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While antibiotics have saved the lives of millions of people since the discovery of the first β-lactam, penicillin, their continued effectiveness is being increasingly threatened by resistant bacteria. Bacterial resistance to β-lactams is mainly achieved through the production of serine-β-lactamases (SBLs) and metallo-β-lactamases (MBLs). Although both types of β-lactamases are commonly isolated in clinical settings, MBLs represent the greatest threat to public health since they are resistant to SBL inhibitors and most β-lactams. However, aspergillomarasmine A (AMA), a fungal natural product synthesized by Aspergillus versicolor, was shown to be a rapid and potent inhibitor against two clinically relevant MBLs: NDM-1 and VIM-2. In bacteria possessing these enzymes, AMA could rescue the activity of meropenem, a broad-spectrum β-lactam that is usually reserved for the treatment of the most severe bacterial infections. However, many questions remain revolving around AMA's inhibitory potency and spectrum. Therefore, the activity of AMA in combination with six β-lactams from three subclasses (carbapenem, penam, cephem) was explored against 19 MBLs from three subclasses (B1, B2, B3). After determining that AMA activity was linked to MBL zinc affinity and that AMA was more potent when paired with a carbapenem, the efficacy of an AMA/meropenem combination was evaluated with and without avibactam, a potent SBL inhibitor. This study used ten Escherichia coli and ten Klebsiella pneumoniae laboratory strains as well as 30 clinical strains producing at least one MBL and one SBL. Once establishing that the AMA/avibactam/meropenem combination was effective against carbapenemase-producing Enterobacterales, new Acinetobacter and Pseudomonas shuttle vectors were created. With these shuttle vectors, it was determined that the AMA/avibactam/meropenem combination was effective against some of the bacteria topping the World Health Organization’s priority pathogen list. / Thesis / Doctor of Philosophy (PhD) / Bacteria are all around us. While some bacteria can promote human health, others can cause serious infections. These infections are typically treated with antibiotics. β-Lactam antibiotics, such as penicillins and cephalosporins, are especially important to medicine. Unfortunately, an increasing number of bacteria employ enzymes, known as β-lactamases, which negate the effects of β-lactam antibiotics. Previous studies demonstrated that a natural product, known as aspergillomarasmine A (AMA), could inhibit some β-lactamase enzymes. Consequently, the inhibitory power of AMA was further explored against a larger number of β-lactamase enzymes and in combination with different β-lactam antibiotics. After discovering that AMA had more inhibitory power when combined with a β-lactam antibiotic known as meropenem, the efficacy of the AMA/meropenem pairing was evaluated against resistant bacteria in the presence and absence of avibactam, another β-lactamase inhibitor. The AMA/avibactam/meropenem combination was shown to be effective against some of the world’s most antibiotic-resistant bacteria.

Syftet: Syftet med denna studie var att kartlägga magnitud och incidens av bakteriemi vid tandbehandling, vid egen munvård samt vid tuggning.Material och metod: En systematisk sökning i PubMed och Cochrane Library genomfördes. Den systematiska sökningen resulterade i 1113 artiklar. Titlar och abstrakt granskades med hjälp av tre oberoende bedömare. Totalt 90 artiklar lästes i fulltext. Granskningen av artiklar i fulltext genomfördes med hjälp av förutbestämda inklusions- och exklusionskriterier. Data extraherades ur utvalda artiklar och sammanställdes i en tabell.Resultat: Totalt inkluderades 42 artiklar. Resultaten visade hög incidens av bakteriemi vid tandextraktion och låg incidens vid avtryck, fastsättning/avlägsnande av ortodontiska band och separationsligatur, låg- och högvarvspreparation, samt suturtagning. Bland egna munaktiviteter hade tuggning och rengöring med tandpetare låg incidens medan tandborstning och rengöring med tandtråd hade högre incidens. Endast 16 artiklar redovisade magnitud och den redovisades med så låg noggrannhet att resultaten inte bedömdes användbara för att dra slutsatser.Slutsatser: Alla dentala ingrepp ingående i studiens sökning kan leda till bakteriemi av varierande grad. Egen munvård och tuggning kan leda till bakteriemi. Egen munvård och tuggning medför mindre risk för uppkomst av bakteriemi jämfört med de dentala åtgärder (tandextraktion, subgingival depuration och dentoalveolär kirurgi), där antibiotikaprofylax rekommenderas enligt Läkemedelsverket. / Aim: The purpose of this study was to identify the magnitude and incidence of bacteremia in dental procedures, at own routine mouth activities and at chewing.Materials and methods: A systematic search in PubMed and Cochrane Library database was done. This resulted in 1113 articles. Titles and abstracts were reviewed by three independent assessors. A total of 90 articles were read in full text applying the inclusion and exclusion criteria. Data were extracted from included articles. Results of bacteremia incidence were charted in a table.Results: Forty-two articles were included. The result showed a high incidence of bacteremia at extraction and low incidence at impression, rubberdam placement, removal or attachment of orthodontic bands and separator, fast and slow drill. Routine mouth activities like chewing and cleaning by toothpicks have low incidence while tooth brushing and flossing have higher incidence. Only 16 articles reported magnitude and it reported with as low accuracy that the results could not be used to draw any conclusion.Conclusions: All dental procedures that were found at the search concerning this study can lead to bacteremia in varying degree. Own routine mouth activities and chewing can lead to bacteremia. Routine daily mouth activities have less risk for the appearance of bacteremia compared with the dental measures (tooth extraction, subgingival scaling and dentoalveolar surgery) when antibiotic prophylaxis is recommended according to recommendations of Läkemedelsverket.

<p>Antibiotic resistance is often linked to human use of antibiotics. However, antibiotics and antibiotic biosynthetic pathways have been evolving for millions of years suggesting that antibiotic resistance is an ancient phenomenon. As of now, there has been no systematic survey of environmental microbes proven to exist in the absence of human influence and Lechuguilla cave offers such environment<em>. </em> Resistance diversity in strains isolated from this cave was analyzed by a phenotypic screen against a panel of 26 different antibiotics. Resistant strains were further investigated through determination of minimal inhibitory concentration (MIC) and inactivation studies. Of particular interest was strain LC044 (<em>Brachybacterium paraconglomeratum</em>), observed to inactivate macrolide antibiotics by phosphorylation. Genome sequencing and bioinformatics (BLAST analysis) identified a putative macrolide phosphotransferase (MPH) in strain LC044 and biochemical characterization of the purified recombinant protein confirmed its macrolide inactivating properties. To investigate if characterized MPH was unique to cave isolate, available terrestrial <em>Brachybacterium faecium</em> DSM 4810 genome was mined for presence of MPH-like protein. The top hit to the MPH from LC044 (a protein with 282 amino acids and 72% identity) was heterologously expressed and purified. Complete biochemical analysis of this enzyme revealed (i) MPH-activity, despite its annotation as aminoglycoside phosphotransferase (APH), and (ii) no significant differences in substrate specificities or kinetic parameters between these two enzymes suggesting that these two enzymes were equally effective resistance enzymes. This work highlights the prevalence of antibiotic resistance in a pristine, cave ecosystem and provides further support for the theory that antibiotic resistance is everywhere. Furthermore, the <em>mph</em> resistance determinant found in cave isolate and closely related terrestrial isolate show homology to clinical<em> mph</em> genes, suggesting that environmental <em>mph</em> genes could have served as reservoir of clinical determinants.</p> / Master of Science (MSc)

Objectives: Aggregatibacter actinomycetemcomitans is major putative bacterial pathogen in human periodontitis, particularly in aggressive periodontitis in younger-aged individuals. Systemic administration of certain antibiotics in combination have been demonstrated to exert synergistic antimicrobial effects against A. actinomycetemcomitans, and to markedly enhance elimination or suppression of A. actinomycetemcomitans from the subgingival dental plaque microbiome of periodontitis patients beyond that attained by periodontal mechanical debridement/surgery. However, studies on antibiotic synergisms against periodontal A. actinomycetemcomitans were conducted over 20 years ago, and only involved subgingival clinical isolates of the organism from periodontitis patients in Europe. Since temporal and geographic changes in antimicrobial susceptibility are documented among periodontitis-associated microorganisms, it is not known whether or not antibiotic synergisms against periodontal A. actinomycetemcomitans are present today among clinical isolates of the organism as recovered from periodontitis patients in the United States. As a result, the purpose of the present study was to assess the potential in vitro antimicrobial synergisms between amoxicillin plus metronidazole, between ciprofloxacin plus metronidazole, and between spiramycin and metronidazole, against clinical periodontal isolates of A. actinomycetemcomitans of United States origin. Methods: Standardized cell suspensions, equivalent to a 1.0 McFarland turbidity standard, were prepared with four fresh clinical isolates of A. actinomycetemcomitans, each recovered from the subgingival microbiota of United States periodontitis subjects, and plated onto to the surfaces of 150-mm diameter culture plates containing Haemophilus test medium. After drying, antibiotic-impregnated, quantitative, gradient diffusion strips (MIC Test Strip, Liofilchem s.r.l., Roseto degli Abruzzi, Italy) for amoxicillin, ciprofloxacin, spiramycin, and metronidazole were placed apart from each other onto the inoculated Haemophilus test medium surfaces, so that two test antibiotics per plate were employed against each A. actinomycetemcomitans clinical isolate for antibiotic susceptibility testing of individual antibiotic drugs. After 48 hours incubation in air + 5% CO2, individual MIC values for each antibiotic against A. actinomycetemcomitans were read in ug/ml at the point where the edge of the bacterial inhibition ellipse intersected with the MIC Test Strip, with the occurrence of antibiotic resistance among the A. actinomycetemcomitans clinical isolates determined using Haemophilus species antibiotic resistance breakpoint concentrations established by the United States Clinical Laboratory Standards Institute (CLSI) and the Société Francaise de Microbiologie. In vitro synergy testing of amoxicillin plus metronidazole, ciprofloxacin plus metronidazole, and spiramycin plus metronidazole was performed after determination of individual antibiotic MIC testing by placing MIC Test Strips for each of the two test antibiotics per combination in a cross formation onto the surfaces of A. actinomycetemcomitans-inculated Haemophilus test medium agar so that there was a 90° angle between the two antibiotic strips at the point where their individual MIC values against A. actinomycetemcomitans intersected on their respective MIC interpretive scales. After 48 hours incubation in air + 5% CO2, the MIC values for each antibiotic in combination against the A. actinomycetemcomitans clinical isolates was read in ug/ml where the edge of the bacterial inhibition ellipses intersected with each of the MIC Test Strips. For each of the three antibiotic combinations tested in vitro against the four A. actinomycetemcomitans clinical isolates, the fractional inhibitory concentration (FIC) index was calculated per each antibiotic in combination. FIC index values of less than or equal to 0.5 indicated the presence of synergistic antimicrobial effects of the antibiotic combination against the test bacterial clinical isolates, whereas FIC index values of greater than 0.5, but less than or equal to 4.0 indicated indifference, and FIC values greater than 4.0 represented the presence of in vitro antimicrobial antagonism between the two antibiotics in combination. Results: All of the four A. actinomycetemcomitans clinical isolates were susceptible in vitro to amoxicillin alone (all MIC values actinomycetemcomitans clinical isolates were resistant in vitro to metronidazole alone, with MIC values of resistant strains ranging between 24.0-48.0 ug/ml (resistance breakpoint threshold MIC value >16 ug/ml), and all were resistant in vitro to spiramycin alone, with MIC values of > 32.0 ug/ml exhibited by each of the four tested periodontal A. actinomycetemcomitans strains. In synergy testing, markedly lower MIC values were found for amoxicillin and metronidazole, as well as with ciprofloxacin and metronidazole, when tested in combination together as compared to being tested alone. FIC index values for the combination of amoxicillin plus metronidazole were all actinomycetemcomitans clinical isolates tested. Similarly, FIC index values for the combination of ciprofloxacin plus metronidazole were all actinomycetemcomitans clinical isolates tested. FIC index values for the combination of spiramycin plus metronidazole were > 0.5 for three of the periodontal A. actinomycetemcomitans strains, ranging from 0.875-2.0, which is indicative of an indifferent in vitro antimicrobial interaction between spiramycin and metronidazole against the three periodontal A. actinomycetemcomitans clinical isolates. The FIC index value of the combination of spiramycin plus metronidazole against a single periodontal A. actinomycetemcomitans clinical isolate was actinomycetemcomitans clinical isolate. Conclusions: Amoxicillin and ciprofloxacin individually were active against all A. actinomycetemcomitans clinical periodontal isolates, whereas most or all strains were resistant to metronidazole and spiramycin by themselves. Antimicrobial synergism was found for the combinations of amoxicillin plus metronidazole, and for ciprofloxacin plus metronidazole, against all four periodontal A. actinomycetemcomitans clinical isolates of United States origin. Spiramycin plus metronidazole generally failed to exhibit antimicrobial synergism against periodontal A. actinomycetemcomitans. These findings confirm and extend European synergism studies conducted in mid-1990s on antibiotic synergisms against periodontal A. actinomycetemcomitans, and are the first to demonstrate antibiotic synergism against United States periodontal A. actinomycetemcomitans clinical isolates. Additional research studies are needed to determine whether these in vitro synergistic effects of amoxicillin plus metronidazole, and of ciprofloxacin plus metronidazole, also occur in vivo and significantly enhance subgingival elimination or suppression of subgingival A. actinomycetemcomitans. / Oral Biology

Enterococcus faecalis is a Gram-positive bacterial species that is typically a member of the human gastrointestinal tract microbiota. However, E. faecalis is also a nosocomial pathogen, which is involved in urinary tract infections, soft tissue infections and endocarditis. In recent times, the occurrence of antibiotic resistance has complicated the treatment of these infections. One of the major differences between commensal and pathogenic strains of E. faecalis is that pathogens contain multiple mobile elements such as plasmids, transposons and integrative conjugative elements (ICE). These elements allow for the acquisition and transfer of virulence factors and resistance genes. Conjugative plasmids are a class of plasmids present in E. faecalis whose transfer to host cells is induced by a small pheromone peptide, cCF10 (LVTLVFV). This peptide is initially encoded as a 22-amino acid precursor (pre-cCF10) from the signal sequence of the chromosomal ccfA gene and is then proteolytically cleaved by signal peptidase II and Eep. Once pCF10 has been transferred a host E. faecalis cell, it is exceptionally stable. A replicon clone is maintained in greater than 85% of host cells over 100 generations in the absence of selection, suggesting the stability of pCF10 is intrinsic to the replicon. Three unique features of the replication initiation protein PrgW may contribute to this stability: (a) the interaction of PrgW with pre-cCF10, (b) disulfide bond formation at three conserved cysteines (C78, C275, and C307) in PrgW, and (c) processing of the nascent PrgW protein. Replication initiation proteins associated with theta replicons, such as pCF10, are often self-contained units. To initiate plasmid replication, the replication initiation protein (PrgW) binds to direct repeats (oriV) in its own coding sequence (prgW). In silico analysis of PrgW suggests the existence of three distinct domains within the protein. The first 122 amino acids are homologous to a conserved domain present in related replication initiation proteins, which includes a Helix-Turn-Helix (HTH) DNA binding domain. This suggests that this domain of PrgW has a DNA-binding function and binds to the oriV site in prgW. The following 61 amino acids are not similar to any known sequence, and are encoded by the DNA sequence containing the direct repeats in the oriV site. This domain may or may not have a distinct function. The remaining sequence forms a domain that contains cysteines C275 and C307, and is also similar to no known structure. It is hypothesized that this domain is related to the stability of pCF10. C307 appears to be critical, as previous experiments indicate that its mutation alone affects plasmid stability. Secondary structure analysis of this domain revealed the presence of multiple alpha-helices that contain distinct hydrophobic domains that possibly contribute to pre-cCF10 binding and PrgW tertiary structure. The positions of the conserved cysteines within these alpha-helices may stabilize a hydrophobic binding pocket that could potentially facilitate interaction with pre-cCF10. PrgW has a predicted molecular weight of 38.6 KDa and can be detected in Western blots as a band with an apparent approximate molecular weight (mw) of 36,000. Previous data from our lab indicates that, when overexpressed in E. faecalis, four bands of PrgW are present with observed molecular weights of 40,000, 36,000, 24,000 and 18,000. Time course experiments revealed that the 40,000 mw form is converted to a 36,000 mw form independent. The 40,000 mw form is unstable (with a complete turnover in 30 minutes) while the 36,000 mw form has a half-life of greater than 4 hours. The 24,000 mw band does not have a DNA binding motif and is likely a turnover product. When the three conserved cysteines (and only cysteines) in PrgW are replaced with alanine, the 40,000 mw form is still processed to the 36,000 mw form. However, the cysteine to alanine mutants accumulate the 36,000 mw form. / Microbiology and Immunology

The current thesis aims to address the prognosis and management of patients who have injuries necessitating orthopaedic surgery. In Chapter 1 I introduce the thesis, and in Chapter 5 I offer conclusions and summarize the contribution of the work. In Chapter 5, I address the scope, rationale, key findings, limitations and implications. Chapter 2 is a systematic review and meta-analysis investigating the effectiveness of antibiotic prophylaxis in patients with open fracture of the extremities. The results demonstrate moderate quality evidence of an important reduction in the infection rate in patients receiving, versus not receiving, antibiotic prophylaxis. We found no difference in infection rate with longer (3 to 5 days) versus shorter (1 day) duration of antibiotics – this finding warrants only low confidence. Chapter 3 is a systematic survey of current practice and recommendations regarding antibiotic prophylaxis in open fracture management. Authors of publications over the last decade strongly support early systemic antibiotics prophylaxis for patients with open fractures of extremities. In practice, most used systemic antibiotics with both gram-positive and gram-negative coverage, and continued the administration for 2 to 3 days. Most recommendations suggested gram-positive coverage for less severe injuries, and administration duration of no more than 3 days (half suggested 1 day). For more severe injuries, most recommendations suggested broad antimicrobial coverage continued for 2 to 3 days. Chapter 4 is a longitudinal study investigating predictors of persistent post-surgical pain after tibia fracture. We found significant independent associations between resolution of pain and male sex, non-smoking and alcohol consumption. Age, obesity, type of fracture (closed versus open), additional injuries, and post-operative weight-bearing status did not predict resolution of pain. Our findings suggest that clinicians should be particularly alert to the possibility of troublesome post-operative pain in female smokers who do not drink alcohol. Clinicians may consider counselling patients to discontinue smoking, inform them that they are at nearly double the risk of incidence of troublesome post-operative pain (in addition to the long-term adverse health consequences of smoking). / Thesis / Doctor of Philosophy (PhD) / Antibiotic prophylaxis reduces infection with 10% fewer event rate than the group without antibiotic prophylaxis (low to moderate confidence in estimates). The optimal antibiotic regimens and duration remain uncertain. There is a higher risk of persistent post-surgical pain in female smokers who do not use alcohol, following tibia fractures.

It is predicted that by 2050 there will be 10 million deaths annually due to super-resistant bacterial infections. Antimicrobial resistance (AMR) is already responsible for nearly 5 million deaths a year. Ribosomes serve as an ideal drug target being frequently targeted by antibiotics and having a highly conserved structure with few options for resistance. However, computer aided drug design (CADD) using ribosome crystal structures presents several challenges and is underutilized in the field. In this work we establish a successful protocol for antibiotic redocking and docking within the high interest sites of the peptidyl transferase center (PTC). Molecular visualization and interaction mapping were used to atomistically delineate binding patterns in the ribosomal PTC that could be used for CADD. Eleven ribosome crystal structures were validated for computational testing, which revealed derivative binding patterns in the A-site and P-site that can be used to increase antibiotic efficacy. Ribosome overlays revealed high interaction frequency nucleotides that were widely conserved throughout the different species and could be used to inform bidentate design to target two pockets at once. This work serves as a basis for methods to computationally explore drug optimization on ribosome targeting antibiotics to help combat the rapid expansion of AMR. / Master of Science in Life Sciences / Antimicrobial resistance (AMR) to antibiotics by bacteria is a rapidly increasing problem. Current trends predict that there will be more death due to super-resistant bacterial strains than cancer by 2050. Ribosomes are essential cellular machinery for bacteria and make an ideal antibiotic target. Using computational tools to optimize antibiotics with available ribosome crystal structural data presents several challenges and is underutilized throughout the field. In this work we establish a successful protocol for determining and exploiting antibiotic binding patterns within the functional center of the ribosome, the peptidyl transfer center (PTC). Nearly a dozen ribosome crystal structures were validated for computational testing, and binding patterns were revealed within the PTC that allowed antibiotic derivatives with increased efficacy to be developed. Ribosome validation also helped inform new drug class design so that multiple drug sites could be targeted at once, which were docked sharing high frequency nucleotide interactions with both parent antibiotics. This work serves as a basis for methods to computationally explore drug optimization on ribosome targeting antibiotics to help combat the rapid expansion of AMR.