The objectives of this study were to evaluate the effect of number of stocker cattle in receiving pens (large: n=150 cattle; small: n=50 cattle) on 1) BRD morbidity/mortality, and performance, 2) antimicrobial use and prevalence of antimicrobial resistance in Mannheimia haemolytica, and 3) profitability of stocker operations. No differences were found for morbidity (p=0.5041). Mortality tended (p=0.0744) to be higher in large groups. BW increased (p LESS THAN 0.0001) over time. A treatment*day interaction (p=0.00592) was found for ADG, with largest gains for both groups from day 14-28. M. haemolytica recovery decreased (p=0.0002) over time. Antimicrobial resistance (p=0.0179) and MDR (p=0.0405) were higher in the small group. Treatment costs were higher in the small group ($1,093.53/hd) compared to large ($1,037.04/hd). Because of the nature of a pilot study, further research are needed to determine the effectiveness of reducing animals in a pen on health, growth, AMR, and profitability associated with stocker cattle.

The antimicrobial resistance (AMR) crisis, where new antibiotic discovery is not keeping pace with the emergence of resistant pathogens, is driven by mobile genetic elements (MGEs). MGEs can autonomously transfer between bacteria, along with AMR genes. The widespread use of antibiotics in the clinic, in agriculture, and animal husbandry, has accelerated the MGE-mediated transfer of AMR genes in the environment. However, despite playing such an important role in the AMR crisis, the dynamics and mechanisms behind the transmission of genes are poorly understood. Furthermore, which natural and man-made compounds inhibit or promote their movement in these environments is unknown. One method to combat the rise in AMR is to identify small molecules as probes to understand the molecular basis of transmission and apply this information to prevent MGE-mediated resistance dissemination. Since conjugation is the main mechanism for AMR gene transfer, targeting MGEs that use conjugation, such as conjugative plasmids (e.g. Tn1549) and conjugative transposons (e.g. F plasmid), has the potential to prevent the emergence of multi-drug resistant pathogens. In this work, a high throughput assay modeled after Tn1549 excision was screened against a library of known bioactive compounds to find modulators of the integrase and excisionase activity. Several fluoroquinolone antibiotics including ciprofloxacin were identified as dose-dependent inhibitors of excision, which acted by changing supercoiling levels in the cell. Ciprofloxacin enhanced conjugation frequency of Tn1549 at sub-MIC concentrations relative to an untreated control and inhibited conjugation frequency at higher concentrations. A second project was focused on a high throughput conjugation assay based on the separation of the lux operon between a donor and recipient cell, such that only transconjugants produce luminescence to reflect active gene transfer. This work furthers our understanding of the development of assays to target MGEs and screening for inhibitors of their movement. / Thesis / Master of Science (MSc) / Antibiotics are small molecules that cure bacterial infections. However, their efficacy is fading as a result of the ability of mobile genetic elements (MGEs) to spread antimicrobial resistance genes between bacteria. Conjugative plasmids (CPs) and conjugative transposons (CTns) are two of the major types of MGEs that contribute to the dissemination of antimicrobial resistance in pathogens. The goal of this research is to search for inhibitors of CTns and CPs in order to prevent the emergence of multi-drug resistant bacteria. High throughput assays were designed to model both a CTn (Tn1549) and a CP (F plasmid) to find small molecules targeting their movement. A screen of the Tn1549 excision assay identified fluoroquinolone antibiotics that inhibit excision in a dose-dependent manner and indirectly inhibit the integrase used to excise the CTn. Ciprofloxacin, a fluoroquinolone, inhibited the conjugation frequency of Tn1549. Future work will focus on identifying new inhibitors of these MGEs and their characterization.

Poultry litter can harbor pathogenic bacteria, including avian pathogenic Escherichia coli (APEC). Applying litter amendments is one strategy to improve bird health and potentially reduce pathogens. Biochar and PLT were applied as litter amendments in a live bird trial to study their effects on E. coli populations, APEC virulence genes (VAG), and antimicrobial resistance (AMR) genes. Samples were collected at days 0, 17, 29, and 41 to enumerate E. coli and store bacterial isolates for antimicrobial-resistance gene analysis. Data analysis showed litter amendments did not significantly affect overall E. coli populations. Grow-out time impacted E. coli populations, with reductions occurring over time. Litter treatment had no impact on the prevalence of VAG or AMR. Time showed VAGs were absent at d 0 while AMR genes were prevalent at d 0. This indicates chicks may have been the source of VAG, while AMR genes were prevalent in used litter.

Yes / Background: Group B Streptococcus (GBS) is a leading cause of morbidity and mortality in infants in both developed and developing countries. To our knowledge, only a few studies have been reported the clinical features, treatment and outcomes of the GBS disease in China. The severity of neonatal GBS disease in China remains unclear. Population-based surveillance in China is therefore required. Methods: We retrospectively collected data of <3 months old infants with culture-positive GBS in sterile samples from three large urban tertiary hospitals in South China from Jan 2011 to Dec 2014. The GBS isolates and their antibiotic susceptibility were routinely identified in clinical laboratories in participating hospitals. Serotyping and multi-locus sequence typing (MLST) were also conducted for further analysis of the neonatal GBS disease. Results: Total 70 cases of culture-confirmed invasive GBS infection were identified from 127,206 live births born in studying hospitals, giving an overall incidence of 0.55 per 1000 live births (95% confidence interval [CI] 0.44–0.69). They consisted of 49 with early-onset disease (EOD, 0.39 per 1000 live births (95% CI 0.29–0.51)) and 21 with late-onset disease (LOD, 0.17 per 1000 live births (95% CI 0.11–0.25)). The incidence of EOD increased significantly over the studying period. Five infants (4 EOD and 1 LOD) died before discharge giving a mortality rate of 7.1% and five infants (7.1%, 2 EOD and 3 LOD) had neurological sequelae. Within 68 GBS isolates from GBS cases who born in the studying hospitals or elsewhere, serotype III accounted for 77.9%, followed by Ib (14.7%), V (4.4%), and Ia (2.9%). MLST analysis revealed the presence of 13 different sequence types among the 68 GBS isolates and ST-17 was the most frequent sequence type (63.2%). All isolates were susceptible to penicillin, ceftriaxone, vancomycin and linezolid, while 57.4% and 51.5% were resistant to erythromycin and clindamycin, respectively. Conclusions: This study gains the insight into the spectrum of GBS infection in south China which will facilitate the development of the guidance for reasonable antibiotics usage and will provide evidence for the implementation of potential GBS vaccines in the future. / Supported by medical and health science and technology projects of Health and Family Planning Commission of Guangzhou Municipality (grant number 20151A010034) and Guangdong provincial science and technology planning projects (grant number 2014A020212520).

Yes / The majority of Mycobacterium tuberculosis isolates resistant to isoniazid harbour a mutation in katG. Since these mutations cause a wide range of minimum inhibitory concentrations (MICs), largely below the serum level reached with higher dosing (15 mg/L upon 15–20 mg/kg), the drug might still remain partly active in presence of a katG mutation. We therefore investigated which genetic mutations predict the level of phenotypic isoniazid resistance in clinical M. tuberculosis isolates. To this end, the association between known and unknown isoniazid resistance-conferring mutations in whole genome sequences, and the isoniazid MICs of 176 isolates was examined. We found mostly moderate-level resistance characterized by a mode of 6.4 mg/L for the very common katG Ser315Thr mutation, and always very high MICs (≥19.2 mg/L) for the combination of katG Ser315Thr and inhA c-15t. Contrary to common belief, isolates harbouring inhA c-15t alone, partly also showed moderate-level resistance, particularly when combined with inhA Ser94Ala. No overt association between low-confidence or unknown mutations, except in katG, and isoniazid resistance (level) was found. Except for the rare katG deletion, line probe assay is thus not sufficiently accurate to predict the level of isoniazid resistance for a single mutation in katG or inhA. / European Research Council (Starting Grant INTERRUPTB 311725 to CM, LR and BdJ), The Damien Foundation

Yes / We investigated whether companion drug resistance was associated with adverse outcome of the shorter MDR-TB regimen in Bangladesh, after adjusting for fluoroquinolone resistance. MDR/RR-TB patients registered for treatment with a standardized gatifloxacin-based shorter MDR-TB regimen were selected for the study. Drug resistance was determined using the proportion method, gatifloxacin and isoniazid minimum inhibitory concentration testing for selected isolates, and whole genome sequencing. Low-level and high-level fluoroquinolone resistance were the most important predictors of adverse outcomes, with pyrazinamide resistance having a significant yet lower impact. In patients with fluoroquinolone-/second-line injectable-susceptible TB, non-eligibility to the shorter MDR-TB regimen (initial resistance to either pyrazinamide, ethionamide, or ethambutol) was not associated with adverse outcome (aOR 1.01; 95%CI 0.4-2.8). Kanamycin resistance was uncommon (1.3%). Increasing levels of resistance to isoniazid predicted treatment failure, also in a subgroup of patients with high-level fluoroquinolone-resistant TB. Our results suggest that resistance to companion drugs of the shorter MDR-TB regimen, except kanamycin resistance, is of no clinical importance as long as fluoroquinolone susceptibility is preserved. Hence, contrary to current WHO guidelines, exclusions to the standard regimen are justified only in the case of fluoroquinolone, and possibly kanamycin resistance. / Damien Foundation Belgium for its financial and logistic support to run the project including its research activities. European Research Council (Starting Grant INTERRUPTB 311725).

No description available.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Harvey Morgan Scott / A randomized controlled field trial was designed to evaluate the effects of two treatment strategies on ceftiofur and tetracycline resistances in feedlot cattle. The two strategies consisted of administering ceftiofur crystalline-free acid administration (CCFA) at either one or else all of the steers within a pen, and subsequent feeding/not feeding of therapeutic doses of chlortetracycline. Both strategies were hypothesized to reduce ceftiofur resistance. The effects of treatment strategies were evaluated via metagenome-based and culture-based assays. In this 26-day study, 176 steers were allocated to 16 pens of 11 steers each. The two strategies were randomly assigned to the pens in a two-way full-factorial manner resulting in four treatment groups. The blaCMY-2, blaCTX-M, tet(A), tet(B), and 16S rRNA gene copies/g feces were quantified using qRT-PCR from fecal community DNA. Antimicrobial susceptibility profiles were determined using microbroth dilution technique from the non-type-specific (NTS) E. coli isolates (n=1,050). The NTS E. coli DNA was screened for the presence of blaCMY-2, tet(A), and tet(B) genes. Pens in which all the steers received CCFA treatment showed an increase in blaCMY-2 and blaCTX-M log10 gene copies/g feces and in the proportion of ceftiofur-resistant and blaCMY-2 positive NTS E. coli. This was in contrast to the pens where only one animal received CCFA treatment. There was a significant decrease in quantities of tetracycline genes in community DNA in pens where all animals received CCFA treatment. In contrast to metagenome-based assay results, culture-based assays indicated an increase in the proportion of tetracycline resistant NTS E. coli upon CCFA treatment. Thereafter, chlortetracycline administration led to rapid expansion both of ceftiofur (blaCMY-2, blaCTX-M) and tetracycline [tet(A) and tet(B)] log10 gene copies/g feces. Chlortetracycline treatment delayed the return of the ceftiofur resistance prevalence to baseline among NTS E. coli and thus did not lead to the hypothesized decrease in ceftiofur resistance. Our data suggest that chlortetracycline use is contraindicated when attempting to avoid expansion of resistance to critically important 3rd generation cephalosporins in feedlot cattle. Further studies are required to better establish the animal-level effects of co-housing antimicrobial-treated and non-treated animals together at varying ratios on the levels of antimicrobial resistance.

Globally, the incidence of infection caused by Neisseria gonorrhoeae is the second highest among the bacterial sexually transmitted infections. In Canada, declining rates during the 1990s suggested progress toward curbing gonorrhea; however, those have been increasing since 1999, with rates in Saskatchewan among the highest in the country. Infection can cause serious complications in men and women, and reported resistance to third-generation cephalosporins could lead to potentially untreatable infections. Increased understanding of gonorrhea transmission dynamics, sexual networks, and predictors of antimicrobial resistance development is needed to inform the development of improved approaches to prevention and treatment. The research presented herein draws upon data from Shanghai, China, and Saskatchewan, Canada, to compare and contrast varying epidemiologic approaches to enhancing understanding of gonorrhea in the two settings. Using traditional statistical approaches, multi-level statistical modeling, social network analysis, and dynamic simulation modeling, questions related to sexual behavior, partner presentation, and antimicrobial resistance development are explored. Each technique is evaluated for its potential contribution to overall understanding of the issues related to the ongoing gonorrhea epidemic, globally, and in Saskatchewan. The relative strengths and limitations of the application of the analytical approaches in the different settings are described. Socio-demographic characteristics provided useful indicators of antimicrobial resistant infection among patients with gonorrhea from Shanghai. Further, socio-demographic characteristics were also useful for predicting presentation of a partner for testing and treatment and the use of condoms during intercourse, among this study population. In Saskatchewan, socio-demographic characteristics were useful in predicting coinfection with gonorrhea and chlamydia at the time of diagnosis as well as repeat infection with gonorrhea. Social network analysis of the Saskatchewan dataset provided little additional understanding of the gonorrhea epidemic in the province. This result was largely related to how STI data are collected and stored in the province. The utility of dynamic simulation modeling to investigate the potential impact of antimicrobial resistance in Saskatchewan was also limited due to the same data constraints. However, the insight gained from the model building process and findings from the working model did offer a starting point for conversations around the best ways to postpone the development of antimicrobial resistance in N. gonorrhoeae in Saskatchewan, as well as contribute additional information about how the ways in which STI data are collected and stored in the province considerably restrict the applicability of otherwise powerful epidemiologic tools. With persistently high rates of disease transmission, and the threat of untreatable infections due to antimicrobial resistance, N. gonorrhoeae remains a substantial public health threat locally and globally. The research presented herein describes various approaches to understanding and controlling this disease, applied in contrasting settings. There are a wide variety of elements that should be considered when choosing the appropriate tool(s) to address gonorrhea in a given population; there is no “one size fits all” solution. The local epidemiology of disease, cultural and behavioural norms, the characteristics of the notifiable disease reporting and information systems, and the availability of suitable data all affect the relative strengths and weaknesses of the available analytic methods and disease control approaches.

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