Objectives: Peri-implantitis is a destructive disease on dental implants where an inflammatory lesion induces bleeding on probing of peri-implant soft tissues, increased peri-implant probing depths, and progressive resorption of surrounding crestal alveolar bone. Treatment of peri-implantitis is controversial, with no universally accepted treatment protocol presently established. Systemic antibiotic therapy has been employed as an adjunct to mechanical implant surface debridement of peri-implantitis lesions, with conflicting outcomes reported with various antibiotics. However, recent clinical trials have found systemic administration of either azithromycin or metronidazole as an adjunct to non-surgical peri-implantitis treatment provided statistically significant additional improvements in peri-implant clinical status than placebo-treated peri-implantitis patients receiving mechanical implant debridement alone. These clinical trial findings suggest potential value of both systemic azithromycin and metronidazole in human peri- implantitis treatment.However, clinical use of azithromycin or metronidazole may be complicated by the presence of pathogenic bacteria in peri-implantitis lesions which are resistant to the antibiotics. Previous studies have reported that peri-implantitis patients in the United States frequently harbor submucosal bacteria resistant to a number of antibiotics commonly prescribed in clinical dental practice, with microbial resistance to metronidazole among major putative bacteria pathogens detected in more than one in five peri-implantitis patients. To date, no data are available on the prevalence of azithromycin resistance in peri-implantitis bacterial populations. Thus, it is not known whether antibiotic resistance of major putative bacterial pathogens in peri-implantitis is greater to metronidazole or to azithromycin. As a result, the purpose of the present study was to determine the prevalence of in vitro resistance of selected bacteria from human peri- implantitis lesions to azithromycin as compared to metronidazole. Methods: Selected data on 65 consecutive peri-implantitis-affected adult patients with in vitro antibiotic testing on their submucosal biofilm samples with azithromycin at a concentration of 4 mg/L, and metronidazole at a concentration of 16 mg/L, were extracted from pre-existing 2021-2022 archived records in the Oral Microbiology Testing Service (OMTS) Laboratory at Temple University School of Dentistry in Philadelphia, Pennsylvania. Qualifying inclusion criteria for the study patient records were 1.) the patient’s submucosal biofilm specimen was culture-positive with one or more of the following bacteria: Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia/nigrescens, Parvimonas micra, Fusobacterium nucleatum, Streptococcus constellatus, Campylobacter rectus, Streptococcus intermedius, or gram-negative enteric rods, 2.) the patient was aged between 35-88 years, 3.) the patient was diagnosed by a periodontist as having peri-implantitis, 4.) the patient’s birth year and gender was reported, and 5.) the total submucosal anaerobic viable count per patient, the total submucosal percent cultivable of P. gingivalis, T. forsythia, P. intermedia/ nigrescens, P. micra, F. nucleatum, S. constellatus, C. rectus, S. intermedius, or gram-negative enteric rods, if present, and whether or not the isolates were resistant in vitro to breakpoint concentrations of either azithromycin (4 mg/L) or metronidazole (16 mg/L), were reported. When a qualifying patient record was identified, the above data were extracted and directly entered into a Microsoft Excel spreadsheet contained in a password-protected and encrypted computer file. Descriptive analysis tabulated de-identified patient data on age, gender, and smoking status, mean total submucosal anaerobic viable count per patient, the prevalence and proportional cultivable recovery of the test bacteria in patients, and the prevalence and subgingival proportions of azithromycin-resistant and metronidazole-resistant test species in patients. Fisher’s exact test evaluated among patients the presence one or more azithromycin-resistant test bacteria as compared to metronidazole-resistant test species. A paired t-test examined mean total cultivable submucosal proportions of the test bacteria per patient resistant in vitro to azithromycin as compared to mean total proportions per patient resistant to metronidazole. Logistic regression statistical modeling was used to assess the relationship between peri- implantitis patients with and without azithromycin-resistant or metronidazole-resistant test submucosal species and patient age of 65 years or older, patient gender, and patient current smoking status. A 2-tailed P-value of ≤ 0.05 was required for all tests of statistical significance. Results: The 65 peri-implantitis patients averaged 64.5 years in age, with females more frequent (52.3%) and only 10.8% with a current smoking habit. All of the peri- implantitis patients yielded one or more of the evaluated putative bacterial pathogens from submucosal biofilm samples, with P. micra, F. nucleatum and P. intermedia/ nigrescens the most frequently isolated test species. No azithromycin-resistant or metronidazole-resistant test bacterial species were found in 20 (30.8%) of the peri- implantitis study subjects. A total of 43 (66.2%) of the 65 peri-implantitis patients harbored azithromycin-resistant test species in submucosal biofilm samples, which was significantly greater than 8 (12.3%) peri-implantitis patients with metronidazole-resistant submucosal species (5.4-fold difference, P < 0.001, Fisher’s exact test). P. micra, S. constellatus, P. intermedia/nigrescens, T. forsythia, and F. nucleatum were test species most frequently resistant to 4 mg/L of azithromycin, whereas a subset of S. intermedius, F. nucleatum and S. constellatus were the only test species resistant to 16 mg/L of metronidazole. Total submucosal proportions of test bacterial species resistant to azithromycin averaged 8.8% per patient, as compared to only 1.4% resistant to metronidazole (P < 0.001, paired t-test). No statistically significant relationships were found between peri-implantitis patients with and without one or more azithromycin- resistant or metronidazole-resistant test species and older patient age (≥ 65 years), gender, or current smoking habit (all P-values > 0.05). Conclusions: Azithromycin in vitro antibiotic resistance was frequently found among predominant cultivable bacteria in human peri-implantitis lesions at levels more than 5-fold more frequent, and at significantly higher total cultivable proportions in submucosal biofilms, than is resistance to metronidazole. The high prevalence of azithromycin microbial resistance in peri-implantitis patients highlights the potential risk of therapeutic failure with empiric prescription of azithromycin in peri-implantitis therapy without guidance from antibiotic resistance testing of putative submucosal bacterial pathogens. / Oral Biology

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The global increase in livestock and poultry production observed in the last decades has led to an increase in animal waste generated. The animal waste contains high levels of nitrogen and may carry antibiotics that can disturb important microbial activities such as denitrification in terrestrial and aquatic ecosystems. Disturbances of microbial denitrification can have detrimental consequences to environmental health. In the terrestrial environment, denitrification is an important source and sink of N2O, a potent greenhouse gas in the atmosphere. In aquatic ecosystems, denitrification is a dominant NO3- removal pathway, contributing to prevent eutrophication. The overall goal of this dissertation is to evaluate the impacts of animal waste and potential antibiotic exposure on microbial communities responsible for denitrification in terrestrial and aquatic ecosystems. To achieve this goal, a combined approach of measuring activity rates and performing a molecular characterization of the microbial communities was used. In Chapter 2, the microbial community changes associated with the impacts of acute antibiotic exposure on denitrification were evaluated in soil microcosms. Antibiotic exposure caused a significant increase in N2O production from denitrification. This increase was paralleled with a greater ratio of fungi:bacteria abundance and lower abundances of particular taxa with N2O reduction capacity. In Chapter 3, the impacts of animal manure and antibiotic contamination on N2O fluxes and the abundance of denitrification genes were investigated in soil mesocosms. N2O fluxes in soils treated with manure fertilizer and tetracycline were considerably higher than in control soils. The manure fertilization and antibiotic exposure had diverse effects on different bacterial taxa responsible for N2O production. In Chapter 4, the denitrification activity and microbial community structure in tidal creek sediments impacted by wastewater discharge from a poultry processing plant were evaluated through a field survey and a microcosm experiment. Denitrification rates were inhibited in the location affected by the wastewater discharge. This decrease in denitrification activity was associated with changes in the microbial community structure, such as a lower relative abundance of bacterial taxa carrying denitrification genes and lower abundance of N2O reducing bacteria. In Chapter 5, the abundance and diversity of antibiotic resistance genes were evaluated in a tidal creek impacted by wastewater discharge from a poultry processing plant. The numbers of antibiotic resistance genes were higher in the location closer to the wastewater discharge, suggesting an historic antibiotic exposure associated with the activity of the poultry processing plant. Overall, this work provides new knowledge of the impacts of animal waste and antibiotics on N2O emissions in terrestrial ecosystems and microbial NO3- removal in aquatic ecosystems. This dissertation emphasizes the functional importance of microbial communities to ecosystem health and their responses to anthropogenic disturbance.