Since microorganisms normally co-exist with other species in nature, they have developed complex metabolic and physiological responses as a result of such inter-species interactions. Biotic elicitation mimics the inter-species interactions in nature by introducing cell extracts, parts of cell wall, or dead cells into the culture of another species thus resulting in complex metabolic responses in the elicited microorganisms. In this thesis we report the exploitation of the interspecies interaction in order to enhance the antibiotic production by the model organism Streptomyces coelicolor. It produces four known antibiotics: actinorhodin, undecylprodigioisn, methylenomycin and the calcium dependent antibiotic. We investigated the production of actinorhodin and undecylprodigiosin only because of the lack of quantitative analytical methods for the other two. The pure cultures of S. coelicolor in a defined medium produce higher concentrations of actinorhodin compared with undecylprodigiosin. However, undecylprodigiosin is more important due to its antitumor activities. We introduced live and dead cells of E. coli, Bacillus subtilis and Staphylococcus aureus, separately, to the S. coelicolor culture. Investigations were performed on Petri dishes, shake flasks and 2 L bioreactors. The suitable amount of each elicitor bacterium was first determined based on its ability to grow in the S. coelicolor medium so that it did not overtake the growth of S. coelicolor. Growth of S. coelicolor and glucose consumption of the elicited cultures were studied and compared with those in the pure culture. Our results revealed an alteration in the antibiotic production pattern by S. coelicolor, such that undecylprodigiosin production was significantly enhanced and actinorhodin decreased. The maximum enhancement occurred in the culture elicited with the live cells of E. coli with an increase of 3.5-fold, whereas the minimum was with elicitation using S. aureus cells (2.1-fold increase). Also, a considerable suppression in the production of actinorhodin was observed upon elicitation with live cells of E. coli or S. aureus. Furthermore, another positive outcome of the elicitation was the earlier onset of undecylprodigiosin production by 24-35h compared to the pure culture of S. coelicolor. Moreover, this study showed that the dead cells of B. subtilis and S. aureus had the same elicitation effects as the live cells, contrary to the heat-killed cells of E. coli that had no such effect. Some optimisation experiments on the amount and the timing of the elicitation were performed and the optimal conditions were chosen that would increase undecylprodigiosin production. Elicitation in the bioreactor resulted in as much as six-fold increase in the production of undecylprodigiosin compared with the pure culture and approximately double that obtained in the shake flasks. The antimicrobial activities of the extracted actinorhodin and undecylprodigiosin on the elicitor bacteria were tested in agar diffusion tests. Undecylprodigiosin always inhibited the growth of the elicitor bacteria whereas actinorhodin was less effective. In addition, our results indicated that the interaction between S. coelicolor and E. coli was mediated via a molecule present in the E. coli culture, while no such evidence was found in the case of interaction with B. subtilis or S. aureus. The results showed that the elicitation with the cells of B. subtilis and S. aureus was not due to peptidoglycan or N-acetyl glucoseamine which is the constituents of the cell wall that may have been released by lyses during the culture process. Such inter-species interactions may form the basis of new strategies in the search for novel antibiotics and other bioactive compounds. They can also be used to increase the productivity of existing processes for antibiotics as it was found in this work.

Biological evolution can be described as a unifying concept in biology. A thorough understanding of evolution is thus important to fully understand different areas of biology. However, learning the concepts of evolution has proven difficult, both to students and teachers. During the last decade, the notion of threshold concepts in learning has emerged. Passing the threshold or grasping the threshold concept is a transformative process, thought to be irreversible and has been described as passing a portal to new areas of understanding. Threshold concepts of importance to understanding evolution has been suggested to be time, spatial scale, complexity, randomness and probability. A hypothesis is therefore that facilitating understanding of those threshold concepts also will lead to a greater understanding of evolutionary mechanisms. Visualisations in science communication and learning has gained increased interest and animations as a form of visualisations has proven to facilitate learning in some situations. Since many (threshoid) concepts in evolution are untangible, such as deep time, small scale (micro and sub micro scale) animations could be a way to make those concepts more tangible for learners. In order to explore the potential for animations in learning evolution by making threshold concepts more tangible an interactive animation was designed and tested with upper secondary students in the course Biology 1. The subject of the animation was development of antibiotic resistance in bacteria. Learning effect was measured as differences in pre and post test scores on a selection of previously used concept questions from the literature, the concept inventory of natural selection (CINS). Open ended questions were also used as well as interview sessions, to gain more insight to the eventual effects of the animation. No statiscally significant improvement in the CINS scores could be observed in total, however improvement on a specific question category (biotic potential) could be observed. The number of misconceptions on evolution seemed unaffected after animation. Indications of conceptual conflicts could also be observed after the animation, indicating a potential for conceptual change with future revisions of the animation.

The number of cases of Clostridium difficile infection (CDI) has been increasing globally. CDI is the main cause of nosocomial diarrhoea, which may be life-threatening in complicated cases, and also costs the health care societies millions of pounds annually. The predominant types and their resistance to antibiotics have been changing and one of the major selective pressures which causes this is antimicrobial use. Although much is known about the role of the toxins in pathogenesis of CDI, the role of immunogenic cell wall components is unclear. They may play a role in colonisation and pathology and a study of these could clarify the infection process. It is therefore important to study the immune responses against these bacterial wall components from different strains and their effects on stimulation of leukocytes to produce cytokines and chemokines. This study was divided into four parts: 1. An epidemiological study to determine frequencies of the predominant types of C. difficile, thus 140 C. difficile isolates from surgical patients and their environment during 2009 were investigated to define their PCR ribotype. This utilised capillary sequencing gel electrophoresis for their analysis. 2. The determination of antimicrobial susceptibility to six antibiotics (ampicillin, erythromycin, tetracycline, metronidazole, moxifloxacin and vancomycin) was assessed and MIC determination by agar dilutions. 3. Investigation of host immunity to molecules with conserved molecular patterns. Surface-layer proteins (SLPs), lipocarbohydrate (LC) and flagellar proteins were separated and purified from five ribotypes of C. difficile (001, 002, 027, 078 and106) predominant in Scotland. a) The immune responses to these molecules were assessed by ELISA by exposing serum of patients and healthy donors and measuring specific IgG levels. b) Innate immunity was investigated by distinguishing responses of a macrophage cell line (THP1) to the above molecules. Induction of interleukins (IL)-1β, IL-6, IL- 8, IL-10 and IL-12 interleukins and TNF-α was detected by ELISA. In this study 15 different ribotypes were identified. The most frequent were 001, 020, 106 ribotypes (52.8%, 7.4% and 5.7%), respectively, while 13 isolates could not be assigned a ribotype. However, all isolates were sensitive to vancomycin, metronidazole and moxifloxacin, but 74.28% of isolates were resistant to erythromycin. The IgG level against bacterial antigens (SLPs, LC and flagella proteins) in donors’ serum showed almost normal distribution to all antigens from the different ribotypes and the sensitivity of the assays was increased by raising the concentration of antigens. Levels to SLPs were generally the highest, but the flagellar protein exceeded the SLPs of the 027 ribotype. The donors, controls, patients and carrier sera gave similar results. The greatest induction of interleukins was obtained using 50μg of antigen with the THP-1 cells activated with 50ng of PMA. The highest induction of all antigens was for IL-10. The highest values for the control LPS was with IL-12. But the best effect for SLPs of 027 was for IL-10 (109.1ng/ml), while the weakest for TNF for SLPs of 027 (4.7ng/ml). In general the IL-1β, IL-6, IL-8 and TNF concentrations ranged from 4.7-60ng/ml for all antigens and in contrast IL-12 and IL-10 average ranged 11- 109.1ng/ml. To conclude, the prevalence of C. difficile and their antibiotic susceptibility are constantly changing. IgG antibodies to SLPs and flagellar proteins from the hypervirulent ribotype 027 were highest in the community and hospitalized individuals. The molecules of conserved molecular patterns are immunogenic with various levels of response in the monocytic THP1 cells. SLPs were best in inducing interleukins. Flagellar proteins from 027 ribotypes accompanied SLPs in IL-10 induction levels. Consequently SLPs and flagellar proteins from 027 ribotypes appeared the best immunogenic bacterial molecules.

Within the last decade antibiotic resistant bacteria have become a major public health concern. A possible major contribution to this problem is thought to be the overuse of antibiotics in food animals. An estimated 70% of antibiotics dispensed yearly throughout the United States are distributed to the livestock industry as growth promoters, prophylactic, and therapeutic treatments, according to the Center for Disease Control and FDA. When food animals are exposed to low doses of antibiotics frequently over a long period of time the bacteria are able to develop resistance to antibiotics. Livestock harbor foodborne pathogens that are generally commensal bacteria in the animals themselves but can cause illness to the people exposed. The problem occurs when treatment becomes difficult, there is some speculation that livestock animals are a main contributor to the increase in antibiotic resistant foodborne pathogens. Salmonella spp. and Staphylococcus aureus are pathogens that can be isolated from livestock and cause serious illness in humans. Objectives of this study include isolating S. aureus and Salmonella from samples collected from food animals, investigating the prevalence of antibiotic resistance in the confirmed S. aureus and Salmonella isolates from animals raised in various areas of Southern New Mexico and Arizona. In this study, samples were collected from various food animals post-harvest at a USDA inspected, non-commercial animal harvest facility in Arizona, and evaluated for the presence of S. aureus and Salmonella. Samples were collected from 129 animals of the following types: Bovine (cow), Caprine (goat), Ovine (sheep), and Porcine (pig). S. aureus and Salmonella were isolated from three different types of samples per animal including hide samples, sub iliac and mesenteric lymph nodes, and nasal swabs. Each sample was cultured separately in enrichment media followed by selective/differential media. Once the pathogen was confirmed via 16s rRNA PCR for S. aureus, invA3 PCR for Salmonella, gel electrophoresis, DNA Sequencing, and other biochemical tests, an antibiotic susceptibility test was performed to check the resistance characteristics of each isolate. The pathogen was exposed to eight different antibiotics- Ampicillin, Cefoxitin, Chloramphenicol, Ciprofloxacin, Erythromycin, Streptomycin, Sulfamethoxazole/Trimethoprim, and Tetracycline; commonly used among animals and humans via the disc diffusion assay. A total of 59 and 60 of 369 samples were confirmed positive for S. aureus and Salmonella, respectively. The animal type that harbored the most Salmonella overall were Bovine/cattle and the sample type that harbored the most Salmonella overall were lymph nodes. The animal type that harbored the most S. aureus overall were porcine/pigs and the sample type that harbored the most S. aureus overall were lymph nodes. 18 out of 129 livestock animals sampled in this study were found to carry both Salmonella and S. aureus and were isolated from: 6-Porcine, 5-Bovine, 5-Caprine, and 2-Ovine. The overall antibiotic resistance prevalence in S. aureus and Salmonella were 22.88% and 32.71%, respectively. Antibiotic resistance patterns were seen in both S. aureus and Salmonella isolated from all different livestock and sample types. Of these S. aureus isolates 43 showed resistance to at least one type of antibiotic, and the most resistance was seen to Ampicillin. 53 Salmonella isolates showed resistance to at least one type of antibiotic, and the most resistance was seen to Erythromycin. The implications of this study indicate that there are antibiotic resistant Staphylococcus aureus and Salmonella found in various food animals and sample types. Most of these Salmonella and S. aureus isolates were resistant to more than one antibiotic. Appropriate control measures are needed to mitigate the problem of antibiotic resistant bacteria among food animals. These control measures could also reduce the spread of resistance from one bacterium to another and possibly lessen the antibiotic resistance problem and infections.

El propóleo es una sustancia resinosa compleja constituida por una gran variedad de compuestos químicos (esteres, flavonoides etc.), su composición no es estable y varía según la fuente de procedencia. Además, una de las propiedades más importantes del propóleo es su actividad antibacteriana, la cual se le atribuye fundamentalmente a los flavonoides. El propóleo se conoce desde la más remota antigüedad y ha sido utilizado por diferentes culturas con diversas finalidades. Con el posterior desarrollo de la farmacéutica y tratamientos fitoterápicos existe un resurgimiento en su uso. Es por esa razón que en los últimos años se han realizado algunas investigaciones acerca de los productos provenientes de las abejas y sus potenciales beneficios para la salud oral. Por lo tanto, la presente revisión de la literatura recolecta la información disponible sobre la composición del propóleo según zona geográfica y la actividad antibacteriana que tiene el propóleo aplicado a la estomatología. / Propolis is a resinous substance complex consisting of a variety of chemical compounds (esters, flavonoids, etc.), it´s composition is not stable and varies depending on the source. In addition, one of the most important properties of propolis is its activity antibacterial, which is attributed mainly to flavonoides. Propolis has been known since ancient times and has been used by different cultures for various purposes. With the subsequent development of pharmaceutical and herbal treatments there is a resurgence in its use. It is for this reason that in recent years have done some research about the products from the bees and their potential health benefits oral. Therefore, this literature review collects the available information on properties of propolis depends on geography and the antibacterial activity propolis has applied to dentistry.

The objective of the work reported in this paper is one of a two-fold nature. The first objective is to develop a disk-plate sensitivity test that is more rapid than that of existing methods. The second requisite is that the materials, techniques, interpretation, and reporting of results be the sane as those required for the disk-plate method described in the Difco Manual.

Burkholderia pseudomallei et Burkholderia mallei sont respectivement les agents biologiques responsables de la mélioïdose et de la morve. Pour déterminer si les échecs thérapeutiques étaient dus à l'émergence d'une résistance acquise durant le traitement antibiotique, nous avons sélectionné des souches de Burkholderia thailandensis, modèle d'étude, B. pseudomallei et B. mallei, avec différents antibiotiques : le chloramphénicol, la doxycycline et le triméthoprime-sulfaméthoxazole. Les Burkholderia ont montré qu'elles étaient capables de développer une multirésistance in vitro en réponse à chaque antibiotique utilisé dans le traitement oral de la mélioïdose ou de la morve. Pour comprendre les mécanismes de résistance impliqués, nous avons étudié les aspects moléculaires et génétiques de la résistance chez B. thailandensis par des méthodes protéomiques et transcriptomiques. Nous avons développé une méthode pour quantifier l'expression des gènes de pompes d'efflux par RT-PCR quantitative après normalisation sur plusieurs gènes de référence. Ces méthodes nous ont permis d'identifier la surproduction séquentielle de trois pompes d'efflux de type RND : BpeAB-OprB, AmrAB-OprA et BpeEF-OprC, toutes induites par le chloramphénicol ou la doxycycline chez les souches multirésistantes. L'étude de mutants déficients en pompe d'efflux nous a permis de mieux appréhender les relations étroites entre ces trois pompes et a confirmé que l'efflux actif était le principal mécanisme impliqué cette résistance induite. / Burkholderia pseudomallei and Burkholderia mallei are respectively the causative agents of melioidosis and glanders. To determine whether treatment failures were due to the emergence of acquired resistance during antibiotic treatment, we selected strains of B. pseudomallei, B. mallei, and Burkholderia thailandensis, used as a study model of these two pathogenic bacteria, with structurally unrelated antibiotics: chloramphenicol, doxycycline and trimethoprim-sulfamethoxazole. We showed that Burkholderia were able to develop multidrug resistance in vitro in response to each of theses antibiotics used in the oral treatment of melioidosis and glanders. To understand the resistance mechanisms involved, we studied the molecular and genetic aspects of resistance in B. thailandensis by proteomic and transcriptomic methods. We have developed a method to quantify efflux pumps gene expression by quantitative RT-PCR after normalization with several reference genes. These methods allowed us to identify sequential overproduction of three RND efflux pumps: BpeAB-OprB, AmrAB-OprA and BpeEF-OprC, all induced by chloramphenicol or doxycycline in multiresistant strains. The study of mutants respectively defective in one of these efflux pumps has allowed us to better understand the close relationship between these three pumps and confirmed that active efflux acted as a major mechanism involved in the induced resistance.

Master of Science / Department of Grain Science and Industry / Bhadriraju Subramanyam / Hulya Dogan / Stored-product insects and stored products from feed mills and swine farms contain antibiotic and potentially virulent Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus hirae. Stored-product insects can serve as potential vectors of these enterococci which possess antibiotic resistance genes that can be spread by horizontal transfer to more serious human pathogens. In the present study, the species and concentration of enterococci from adults and larvae of key stored-product insects and insect diets and their antibiotic resistance profile were characterized. Adults of five species out of the 15 stored-product insects were tested positive for enterococci, and these included Callosobruchus maculatus (F.), Sitophilus granarius (L.), Stegobium paniceum (L.), Lasioderma serricorne (F.), and Sitophilus zeamais Motschulsky. Three enterococcal species (E. casseliflavus, E. faecalis, and E. faecium) were found in 53 to 97% of the 30 adults screened for each insect species, and the enterococcal concentrations ranged from 1.4 x 10³ to 3.1 x 10⁶ CFU/adult. About 10 to 100% of the mature larvae of the respective five insect species had these three enterococcal species with concentrations ranging from 0.3 x 10¹ to 1.4 x 10⁵ CFU/larvae. Only three of the eight insect diets screened had the same three enterococci species in addition to E. gallinarum and E. hirae at concentrations of 0.2 x 10¹ to 5.9 x 10³ CFU/g. The greatest enterococcal concentration was found in C. maculatus adults but not in their larvae or diet (cowpeas). In C. maculatus during a nine-day period after adult eclosion, the enterococcal concentrations increased exponentially from 0.6 x 10¹ to a maximum of 4.1 x 10⁷ CFU/adult. Enterococci were detected in the fecal material of C. maculatus during a four-day period with a maximum concentration of 3.3 x 10³ CFU/adult on the fourth day. A total of 298 enterococcal isolates from adults, larvae, and diets were represented by E. faecalis (51.7% of the total), E. faecium (19.1%), E. casseliflavus (18.8%), E. gallinarum (5.7%), and E. hirae (4.7%). Enterococci were phenotypically resistant to quinupristin (51.3% of the total), erythromycin (38.9%), tetracycline (30.1%), enrofloxacin (29.2%), doxycycline (11.5%), and tigecycline (2.7%). All isolates were susceptible to ampicillin and vancomycin.

Cystic fibrosis (CF) is the most common fatal genetic disorder that afflicts young Canadians. The major cause of morbidity and mortality in patients with CF is chronic pulmonary infection with the opportunistic Gram-negative pathogen Pseudomonas aeruginosa. Once established, P. aeruginosa lung infections cannot be cleared despite sustained and aggressive antimicrobial therapy. Treatment failure of P. aeruginosa lung infections is caused by a combination of antibiotic resistance and tolerance mechanisms. Antibiotic resistance is mainly mediated by multidrug efflux pumps such as MexAB-OprM. Antibiotic tolerance has been attributed to biofilms and to nutrient starvation. In this thesis, I present an analysis of three transcriptional regulators (PA3225, RpoS, and RpoN) and their contributions to resistance and tolerance in P. aeruginosa. PA3225 is a transcriptional regulator that I initially identified as a candidate regulator of a type VI secretion system (T6SS) that had been previously implicated in biofilm tolerance. While a ΔPA3225 deletion mutant did not, unfortunately, have dysregulated expression of the T6SS, I fortuitously discovered that the mutant displayed increased resistance to various antibiotics from different functional classes. I linked the increased antibiotic resistance of ΔPA3225 to upregulation of MexAB-OprM and provided evidence that PA3225 may be a direct repressor of mexAB-oprM. Next, I sought to identify a transcriptional regulator of ndvB, which is another gene that plays a role in biofilm tolerance. I found that the stationary phase sigma factor, RpoS, was essential for expression of ndvB in stationary phase and biofilm cells. Moreover, RpoS was important for tolerance of stationary phase cells to tobramycin (TOB), an aminoglycoside antibiotic that is used to treat CF patients. In recent years, several groups have sought to identify novel treatments to combat antibiotic tolerance in P. aeruginosa. A popular strategy is metabolic potentiation, which involves co-administration of an antibiotic with a metabolite to reverse tolerance due to nutrient starvation. For example, one group found that fumarate (FUM) combined with TOB (TOB+FUM) was highly effective at killing tolerant P. aeruginosa. FUM uptake depends on C4-dicarboyxlate transporters, which are transcriptionally regulated by the alternative sigma factor, RpoN. Importantly, rpoN loss-of-function mutations are a recognised mechanism of pathoadaptation in CF clinical isolates. I demonstrated that TOB+FUM was unable to kill ΔrpoN stationary phase and biofilm cells due to loss of FUM uptake and that rpoN alleles from CF clinical isolates were unable to complement the ΔrpoN mutant. These findings could have important implications for TOB+FUM as a treatment modality in CF patients with a high burden of rpoN mutants. Overall, my work has provided interesting and, in the case of RpoN, clinically relevant insights into the regulatory networks that determine antibiotic susceptibility in P. aeruginosa.

Thesis advisor: Jianmin Gao / With the rapid evolution of antibiotic resistance, the need for more effective antibiotics is imminent. Bacterial membranes are an appealing target due to their accessibility and relatively conserved structures. Membrane targeting antibiotics, especially cationic antimicrobial peptides (CAMPs) such as host defense peptides, have been increasingly explored as novel antibiotics and tunable innate antimicrobials. The latter could be achieved by treatment with an antibiotic adjuvant: a compound that would increase the potency of host CAMPs without killing the bacteria on its own. Boosting the host’s own immune system with an adjuvant is beneficial over using antibiotics and would theoretically avoid triggering bacterial resistance. One mechanism of bacterial resistance is increasing the cationic charge of the membrane. As CAMPs are electrostatically attracted to anionic bacterial membranes, making the membrane more cationic decreases that attraction, rendering CAMPs less effective. To target this resistance mechanism chemically, two antibiotic adjuvant strategies were explored as co-treatments with various CAMPs: membrane targeting peptides used to bind and block surface amines, and small molecules used to either acetylate surface amines or convert a cationic membrane phospholipid to an anionic phospholipid. Co-treatment of the Staphylococcus aureus (S. aureus) membrane targeting peptide KAM-CT and various CAMPs increased S. aureus susceptibility to those CAMPs. Bacterial surface acetylation using sulfo-NHS-acetate followed by CAMP treatment caused up to 10 times increased CAMP potency. Hydrazine and hydroxylamine were shown to cleave the lysine moiety from the lysyl-phosphatidylglycerol (Lys-PG) phospholipid to generate phosphatidylglycerol (PG) in liposome models. S. aureus was treated with a hydroxylamine-CAMP conjugate, but it showed decreased antibiotic activity compared to the CAMP alone. To better understand what was happening in the bacteria, a novel Lys-PG quantification protocol was created by fluorophore labeling Lys-PG and quantifying the labeled Lys-PG via normal phase high-performance liquid chromatography (NP-HPLC). Cyclic peptides, such as KAM-CT, represent complex yet synthetically attainable moieties that could be used as novel antibiotics adjuvants. Expanding the repertoire of reversible covalent chemistries, especially those applied to peptide cyclization, is desirable due to the high potency and selectivity of such interactions. Herein, we also describe a novel reversible covalent chemistry between 2-formylphenylboronic acid (FPBA) and 2,3-diaminopropionic acid (Dap): the imidazolidino boronate (IzB) conjugate. It was found to be potent (Kd = 100 μM) and quickly reversible (t1 = ~6 sec) under physiological conditions. IzB formation was successfully employed as a peptide cyclization strategy as there was little interference from biologically relevant small molecules, except cysteine. Cysteine interference was utilized to create “smart” peptides that can linearize upon increasing cysteine concentrations via thiazolidino boronate (TzB) formation with the FPBA moiety in the peptide. Such “smart” peptides could be used as pH-responsive peptides or cysteine sensors able to report on the cysteine concentration in complex media. / Thesis (MS) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.