Present throughout all classes of life, antimicrobial peptides (AMPs) confer defense against bacteria, viruses, fungi, and insects. Identifying maize AMPs would provide breeders with a new defense resource. Here, the investigation of maize AMPs is reported. The distribution of AMPs within a panel of ten Mississippi maize inbred lines with varying resistance to Lepidoptera larvae feeding and Aspergillus infection is explored to characterize their observed resistances. Homology data-mining with two comprehensive AMP databases revealed 88 unique maize AMP protein sequences across 81 genes in the MaizeGDB B73 genome assembly. AMP-related polymorphic sites were identified using genomic primers. Analyses with qRT-PCR revealed 8 differentially expressed maize AMP genes. Computational 3D models of AMPs are limited, and models of these eight maize AMP genes were predicted. Two-dimensional electrophoresis gels were used to contrast protein profiles of inbreds with varying resistance to identify regions related to AMPs and other defense-related protein.

Prevalence of antimicrobial resistance in fecal Escherichia coli isolates from feedlot cattle was characterized. Tetracycline resistance in isolates from newly weaned, auction market derived calves on arrival at the feedlot in a clinical trial was 9.8% and resistance to three or more antimicrobials was 2.1% compared to 17.6 % and 5.9% in a cohort study. The prevalence of tetracycline resistance at 78.3% and resistance to three or more antimicrobials at 52.5 % in isolates from spring calves submitted to a regional diagnostic laboratory were higher than those found on arrival at the feedlot. Of isolates from composite feedyard pen samples late in the feeding period, 39.4% were tetracycline resistant and 7.6% were resistant to three or more antimicrobials, somewhat higher than on arrival. Use of oxytetracycline in the feed for disease prophylaxis and the metaphylactic use of long-acting injectable oxytetracycline were associated with increased proportions of cattle with one or more resistant E. coli isolates early in the feeding period, while the use of individual animal treatments was not. The proportion of animals with one or more tetracycline resistant E. coli isolates was not different between the control, metaphylactic treatment and prophylactic treatment groups preslaughter; however, there were significantly more resistant animals in all groups preslaughter than at arrival. There were also no associations found between the total volume of parenteral antimicrobials used for disease treatment in individual animals and antimicrobial resistance in the cohort study. In addition, no strong associations were found between pen-level prevalence of antimicrobial resistance antimicrobial use or other variables. There was no significant difference between the proportion of isolates per pen resistant to tetracycline, one or more, two or more antimicrobials, or three or more antimicrobials when using 20, 15, 10 or 5 isolates from composite pen-level fecal samples. Variance for isolates resistant to three or more antimicrobials was partitioned as 12.7% at the feedyard-level and 28.7% at the pen-level. The use of diagnostic laboratory data for AMR surveillance was also discussed, and alternatives to antimicrobial treatment in the feedlot were also investigated. Overall a significant contribution to our understanding of antimicrobial resistance in feedlot cattle was achieved.

This thesis summarizes an investigation of antimicrobial resistance (AMR) and antimicrobial use (AMU) in cow-calf herds. The specific objectives of this project were to describe common reasons for treatment and the types of antimicrobials used in cow-calf herds, to describe the frequency of AMR in generic fecal Escherichia coli isolated from various age groups commonly found on cow-calf farms, to determine risk factors associated with the occurrence of AMR, and finally to investigate the underlying molecular mechanisms of AMR in cow-calf herds. At least 86% of the herds treated one or more calves or cows during the study period; however, the overall proportion of both calves and cows reported as treated was less than 14% for calves and 3% for cows. The majority of antimicrobials reported as used in cow-calf operations were for individual therapeutic use rather than prophylaxis, metaphylaxis, or growth promotion. Injectable formulations were the most commonly reported method of antimicrobial administration on cow-calf farms. Cow-calf herds in Wetern Canada are not a significant reservoir for resistance to antimicrobials classified as very important to human medicine such as ciprofloxacin and ceftiofur. The three most common resistances detected were to tetracycline, sulphamethoxazole, and streptomycin regardless of age group. Young calves sampled in the spring of the year were more likely to be shedding AMR E. coli than older calves sampled in the fall of the year or than cows sampled in the spring of the year. The cow-calf pair relationship was not an important factor in transfer of AMR from the individual cow to her calf, but the presence of AMR in the general cow herd was associated with AMR in the calf population. The potential importance of co-selection for AMR at the molecular level was demonstrated by both the risk factor analysis and the molecular work. Phenotypic resistance to streptomycin, tetracycline, and sulphamethoxazole were each associated with the presence of resistance genes from all six families of antimicrobials examined in this study. Several statistically significant associations were also detected between the resistance genes considered. No significant associations were detected between any of the AMR phenotypes or genotypes and the STEC virulence factors stx1, stx2 and eae.

Prevalence of antimicrobial resistance in fecal Escherichia coli isolates from feedlot cattle was characterized. Tetracycline resistance in isolates from newly weaned, auction market derived calves on arrival at the feedlot in a clinical trial was 9.8% and resistance to three or more antimicrobials was 2.1% compared to 17.6 % and 5.9% in a cohort study. The prevalence of tetracycline resistance at 78.3% and resistance to three or more antimicrobials at 52.5 % in isolates from spring calves submitted to a regional diagnostic laboratory were higher than those found on arrival at the feedlot. Of isolates from composite feedyard pen samples late in the feeding period, 39.4% were tetracycline resistant and 7.6% were resistant to three or more antimicrobials, somewhat higher than on arrival. Use of oxytetracycline in the feed for disease prophylaxis and the metaphylactic use of long-acting injectable oxytetracycline were associated with increased proportions of cattle with one or more resistant E. coli isolates early in the feeding period, while the use of individual animal treatments was not. The proportion of animals with one or more tetracycline resistant E. coli isolates was not different between the control, metaphylactic treatment and prophylactic treatment groups preslaughter; however, there were significantly more resistant animals in all groups preslaughter than at arrival. There were also no associations found between the total volume of parenteral antimicrobials used for disease treatment in individual animals and antimicrobial resistance in the cohort study. In addition, no strong associations were found between pen-level prevalence of antimicrobial resistance antimicrobial use or other variables. There was no significant difference between the proportion of isolates per pen resistant to tetracycline, one or more, two or more antimicrobials, or three or more antimicrobials when using 20, 15, 10 or 5 isolates from composite pen-level fecal samples. Variance for isolates resistant to three or more antimicrobials was partitioned as 12.7% at the feedyard-level and 28.7% at the pen-level. The use of diagnostic laboratory data for AMR surveillance was also discussed, and alternatives to antimicrobial treatment in the feedlot were also investigated. Overall a significant contribution to our understanding of antimicrobial resistance in feedlot cattle was achieved.

This thesis summarizes an investigation of antimicrobial resistance (AMR) and antimicrobial use (AMU) in cow-calf herds. The specific objectives of this project were to describe common reasons for treatment and the types of antimicrobials used in cow-calf herds, to describe the frequency of AMR in generic fecal Escherichia coli isolated from various age groups commonly found on cow-calf farms, to determine risk factors associated with the occurrence of AMR, and finally to investigate the underlying molecular mechanisms of AMR in cow-calf herds. At least 86% of the herds treated one or more calves or cows during the study period; however, the overall proportion of both calves and cows reported as treated was less than 14% for calves and 3% for cows. The majority of antimicrobials reported as used in cow-calf operations were for individual therapeutic use rather than prophylaxis, metaphylaxis, or growth promotion. Injectable formulations were the most commonly reported method of antimicrobial administration on cow-calf farms. Cow-calf herds in Wetern Canada are not a significant reservoir for resistance to antimicrobials classified as very important to human medicine such as ciprofloxacin and ceftiofur. The three most common resistances detected were to tetracycline, sulphamethoxazole, and streptomycin regardless of age group. Young calves sampled in the spring of the year were more likely to be shedding AMR E. coli than older calves sampled in the fall of the year or than cows sampled in the spring of the year. The cow-calf pair relationship was not an important factor in transfer of AMR from the individual cow to her calf, but the presence of AMR in the general cow herd was associated with AMR in the calf population. The potential importance of co-selection for AMR at the molecular level was demonstrated by both the risk factor analysis and the molecular work. Phenotypic resistance to streptomycin, tetracycline, and sulphamethoxazole were each associated with the presence of resistance genes from all six families of antimicrobials examined in this study. Several statistically significant associations were also detected between the resistance genes considered. No significant associations were detected between any of the AMR phenotypes or genotypes and the STEC virulence factors stx1, stx2 and eae.

Antimicrobial-resistant bacteria are a major source of ailment in modern-day nosocomial settings, with numerous risks including leading to possible further drug resistance or spreading to those who cannot fight off the infection due to immune suppression or dysfunction. Previous work in our laboratory has determined that Klebsiella pneumoniae possesses inhibitory effects on the growth of a variety of bacteria that contain antimicrobial-resistant properties in the Enterobacteriaceae family, a major source of nosocomial antimicrobial-resistance. This novel property of K. pneumoniae inhibits the growth of Citrobacter freundii, Enterobacter aerogenes, and Enterobacter cloacae through an anti-microbial protein. The antimicrobial protein secreted from K. pneumoniae has been shown to reduce the density and growth of C. freundii, E. aerogenes, and E. cloacae in both biofilm and planktonic forms. The work performed in this thesis has shown that the antimicrobial protein is plasmid mediated by introducing a transposon (Tn5) to the plasmid to provide resistant selection and a possible way to create a mutant knockout to find the exact location of the gene in the plasmid. Upon transposon mutagenesis of the plasmid, it was electroporated into Rec- E. coli. The E. coli were then able to produce the antimicrobial protein allowing the formation of zones of inhibition when screened on C. freundii, E. aerogenes, and E. cloacae lawns. Upon confirmation that the plasmid mediates the anti- microbial protein, the plasmid was sent for sequencing to further characterize the gene responsible for coding the anti-microbial protein. This novel antimicrobial protein has high sequence similarity to bacteriocins and, thus far, is a novel and uncharacterized protein of plasmid origin found in only in this particular strain of K. pneumoniae. Further research involving this new bacteriocin could aid in the development of treatments for the highly drug resistant Enterobacteriaceae family members.

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