The ubiquitous nature of antibiotic resistance and antibiotic resistance genes (ARGs) among environmental pathogens from a variety of wastewater effluents, suggests that the aquatic environment, and specifically alternative wastewater treatment systems, may act as reservoirs for drug resistant bacteria and ARGs, thereby contributing to the widespread dissemination of antibiotic resistance. More research is necessary to contribute to our understanding of the occurrence, fate, and mobility of antibiotic resistance and ARGs among bacterial indicators of faecal contamination as well as pathogenic bacteria within Canadian wastewater treatment systems. The primary objective of this research was to determine the prevalence, fate, and potential transfer of bacterial resistance and ARGs among selected environmental pathogens exposed to alternative wastewater treatment systems, while considering the impact of treatment strategies on the expression of antibiotic resistance. A detailed analysis was initially conducted with respect to the characterization and quantification of microbial populations (including antibiotic resistant bacteria) in a variety of treatment systems and waste effluent sources. Traditional culture-based screening techniques in combination with molecular characterization (through colony or multiplex PCR), and molecular quantification using real-time quantitative PCR were utilized in order to help establish a preliminary environmental assessment of selected pathogens (Escherichia coli, Enterococcus spp., Salmonella spp.) and ARGs (tetA, blaSHV, & ampC) within a variety of wastewater treatment systems (lab-scale mesocosms, constructed wetland, constructed lagoon system, and pilot-scale biological nutrient removal (BNR) system). Overall, the level of multiple antibiotic resistance (MAR) among culturable indicator (E. coli & Enterococcus spp.) and environmental bacteria was high (reaching 100% in several instances) within different types of wastewater treatment systems and effluent sources (poultry waste effluent, municipal wastewater, aquaculture wastewater). Common antibiotic resistance profiles among E. coli isolates included simultaneous resistance to between three and five antimicrobials, whereas common MAR profiles among Enterococcus spp. isolates showed resistance to ten or more antibiotics. Real time quantitative PCR was used to determine the concentration of three bacterial pathogens; E. coli, Enterococcus faecalis, and Salmonella spp., and three ARGs; tetA, ampC, and blaSHV, within a variety of wastewater samples. Based on the results, it was concluded that high concentrations of ARGs were present in the treated effluent (10⁴- 10⁶ target gene copies/100 mL), regardless of system type (i.e. constructed lagoon, pilot-scale BNR, or constructed wetland), which may ultimately serve as a potential route for entry of ARGs and antibiotic resistant bacteria into the natural environment. Water is considered an important medium for transfer of resistance genes and resistant bacteria to the broader environment. Few studies have examined the transferability via conjugation of ARGs in E. coli and Salmonella spp. isolated from wastewater. Identification of three resistance determinants (tetA, strA, strB) conferring resistance to tetracycline and streptomycin was performed on selected multi-drug resistant Salmonella spp. and E. coli isolates. The potential for transfer of tetracycline and streptomycin resistance genes was demonstrated through broth conjugation experiments using multi-drug resistant Salmonella spp. and E. coli isolates as donors, and E. coli K12 as the recipient. Conjugation was successfully observed in 75% (9/12) of donor isolates, occurring in both Salmonella spp. and E. coli isolates. Six strains (50%) were capable of transferring their tetA, strA, and strB genes to the recipient strain, resulting in 58.5% (38/65) of total transconjugant strains acquiring all three resistance determinants. The results confirm the role of environmental bacteria (isolated from wastewater treatment utilities) as a reservoir of antibiotic resistance and ARGs, containing mobile genetic elements, which are capable of disseminating and transferring ARGs. As concerns about water quality and environmental contamination by human and agricultural effluents have increased, it has become increasingly more important to consider the prevalence and transferability of ARGs to opportunistic and human pathogens. As observed in this research, the ubiquitous nature of multi-drug resistant bacteria in water and wastewater effluents, the presence of diverse ARGs of human and veterinary health significance, as well as the transfer of resistance determinants through conjugative plasmids to recipient bacteria, suggests that environmental exposure through contact or consumption with contaminated water is probable. However, a lack of critical information still exists regarding the movement of resistance genes within and between microbial populations in the environment. In addition, the extent of human exposure to ARGs and antibiotic resistant bacteria is still not well understood, and future studies on human exposure to these resistant contaminants are necessary.

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

Since penicillin was discovered in 1928, antibiotic usage in human and veterinary medicine and prevalence of antibiotic resistant bacteria (ARB), has been increasing. While antibiotics and antibiotic resistance genes (ARGs) naturally occur in soils, increasing abundances of ARGs correlate with increased antibiotic usage in agricultural settings. When livestock are treated with antibiotics, the antibiotic compounds, ARB, and ARGs can enter soil via manure excreted onto pastures or applied to other fields as fertilizer, thereby spreading antimicrobial resistance (AMR) in the environment. In addition to human health implications, increased AMR has negative impacts on ecosystem services such as carbon and nitrogen cycling. While many studies have researched antibiotic persistence in agricultural systems and their impacts on soil microbial communities, there are still significant knowledge gaps around the long-term effects of antibiotic exposure in soils, how those impacts differ among soils, and how elements of AMR may differentially transport through soil. To address these knowledge gaps, our objectives were to 1) examine the impact of multi-year repeated additions of manure from cattle administered antibiotics on soil microbial communities, 2) determine the interactive effects of soil moisture and type on soil microbial communities exposed to antibiotics and manure, and 3) differentiate between vertical transport of AMR in the form of viable ARB or ARGs in extracellular plasmids. Our results demonstrate that soil bacterial community structures were consistently altered by 3-year additions of manure from cattle administered antibiotics compared to soil amended with antibiotic-free manure. Furthermore, ARG abundances were higher in soils with manure additions compared to soil without manure, although this was true regardless of whether the cattle were administered antibiotics, suggesting that manure and antibiotic impacts on soil microbial communities can persist over multi-year of repeated manure applications. Additionally, in microcosms, effects of manure from cattle administered antibiotics on ARG abundances, microbial community structures, respiration, and nitrogen pools in soil were seen across multiple soil types and moisture contents, suggesting environmental conditions can alter how manure and antibiotics impact microbial community structure and nutrient cycling. Finally, ARB flowed readily through saturated soil, but were also detectable in the top 5 cm of soil columns. However, ARGs on extracellular plasmids did not flow through soil columns and were not detected in soil, indicating that extracellular DNA does not persist or transport through the soil to any meaningful degree. Overall, these results indicate a nuanced approach is required to mitigate the environmental spread of AMR. Soil management strategies for addressing the AMR crisis should consider the broader context of manure management, as high ARG abundances can come from application of manure from antibiotic-free cattle, and soil microbial communities in individual environments may have varied responses to manure antibiotic exposure. Furthermore, the transport of AMR through soil is complex and dynamic, as elements of AMR may transport differently through soil and require separate consideration in modeling and management. Future AMR management practices that consider diverse factors that affect persistence and spread of AMR in the environment can help protect livestock productivity and maintain the efficacy of antibiotics to protect human and animal health. / Doctor of Philosophy / Antibiotics are an important tool used to fight infections in humans, pets, and livestock. As antibiotics are used more frequently, the bacteria they target are more likely to develop resistance to the antibiotics, leading to increasing cases of infections that are harder to treat and higher risk. Antibiotic resistance can persist and spread in multiple forms, including the antibiotic compounds themselves, as antibiotic resistant bacteria (ARB), or as the genetic material that encodes for antibiotic resistance genes (ARGs). In agricultural systems, when livestock are treated with antibiotics they can excrete the antibiotics, along with ARB and ARGs, in the manure, which is then applied to land as fertilizer. In addition to the associated health risks, the spread of antibiotic resistance impacts microscopic bacteria and fungi in the soil, which are important for recycling nutrients for plants and maintaining ecosystem health. The overall goal of this dissertation was to gain a better understanding of how manure from cattle given antibiotics impacts these bacteria and fungi when manure is applied to the soil. The specific objectives were to 1) look impacts after long-term (multiple years) of manure addition, 2) examine how bacteria and fungi might respond differently to antibiotics in soils of different type or with different amounts of water, and 3) determine if ARGs that exist as free genetic material outside of living bacteria can be moved through the soil with flowing water in the same way as living bacteria. Results showed that while the composition of bacterial and fungal communities in the soil vary from year to year, adding manure with and without antibiotics had both caused different and consistent changes on the composition of bacterial communities. There were also higher concentrations of ARGs in soil that had manure added, however antibiotics in the manure did not cause ARGs to increase further, suggesting that even antibiotic-free manure can impact the spread of antibiotic resistance. Experimental work also demonstrated that the soil type and water content of soil can alter how bacteria and fungi respond to antibiotics in manure. The composition of bacterial and fungal communities, their activity rates, and the amount of nitrogen – an important plant nutrient with availability that is strongly affected by microbial activity – all differed with soil type and water content. Thus, while antibiotic resistance antibiotic resistance can cause measurable changes in soil across a range of environmental conditions, it is also likely to persist and spread in different ways in different environments. Finally, when water containing elements of AMR was added to soil, ARB were shown to both move through the soil easily and remain near the top of soil. In contrast, ARGs contained on genetic material outside of living cells did not move through the soil and were broken down within a few days, suggesting that antibiotic resistance likely spreads through living bacteria more than genes outside of cells. Overall, this work highlights the complexity of understanding the role of environmental transmission in the antibiotic resistance crisis and demonstrates the need for nuanced management approaches that take specific environments and conditions into account.

Antibiotic resistance is a growing human health threat, making infections more difficult to treat and increasing fatalities from and cost of treatment of associated diseases. The rise of multidrug resistant pathogens threatens a return to the pre-antibiotic era where even the most common infections may be impossible to treat. It is estimated that the majority of global antibiotic use, and use in the U.S., is dedicated towards livestock, where they are used to promote growth, treat, or prevent disease. Given that exposure to antibiotics selects for antibiotic resistant bacteria (ARBs) and can stimulate the horizontal transfer of their associated antibiotic resistance genes (ARGs), it is important to examine livestock operations as a reservoir of resistance. Correspondingly, there is growing interest in identifying how agricultural practices can limit the potential for spread of antibiotic resistance through the "farm to fork continuum," starting with antibiotic use practices, manure management and land application and ending with the spread of ARBs and ARGs present onto edible crops and serving as a route of exposure to consumers. This study focused specifically on the effect of composting on the prevalence of ARBs and ARGs in cattle manure. Three composting trials were performed: small-scale, heat-controlled, and large-scale. The small-scale composting trial compared dairy and beef manures, with or without antibiotic treatment (treated beef cattle received chlortetracycline, sulfamethazine, and tylosin while treated dairy cattle received cephapirin and pirlimycin), subject to either static or turned composting. The heat-controlled composting trial examined only dairy manure, with or without antibiotic treatment, subject to static composting, but using external heat tape applied to the composting tumblers to extend the duration of the thermophilic (>55°C) temperature range. The large-scale composting trial examined dairy manure, with or without antibiotic treatment, subject to static composting at a much larger scale that is more realistic to typical farm practices. Samples were analyzed to assess phenotypic resistance using the Kirby Bauer disk diffusion method and by diluting and plating onto antibiotic-supplemented agar. Genetic markers of resistance were also assessed using quantitative polymerase chain reaction (qPCR) to quantify sul1 and tet(W) ARGs; metagenomic DNA sequencing and analysis were also performed to assess and compare total ARG abundance and types across all samples. Results indicate that composting can enrich indicators of phenotypic and genetic resistance traits to certain antibiotics, but that most ARGs are successfully attenuated during composting, as evidenced by the metagenomic sequencing. Maintaining thermophilic composting temperatures for adequate time is necessary for the effective elimination of enteric bacteria. This study suggests that indicator bacteria that survive composting tend to be more resistant than those in the original raw manure; however, extending the thermophilic stage of composting, as was done in the heat-controlled trial, can reduce target indicator bacteria below detection limits. Of the two ARGs specifically quantified via qPCR, prior administration of antibiotics to cattle only had a significant impact on tet(W). There was not an obvious difference in the final antibiotic resistance profiles in the finished beef versus dairy manure composts according to metagenomics analysis. Based on these results, composting is promising as a method of attenuating ARGs, but further research is necessary to examine in depth all of the complex interactions that occur during the composting process to maximize performance. If not applied appropriately, e.g., if time and temperature guidelines are not enforced, then there is potential that composting could exacerbate the spread of certain types of antibiotic resistance. / Master of Science

Widespread use of antibiotics in livestock production can result in the dissemination of bacteria carrying antibiotic resistance genes (ARGs) to the broader environment. Within agroecosystems, ARGs can pose a risk to livestock handlers, farmers, and ultimately consumers. The overall goals of this dissertation are to examine the presence of resistance (antibiotic, metal) in agricultural soils and evaluate the most critical potential points of best management control of antibiotic resistance spread along the agricultural production chain. The relative impacts of agricultural practices, manure management, native soil microbiota, and type of crop grown and harvested on the agricultural resistome are multi-dimensional and cannot be captured via a single analytical technique or by focusing on one specific point in the agricultural process. Culture-, molecular "indicator"-, and next-generation sequencing- based methods were employed to characterize antibiotic resistance via taxonomic and functional profiles on the broader manure, soil, and vegetable surface microbial communities through 16S rRNA amplicon sequencing and shotgun metagenomics. Although antibiotic concentrations dissipated in the soil after 28 days after amendment application, antibiotic resistance presence was recoverable throughout the entire 120d growing season in the compost and manure amendments, the amended soil, and on vegetable surfaces. The addition of organic fertilizers increased antibiotic resistance presence compared to background levels. Further, metals and metal resistance were also measured in the amended soils and were found to be at greater levels in the inorganically fertilized soils compared to the manures and compost amended soils. Analysis of the widespread agroecosystem microbial community composition and broader metagenome has characterized varying genera profiles in the soil and on the vegetable surfaces and specific ARG and mobile genetic element (plasmid) co-occurrences. These co-occurrences highlight which ARGs may be most critical for future antibiotic resistance dissemination research. It is imperative to employ multiple methods when measuring agricultural resistance, as one method alone may miss significant patterns and lead to different best management recommendations. Linking the livestock manure, soil, and vegetable surface-associated ARBs, ARGs, resistomes, and microbiomes will help identify critical control points for mitigation of agricultural dissemination of antibiotic resistance to the environment and food production. / Doctor of Philosophy / By 2050, it is estimated that antibiotic resistant infections will be the leading cause of death worldwide. It is important to consider human, animal, and environmental health when researching antibiotic resistance, which is known as a "One Health" approach. In this dissertation work, I focus on the environmental side of antibiotic resistance in our agricultural systems. Agriculture is a known source of antibiotic resistance due to its use of antibiotics in livestock as a treatment for illness, and in some instances, as a growth promoter. Over one growing season, I measured antibiotic resistance in an agricultural setting using many techniques. First, I analyzed the effects of inorganic (chemical) versus organic (manure and compost) fertilization on antibiotic resistance in the soil. I measured antibiotic resistance by growing antibiotic resistant bacteria, quantifying specific antibiotic resistant genes (ARGs) using DNA amplification, and quantifying all the ARGs in the soil using a next-generation sequencing (NGS) technique called shotgun metagenomics. I found that adding manure to the soil increases ARGs compared to background soil levels, and that composting in an effective management strategy in decreasing ARGs in the soil over time. Second, I analyzed the same effects of fertilization on metal resistance in the soil. I was able to use the same NGS dataset to measure metal resistance genes (MRGs). I found that adding inorganic chemical fertilizer increases MRGs in the agricultural soils compared to the organic (manure or compost) fertilizer. Additionally, I studied the microbes that live in the agricultural soils using another kind of NGS data specific for microbial identification. I found that although there were small differences between the microbial populations in the soil when fertilizers were added, they returned to similar composition over the growing season. Lastly, I measured antibiotic resistance and microbes throughout the entire agricultural system. I picked the point of fertilization (manure management), soil, and the lettuce surface to evaluate if antibiotic resistance spreads from the farm to the vegetable that ends up on a consumer's plate. I found that at each point antibiotic resistance is present, but at different levels. Composting reduces ARGs compared to raw manure. Agricultural soils may act as a natural buffer to antibiotic resistance. Lettuce plants grown in compost fertilized soils have less ARGs than lettuce plants grown in manure. There are many agricultural management practices that effectively reduce antibiotic resistance and using all of them plus many measurement methods will ultimately help farmers and consumers reduce antibiotic resistance in our agricultural systems.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The arsenic resistance operon (ars operon) of the Gram-positive, iron-oxidizing, acidophilic, moderately thermophilic bacterium, Sulfobacillus thermosulfidooxidans VKM B-1269T (Sb. t. VKM B-1269T), was isolated and characterised. The ars operon was chromosomally located and consisted of an arsR (codes for a transcriptional regulator) and an arsB (codes for a membrane located arsenic/antimony efflux pump). The arsRB genes were transcribed in the same direction. An arsC (codes for an arsenate reductase), usually associated with ars operons, was absent from this ars operon. PCR and Southern-hybridization experiments revealed that no arsC, representative of either the Grx/GSH or Trx ArsC families was present in the genome of Sb. t. VKM B-1269T. An interesting feature of the ars operon was the presence of a gene encoding a 525 amino acid (60.83 kDa) kumamolisin-As precursor located upstream of the arsRB operon. The intergenic region between the termination end of the kumamolisin-As precursor gene and the transcriptional start of the arsR gene was only 77 bp, suggesting that this ars operon might consist of three genes. RT-PCR analysis showed that the ars operon of Sb. t. VKM B-1269T, was not co-transcribed with the kumamolisin-As precursor gene in its native Sulfobacillus host. The ars operon of Sb. t. VKM B-1269T did not complement an Escherichia coli arsenic sensitive mutant. mRNA transcript analysis and promoter expression studies confirmed that processes involved in the production of functional proteins from the ars operon transcript were likely to be responsible for the inability of the arsRB operon of Sb. t. VKM B-1269T to confer resistance to arsenic in the heterologous E. coli host. Eight Sulfobacillus strains isolated from different geographical areas were subjected to amplified ribosomal DNA restriction enzyme analysis (ARDREA) using the restriction endonuclease Eco1015 (SnaBI) and revealed that they could be divided into the proposed Sulfobacillus spp. subgroup I and subgroup II, respectively (Johnson et al., 2005). The presence, distribution and relatedness of the ars genes among members of genus Sulfobacillus was determined. Phylogenetic sequence comparisons revealed two clearly defined arsB clusters within genus Sulfobacillus and showed that the arsB of a specific Sulfobacillus sub specie is distinctive of that specific Sulfobacillus sub specie. Futhermore, sequence analysis of the isolated arsB homologue fragments from the respective Sulfobacillus spp. showed that four distinctive profiles could be identified based on differences in the location of restriction endonuclease recognition sites. / AFRIKAANSE OPSOMMING: Die arseen weerstandbiedendheidsoperon (ars operon) van die Gram-positiewe, ysteroksiderende, asidofiliese, matige termofiliese bakterium, Sulfobacillus thermosulfidooxidans VKM B-1269T (Sb. t. VKM B-1269T), was geïsoleer en gekarakteriseer. Die ars operon was op die chromosoom geleë en het uit ‘n arsR (kodeer vir ‘n transkripsionele reguleerder) en ‘n arsB (kodeer vir ‘n membraan geleë arseen/timien uitskeidings pomp) bestaan. Die arsRB gene word in dieselfde rigting getranskribeer. ‘n arsC (kodeer vir ‘n arsenaat reductase), wat gewoontlik geassosïeer word met ars operons, was afwesig van hierdie ars operon. PKR en Southern-hibridisasie eksperimente het aangedui dat geen arsC, verteenwoordigend van beide die Grx/GSH of Trx ArsC families, nie teenwoordig was in die genoom van Sb. t. VKM B-1269T, nie. ‘n Interressante eienskap van hierdie ars operon was die teenwoordigheid van ‘n geen wat stroom-op van die arsRB operon geleë is en ‘n 525 amino suur (60.83 kDa) kumamolisin-As voorloper kodeer. Die intergeniese gedeelte tussen die terminerings einde van die kumamolisin-As voorloper en die transkriptionele begin van die arsR geen was slegs 77 bp, wat voorgestel het dat die ars operon moontlik uit drie gene bestaan. RT-PKR analiese het bewys dat die ars operon van Sb. t. VKM B-1269T, nie geko-getranskribeer word met die kumamolisin-As voorloper in sy oorspronklike Sulfobacillus gasheer nie. Die ars operon van Sb. t. VKM B-1269T, het nie ‘n Escherichia coli arseen sensitiewe mutant gekomplimenteer nie. mRNA transkrip-analiese en promoter uitdrukkings eksperimente het bevestig dat prosesse wat betrokke is in die produksie van funksionele proteïene vanaf die ars operon transkrip, moontlik vir die onvermoë van die arsRB operon van Sb t. VKM B-1269T verantwoordelik was om weerstandbiedendheid teen arseen in die heteroloë E. coli gasheer te verleen. Agt Sulfobacillus stamme wat geïsoleer is vanuit verskillende geografiese areas, was onderhewig aan geamplifiseerde ribosomale DNA restriksie-ensiem-analiese (ARDREA) deur gebruik te maak van restriksie endonuklease Eco1015 (SnaBI) en het aangedui dat hulle in die voorgestelde Sulfobacillus spp. subgroup I en subgroup II ingedeel kan word (Johnson et al., 2005). Die aanwesigheid, verspreiding en verwantskappe van die ars gene tussen lede van genus Sulfobacillus was bepaal. Filogenetiese DNA volgorde vergelykings het aangedui dat twee duidelik definïeerbare arsB groepe van mekaar onderskei kan word en dat die arsB van ‘n spesifieke Sulfobacillus sub spesie uniek tot daardie spesifieke Sulfobacillus subspesie is. Bykomend, DNA volgorde analiese van die geïsoleerde arsB homoloog fragmente van die Sulfobacillus spp. het gewys dat vier unieke profiele, op grond van verskille in die ligging van restriksie ensiem herkenning setels, geïdentifiseer kan word.

Copper cast alloys de-activate antibiotic-resistant bacteria on contact and could be very effective in decreasing potentially harmful microorganisms in the environment. In this study copper alloys with varying copper contents were utilized to evaluate their antimicrobial effects on Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecium in changing environmental conditions. The survival rates of P. aeruginosa wild type and its derivative transposon mutants of the cin operon on copper cast alloys were investigated in order to demonstrate the influence of genes involved in copper resistance. The gene disruption of the response regulator of the cin operon resulted in shorter survival rates on copper alloys, which was also influenced by temperature and method of exposure. Bacteria often have acquired copper resistance mechanisms in order to withstand higher copper concentrations in their surroundings, which may be a factor in their survival rates on copper surfaces. Copper- and antibiotic-resistant E. coli and E. faecium strains were obtained from pigs raised on feeds containing copper sulfate and antibiotics. Survival rates of these bacteria were influenced by the percentage of copper in the alloys, varying moisture conditions on copper surfaces, suspension media used, and differences between strains. Survival was also dependent on copper corrosion rates since corrosion inhibition with benzotriazole or a thick surface layer of thermal oxide resulted in prolonged survival on copper surfaces. Corrosion of copper surfaces releases copper ions which directly affect bacterial survival on copper alloys. The results obtained in this study emphasize that copper alloys are effective as antimicrobial materials but changing environmental conditions can significantly influence bacterial survival on copper surfaces. These findings can be applied to a better utilization of copper alloys in water, food, and healthcare environments. Antibiotic- and copper ion-resistant bacteria can be killed on contact with copper alloys which makes the proliferation of these microorganisms less likely and reduces the risk to human health. However, in a very different environment copper ion-resistant microbes can be useful for plant-microbe associations in bioremediation of copper mining wastelands.

Gene orf18, which is situated in the conjugative transposon Tn916 from the bacterial pathogen Enterococcus faecalis, encodes a putative ArdA (alleviation of restriction of DNA) protein. ArdA from Tn916 may be responsible for the apparent immunity of the transposon to DNA restriction and modification (R/M) systems and for ensuring that the transposon has a broad host range. The orf18 gene was engineered for overexpression in Escherichia coli and the recombinant ArdA protein was purified to homogeneity. Biophysical characterisation of ArdA demonstrated tight association between ArdA and the M.EcoKI. Also, ArdA was shown to efficiently inhibit restriction and modification by all four major classes of Type I R/M enzymes in vivo. Thus, ArdA can overcome the restriction barrier following conjugation and so helps to increase the spread of antibiotic resistance genes by horizontal gene transfer. The amino acid sequence of KlcA, from the incompatibility plasmid pBP136 from Bordetella pertussis, showed a high degree of similarity with the antirestriction protein ArdB from the IncN plasmid pKM101. In this study the solution structure of KlcA was solved with high-resolution NMR and its antirestriction function demonstrated. The structure of KlcA showed a rigid globular molecule with a novel fold. No antimodification function was observed for KlcA in vivo and the antirestriction function of KlcA has been successfully shown in vivo but not in vitro. Because no direct binding of KlcA to EcoKI was observed in vitro, the mechanism of the endonuclease blocking was assumed to be different from that of ArdA. Preliminary experiments including coimmunoprecipitation assays were conducted in order to elucidate the antirestriction mechanism of KlcA.

La rouille orangée du caféier causée par le champignon biotrophe Hemileia vastatrix (Berk et Br.) est la maladie la plus dévastatrice du caféier (Coffea arabica L.). La résistance à la rouille dans l'espèce allotétraploïde C. arabica semble jusqu'à présent conditionnée principalement par 9 facteurs de résistance majeurs (SH1-SH9), seuls ou en combinaison. Les variétés d'Arabica contenant le facteur de résistance SH3 introgressé d'une espèce de caféier sauvage (C. liberica) ont démontré une résistance durable au champ. L'objectif principal de ce travail était d'étudier l'organisation génomique, l'évolution et la diversité du locus SH3 chez le caféier et parallèlement de développer des outils permettant la validation fonctionnelle des gènes candidats pour la résistance à la rouille. Comme Une carte physique couvrant la région SH3 a tout d'abord été construite chez C. arabica et la position du locus de résistance a été délimitée dans un intervalle de 550 kb. La région a été séquencée chez trois génomes de caféier, Ea et Ca sous-génomes de C. arabica et Cc génome de C. canephora. L'analyse des séquences a révélé la présence d'un nombre variable de membres de la sous-classe CC-NBS-LRR ; ces gènes semblant être exclusivement présents à ce locus chez C. arabica. L'analyse génomique comparative indique que i) l'origine de la plupart des copies SH3-CNL est antérieure à la divergence entre les espèces de Coffea, ii) la copie ancestrale SH3-CNL a été insérée dans le locus SH3 après la divergence entre les Solanales et Rubiales. En outre, les mécanismes de duplication, délétion, conversion génique et de sélection positive semblent être les principales forces qui déterminent l'évolution des membres de la famille SH3-CNL. Différentes approches ont été entreprises pour le clonage des gènes candidats de résistance à la rouille. En parallèle, un protocole très efficace de transformation par Agrobacterium tumefaciens a été développé chez le caféier. Des plantes transgéniques contenant l'un des gènes R candidats ont été produites et acclimatées. Le bio-essai pour évaluer la résistance à H. vastatrix a été mis au point. La mise en place de protocoles efficaces pour le clonage des gènes de résistance et la transformation génétique ouvre la voie à la génomique fonctionnelle en routine chez le caféier. L'amélioration des connaissances sur la diversité des gènes de résistance à la rouille permettra l'optimisation des schémas de sélection pour la résistance durable à cette maladie majeure du caféier. / Coffee leaf rust (CLR) caused by the biotrophic fungus Hemileia vastatrix (Berk and Br.) is the most devastating disease of Arabica coffee (Coffea arabica). Resistance to CLR in its allotetraploid species appears so far conditioned primarily by 9 major resistance factors (SH1-SH9) either singly or in combination. Arabica varieties harboring the SH3 resistant factor introgressed from a wild coffee species (C. liberica) have demonstrated agronomical acceptable durable resistance in field conditions. The main objective of this work was to study the genomic organization, evolution and diversity of SH3 locus in coffee and in parallel to develop tools for functional gene validation of resistance candidate genes to CLR. As the first step, a physical map spanning the SH3 region in C. arabica was constructed and the position of the resistance locus was delimited within an interval of 550 kb. The region was completely sequenced in three coffee genomes, Ea and Ca subgenomes from C. arabica and Cc genome from C. canephora. Sequence analysis revealed the presence of a variable number of members CC subclass of NBS-LRR genes thatappeared to be exclusively present at this locus in C. arabica. Comparative genomic analysis indicated that i) the origin of most of the SH3-CNL copies predates the divergence between Coffea species ii) the ancestral SH3-CNL copy was inserted in the SH3 locus after the divergence between Solanales and Rubiales lineages. Furthermore, duplications, deletions, gene conversion and positive selection appeared as the main forces that drive SH3-CNL evolution. Different approaches have been undertaken to clone candidate genes to CLR. In parallel, a highly efficient and reliable Agrobacterium tumefaciens-mediated protocol was established for coffee. Transgenic plants containing one of the candidate gene were successful produced and acclimated into the greenhouse. The preliminary bioassay against H. vastatrix was achieved. The setting-up of efficient protocols for cloning resistance genes and for genetic transformation paves the way for routine functional genomics in coffee. The better knowledge on CLR resistance gene diversity would allow optimization of breeding schemes for durable resistance to this major coffee disease.

IIntrodução A utilização excessiva de antimicrobianos na medicina humana, veterinária e agricultura resultou no aparecimento da resistência bacteriana. Este fenômeno gera problemas de saúde pública que podem resultar na reemergência de doenças infecciosas. O uso de antibióticos, principalmente de forma profilática, tornou-se prática na aquicultura, como ocorre no Brasil, onde a regulamentação para o uso de medicamentos veterinários é ineficiente. Além disso, há evidências da transferência de organismos resistentes para humanos por meio do consumo de produtos de origem animal, quando, durante a fase de criação e produção destes foram administrados antibióticos. Objetivo Pesquisar a ocorrência de genes de resistência a antibióticos em filés de tilápia comercializados em supermercados do município de São Paulo SP. Material e Métodos Foram coletadas 10 amostras de filé de tilápia e realizada a pesquisa de coliformes termotolerantes como indicadores das condições higiênico-sanitárias do alimento. Em seguida, as amostras foram inoculadas em Caldo Lúria 0,5 por cento e o DNA total das bactérias cultivadas nesse meio foi extraído por meio de choque térmico para pesquisa de genes de resistência aos antibióticos -lactâmicos e tetraciclinas pela PCR. Os genes identificados pela PCR foram confirmados pelo sequenciamento. Resultados Em 100 por cento das amostras analisadas o resultado para coliformes termotolerantes foi < 3 NMP.g-1. Na pesquisa de genes de resistência a -lactâmicos, o gene blaOXY-5 foi detectado em 90 por cento das amostras, blaTEM-1b em 20 por cento , blaLEN-16 em 10 por cento , blaSHV-28 em 10 por cento , blaKPC-2 em 20 por cento , blaMOX-6 em 10 por cento e blaCphA em 60 por cento . Os genes de resistência a tetraciclinas identificados foram tetB em 10 por cento das amostras, tetC em 20 por cento , tetD em 80 por cento , tetE em 50 por cento , tetG em 60 por cento , tetO e tetS em 10 por cento cada e tetW em 20 por cento . Conclusões Em 90 por cento das amostras foi identificada a presença de genes de resistência aos antibióticos -lactâmicos e tetraciclinas, demonstrando uma grande circulação de resistência bacteriana na aquicultura e verificando a necessidade de legislação e fiscalização mais atuantes no controle do uso de antibióticos na aquicultura / IIntroduction The excessive use of antimicrobials in human medicine, veterinary medicine and agriculture has resulted in the emergence of bacterial resistance. This phenomenon creates public health problems that may result in the re-emergence of infectious diseases. The use of antibiotics, mainly prophylactically, has become a practice in aquaculture, including Brazil, where the legislation for the use of veterinary drugs is inefficient. Furthermore, there is evidence of transmission of resistant organisms to humans through consumption of animal products, especially when the antibiotics have been administered during the raising and production of these animals. Objective To search for the occurrence of antibiotics resistance genes in tilapia fillets commercialized in supermarkets in São Paulo city - SP. Material and Methods 10 tilapia fillet samples were collected and submitted to fecal coliform search as an indicator of the sanitary conditions of the food. Then, the samples were inoculated into Luria broth 0,5 per cent and the total DNA was extracted from growing bacteria by thermal shock for the search of resistance genes to -lactam and tetracyclines antibiotics by PCR. The genes identified by PCR were confirmed by sequencing. Results In 100 per cent of samples the result was < 3 NMP.g-1 for fecal coliform organisms. In the study of -lactam resistance genes, blaOXY-5 gene was detected in 90 per cent of samples, blaTEM-1b in 20 per cent , blaLEN-16 in 10 per cent , blaSHV-28 in 10 per cent , blaKPC-2 in 20 per cent , blaMOX-6 in 10 per cent and blaCphA in 60 per cent . The tetracyclines resistance genes identified were tetB in 10 per cent of samples, tetC in 20 per cent , tetD in 80 per cent , tetE in 50 per cent , tetG in 60 per cent , tetO and tetS in 10 per cent each and tetW in 20 per cent . Conclusions 90 per cent of the samples showed the presence of -lactam and tetracyclines resistance genes, demonstrating a high circulation of bacterial resistance in aquaculture and verifying the need for more active law and surveillance in controlling the use of antibiotics in aquaculture.