No description available.

No description available.

Prevotella intermedia/nigrescens group bacteria are Gram-negative, non-motile, anaerobic rods abundant in the subgingival microbiome of human periodontitis patients, and relatively sparse in persons with periodontal health. P. intermedia/nigrescens may be inadequately suppressed in periodontal pockets with conventional mechanical-surgical forms of periodontal therapy. Therefore, short-term systemic antibiotic therapy is often recommended in the treatment of recalcitrant (refractory) severe periodontitis patients where high numbers of P. intermedia/nigrescens persist in the subgingival microbiota. Limited available data suggests that the antibiotic sensitivity profile of periodontal P. intermedia/nigrescens has changed over time among severe periodontitis patients in the United States, with increasing levels of antibiotic resistance reported. These findings have potentially important clinical implications for dental professionals and their severe periodontitis patients which need further confirmation and clarification. To further expand knowledge on this clinically relevant issue, the purpose of the present study was to determine and compare over a 10-year period (2011 versus 2021) the prevalence of in vitro resistance of periodontal P. intermedia/nigrescens to the antibiotics amoxicillin, metronidazole, clindamycin, and doxycycline. / Oral Biology

Control of antibiotic resistance in bacteria is based on the concept that resistance incurs a fitness cost in non-selective conditions. Fitness costs were assessed for low- and high-level mupirocin resistance in locally-derived Staphylococcus aureus and S. epidermidis. Costs of resistance were assessed in pure cultures by comparing growth curve characteristics and in mixed culture as the proportion of resistant cells surviving. Costs were not present in comparisons of growth rates among groups of naturally-occurring isolates from the different resistance categories. However, in S. aureus, growth rates within resistance categories differed by approximately 30 – 90%. Among near-isogenic pairs of strains, fitness costs ≥10% were present in three of eleven pairs under pure culture and in six of eleven pairs under competition in mixed culture. Differences in intrinsic growth rates could easily mask fitness costs of the magnitudes observed. Thus, clinical outcomes also depend on whether there is a mixed infection and if so, on the growth rates of strains present.

An alarming problem has plagued both human and veterinary healthcare for decades: the ever-increasing presence of antibiotic resistant bacteria. Specifically, Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-resistant Staphylococcus pseudintermedius (MRSP) are of great concern. A new compound, aptly named, LavenGel has recently been developed and demonstrates treatment potential to effectively inhibit the growth of at least two Staphylococcus species: S. aureus and S. pseudintermedius. LavenGel has been previously shown in our lab to inhibit a variety of microbes, particularly S. aureus. However, the molecular pathway that LavenGel utilizes to inhibit S. aureus and whether this inhibition could be translated to other Staphylococcus species, particularly in animals, has yet to be investigated. The major aims of this study are to demonstrate and quantify the efficacy of LavenGel in preventing S. aureus and S. pseudintermedius growth and to understand the specific S. aureus cellular mechanisms that LavenGel impacts. In order to quantitatively represent the effectiveness of LavenGel for veterinary purposes, biofilms of S. pseudintermedius were treated at different phases of development. LavenGel inhibited both the attachment of cells to form biofilms, as well as the eradication of pre-existing biofilms. Minimum inhibitory concentrations and minimum bacterial concentrations were determind for S. pseudintermedius. To better understand the impact that LavenGel may have in human healthcare, a panel of genes expressed under LavenGel treatment were examined. LavenGel does not induce the typical SOS response in S. aureus that is seen when using other leading bactericidal treatments for Staphylococcus infections which have also been shown to induce resistance. LavenGel could potentially help solve the bacterial resistance issue by working against the bacterial cell membrane instead of inducing the typical SOS response. The threat of antibiotic resistant bacteria is a constant concern in the scientific and healthcare community. The implications of this study dictate LavenGel is a highly effective, all-natural, unique option for treating common Staphylococcus infections in both veterinary and human healthcare and shows promise as a treatment that, as of yet, does not induce bacterial resistance. LavenGel could prove to be a powerful tool in the future of medical management of bacterial infections.

No description available.

Antibiotic resistant infections are a growing health care concern, with many cases reported annually. Infections can cause irreversible bodily damage or death if they are not diagnosed in a timely matter. To rapidly diagnose antibiotic resistance in infections, it is important to be able to capture and isolate the DNA coding for the resistance genes. This is challenging because bacteria are present in blood in minute concentrations. To enrich the DNA to detectable levels, I modified magnetic microbeads with ssDNA sequences complementary to the target DNA to capture the DNA via hybridization. I compared DNA capture efficiency in three different methods: Co-flow, packed bead bed, and pre-hybridization. The pre-hybridized method worked better than the other two. Since pre-hybridization involved mixing, I chose to study mixing in a microfluidic device. The mixing chamber was a well carved out of PMMA placed between two electromagnets. To test the mixing well, beads and capture DNA were placed in it, and the electromagnets were subjected to different frequencies, including symmetric or asymmetric magnetic fields. For each condition the capture efficiency was determined by measuring the relative fluorescence units (RFU). A 100 Hz asymmetric magnetic field had the best capture efficiency out of all conditions. These results demonstrate a path for enriching low concentrations of DNA to detectable levels, and future work should be done to develop electromagnetic mixing in microfluidic devices.

The upper water column (<1 m depth) of freshwater lakes, which includes the surface microlayer (SML; <1 mm depth), is an important microbial habitat as well as an accumulation and dissemination site for chemical and microbial contaminants. This doctoral thesis reports novel insights into how the physical structure and functional capabilities of microbial communities can influence the presence of trace metals and health-relevant bacteria in the upper waters (SML and 0.5 m depth) of freshwater lakes. Two physically and geochemically contrasting lake environments, a remote sheltered boreal lake and a higher energy urban beach on Lake Ontario, were investigated to identify system-dependent physical and biogeochemical factors controlling contaminant-relevant microbial characteristics. The SML was identified as a major site for generation of contaminant-sequestering suspended flocs from a distinct biofilm-forming microbial community over diurnal timeframes via wind and sunlight exposure, with this process being enhanced at the higher energy beach site. More generally, upper waters including the SML were demonstrated to be inhabited by a diverse group of atypical facultative Fe(III)-reducing bacteria (IRB) that exhibited a SML- and lake-specific capacity for solid Fe(III) reduction directly related to floc and Fe(III) availability. Although IRB were hypothesized to be highly resistant to metals and antibiotics relative to other bacteria due to their ability to dissolve metal-rich Fe(III) minerals, this was not found to be the case. Nevertheless, IRB enriched from the SML demonstrated higher antibiotic resistance compared to those from 0.5 m depth and enriched Fe(III)-reducing communities from both depths harboured resistance-mobilizing genetic elements and included potentially pathogenic bacteria. Results of this thesis represent new knowledge concerning how microbial communities regulate the presence of contaminants in the upper waters of lakes. This has important implications for assessing the ecological and human health impacts of contaminants in freshwater systems. / Thesis / Doctor of Philosophy (PhD)

Hospitals worldwide are becoming increasingly plagued by antibiotic-resistant pathogens; concomitantly, the number of patients who die from antibiotic-resistant pathogens is increasing. The rise of multi-drug resistant (MDR) pathogens has rendered many antibiotics obsolete. The streptogramin and aminoglycoside antibiotics are drugs of last resort against life-threatening, MDR Gram-positive (e.g., methicillin resistant Staphylococcus aureus (MRSA)) and -negative (e.g., Pseudomonas aeruginosa) pathogens, respectively; however, as microbial drug resistance continues to emerge, the efficacy of these two important drug classes is decreasing. One of the most common mechanisms by which pathogens become resistant to streptogramin and aminoglycoside antibiotics is enzymatic inactivation: the Vat and AAC(3) acetyltransferases are employed by pathogens to inactivate streptogramin and aminoglycoside antibiotics, respectively. There currently is a dire need to not only develop new antibiotics, but to find new, creative strategies to outwit microbial resistance mechanisms. One of these strategies is to rescue the activity of antibiotics through the discovery of antibiotic adjuvants. In the current study, adjuvants which rescue the activity of streptogramin and aminoglycoside antibiotics through inhibition of the resistance acetyltransferases, VatD and AAC(3)-Ia, have been discovered—through the development of a cell-based screening method, we have found the first inhibitors of VatD, as well as of AAC(3)-Ia and its homologues, AAC(3)-Ib, AAC(3)-Ic, and AAC(3)-Id. We have demonstrated that streptogramin and aminoglycoside resistance can be reversed both in vitro and in vivo by the protein kinase inhibitors, GW5074 and rottlerin. Steady state kinetics revealed these compounds to inhibit VatD and AAC(3) enzymes mainly through noncompetitive or mixed mechanisms. This study has also demonstrated that eukaryotic kinase inhibitor libraries may be repurposed for the discovery of not only new antibiotic adjuvants, but also new antimicrobial targets. The inhibitors described herein may someday serve as effective adjuvants of streptogramin and aminoglycoside antibiotics. / Thesis / Doctor of Philosophy (PhD)

Antimicrobial resistance (AMR) is a One-Health (human, animal, environment) challenge that requires collaborative, interdisciplinary action. Comparable surveillance data are needed to effectively inform policy interventions aimed at preventing the spread of AMR. Environmental monitoring lags behind that of other One Health sectors and is in need of agreed upon targets and standardized methods. A challenge is that there are numerous microorganisms, antibiotic resistance genes (ARGs), and mobile genetic elements and corresponding methods that have been proposed. In this dissertation, a framework for AMR monitoring of aquatic environments was developed through a combination of literature review and stakeholder input, via surveys and a workshop. Through this process, three targets were selected for standardization: the sulfonamide resistance gene (sul1), the class 1 integron integrase gene (intI1), and cefotaxime-resistant Escherichia coli. Quantitative polymerase chain reaction (qPCR)- and culture-based protocols were developed and pilot tested in two independent laboratories on a set of six water matrices: wastewater, recycled water, and surface water from six different wastewater utilities engaging in water reuse located in five states across the USA. The impact of wastewater treatment and advanced water treatment processes was examined in terms of removal of these targets. Finally, qPCR and culture methods were used to examine the relationship between sul1, intI1, E. coli, and fecal indicators in private household wells across four states in the Southern USA that were identified as susceptible to storm events. The overall findings provide a useful baseline occurrence of the proposed AMR monitoring indicators across a range of water types and protocols that are accessible to water utilities. / Doctor of Philosophy / Life-saving drugs and treatments are failing at an increasing rate because of antimicrobial resistance (AMR). Antimicrobials, such as antibiotics, are a double-edged sword, because they are an effective weapon for killing disease-causing pathogens, but the more they are used the greater the likelihood that microbes that are resistant to them will survive, reproduce, and spread. National action plans for AMR have been created by a majority of countries, emphasizing the importance of antibiotic stewardship and other mitigation strategies. However, numerous data gaps need to be addressed in order to identify strategies that are most likely to be effective and to implement them. Environmental surveillance, including wastewater influent, wastewater effluent, and surface water, could prove an informative means to track AMR trends with time and relate them to human activities and corresponding mitigation efforts. The purpose of this dissertation was to develop a framework for AMR surveillance of aquatic environments and to test it across an array of sample types. We considered an array of possible culture- and DNA-based targets from available scientific literature and engaged experts and stakeholders in narrowing down the list to options that were both informative and feasible. We developed protocols for quantifying an antibiotic resistance gene (sul1), a mobile genetic element that has been implicated in the spread of multi-antibiotic resistance (intI1), and an extended spectrum beta-lactamase (ESBL) producing form of Escherichia coli. We compared the methods between two independent laboratories on untreated wastewater, treated wastewater, recycled water, and surface water collected from six locations across five states. We additionally did a survey of private household well water that was hypothesized to be vulnerable to contamination due to storms and lack of resources for maintenance. The results of this research can help to support environmental monitoring of AMR across the US and globally.