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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

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The prevalence of antibiotics on farms for therapeutic and prophylactic use in animals can cause negative effects on biomethane production during anaerobic digestion. Previous literature has found decreased biomethane production rates from a variety of antibiotics, but biogas inhibition differs between studies of continuous and batch reactors and the type of antibiotic studied. Cephalosporin drugs are the most common antibiotic class used to treat mastitis in dairy cows and can retain most of their bioactivity after excretion. Ceftiofur is a commonly used cephalosporin drug but no previous study investigating the effect of Ceftiofur on biomethane during continuous anaerobic digestion has been performed. The aim of this study was to examine the effect on biomethane production when manure from cows treated with Ceftiofur was anaerobically digested. Laboratory sized anaerobic digesters (AD) were run at thermophilic (55°C) temperatures and a 10 day hydraulic retention time. Manure from cows treated with Ceftiofur were fed to the antibiotic treatment reactors for 50 days. The reactor performance was measured by i) biomethane production, ii) waste stabilization in terms of solids and chemical oxygen demand, iii) change in mass of Ceftiofur and iv) change in concentration of antibiotic resistant genes, specifically cfx(A), mef(A), and tet(Q). There was statistically significant decrease in cumulative gas production due to the addition of Ceftiofur into the reactors, but no significant difference between treatments in waste stabilization in terms of percent volatile solids (VS) and total chemical oxygen demand (TCOD) reduction. Anaerobic digestion decreased the amount of Ceftiofur in manure, and the amount of Ceftiofur in the reactors reduced over the time of the experiment. Change in antibiotic resistant genes (ARGs) were gene dependent over time. Concentrations of tet(Q) reduced significantly between feed and effluent of both treatments, and cfx(A) reduced significantly for the control treatment but not the Ceftiofur treatment. Concentrations of mef(A) increased over time in both treatments. Overall, the addition of Ceftiofur in continuously operated anaerobic digesters negatively affected biomethane production, a value-added product responsible for on-farm renewable energy. However, anaerobic digestion does decrease the mass of Ceftiofur within manure, thereby reducing the environmental loading from run-off from farms. / Master of Science / Anaerobic digestion is a biological treatment technology used on farms to treat manure. It can be used to reduce potential environmental damage from contaminants and manure, homogenize manure for fertilizer, and produce methane gas for renewable energy. An emerging challenge in manure management is the presence of antibiotics such as ceftiofur used in animal production to prevent and treat illnesses. When antibiotics are used on livestock, they are excreted from the animal in manure. When the manure is added to the digester, the antibiotic molecules within the manure can kill the bacteria responsible for manure homogenization and gas production. Ceftiofur is a type of cephalosporin antibiotic used to treat dairy cows for mastitis, a bacterial infection of the udder. When the cows are treated with Ceftiofur, it can remain in the excreted manure and enter the digester. The use of antibiotics on farms is also leading to a global phenomenon known as antibiotic resistance. The bacteria that are exposed to antibiotics can develop mutations to become immune to the antibiotic, and can spread the mutations through antibiotic resistant genes (ARGs). ARGs can spread to bacteria which have never been exposed to antibiotics, making them resistant. This causes a significant concern in regards to disease treatment across the world as the efficacy of antibiotics is threatened. Understanding how ARGs move and how they can be eliminated is crucial to preventing global antibiotic resistance. The purpose of this study was to assess the effect of anaerobic digestion on Ceftiofur and ARGs. Four continuous lab-scale anaerobic digesters, two using control manure and two using manure from cows treated with Ceftiofur, were run at 55˚C for a period of 50 days. Over time, the reactor with manure from cows treated with the Ceftiofur antibiotic produced less gas as compared to the control digesters. The amount of Ceftiofur within the digesters decreased over time, demonstrating anaerobic digestion’s ability to degrade the antibiotic molecule. The effect of anaerobic digestion on the ARG concentration was gene specific. The concentration of the tet(Q) gene, a gene responsible for resistance against the very common antibiotic tetracycline, was reduced by anaerobic digestion. These results demonstrate that anaerobic digestion is a technology which can reduce the environmental impact of manure from Ceftiofur-treated cows. This shows that manure treatment can be a first step in combating antibiotic resistance across the globe.

Rising global antibiotic resistance has caused concerns over sources and pathways for the spread of contributing factors. Majority of the antimicrobials used in the U.S. are involved in veterinary medicine, primarily with livestock rearing. Animal manure land application integrates livestock farming and agroecosystems. This manure contains antibiotic resistant elements (AREs) (resistant bacteria, resistance genes, and veterinary antibiotics) that contribute towards antimicrobial resistance. Altering manure application techniques can reduce surface runoff of other contaminants such as excess N and P, pesticides, and hormones, that can impact water quality. Conventional tillage practices in the U.S. has reduced or stopped, making subsurface injection of manure a promising option when compared to surface application. Our research compared manure application methods, manure application seasons, cropping system, and manure-rainfall time gaps to gauge the impact on AREs in the environment. Two field-scale rainfall simulation studies were conducted along with one laboratory study. Using the injection method lowered concentrations of manure associated AREs entering surface runoff. When manure was surface applied and rainfall occurred 7 d after application, 9-30 times less resistant fecal coliform bacteria (FCB) entered surface runoff when compared to 1 d time gap for that broadcast method. Within a day of manure application, antibiotic resistance gene (ARG) profiles in soil began to differ from each other based on manure application and soil ARG richness in all manure-amended soil increased compared to the background. Runoff from injection plots contained 52 ARGs with higher abundance compared to runoff from surface applied plots. ARGs in the former were more correlated to soil and more correlated to manure in the latter. The highest antibiotic concentrations were in the injection slit soil of those plots. Antibiotic concentrations in samples corresponded positively to concentrations of resistant FCB and ARGs, and there was a positive correlation between resistant FCB and their associated ARGs (Spearman's ρ = 0.43-0.63). A CRIISPR-Cas12a assay for quantification of ARGs in environmental samples was just as precise as conventional methods. There is also potential for in-situ detection. These combined results can hopefully help farmers improve manure management practices that mitigate spread of AREs to surrounding water, crops, and soil. / Doctor of Philosophy / Rising global antibiotic resistance cause concerns over sources and pathways for the spread of contributing factors. Most of the antimicrobials used in the U.S. are involved in veterinary medicine, especially with livestock rearing. Overuse of antibiotics that are medically important to human medicine compromises the effectiveness of our medicines. Animal manure contains antibiotic resistant elements (AREs) such as resistant bacteria, resistance genes, and antibiotics) that contribute towards resistance issues. Once these AREs enter the environment, they can be taken up by crops, runoff into surface water or leached into ground water, or even reside within the animal products we consume. Altering manure application techniques is beneficial for nutrient conservation but also potentially for reducing ARE spread. With our research, we compared manure application methods, manure application seasons, cropping systems, and manure-rainfall time gaps to find ways to balance the need for manure application and the spread of resistance. We used two field-scale rainfall simulation studies along with one laboratory study. Overall, using the injection method resulted in significantly lower concentrations of manure associated AREs entering surface runoff. When manure was surface applied and rainfall occurred 7 d after application, less resistant fecal coliform bacteria (FCB) entered surface runoff when compared to the 1 d time gap for broadcast methods. Within a day of manure application, antibiotic resistance gene (ARG) profiles in soil began to differ from each other and soil ARG totals in all manure applied soil increased compared to the background. Runoff from injection plots contained more soil ARGs and runoff from surface applied plots containing more manure associated ARGs. The subsurface injection method also caused highest antibiotic concentrations in the injection slit soil of those plots. High antibiotic concentrations in samples generally meant high concentrations of resistant FCB and ARGs, and resistant FCB were also found with their associated ARGs as well. A CRISPR-Cas12a assay for quantification of ARGs in environmental samples was just as precise as conventional methods. There is also potential for onsite detection. These combined results can hopefully help farmers improve manure management practices that mitigate spread of AREs to surrounding water, crops, and soil.