Secondary plant metabolites have recently been gaining interest in livestock production systems following the ban of in-feed antibiotics within the European Union. The rise in antimicrobial resistance found in pathogenic and non-pathogenic bacteria has lead to increased interest in the research community regarding the use of phythochemicals as an alternative to antibiotics. The purpose of this research was to evaluate the impact of including phytochemicals in a livestock production system. Specifically, a high tannin-containing forage, sainfoin (Onobrychis viciifolia), was evaluated in vitro for its antimicrobial effect on Escherichia coli. We determined that phytochemicals alone are not as inhibitory as synthetic antibiotics. Thus, the use of combination therapy to deter the development of antimicrobial resistance was evaluated. A myriad of plant compounds were screened for their synergistic interactions with ciprofloxacin. Geraniol, an essential oil, was identified to possess good antimicrobial activity and synergistic interactions with ciprofloxacin. Therefore the effect of long term exposure to both ciprofloxacin and geraniol were examined. Results demonstrated that once an antimicrobial concentration threshold was reached, resistance to ciprofloxacin increased markedly in the presence of both geraniol and ciprofloxacin. Finally, an in vivo trial was conducted in which forty steers were fed sainfoin or alfalfa over a 9-week period to evaluate its ability to reduce E. coli shedding and its impact on gut microbiota in the context of popular theoretical ecology concepts. Results from the in vivo study indicate that sainfoin was able to promote a slight decrease in generic E. coli shedding which could be maintained throughout the trial. Using high-throughput sequencing, the effect of sainfoin on the microbial ecosystem of the ruminant gut was evaluated. Sainfoin induced a significant shift in the microbial community structure of the rumen and to a lesser extent in the hindgut. Using ecology theories, a hypothesis was formulated regarding the mechanisms that mediate the development of tolerance and the fundamental ecological processes controlling microbial population shifts. Understanding how the gut ecosystem functions and predicting its behaviour in the presence of various fluctuating environmental conditions will enable more efficient manipulation of the rumen and promote best management practices in livestock production.

Staphylococci are important opportunistic pathogens often found in the microflora of skin and mucosal surfaces of the upper respiratory tract of man and animals. The coagulase-positive species such as Staphylococcus aureus are capable of causing invasive (eg furuncles and bacteraemia) and non-invasive (food poisoning and toxic shock syndrome) conditions in humans. In animals, S. intermedius and S. aureus have been implicated in a variety of conditions including pyoderma in dogs, mastitis in cows and skin infections in horses with S. intermedius being responsible for more than 95% of staphylococcal infections in dogs. The emergence of antibiotic-resistant bacteria, in humans, animals and the environment, has become a cause for concern following the introduction of antimicrobial agents in clinical practice. Staphylococcal species, in particular, have developed or acquired antibiotic-resistance determinants to almost all the antimicrobial agents in clinical use today. In recent years there has been an increase in reports of the isolation of antibiotic-resistance staphylococci especially methicillin-resistant staphylococci (MRS), from cats and dogs. Cats and dogs are in close contact with humans, especially in advanced countries and therefore the possibility for transfer of antibiotic-resistant staphylococci from these animals to humans or vice versa may exist. The aims of this study were; to determine the species distribution and antibiotic sensitivity of staphylococci obtained from cats and dogs, to investigate the molecular basis of resistance and to examine the genetic relatedness of specific resistant isolates. Many studies have shown that S. intermedius and S. simulans (S. felis) are the predominante species on healthy dogs and cats, respectively, and lesions in these animals are caused by S. aureus and S. intermedius. In this study, a diverse range of coagulase-negative staphylococci was isolated from healthy animals but staphylococci from skin lesions of cats and dogs were identified mainly as S. intermedius as reported in the literature. A limited number of resistant isolates (~20%) were observed in this study and were mostly isolated from dogs. Investigations into the molecular basis of resistance to beta-lactam, macrolide and tetracycline antibiotics were carried out. The resistant isolates were analysed by polymerase chain reaction (PCR) and DNA sequencing techniques. MRS were analysed for the presence of the mecA gene and the staphylococcal cassette chromosome mec (SCCmec). The recombinase genes, cassette chromosome recombinase (ccr) on SCCmec elements were also examined. The SCCmec elements detected were as diverse as those reported in human staphylococcal strains. Comparative analysis of nucleotide and amino acid sequences of mecA and ccr gene complexes revealed that the genes are conserved among MRS of cat and dog orgin. Multilocus sequence typing (MLST) of methicillin-resistant S. aureus (MRSA) and S. epidermidis isolates showed that, the MRSA were of human origin but the S. epidermidis isolates were unique to cats and dogs. In addition, the blaZ gene which codes for ??-lactamases production, the erm genes responsible for erythromycin resistance and the tet genes which encode tetracycline resistances, were found to be identical to those observed in humans and other animal staphylococci and demonstrated similar diversity. The study has provided important information about the molecular basis of resistance in beta-lactamase producing staphylococci as well as the molecular epidemiology of MRS of cat and dog origin and identifies the risk of spread of MRS between humans and pets and vice versa. These findings should form part of a larger surveillance study on staphylococci of cat and dog origin for a better understanding of the epidemiology of antibiotic-resistance genes for improved management and control of resistant staphylococci in the community and in health care settings. / PhD Doctorate

Thesis (Ph.D)--Malmö University, 2009. / Includes bibliographical references. Also available on the Internet.

Antibiotics are commonly used in animal agriculture to treat and prevent diseases and promote growth. Unfortunately, large amounts of antibiotics are not metabolized, but instead are excreted in urine and feces. Rainfall simulation studies were used to investigate the transport of the antibiotic oxytetracycline and various constituents in runoff and the ability of alum to reduce pollutant transport. Runoff samples were collected at several points during the simulated storm event from each of four treatments: control (C), manure only (M), manure and antibiotics (MA), and manure, antibiotics and alum (MAA). Flow-weighted composite samples were created and compared to the flow weighted mean concentration (FWMC). Constituents with concentrations well-above the detection limits (E. coli, NH4-N, turbidity, TSS, TOC, and EC) showed a strong correlation between flow-weighted composite samples and FWMC. When constituent concentrations were at or near the detection limits, errors associated with the composite samples were magnified. Oxytetracycline concentrations had the strong correlation to E. coli, Cl, TOC, TSS, and turbidity suggesting that a BMP effective at trapping sediment or particulates may work best for reducing oxytetracycline concentrations in runoff. Alum (1%) did not reduce levels of oxytetracycline in runoff. It is recommended that higher doses of alum be tested.

In this project we set out to find when the resistance level against first line antibiotics would reach 20%. This was executed by first defining relevant bacteria and antibiotics for urinary and respiratory tract infections (UTI's, RTI's). The data was collected from the European Center for Disease Control (ECDC) and the Center for Disease Dynamics, Economics & Policy (CDDEP). The data included the level of resistance for specific years for countries in Europe, as well as for the USA. A prediction model was made using the programming language R. A linear model was used to make a five and ten year prediction. The accuracy was tested. The results were then visualized using R and MATLAB. The results show a big variation between different bacteria and antibiotic combinations. For the two E. coli combinations the resistance is already near 20% for many countries and the resistance is increasing. For the three K. pneumoniae combinations the resistance is high in Southern Europe, meaning many countries have reached or are near 20%. For the two P. aeruginosa combinations there is also a higher resistance in Southern Europe but the resistance is decreasing in most countries. The resistance for E. faecalis is also decreasing and is generally very low in all of Europe. For the only RTI relevant combination, S. pneumoniae and penicillins, the resistance is low and many countries except for Sweden show a decrease in resistance. The USA did not have data for the same time span as Europe and was therefore analyzed separately. For many combinations the USA are near the 20% limit. Only for two combinations the USA showed a decrease in resistance level, and for one of those combinations the prediction is too uncertain to make any assumptions about. For the USA there were two more combinations for RTI than for Europe. For the S. pneumoniae and penicillins combination they have, just as most of Europe, a decreasing resistance. The two combinations with Acinetobacter spp. have a high resistance that is increasing. The main challenge during this project was finding relevant data with a long timespan and with high certainty. The data found is based on invasive isolates which means that the disease which the samples are taken from is not known. The timespan and the certainty of the data affected the accuracy of the prediction model and how long period that could be predicted. The prediction model generated 202 predictions that were visualized. An ethical analysis was made concerning both research ethics and general ethics on the topic of antibiotic resistance. This analysis is meant to acknowledge these questions since we believe they are important when discussing antibiotic resistance. The objective of this project turned out to be more difficult to attain than first believed. This was because of the lack of quality data. Even though we cannot give a clear answer when each country will reach a resistance of 20% this report gives a good understanding of how the situation looks for UTI and RTI relevant bacteria.

Orientador: Antonio Celso Pezzato / Resumo: RESUMO. Para avaliar a inclusão de diferentes combinações e níveis de prebióticos em substituição aos antibióticos melhoradores de desempenho, dois experimentos foram delineados. No primeiro estudo, 1250 pintos de corte machos, linhagem ROSS AP95, foram casualmente distribuídos em 5 tratamentos, com 10 repetições cada, objetivando testar diferentes proporções de mananos com β-glucanos e frutoligossacarídeos com galactoligossacarídeos, combinados e incluídos em dietas de frangos de corte. Os tratamentos consistiram de: CP, dieta basal (DB) +AMD; DB+BUT: DB + Butirato de Cálcio; GM1⁄3+FG2⁄3: DB + GLUCANMOS (1/3 da mistura) : FOS:GOS (2/3 da mistura); GM1⁄2+FG1⁄2: DB + GLUCANMOS (1/2 da mistura) : FOS:GOS (1/2 da mistura); GM2⁄3+FG1⁄3: DB + GLUCAN MOS (2/3 da mistura) : FOS:GOS (1/3 da mistura). No segundo estudo, 1250 pintos de corte machos, linhagem ROSS AP95, foram casualmente distribuídos em 5 tratamentos, com 10 repetições cada, objetivando testar diferentes níveis de inclusão de GLUCANMOS (67% da mistura) associado a FOS:GOS (1:1). Os tratamentos consistiram de: CP, dieta basal (DB) +AMD; 0,1%; 0,2%; 0,3% e 0,4% de inclusão da mistura prebiótica. Foram avaliados o desempenho, peso relativo de órgãos, barreiras físicas e imunológicas dos segmentos do intestino delgado (integridade de vilosidades, células caliciformes e linfócitos intraepiteliais), histomorfometria de Bursa de Fabricius e contagem diferencial de leucócitos no sangue, ainda, foram quantificados os ácidos grax... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Hyperbaric Oxygen Therapy (HBOT) is an old technology which has acquired value in chronic wound care. HBOT is known to promote local and systemic healing effects by improving the oxygenation of the wound tissue. The increased tissue oxygenation hastens removal of the bacterial bioburden, which allows resolution of inflammation and facilitates matrix production, cell division, and ultimately wound closure. Staphylococcus aureus is the most frequently isolated organism from Diabetic Foot Infections (DFI). Therefore, our lab chose to use the treatment paradigm of HBOT to initially look at the single species level as to how HBOT affects S. aureus. DFI are primarily polymicrobial, so the responses of bacterial communities to this therapy were also considered. Previous research focused solely on host response to HBOT, but our pilot testing indicates that HBOT also exhibits a bacterial response. Initial testing with S. aureus indicated that HBOT can create growth defects in bacteria in vitro. In preliminary experiments, our lab discovered that bacterial culture on solid medium is greatly altered under the pressure of hyperbaric oxygen. Normal robust growth and pigmentation are seen in S. aureus cultured in ambient conditions. However, when the same strain is cultured under HBOT conditions, there is a marked decrease in pigmentation and colony size. When other species were exposed to HBOT conditions, growth on solid media was significantly diminished. Interestingly, K. pneumoniae is able to grow normally under HBOT conditions. Normal air mixtures at the increased pressure do not have any discernable effect on bacterial growth, and the limiting effects of oxygen are not seen unless used at the increased pressure. In a broth macrodilution MIC assay, various antibiotics show an increase in susceptibility after exposure to HBOT. Lastly, biofilm formation is altered under HBOT conditions, further supporting a bacterial adjustment to HBOT and an altered mode of growth. In order to better understand the effects of a high pressure high oxygen environment on the bacterial bioburden, this study investigates the effects of HBOT on bacterial species comprising a chronic wound. Primary data has suggested that HBOT increases susceptibility of antibiotics, and can alter bacterial transcription to hinder growth of many organisms. We hypothesize that Hyperbaric Oxygen Therapy affects diabetic foot infections by changing the healing process via transcriptional alteration of bacterial species in the wound. Furthermore, we hypothesize that HBOT alters the efficacy of some antibiotics as well as affecting the biofilm capacity of many bacterial species.

It has long been appreciated that genetic diversity can be stably maintained in spatially structured environments depending on the method by which subdivided populations are regulated. Theory suggests diversity in spatially heterogeneous environments can be stably maintained when populations are regulated at the level of each individual subpopulation (soft selection) rather than at the level of the total mixed population (hard selection). Although mathematically established decades ago, direct empirical tests of these predictions remain scarce, and its robustness to prolonged selection on evolutionary timescales remain unclear. Here, we track the emergence and fate of diversity in replicate laboratory populations of the opportunistic human pathogen, Pseudomonas aeruginosa, evolving in spatially structured environments composed of patches with or without antibiotic selection. We manipulate the scale of population regulation by transferring either a fixed number (soft selection) or volume (hard selection) of cells. We find that, while both forms of population regulation can lead to prolonged coexistence between resistant and sensitive strains, diversity is eventually lost by the evolution of broadly adapted resistant strains that pay lower costs of resistance. Notably, coexistence on average persists longer under soft selection than hard selection, consistent with the idea that soft selection is more effective at maintaining diversity than hard selection. Our results suggest that, in line with theory, the manner of population regulation can impact the maintenance of diversity and that the ecological conditions supporting diversity in the short term can be readily undermined by evolutionary processes in the long term.

La résistance aux antibiotiques est une menace non négligeable pour la santé publique. Ces résistances peuvent être portées par différents supports dont les îlots génomiques. Il a été démontré que les îlots génomiques Salmonella Genomic Island 1 (SGI1) et Proteus Genomic Island 1 (PGI1) sont des acteurs importants de la multirésistance aux antibiotiques. Quelques variants de SGI1 et PGI1 ont déjà été décrits au sein de l’espèce P. mirabilis. Dans ce contexte, ce projet de thèse se proposait d’approfondir notre connaissance de la situation épidémiologique de la diffusion de SGI1 et PGI1 chez P. mirabilis chez l’homme et l’animal en France, en ce qui concerne la diversité des isolats, mais aussi celles des variants de SGI1/PGI1. En parallèle, une autre volonté a été d’identifier d’autres facteurs et acteurs permettant l’acquisition de gènes de résistances d’intérêt au sein des Morganellaceae (β-Lactamases à Spectre Etendu, céphalosporinase AmpC, Plasmid-mediated Quinolone Resistance...). Au final, cette étude a permis en outre de révéler les premiers cas de SGI1 et PGI1 chez P. mirabilis chez l’animal en France. De nouveaux variants de SGI1 ont également été mis en évidence. Et pour la première fois, SGI1 a été décrit chez M. morganii, une autre espèce d’entérobactérie. / The antibiotic resistance is a major treat for public health. These resistances can be hold by different element and genomic islands are one of them. Salmonella Genomic Island 1 (SGI1) and Proteus Genomic Island 1 (PGI1) are important genetic elements for the antibiotic resistance. A few SGI1 and PGI1 variants were already described in P. mirabilis. It is in this context that this thesis project aimed to improve our knowledge about the epidemiological spread of SGI1 and PGI1 in P. mirabilis in humans but also in animals in France (diversity of isolates and SGI1/PGI1 variants). Moreover, another wish was to identify other factors and actors for the acquisition of antibiotic resistance in the Morganellaceae tribe (Extended-Spectrum β-Lactamases, AmpC cephalosporinase, Plasmid-mediated Quinolone Resistance…). Finally, this study revealed the first cases of SGI1 and PGI1 in P. mirabilis in animals in France. New SGI1 variants were also described. And for the very first time, SGI1 was found in M. morganii, another entrobacterial species.

The continuing rise of antibiotic resistance is threatening a return to the world of pre-antibiotic medicine. Multi-drug resistant pathogens are already claiming lives and causing economic losses in developing and developed countries alike. We need, therefore, to understand what allows resistant strains to spread; what makes them evolutionarily competitive in and amongst other strains and species. The majority of laboratory studies of antibiotic resistance focus on simple growth in liquid culture. By contrast, microbes commonly grow as surface-associated communities, in which interactions between neighbouring cells have strong consequences for competition and evolution. My first goal was to understand how growth in such environments affects the success of a resistant strain. By competing an antibiotic resistant and susceptible strain of the pathogenic bacterium Pseudomonas aeruginosa, I found that growth in dense colonies on agar allowed a resistant strain to protect susceptible strains, to the extent that the susceptible strain may even prevail under antibiotic treatment. This effect was specific to a cooperative mechanism of antibiotic resistance, however; a β-lactamase enzyme that digests the antibiotics surrounding a resistant cell. A further, unexpected reason that susceptible cells could prevail was that they elongate under antibiotic treatment, allowing them to push shorter resistant cells aside in the competition for the growing edge of a colony. My work suggests that the rise of cooperative resistance mechanisms should be more easily suppressed than for non-social mechanisms. However, one major strategy to overcome antibiotic resistance is the use of antibiotic-adjuvants, drugs which inhibit a mechanism of antibiotic resistance. It is not clear if these adjuvants will tend to suppress or promote cooperative resistance mechanisms. I performed experiments to test the effects of inhibitory adjuvants on cooperative resistance. These revealed that the effects of adjuvants are varied. In liquid culture, an adjuvant inhibited resistance evolution, while, in colony experiments, it promoted resistance evolution by removing the cross protection of susceptible strains. Given the complexity and importance of antibiotic adjuvants, I developed an eco-evolutionary model to dissect these complexities associated with the combination of interacting microbial and molecular species. As in my experiments, the models identified conditions where an inhibitory adjuvant can increase selection for resistance. However, the theory also identifies scenarios for which adjuvants will delay resistance evolution by shutting down the associated evolutionary pathway. Broadening the modelling framework to include the stochastic effects of rare mutation, I found that early administration of adjuvant inhibitors can be a powerful way to suppress the emergence of antibiotic resistance. Microbial interactions are complex and affected by the growth environment. My thesis underlines that the study of antibiotic resistance will benefit from greater consideration of how bacteria interact and, more broadly, how their ecology and evolution determine the rise, or fall, of resistance.