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.

Tesis por compendio / [ES] La concienciación social con respecto al bienestar animal, la seguridad alimentaria, las resistencias antimicrobianas y la salud medioambiental ha incrementado en los últimos años, promoviendo la implementación de sistemas de producción que incluyan el concepto 'One Health' en su diseño. Por ello, en el sector avícola se han propuesto alternativas a nivel de campo centradas en la mejora de la bioseguridad, el uso de estirpes rústicas y de crecimiento lento y la implementación de ganadería de precisión. En este contexto, la microbiota intestinal tiene un papel importante, tanto en la salud de los animales, como en la diseminación de resistencias antimicrobianas y la transmisión de patógenos zoonósicos a lo largo de la cadena alimentaria. El bienestar animal promueve la presencia de microbiota beneficiosa y la integridad del epitelio intestinal, reduciendo las interacciones con las bacterias ambientales. Por ello, sería posible conseguir una reducción en la administración de antibióticos y reducir la presencia de patógenos en la cadena alimentaria. Salmonella spp. es la principal causa de brotes alimentarios en la Unión Europea, y la principal fuente de infección son los productos avícolas. Entre los principales serotipos se encuentra S. Infantis, actualmente muy prevalente en pollos de engorde. Por todo ello, el objetivo general de esta tesis doctoral fue evaluar el efecto de sistemas alternativos de producción avícola sobre el desarrollo y la composición de la microbiota, la evolución de las resistencias antimicrobianas y la epidemiología de Salmonella. En el primer experimento, se estudió el efecto de la estirpe genética, comparando una estirpe comercial de crecimiento rápido frente a una estirpe alternativa de crecimiento lento. Los objetivos fueron caracterizar la microbiota cecal e investigar la dinámica de las resistencias y multirresistencias antimicrobianas a lo largo del ciclo. Respecto a la composición de la microbiota, los resultados mostraron que Firmicutes fue el filo dominante y los géneros predominantes fueron Oscillospira spp., Ruminococcus spp., Coprococcus spp., Lactobacillus spp. y Bacteroides spp. Por otro lado, los resultados obtenidos en el estudio de resistencias antimicrobianas mostraron que, al inicio del ciclo, los animales de la estirpe de crecimiento rápido presentaron un mayor porcentaje de resistencia, pero al final del periodo no se encontraron diferencias. En el segundo experimento, se evaluó el efecto de las condiciones de manejo, comparando las condiciones comerciales de densidad y ventilación, frente a condiciones mejoradas. Los objetivos fueron caracterizar la microbiota cecal, evaluar la evolución de las resistencias y multirresistencias antimicrobianas, e investigar el desarrollo de S. Infantis y sus resistencias antimicrobianas a lo largo del periodo de engorde. Los resultados obtenidos en la evolución de la microbiota, mostraron un mayor nivel de diversidad en el grupo producido bajo condiciones mejoradas. Además, Firmicutes fue el filo dominante durante todo el ciclo y los géneros predominantes fueron Oscillospira spp., Ruminococcus spp., Bacteroides spp. y Coprococcus spp. Los resultados obtenidos en el estudio de resistencias antimicrobianas mostraron altas tasas de resistencia a lo largo de todo el ciclo, sin diferencias entre grupos. Por último, los recuentos más altos de excreción de Salmonella se observaron el día de sacrificio en ambos grupos. Además, el 100% de las cepas aisladas fueron multirresistentes tras la primera semana post-infección. En conclusión, los principales resultados obtenidos en esta tesis doctoral incluyen que la diversidad y la composición de la microbiota están en constante desarrollo a lo largo del periodo de engorde, viéndose afectadas por los factores de manejo estudiados. Además, las resistencias antimicrobianas están presentes en las bacterias comensales desde el día de llegada, y aumenta hasta el final del ciclo, destacando la necesidad de controlar la administración de antibióticos en todas las etapas de la producción avícola. En cuanto a la epidemiología de S. Infantis, la continua excreción durante todo el periodo de engorde y su capacidad de adquirir resistencias, independientemente de las condiciones de manejo en granja, sugieren la necesidad de realizar más estudios para poder establecer mejores programas de control de la bacteria a lo largo de la cadena alimentaria. / [CA] La conscienciació social amb respecte del benestar animal, la seguretat alimentaria, les resistències antimicrobianes i la salut mediambiental han incrementat en els últims anys, promovent la implementació de sistemes de producció alternatius que incloguen el concepte 'One Health'. Per aixó, en el sector avícola s'han proposat diverses alternatives en granja, centrades en la millora dels protocols de bioseguretat, l'ús d'estirps més rústiques i de creixement lent, així com la implementació de la ramaderia de precisió. En aquest context, la microbiota intestinal té un paper clau en la salut dels animals, la diseminació de resistències antimicrobianes i la transmissió de patògens zoonòsics al llarg de la cadena alimentària. El benestar animal promou la presència de microbiota intestinal beneficiosa i la integritat de l'epiteli intestinal, reduïnt les interaccions amb els bacteris ambientals. D'aquesta manera, es pot aconseguir una reducció de l'administració d'antibiòtics i la presència de patògens en la cadena alimentària. Salmonella spp. és la principal causa de brots alimentaris en la Unió Europea, i la principal font d'infecció són els products avícoles. Entre els principals serotips relacionats amb aquestos brots es troba S. Infantis, actualment molt prevalent en pollastres. Per tot açò, l'objectiu general d'aquesta tesi doctoral va ser avaluar l'efecte de sistemes alternatius de producció avícola sobre el desenvolupament i la composició de la microbiota, l'evolució de les resistències antimicrobianes i l'epidemiologia de Salmonella. En el primer experiment, es va estudiar l'efecte de l'estirp genètica, comparant una estirp comercial de creixement ràpid front a una estirp alternativa de creixement lent. Els objectius van ser caracteritzar la microbiota fecal i investigar la dinàmica de les resistències i multirresistències antimicrobianes al llarg del cicle productiu. Els resultats de la composició de la microbiota mostraren que Firmicutes va representar el fil dominant i els gèneres predominants van ser Oscillospira spp., Ruminococcus spp., Coprococcus spp., Lactobacillus spp. i Bacteroides spp. D'altra banda, els resultats obtinguts en el estudi de resistències antimicrobianes van mostrar que, a l'inici del cicle, els animals de l'estirp de creixement ràpid van presentar un major percentatge de resistència, però al final del període no es van encontrar diferències. En el segon experiment, es va avaluar l'efecte de les condicions de maneig de la granja, comparant les condicions comercials de densitat i ventilació, front a condicions millorades. Els objetius van ser caracteritzar la microbiota fecal, avaluar l'evolució de les resistències i multirresistències antimicrobianes, i investigar el desenvolupament de S. Infantis i les seues resistències antimicrobianes al llarg del cicle productiu. Els resultats obtinguts en la evolució de la microbiota van mostrar un major nivell de diversitat en el grup produït davall condicions de maneig òptimes. A més, Firmicutes va ser el fil dominant i els gèneres predominants van ser Oscillospira spp., Ruminococcus spp., Bacteroides spp. i Coprococcus spp. Els resultats obtinguts en el estudi de resistències antimicrobianes, van mostrar altes tases de resistència al llarg del període, sense diferències entre grups. Per últim, els recomptes més alts d'excreció de Salmonella es van observar en la en el dia de sacrifici en ambdós grups. A més, el 100% dels ceps aïllats van ser multirresistents després de la primera setmana post-infecció. En conclusió, els principals resultats obtinguts en aquesta tesi doctoral inclouen que la diversitat i la composició de la microbiota es troben en constant desenvolupament al llarg del període d’engreixament, veient-se afectades per els factors de maneig estudiats. A més a més, les resistències antimicrobianes es troben presents en els bacteris comensals des del dia d’arrivada, i augmenta fins al final del cicle, destacant la necessitat de controlar l’administració d’antibiòtics en totes les etapes de la producció avícola. Quant a l’epidemiologia de S. Infantis, la contínua excreció durant tot el període d’engreixament i la seua capacitat d’adquirir resistències, independentment de les condicions de maneig en granja, sugereixen la necessitat de realitzar més estudis per poder establir millors programes de control del bacteri al llargo de la cadena alimentària. / [EN] Social awareness regarding animal welfare, food safety, antimicrobial resistance and environmental health has increased, promoting the implementation of alternative sustainable production systems that include the 'One Health' concept. For this reason, in the poultry sector different alternatives at field level have been proposed, centred on the improvement of biosecurity protocols, the use of rustic slow-growing breeds and the implementation of precision livestock farming. In this context, intestinal microbiota play an important role in poultry health, in the spread of antimicrobial resistance and in the transmission of zoonotic pathogens throughout the poultry production chain. Animal welfare promotes the presence of beneficial microbiota and the integrity of the intestinal epithelium, reducing the interactions between environmental and intestinal bacteria. This way, it could be possible to achieve a reduction in antibiotic administration at field level, and also the presence of zoonotic pathogens in the food chain. Salmonella spp. is the main cause of human foodborne outbreaks in the European Union, and the main sources of infection are poultry products. Between the main serovars related to these outbreaks is S. Infantis, which is currently the most prevalent serovar isolated in broiler chickens. Therefore, the general objective of this doctoral thesis was to evaluate the effect of alternative production systems of poultry production on the microbiota composition development, antimicrobial resistance dynamics and Salmonella epidemiology. In the first experiment, the effect of the genetic breed was studied by comparing a commercial fast-growing breed vs. an alternative slow-growing breed. The objectives were to characterise the caecal microbiota and to investigate antimicrobial resistance and multidrug-resistance dynamics throughout the growing period. Regarding microbiota composition, results showed that Firmicutes represented the dominant phylum for both systems, and the most predominant genera were Oscillospira spp., Ruminococcus spp., Coprococcus spp., Lactobacillus spp. and Bacteroides spp. On the other hand, results obtained in the study of antimicrobial resistance showed that at the onset of the cycle, fast-growing day-old-chicks showed higher antimicrobial resistance rates. However, at the end of the period no significant differences were found. In the second experiment, the effect of the farm management conditions was evaluated by comparing commercial density and ventilation conditions vs. improved conditions. The objectives were to characterise the caecal microbiota, to evaluate antimicrobial resistance and multidrug-resistance dynamics, and to investigate the development of S. Infantis and its antimicrobial resistance throughout the growing period. Results obtained in microbiota development showed a higher level of microbiota complexity in the group reared under optimal farm conditions at the end of rearing. Moreover, Firmicutes was the dominant phylum during all the growing period, and the predominant genera were Oscillospira spp., Ruminococcus spp., Bacteroides spp. and Coprococcus spp. Results obtained in the study of antimicrobial resistance showed high antimicrobial resistance rates throughout rearing, and no statistical differences were observed between groups. Finally, Salmonella shedding showed that the highest counts were observed at slaughter day for both groups. Moreover, 100% of the isolates were multi-resistant after the first week post-infection. In conclusion, the main results obtained include that microbiota diversity and composition are in constant development throughout the growing period, and antimicrobial resistance is present as of the arrival day and increases until the end of rearing. Regarding S. Infantis epidemiology, it has been demonstrated the continuous shedding throughout the growing period and its ability to gain antimicrobial resistance. / Montoro Dasí, L. (2021). Effect of Farm Management on Antimicrobial Resistance and Intestinal Microbiota in Poultry Production [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172610 / Compendio

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).

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.

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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