that a prophylactic dose may have some impact on the flora at a surgical site. One may speculate that because streptococci are probably the predominant bacteria, both at the surgical site and in bacteremia, that may follow a surgical incision, an effect might be achieved by a single dose of amoxicillin as prophylaxis prior to surgical interventions in the oral cavity. CONCLUSIONS A single dose given as prophylaxis to prevent a surgicalsite infection results in a significant reducing effect on the oral streptococcal flora in the gingival crevice but not in saliva or on the dorsum of the tongue. As

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streptococci constitute the absolute majority of microorganisms on mucosal membranes, in the gingival crevice and in saliva and the finding that efficient plasma concentrations are reached after 1 and 4 hours, the prophylactic administration of amoxicillin may be recommended for dentoalveolar surgery in patients with systemic diseases, e.g., cancer or autoimmune diseases and patients treated with, e.g., antiresorptives. ACKNOWLEDGMENTS This study was supported by the Local Research and Development Board for Gothenburg and Södra Bohuslän, the Swedish Dental Association, the Gothenburg Dental Association and the Local Research and Development Board for Södra Älvsborg. We are grateful to Georgios Charalampakis for the statistical calculations. REFERENCES 1. WHO. Antimicrobial resistance: global report on surveillance 2014. http://www.who.int/drugresistance/documents. 2. Llor C, Bjerrum L. Antimicrobial resistance: risk associated with antibiotic overuse and initiatives to reduce the problem. Ther Adv Drug Saf 2014; 5:229–241. 3. Kreutzer K, Storck K, Weitz J. Current evidence regarding prophylactic antibiotics in head and neck and maxillofacial surgery. Biomed Res Int. 2014; 2014:7. 4. Lodi G, Figini L, Sardella A, Carrassi A, Del Fabbro M, Furness S. Antibiotics to prevent complications following tooth extractions. Cochrane Database Syst Rev 2012; (11):CD003811. 5. Esposito M, Grusovin MG, Worthington HV. Interventions for replacing missing teeth: antibiotics at dental implant placement to prevent complications. Cochrane Database Syst Rev 2013; (7):CD004152. 6. Ata-Ali J, Ata-Ali F. Do antibiotics decrease implant failure and postoperative infections? A systematic review and metaanalysis. Int J Oral Maxillofac Surg 2014; 43:68–74. 7. Bafail AS, Alamri AM, Spivakovsky S. Effect of antibiotics on implant failure and postoperative infection. Evid Based Dent 2014; 15:58. 8. Hoefert S. Eufinger H. Relevance of a prolonged preoperative antibiotic regime in the treatment of bisphosphonaterelated osteonecrosis of the jaw. J Oral Maxillofac Surg 2011; 69:362–380. 9. Dahlen G. Bacterial infections of the oral mucosa. Periodontol 2000 2009; 49:13–38.

10. Chardin H, Yasukawa K, Nouacer N, et al. Reduced susceptibility to amoxicillin of oral streptococci following amoxicillin exposure. J Med Microbiol 2009; 58(Pt 8):1092– 1097. 11. Krizek TJ, Robson MC. Biology of surgical infection. Surg Clin North Am 1975; 55:1261–1267. 12. Dahlen G, Pipattanagovit P, Rosling B, Moller AJ. A comparison of two transport media for saliva and subgingival samples. Oral Microbiol Immunol 1993; 8:375–382. 13. Moller AJ. Microbiological examination of root canals and periapical tissues of human teeth. Methodological studies. Odontol Tidskr 1966; 74(Suppl 5):1–380. 14. Colin P, De Bock L, T’Jollyn H, Boussery K, Van Bocxlaer J. Development and validation of a fast and uniform approach to quantify beta-lactam antibiotics in human plasma by solid phase extraction-liquid chromatography-electrospraytandem mass spectrometry. Talanta 2013; 103:285–293. 15. Wen A, Hang T, Chen S, et al. Simultaneous determination of amoxicillin and ambroxol in human plasma by LC-MS/ MS: validation and application to pharmacokinetic study. J Pharm Biomed Anal 2008; 48:829–834. 16. Khuroo AH, Monif T, Verma PR, Gurule S. Simple, economical, and reproducible LC-MS method for the determination of amoxicillin in human plasma and its application to a pharmacokinetic study. J Chromatogr Sci 2008; 46:854– 861. 17. Goddard AF, Jessa MJ, Barrett DA, et al. Effect of omeprazole on the distribution of metronidazole, amoxicillin, and clarithromycin in human gastric juice. Gastroenterology 1996; 111:358–367. 18. Ragazzi E, Fille M, Miglioli PA. Saliva concentration of amoxicillin, erythromycin, and ciprofloxacin in outpatients: a comparison between the young and the elderly. J Chemother 2013; 25:126–128. 19. Ortiz RA, Calafatti SA, Corazzi A, et al. Amoxicillin and ampicillin are not transferred to gastric juice irrespective of Helicobacter pylori status or acid blockade by omeprazole. Aliment Pharmacol Ther 2002; 16:1163–1170. 20. Baglie S, Del Ruenis AP, Motta RH, et al. Plasma and salivary amoxicillin concentrations and effect against oral microorganisms. Int J Clin Pharmacol Ther 2007; 45:556–562. 21. Tenenbaum H, Jehl F, Gallion C, Dahan M. Amoxicillin and clavulanic acid concentrations in gingival crevicular fluid. J Clin Periodontol 1997; 24:804–807. 22. Walker CB. The acquisition of antibiotic resistance in the periodontal microflora. Periodontol 2000 1996; 10:79–88. 23. Socransky SS, Haffajee AD. Dental biofilms: difficult therapeutic targets. Periodontol 2000 2002; 28:12–55. 24. Hoiby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O. Antibiotic resistance of bacterial biofilms. Int J Antimicrob Agents 2010; 35:322–332.

Antimicrobial Effect of a Single Dose of Amoxicillin on the Oral Microbiota.

Amoxicillin is commonly used in oral surgery for antimicrobial prophylaxis against surgical-site infection and bacteremia because of its effect on ora...
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