Methicillin-Resistant Staphylococcus aureus (MRSA) began as a nosocomial infection due to overuse of antibiotics. Several previous studies have reported an increase in this infection in adult patients who have not been hospitalized. It has also been reported that there is an increase in MRSA in children. Some of these children became infected even though they were not at high risk for the infection. After approval from the All Children's Hospital Institutional Review Board (IRB), a cross sectional study was conducted with pediatric admissions and pediatric emergency room visits to determine the characteristics of Methicillin-Sensitive Staphylococcus aureus and MRSA. During this study, a review of 672 medical charts was conducted. The study participants ranged in age from newborns to 18 years of age. In order to be enrolled in the study, the subjects' cultures were collected either as outpatients or within 72 hours of admission. The data that was collected from each chart included age, race/ethnicity, gender, type of infection, preexisting medical conditions, and risk factors for infection. The potential risk factors include antibiotic use, previous surgery or outpatient procedure, previous MRSA infection, immunotherapy, community worn device, and residence in a facility. Statistical analysis was conducted using Epi Info and SAS software packages. In regards to demographic characteristics, black children are 2.98 times more likely to have an MRSA infection than white children. Gender and age were not risk factors for the development of the infection. The risk factors that were significant in whites were home health care (OR= 6.12, CI= 5.16, 7.08), community worn device (OR= 2.28, CI= 1.67, 2.89), previous hospitalization (OR= 2.43, CI= 1.95, 2.91), previous MRSA infection (OR= 3.69, CI= 2.90, 4.48), and previous surgery (OR= 2.02, CI= 1.51, 2.53). In blacks, females were more likely to have MRSA (OR= 2.57, CI= 1.73, 3.41). This finding may be due to the small sample size of black children in the study. Of the analyzed risk factors, home health care (OR= 2.95, CI= 1.11, 4.79), community worn device (OR= 2.85, CI= 1.71, 4.01), previous hospitalization (OR= 1.98, CI= 1.13, 2.83), previous surgery (OR= 2.79, CI= 1.79, 3.79), and previous antibiotic (OR= 5.60, CI= 4.66, 6.54) use were all significant risk factors in blacks. Effect modification was tested between race and all risk factors. Race was an effect modifier only for the risk factor of previous antibiotic use (pvalue =.02). Adjustment of confounding was performed for each race due to the presence of effect modification. After the adjustment for confounding in whites, only home health care (OR=4.37 CI= 1.55, 12.32), previous MRSA infection (OR= 2.86 CI= 1.16, 7.05), and previous hospitalization (OR= 2.00 CI= 1.14, 3.50) remained statistically significant. In blacks, after adjustment of confounding, only previous antibiotic use (OR= 5.13 CI= 1.75, 15.08) remained significant. Adjustment for confounding was also preformed on the total risk factors model. A dose response relationship was present with increasing risk factors present.

Bacterial source tracking (BST) methods often involve the use of phenotypic or genotypic fingerprinting techniques to compare indicator bacteria such as Escherichia coli isolated from unknown sources against a library of fingerprints from indicator bacteria found in the feces of various known source animals. The predictive capability of a library is based in part on how well the library isolates reflect the true population diversity of indicator bacteria that can potentially impact a water body. The purpose of this study was to compare the behavior of E. coli population structures in the feces of humans, beef cattle and horses across different parameters. Ribotyping and antibiotic resistance analysis were used to "fingerprint", or subtype E. coli isolates. Significantly greater diversity was observed in the E. coli population of horses compared to the human or beef cattle sampled. Subtype sharing between individuals from all host categories was infrequent, therefore the majority of E. coli subtypes were sampled from a single individual. The dominant E. coli populations of nine individuals (three per host source category) were monitored over time, which demonstrated that E. coli subtypes within a host individual vary on a monthly time frame, and an increase in the frequency of subtype sharing was noted between individuals within the same source group over time. The E. coli population of a single human that had just finished antibiotic treatment was studied on a daily basis for one month. The loss of an E. coli subtype with high antibiotic resistance was observed over time, however there was a single dominant E. coli subtype that was present at every sampling event during the entire month. Geographic distinctiveness of E. coli populations was investigated by sampling four herds located in different geographical regions. We observed that E. coli populations are not geographically distinct, but are somewhat individual-specific, as most E. coli isolates had a subtype that was found in a single individual. This study defines factors that should be considered when constructing a successful BST library, and suggests that E. coli may not be the appropriate indicator organism for BST.

>Magister Scientiae - MSc / The continued emergence of bacterial resistance to the antibiotics currently employed to treat several diseases has added to the urgency to discover and develop novel antibiotics. It is well established that natural products have been the source of the most effective antibiotics that are currently being used to treat infectious diseases and they remain a major source for drug production. Natural products derived from marine microorganisms have received much attention in recent years due to their applications in human health. One of the biggest bottlenecks in the drug discovery pipeline is the rediscovery of known compounds. Hence, dereplication strategies such as genome sequencing, genome mining and LCMS/MS among others, are essential for unlocking novel chemistry as it directs compound discovery away from previously described compounds. In this study, the genome of a marine microorganism, Thalassomonas viridans XOM25T was mined and its antimicrobial activity was assessed against a range of microorganisms. Genome sequencing data revealed that T. viridans is a novel bacterium with an average nucleotide identity of 81% to its closest relative T. actiniarum. Furthermore, genome mining data revealed that 20% of the genome was committed to secondary metabolisms and that the pathways were highly novel at a sequence level. To our knowledge, this species has not previously been exploited for its antimicrobial activity. Hence, the aim of this study was to screen for bioactivity and identify the biosynthetic gene/s responsible for the observed bioactivity in T. viridans using a bioassay-and-genome- guided isolation approach to assess the bioactive agent. The bioassay-guided fractionation approach coupled to LCMS/MS led to the identification of a novel antimicrobial protein, TVP1. Bioinformatic analyses showed that TVP1 is a novel antimicrobial protein that is found in the tail region of a prophage in the T. viridans genome. Phage-derived proteins have previously been shown to induce larval settlement in some marine invertebrates. Since the mechanism of action of TVP1 remains unknown, it remains a speculation whether it may offer a similar function. More research is required to determine the biotechnological application and the role of TVP1 in its host and natural environment.

Biofilms are a crucial adaptation for bacterial survival against stresses from external environments. Biofilms are adherent colonies of sessile bacteria embedded within a self-produced matrix. Bacterial control over formation, maintenance, and response to external stresses are strictly regulated. However, complexities of intracellular signaling for biofilm regulation are still not fully understood. In this thesis, I report on two distinct regulatory systems important for biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. The first regulatory system I report on is the two-component system TctD-TctE. This system is involved in regulating the uptake of tricarboxylic acids such as citric acid and is involved in biofilm-specific susceptibility to aminoglycoside antibiotics. Here I describe work I performed characterizing the involvement of TctD-TctE in biofilm development when citric acid is present as a carbon source in nutrient media. In further characterizing a previously observed aminoglycoside susceptibility, I found that a strain with a deletion of TctD-TctE (ΔtctED) has a heightened accumulation of tobramycin in its biofilms when grown in the presence of citric acid. In ΔtctED, I determined that there was an inhibition of overall cell growth when citric acid was present in nutrient media. Additionally, in the presence of citric acid, ΔtctED displayed high levels of biofilm formation. This contrasted with normal biofilm development observed in the PA14 wild type strain where biofilm mass was reduced in the presence citric acid. The second project of this thesis reports on a novel regulatory small RNA, the Small RNA Regulator of Biofilms (SrbA). SrbA was found to be unique to P. aeruginosa and displayed no homology with any other sequenced bacterial species. I found that loss of SrbA resulted in a significant reduction in biofilm mass. Subsequently, loss of SrbA also leads to attenuation of P. aeruginosa pathogenicity in Caenorhabditis elegans nematodes. Bacterial biofilms possess specific regulatory programs that are still just being appreciated for their complexity. This thesis work adds to our understanding of biofilm regulation by studying roles of the two-component system TctD-TctE and the small RNA SrbA in P. aeruginosa.

This study was conducted to examine the rate of contamination and molecular characteristics of enteric bacteria isolated from a selection of food sources in Vietnam. One hundred and eighty raw food samples were tested and 60.8% of meat and 18.0% of shellfish samples were found to be contaminated with Salmonella spp. which belonged to variety of serogroups and serotypes. More than 90% of all food sources contained Escherichia coli and 32% of 50 shellfish samples were contaminated with Vibrio parahaemolyticus. PFGE was used to determine the degree of relatedness of Salmonella spp. There were 33 distinct PFGE patterns from 51 Salmonella spp. isolates tested, indicating that PFGE could be used as an alternative method for serotyping for use in epidemiology of Salmonella spp. Susceptibility of the isolates to 15 antimicrobial agents was investigated. Moderate to high frequencies of resistance to antibiotics were observed in Salmonella spp. and E. coli isolates and multi-resistance, defined as resistance to at least 4 antibiotics, was observed. All of the V. parahaemolyticus isolates were resistant to ampicillin/amoxicillin but not to other antibiotics. Betalactam TEM gene and tetracycline resistance tetA, tetB genes were widely distributed in both E. coli and Salmonella spp. isolates. Other resistance genes, including sulI, cmlA, aadA, aphA1, dhfrV, and aac(3)-IV were also present at high to moderate levels. Identification and characterisation of the mobile genetic elements, including identification of class 1 integrons and plasmids were carried out for multi-resistance isolates. The integrons harboured varying gene cassettes, including aadA1, aadA2, aadA5, aacA4, dhfrXII, drfA1 and dhfrA17, blaPSE1 and catB3. Thirty-five percent of Salmonella spp. isolates and 76% of E. coli isolates harboured plasmids of more than 95 kb. Transfer of resistance phenotypes between the isolates via conjugation and phage transduction was also demonstrated. Salmonella genomic island 1 (SGI1), a 43-kb genomic region contains a 13-kb antibiotic resistance gene cluster, has been identified in an isolate of S. Albany from chicken. The presence of Salmonella spp. virulence genes was investigated to examine the pathogenicity potential of the isolates. The invA gene was present in all Salmonella spp. isolates and the plasmid virulence gene spvC was detected in one S. Typhimurium isolate only, on a 95 kb virulence plasmid. Invasion assays performed in vitro demonstrated that all Salmonella isolates were capable of invading human intestine INT407 cells. In addition, the investigation for the presence of 58 selected virulence genes showed that all the tested isolates contained at least one virulence gene and there were 16 genes which are associated with different pathotypes detected. The data obtained in this study indicates that raw food in Vietnam is a potential reservoirs for many pathogenic organisms, and confirms the role of food animals as a reservoir of multidrug resistant E. coli and Salmonella spp.

<p>Objective: To explore views of medical doctors, veterinarians and drug sellers on use of antibiotics on humans and nonhumans and on factors that influences the development of resistance to antibacterial agents. Further, to look at the bi-directional relationship between antibiotic use and environment. </p><p>Methods: The study was a qualitative explorative interview study, analysed using conventional content analysis. It was conducted in Orissa, India. Data were collected by face to face semi structured interview. The interviews were tape recorded and transcribed into Oriya, then translated in to English. Each paragraph or sentence was coded. Similar codes were clustered together and collapsed into sub categories and categories. The main themes were allowed to emerge, based on the relationship between categories.</p><p>Findings: The main finding of the study was mishandling and abuse of antibiotics in patients as well as at professional level due to weak implementation of legislation, which appears to be the major cause of antibacterial agent resistance. Incomplete course or dose due to poverty in rural area and self medication in urban area are more common. The study also showed that climatic factors, pollution and population density are the major ecological factors which influence antibiotic prescriptions. Another major finding of this study was that, due to improper disposal system of pharmaceuticals; antibiotics are contaminating air, water and terrains which can cause major risk to aquatic and grazing animals. </p><p>Conclusion: This study emphasises the need for comprehensive actions including information, training, legislation and education at all levels of drug delivery system to rationalize antibiotic use by improving prescribing pattern and creating awareness among consumers. Proper disposal of pharmaceutical wastes is required to prevent the contamination of environment from pharmaceutical pollutants. Further study is essential concerning environmental impact of antibiotics.</p><p>Key words: Antibiotic use; antibiotic resistance; environment; qualitative; conventional; content analysis; veterinarians; medical doctors; drug sellers; Orissa; India.</p>

<p>The worldwide increase in antibiotic resistance is a concern for public health. When the appropriate antibiotic dosage is determined, the priorities are efficacy and toxicity. The aim of this thesis was to gain knowledge about the most efficient dosing regimens in order to minimize the emergence and selection of antibiotic-resistant mutants. We also wanted to assess the impact of antibiotic selective pressure and host to host transmission for the dissemination of resistance.</p><p><i>Escherichia coli </i>bacteria with different levels of cefotaxime susceptibility were competed in an in vitro kinetic model, demonstrating a complex selection of low-level resistance influenced e.g. by the time duration of selective concentrations and the rise of new mutants. We also constructed a mathematical model incorporating biologically relevant parameters and showed its usefulness when assessing the risks of resistance development.</p><p>When <i>E. coli </i>populations with pre-existing fluoroquinolone-resistant mutants were exposed to simulated serum concentrations, several currently used doses of fluoroquinolones clearly enhanced the development and selection of resistance. </p><p>The mutant prevention concentration (MPC) was measured for several <i>E. coli</i> isolates with different fluoroquinolone susceptibilities, and because of fluctuating antibiotic concentrations in the human body, the pharmacokinetics was considered when evaluating MPC. Results indicate that the area under the serum concentration time curve in relation to the MPC may be a useful predictor for emergence of resistance.</p><p>In the commensal flora of healthy human couples we noted a high frequency of trimethoprim-resistant <i>E. coli.</i> There was also an extensive sharing and transmission of <i>E. coli</i> clones. Treating the female with trimethoprim reduced the number of intestinal <i>E. coli</i> which might have facilitated the transmission from the male partner. These findings suggest that the rate of transmission is high and effectively contributes to the spread of both susceptible and antibiotic-resistant <i>E. coli</i> in intrafamilial settings.</p>

Plants, more precisely their chloroplasts with their bacterial-like expression machinery inherited from their cyanobacterial ancestors, can potentially offer a cheap expression system for proteinaceous pharmaceuticals. This system would be easily scalable and provides appropriate safety due to chloroplasts maternal inheritance. In this work, it was shown that three phage lytic enzymes (Pal, Cpl-1 and PlyGBS) could be successfully expressed at very high levels and with high stability in tobacco chloroplasts. PlyGBS expression reached an amount of foreign protein accumulation (> 70% TSP) that has never been obtained before. Although the high expression levels of PlyGBS caused a pale green phenotype with retarded growth, presumably due to exhaustion of plastid protein synthesis capacity, development and seed production were not impaired under greenhouse conditions. Since Pal and Cpl-1 showed toxic effects when expressed in E. coli, a special plastid transformation vector (pTox) was constructed to allow DNA amplification in bacteria. The construction of the pTox transformation vector allowing a recombinase-mediated deletion of an E. coli transcription block in the chloroplast, leading to an increase of foreign protein accumulation to up to 40% of TSP for Pal and 20% of TSP for Cpl-1. High dose-dependent bactericidal efficiency was shown for all three plant-derived lytic enzymes using their pathogenic target bacteria S. pyogenes and S. pneumoniae. Confirmation of specificity was obtained for the endotoxic proteins Pal and Cpl-1 by application to E. coli cultures. These results establish tobacco chloroplasts as a new cost-efficient and convenient production platform for phage lytic enzymes and address the greatest obstacle for clinical application. The present study is the first report of lysin production in a non-bacterial system. The properties of chloroplast-produced lysins described in this work, their stability, high accumulation rate and biological activity make them highly attractive candidates for future antibiotics. / Lytische Enzyme aus Bakteriophagen bieten Eigenschaften, die sie zu vielversprechenden Medikamenten im Einsatz gegen bakterielle Krankheiten machen. Obwohl sie speziell beim Einsatz gegen bakterielle Infektionen, welche durch Antibiotika resistente Erreger hervorgerufen werden, eine maßgebende Rolle spielen könnten, waren bisher die hohen Produktionskosten ein Hindernis für die medizinische Anwendung. Ein kostengünstiges und einfach zu handhabendes System, wie beispielsweise Chloroplasten in Pflanzen, würde diese lytischen Enzyme zu einer effizienten Alternative zu herkömmlichen Antibiotika machen. In dieser Arbeit wird erstmals die erfolgreiche Produktion von lytischen Enzymen in Tabak-Chloroplasten vorgestellt, welche mit einem Fremdproteingehalt von mehr als 70% des gesamtlöslichen Proteins der Pflanze eine Menge beschreibt, die bisher mit diesem Verfahren noch nicht erreicht wurde. Alle in Chloroplasten hergestellten lytischen Enzyme zeigten hohe spezifische bakteriolytische Aktivität gegen die gewählten Humanpathogene und waren innerhalb von Minuten in der Lage diese Bakterien abzutöten. Zur Herstellung von zwei lytischen Enzymen wurde in dieser Arbeit ein spezieller Shuttle-Vektor entworfen, der die Expression von toxischen Genen innerhalb von E. coli Zellen im Zuge der DNA Replikation vermeidet, jedoch die Herstellung einer ungehinderten Expression der toxischen Gene in den Chloroplasten nach Beseitigung des Selektionsmarkers erlaubte. Ein Vergleich zwischen einem herkömmlich verwendeten Transformationsvektor und dem Shuttle-Vektor mittels eines Reportergens zeigte, dass das neu entwickelte System bis zu 4 mal mehr Protein produzierte. Diese Ergebnisse zeigen das Potential von Chloroplasten als kostengünstige und leicht zu handhabende Produktionsplattform für lytische Enzyme, welche als neue Generation von Antibiotika attraktive Alternativen zu herkömmlichen Therapien bieten.

Determining the properties of mutations is fundamental to understanding the mechanisms of adaptive evolution. The major goal of this thesis is to investigate the mechanisms of bacterial adaptation to new environments using experimental evolution. Different types of mutations were under investigations with a particular focus on genome rearrangements. Adaptive evolution experiments were focused on the development of bacterial resistance to antibiotics. In paper I, we performed stochastic simulations to examine the role of gene amplification in promoting the establishment of new gene functions. The results show that gene amplification can contribute to creation of new gene functions in nature. In paper II, the evolution of β-lactam resistance was studied by evolving S. typhimurium carrying a β-lactamase gene towards increased resistance against cephalosporins. Our results suggest that gene amplification is likely to provide an immediate solution at the early stage of adaptive evolution and subsequently facilitate further stable adaptation. In paper III, we isolated spontaneous deletion mutants with increased competitive fitness, which indicated that genome reduction could be driven by selection. To test this hypothesis, independent lineages of wild type S. typhimurium were serially passaged for 1000 generations and we observed fixation of deletions that significantly increased bacterial fitness when reconstructed in wild type genetic background. In paper IV, we developed a new strategy combining 454 pyrosequencing technology and a ‘split mapping’ computational method to identify unique junction sequences formed by spontaneous genome rearrangements. A high steady-state frequency of rearrangements in unselected bacterial populations was suggested from our results. In paper V, the rates, mechanisms and fitness effects of colistin resistance in S. typhimurium were determined. The high mutation rate and low fitness costs suggest that colistin resistance could develop in clinical settings. In paper VI, a novel Metallo-β-lactamase (MBL) with low resistance against β-lactam antibiotics was employed as the ancestral protein in a directed evolution experiment to examine how an enzyme evolves towards increased resistance. For most isolated mutants, in spite of their significantly increased resistance, both mRNA and protein levels were decreased as compared with the parental protein, suggesting that the catalytic activity had increased.

Vibrio spp., natural inhabitants of aquatic environments, are one of the most common causes of bacterial gastroenteritis in the world, being spread to humans via the ingestion of seafood, contaminated drinking water or exposure to seawater. The majority of Vibrio spp. are avirulent, but certain strains may sporadically be human pathogenic. Vibrio cholerae may cause cholera and fatal wound infections, Vibrio parahaemolyticus may cause gastroenteritis and Vibrio vulnificus may cause wound infections and sepsis. To expand current knowledge of the occurrence, ecological niche and persistence of potential human pathogenic Vibrio spp. in aquatic environments, occurrence and laboratory studies were performed. The seasonal variation of Vibrio spp. in clams and mussels from Mozambique and Sweden were studied, with isolated strains characterized and compared with those isolated from water samples collected in India. Results showed that the numbers of Vibrio spp. in Mozambican clams peaked during the warmer rainy season and that the dominating species was V. parahaemolyticus. Biochemical fingerprinting and virulence screened by PCR revealed a high similarity among strains from the different aquatic environments. However, isolate functional hemolytic analyses and antibiotic resistance patterns differed between strains; Swedish and Indian strains were less sensitive to the tested antibiotics and had a lower hemolytic capacity than those from Mozambique. Molecular analysis of bacterial DNA from Swedish mussels showed the presence of the three Vibrio spp. most commonly linked with human illness, as well as their associated virulence genes. The strains isolated from marine and clinical environments were equally and highly harmful to the tested eukaryotic cells. The persistence of clinical V. cholerae in aquatic environments was investigated in vivo. Strains were exposed to mussels, with bacterial uptake and elimination then examined. The mussels were able to avoid the most potent strain by complete closure of shells. The less potent strain was accumulated in mussel tissue in low levels and one marine control strain to a higher degree. Mussels eliminated the pathogenic strain less efficiently than they did the marine strain. One clinical and one marine strain were then exposed to 4°C for 21 days, with the temperature then increased to 20°C. The clinical strain was more prone to lose culturability than the marine strain at 4°C, the former performed significantly better in regaining culturability after the temperature up-shift. Subsequently, the persistence of the clinical strain in natural bottom sediment, incubating as above, was studied and results showed a similar decrease in culturable numbers in the sediment as in the water. As the clinical V. cholerae strains did not carry any of the standard set of virulence genes, the ability to change from non-culturable to culturable may be of great importance to strain pathogenicity. The results also show that natural bottom sediment may be a potential reservoir of human pathogenic Vibrio spp.