This cross-sectional study described antimicrobial resistance (AMR) in healthy pigs in 20 Alberta and Saskatchewan herds. All herds used antimicrobials; the daily probability of antimicrobial exposure was 0.8 for nursery pigs and 0.3 for grow-finish pigs. Salmonella spp. (n = 468) were isolated from nursery, grow-finish pigs and sows while <i>Escherichia coli</i> (n = 1439) and <i>Campylobacter</i> spp. (n = 405) were isolated from grow-finish pigs. <p>Fifty-nine percent of the Salmonella were pansusceptible. Isolates from sows were more likely to be pansusceptible than those from other production phases, while Salmonella from nursery pigs were more likely to be multiresistant. All Salmonella and E. coli were susceptible to ceftriaxone and ciprofloxacin, drugs critically important to human medicine, while one E. coli was resistant to ceftiofur. Resistance was most common to tetracycline and sulfamethoxazole (Salmonella, 35% and 27%; E. coli, 68% and 46%). Although often considered an indicator organism, <i>E. coli</i> AMR was a poor sentinel for Salmonella AMR at the herd-level. <p>Antimicrobial resistance genes, described in 151 <i>E. coli</i>, were associated in two sets: aadA1 / sul1 / tetA and blaTEM / strA strB / sul2 / tetB. Associations between genes consistently matched associations between phenotypes suggesting phenotype data may be useful for predicting co-selection. Demonstrating dose-response relationships between various antimicrobial exposures and resistance phenotypes in E. coli reiterated the importance of co-selection. Significant predictors included exposures in other production phases and to unrelated drugs. Four <i>E. coli </i> resistance-phenotypes were associated with macrolide exposure; the most commonly used antimicrobial class in study herds. Additionally, 70% of the Campylobacter were resistant to a macrolide and this resistance was associated with macrolide exposure in nursery pigs. Study herds did not use quinolones. Despite this, 15% of Campylobacter were resistant to a quinolone. Both Campylobacter and <i>E. coli</i> AMR clustered within herds, indicating on-farm interventions could mitigate AMR in pigs.<p>This study described AMR in enteric bacteria from healthy pigs. Identifying dose-response relationships between antimicrobial resistances and exposures to unrelated drugs, and exposures of pigs in different production phases, emphasize the importance of judicious antimicrobial use in pig production.

The effects of selected antimicrobials, applied singularly or in combination, and frozen or refrigerated storage conditions on the survival of Listeria spp. on inoculated shrimp was evaluated in this study. A combination of 0.5% CPC (Cetylpyridinium Chloride) with a water wash at room temperature and freezing of the shrimp at -22.3 degrees C was the only treatment that had a significant antimicrobial effect on the Listeria spp. Antimicrobial effects and the mode of action of PEF (Pulsed Electric Field) and CPC on Listeria cells were evaluated in detailed studies. PEF in 0.1% sodium chloride had a bacterostatic effect toward Listeria spp. during refrigerated storage, but no immediate or bacteriostatic effect was caused by freezing the samples. A concentration of 1% sodium chloride reduced the Listeria spp. population after freezing by 1.1 log; however, the pungent chlorine odor that was generated during treatment might cause discomfort for employees in shrimp processing facilities. Also, chlorine might cause corrosion of metal surfaces of processing equipment. There was no difference in the antimicrobial effects on the survival of Listeria spp. by PEF between the exposure times of 1 or 2 min, as well as in the sodium chloride concentrations of 0.1 and 0.5%. PEF treatment in the presence of 0.1% sodium chloride is recommended. A solution of 0.5% CPC effectively inhibited all of the strains of Listeria spp. in the cell suspensions. A treatment of 0.5% CPC combined with PEF treatment in a sodium chloride concentration of 0.1% caused a delayed effect on the Listeria spp. after 2 d of refrigerated storage. After 2 d of frozen storage, the formation of ice crystals was decreased in the number of Listeria spp. when contaminated samples were treated with water. The results indicated chemicals (e.g. CPC and NaCl) might protect Listeria spp. from the formation of ice crystals. TEM (Transmission Electron Microscopy) micrographs revealed that cell membranes were damaged by PEF treatment and that cells were ruptured by CPC treatment. A maximum reduction of 2.76 log10 CFU/g of Listeria spp. on shrimp was achieved by a combination of PEFCPC.

Many studies have attempted to link antimicrobial use in food animal agriculture with an increased risk of antimicrobial-resistant (AR) bacterial levels in humans. Our data arise from longitudinal aggregated fecal samples in a 3-year cohort study of vertically integrated populations of human workers and consumers, and swine. Human and swine E. coli isolates (N = 2130 and 3485, respectively) were tested for antimicrobial susceptibility using the SensititreTM broth microdilution system. The associations between AR prevalence for each antimicrobial agent, multi-drug resistant E. coli, or multivariate AR E. coli, and the risk factors (host species, production type (swine), vocation (human swine worker versus non-worker), and season) in the study were assessed using generalized estimating equations (GEE), GLM with multinomial distribution, or GEE in a multivariate model using a SAS® macro to adjust for the correlated AR phenotypes. There were significant (p < 0.05) differences in AR isolates: 1) between host-species with swine at higher risk for ceftiofur, chloramphenicol, gentamicin, kanamycin, streptomycin, sulfisoxazole, and tetracycline. The prevalence of ciprofloxacin, nalidixic acid, and trimethoprim/sulfamethoxazole resistance were higher among human isolates, 2) swine production group was significantly associated with AR with purchased boars, nursery piglets, and breeding boars at a higher risk of resistance to streptomycin and tetracycline, and 3) human swine worker cohorts exhibited an elevated tetracycline prevalence, but lowered sulfisoxazole prevalence when compared to nonworkers. High variability among seasonal samples over the 3-year period was observed. There were significant differences in multiple resistance isolates between host species, with swine at higher risk than humans of carrying multi-resistant strains; however, no significant differences in multiple resistance isolates within humans by vocation or within swine by production group. The odds-ratios, adjusted for multivariate dependence of individual AR phenotypes, were increased relative to unadjusted oddsratios among 1) swine as compared to human for tetracycline (OR = 21.8 vs. 19.6), and 2) increased significantly among swine-workers as compared to non-workers only for tetracycline (OR = 1.4 vs. 1.3). Occupational exposure to swine-rearing facilities appears to be associated with an increased relative odds for the prevalence of tetracycline resistance compared to non-workers.

To effectively minimize biofilms, which are prevalent in chlorinated drinking water distribution systems, the effect of biofilm age on antimicrobial tolerance and resistance must be investigated. It was our hypothesis that antimicrobial tolerance emerges quickly during biofilm development and that both antimicrobial tolerance and resistance increase as the biofilm ages. We further hypothesized that antimicrobial tolerance and resistance vary among the individual community members. In this study, young and mature biofilms of Pseudomonas aeruginosa, a model biofilm organism, were grown and exposed to antimicrobial agents in several different treatments. Results showed that the increased antimicrobial tolerance of intact biofilms compared to planktonic cells arises early (i.e., within hours) in biofilm development. The short-term tolerance of resuspended mature biofilm cells to antimicrobial agents peaked at a biofilm age of 14 days and subsequently declined; the peak and decline in antimicrobial tolerance may be related to periodic detachment events in the biofilm. The antimicrobial resistance of resuspended mature biofilm cells continuously exposed to antimicrobial agents increased with biofilm age. Furthermore, individual members in mature biofilm communities exhibited variation in antimicrobial tolerance, thereby highlighting divergence of the biofilm community from the original parent strain.

<p>Antimicrobial peptides (AMPs) are part of the innate immune system that is widely distributed in nature, acting as a defense mechanism against invading microorganisms. AMPs have potent antimicrobial activity against a range of microorganisms including fungi, bacteria and viruses. In view of growing multidrug resistance, AMPs are increasingly being viewed as potential therapeutic agents with a novel mechanism of action. Mastoparan is a natural, highly positively charged AMP derived from the venom of wasps. It was originally of interest based on its inherent mast cell degranulation activity. Previously, mastoparan has been shown to exhibit antimicrobial activity in vitro however these studies have been limited in scope. Here we hypothesize that mastoparan possess the capacity to be a potent broad spectrum antibacterial agent including activity against multidrug resistant bacteria. </p><p>We examined the scope of antibacterial activity exhibited by mastoparan using a variety of antimicrobial susceptibility tests and have utilized a bacterial skin infection (S. aureus) model to determine the potential of mastoparan to serve as a therapeutic agent. We tested mastoparan against 4 Gram-positive clinical isolates (e.g., S. aureus, and E. faecium), 9 Gram-negative clinical isolates (e.g., E. coli, P. aeruginosa, and B. cepacia), and 4 multidrug resistant clinical isolates (e.g., MRSA, ESBL E.coli, and ESBL K. pneumonia). These studies reveal that mastoparan exhibits broad spectrum activity against both Gram-negative (MIC: 1.9 - 125 &mug/ml) and Gram-positive (MIC: 15.6 - 125 &mug/ml) bacteria and against multidrug resistant bacteria (MIC: 7.8 - 125 &mug/ml). We also demonstrated that mastoparan disrupts the bacterial membrane, exhibits fast acting antibacterial activity and is highly effective against both multiplying and non-multiplying bacteria. Furthermore, we have shown that mastoparan demonstrates efficacy as a topical antimicrobial agent reducing lesion size by up to 79% and the amount of bacteria recovered from skin lesions by up to a 98% reduction. Based on these results we conclude that mastoparan is a highly effective antibacterial agent and is therefore a potential alternative to currently antibiotics. Mastoparan offers a promising new therapeutic option for treating bacterial infections.</p> / Dissertation

Biomaterial-associated infections remain a significant complication of medical implants. Of the different strains of bacteria associated with nosocomial infections, 70% are resistant to at least one of the drugs used for treatment (Bren 2002). In 2000 the Center for Disease Control ranked microbial agents as the 4th leading actual cause of death in the United States of America (Mokdad et al. 2004).In an effort to improve the prevention and treatment of infections, this research has three objectives: the development of an alternative sterilization method for medical devices; assessing a new antimicrobial material for the prevention of infections in situ; and assessing mechanisms of acquired microbial resistance. The biocide being investigated in this body of work is chlorine dioxide gas.While multiple sterilization methods are available, there are limitations to all of these technologies. For example, chemical sterilization can leave residues on the surface of the material. These residuals can be toxic, causing sensitization reactions when the materials are implanted in the body (Dolovich et al. 1984; Marshall et al. 1985; Chapman et al. 1986; Dolovich et al. 1987). Research has shown that materials sterilized with increasing concentrations of the chemical sterilizing agent have increasing quantities of residuals (Lyarskii et al. 1984). The studies presented here will ascertain the environmental parameters required for sterilization of biomaterials with low concentrations of chlorine dioxide gas and assess polymers sterilized using these conditions for the cytotoxicity of possible chemical residuals.Investigations into preventing biomaterial-based infections in situ have focused on changing the biomaterial properties. Materials with altered physicochemical characteristics to prevent bacterial adhesion have been developed, and antibiotics and silver have been incorporated into the biomaterials to inhibit bacterial colonization. Unfortunately, the rapid depletion of incorporated antimicrobial agents, altered bactericidal activity in vivo, and the development of antibiotic resistance, have all limited the effectiveness of current technologies. In these studies a chlorine dioxide generating material was assessed using in vitro and in vivo assays.While assessing the bactericidal efficacy of a selection of chlorine dioxide generating materials, a spontaneous bacterial mutant with a reduced susceptibility to chlorine dioxide was isolated. The final section of this work will investigate a potential mechanism of resistance to chlorine dioxide.

Cationic antimicrobial peptides (CAPs) are small peptides that constitute an important defence against microbial pathogens. Certain CAPs also possess anticancer properties. NRC-03 and NRC-07 are pleurocidins derived from winter and yellowtail flounder, respectively. The purpose of this investigation was to study the anticancer properties of NRC-03 and NRC-07. NRC-03 and NRC-07 killed breast cancer cells, including P-glycoprotein-overexpressing cells, in a time-dependent manner that peaked at 4 h. NRC-03 and NRC-07 lysed breast cancer cells by a mechanism that involved cell binding, mitochondrial destabilization, nuclear localization, and significant membrane damage. Interestingly, NRC-07, but not NRC-03, caused DNA fragmentation. NRC-03 and NRC-07 killed normal human epithelial cells, but did not kill endothelial cells or fibroblasts, or lyse human erythrocytes. NRC-03, and to a lesser extent NRC-07, had chemo-sensitizing properties, suggesting promise for their inclusion in combinational treatment regimens. Importantly, intratumoural injections of NRC-03 or NRC-07 inhibited tumour growth in a mouse model of breast cancer. Fetal bovine serum dose-dependently reduced cell killing by NRC-03. NRC-03 was degraded in human and mouse serum, which limited its potency. NRC-03- and NRC-07-induced cytotoxicity correlated with expression of several different negatively-charged molecules, rationalizing the generation of [D]-NRC-03, which carries the same positive charge as NRC-03, and was more potent but less selective for cancer cells than NRC-03. [D]-NRC-03 was also cytolytic and exhibited in vivo anticancer properties. To further test the clinical potential of NRC-03- and NRC-07-resistant cells were generated. NRC-03 and NRC-07 bound to resistant cells to a lesser extent than parental cells and were phenotypically distinct. Importantly, NRC-03- and NRC-07-resistant cells were killed by chemotherapeutic drugs, as well as [D]-NRC-03. These studies demonstrate that NRC-03, NRC-07, and [D]-NRC-03 are cytolytic peptides that kill breast cancer cells in vitro and in vivo. While more potent than NRC-03, [D]-NRC-03 requires further modification to minimize its toxicity toward normal cells. Although cancer cells may become resistant to NRC-03 and NRC-07 over time, resistant cells are still killed by other cytotoxic drugs, thereby reinforcing the value of adding these peptides to combinational regimens for the treatment of breast cancer.

No description available.

Antimicrobial peptides (AMPs) are relatively-short chain molecules that living organisms use to defend themselves against a wide range of invading microorganisms such as bacteria and viruses. They selectively bind to and kill microbes over host cells by permeabilizing cell membranes or by inhibiting the biological functions of intra-cellular components. Despite its significance in determining their cell selectivity, however, the cell-concentration dependence of AMP's membrane-perturbing activity has not been criticality examined. In this thesis, we present a physical model for cell selectivity of AMPs, especially its cell-concentration dependence. To this end, we use a coarse-grained model that captures essential molecular details such as lipid composition (e.g., fraction of anionic lipids) and peptide amphiphilicity and charge. In particular, we calculate the surface coverage of peptides in the membrane-perturbing mode as a function of peptide and cell densities: those that bind to the interface between lipid headgroups and tails. This allows us to extract the minimum inhibitory concentration (MIC) and the minimum hemolytic concentration (MHC) of the peptides. Our results show that both MIC and MHC increase as the cell density increases so that the peptide selectivity (given by MHC/MIC) decreases with increasing cell density. Our results will help resolve conflicting interpretations of peptide-selectivity experiments.

Public health concerns associated with high prevalence of foodborne salmonellosis, the emergence of antibiotic-resistant organisms and the identification of poultry meat and products as one of the most common sources of Salmonella support the need for new pathogen control strategies in the poultry industry. Scientific research has focused on the use of bacteriophages as therapeutic agents for humans and animals; however, limited studies have been conducted on bacteriophage application on food safety, especially on poultry. Therefore, the objective of this study was to evaluate the phage density and exposure time required to reduce Salmonella load on experimentally inoculated chicken meat. In Experiment 1, serovars of Salmonella were tested for antimicrobial susceptibility and rifampicin-resistant isolates were generated. Cocktails of the serovars Enteritidis, Kentucky and Typhimurium (EKT), and Hadar and Heidelberg (HH), were inoculated on chicken breast samples to a target of 104 CFU/g. A mixture of three lytic bacteriophages, active against multiple Salmonella serovars was applied to chicken samples. A total of 84 samples (25 +/- 2 g) per each cocktail were distributed among a negative control, Salmonella-inoculated positive control, Salmonella-inoculated samples treated with the phage mixture at differing titers (105, 106, 107, 108, and 109 PFU/ml) with two identical samples at 0, 15, 30, 60, 120, 360 min at 4 degrees C. Experiment 2 evaluated nalidixic acid-resistant Salmonella Typhimurium among negative control, Salmonella-inoculated control (positive control), Salmonella with two phage titers (105 and 109 PFU/ml) at 0, 30, 60 and 120 min at 25 degrees C and 4 degrees C. Results showed differences in means for Salmonella cocktail EKT ranged from 0.1 to 0.7 log10 CFU/g with 0.7 log10 for 108 PFU/ml, 30 min, 4 degrees C. For Salmonella cocktail HH, reductions ranged from 0.1 to 0.4 log10 CFU/g with 0.4 log10 on samples treated with 108 PFU/ml, 120 min, 4 degrees C. For the Experiment 2, a higher phage concentration (109 PFU/ml) at 120 min post-inoculation storage at 25 degrees C was required to yield a 0.9 log10 difference in means. These findings showed that higher concentrations of bacteriophage were more effective controlling Salmonella than lower ones at both temperatures. In addition, temperature, time and bacterial attachment may influence phage efficacy.