Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with a great ability to adapt to stress, in particular, to the selective pressure of antibiotics in the hospital environment. This pathogen constitutes a real public health concern, especially in low- and middle-income countries. In Cameroon, little is known about the drug resistance patterns of Pseudomonas aeruginosa. This study sought to determine the prevalence of Pseudomonas aeruginosa strains producing carbapenemases in six health facilities in the center, littoral, and west regions of Cameroon. An analytical cross-sectional study was conducted over a four-month period from July to October 2021. All Pseudomonas aeruginosa or suspected strains isolated from pathological products at the bacteriology laboratory of different health facilities were systematically collected and underwent a re-identification. After growing on cetrimide agar and successfully subculturing on nutrient agar, an oxidase test was performed on pure colonies, followed by biochemical identification (API 20NE system) of the bacterial suspension (0.5McFarland standard). Drug susceptibility testing for the detection of extended-spectrum beta-lactamases of overproduced inducible cephalosporinases and carbapenemases was performed according to adequate standard procedures. Of the 468 isolates collected, 347 (74.14%) were confirmed Pseudomonas aeruginosa after re-identification, of which 34.49% (120/347) produced inducible cephalosporinases (CAZR and C/TS) and 32.26% (112/347) extended-spectrum beta-lactamases. The prevalence of carbapenemase-producing P. aeruginosa (IMPR and C/TR) was 25.07% (87/347), with 17.24% (15/87) class A and 82.76% (72/87) class B. A high rate of resistance to penicillin (piperacillin: 70.58% and ticarcillin: 60.24%) was observed. We also noted a 34.49% resistance to ceftazidime, 30.22% to imipenem against 37.02% to meropenem, and 25.1% to ceftolozane/tazobactam (C/T). These strains also exhibited 79.57% resistance to quinolones and about 26% to aminoglycoside families. Multivariate analysis revealed that carbapenemase-producing Pseudomonas aeruginosa-related infections were significantly associated with hospitalization (p = 0.04), maternity (p = 0.03), surgery (p = 0.04), and intensive care wards (p = 0.04). This study highlighted a high burden of resistant strains of carbapenemase-producing Pseudomonas aeruginosa. Surveillance should be intensified to prevent the dissemination and spread of these strains.

Introduktion: Världshälsoorganisationen (WHO) har förklarat att antibiotikaresistens är en global folkhälsokris som finns i både låginkomstländer och höginkomstländer. Faktorer som sjukvård, förskrivarnas kunskap om infektionssjukdomar och deras kommunikation påverkar antibiotikaanvändningen och dess förskrivning. Därför metoder som påverkar kommunikationsstrategier för en rationell användning av antibiotika kan vara fördelaktiga i samhället då förökad kunskap kan bidra till bättre användning av antibiotika . Syfte: Syftet med denna studie var att granska informationsaktiviteter i svenska regionala läkemedelskommittéer för att förebygga antibiotikaresistens: lärdomar och områden för förbättring. Metod: 21 läkemedelskommittéer i Sverige fick 5 frågor via e-post att besvara på gällande antibiotika kommunikation i kommunen. Detta för att sedan analysera svaren med hjälp av kvalitativ tematisk analys. Resultat: 18 läkemedelskommittéer besvarade på frågorna därav tre teman och ett undertema identifierades; Nationell reglering och övervakningsverktyg är viktiga för länen, undertema, frekvens av informationsutbyte om antibiotikaförskrivning, Covid-19-pandemins påverkan på informationsaktivitet och Patienternas medvetenhet – ett område att fokusera på. Slutsats: I Sverige har länen en viktig roll att förmedla information, uppdateringar av riktlinjer och följa upp antibiotikaförskrivningar på förskrivarnivå. Covid-19 pandemin har ökat kunskapen om att fysiska möten är mer efektiva för att förmedla information. Det finns även ett behov av att utbilda allmänheten även om regionerna är fokuserade på denna aspekt, de flesta län måste fortfarande uppmärksamma detta specifika område. / Introduction: The World Health Organization (WHO) has declared that antibiotic resistance is a global public health emergency that exists in both low income countries and high income countries. Factors like healthcare, prescribers’ knowledge in infectious diseases and their communication affect antibiotic use and its prescription. Therefore methods that impact communication strategies enhance rational use of antibiotics. Aim: The purpose of this study was to explore information activities of Swedish Regional Pharmaceutical Committees for preventing antibiotic resistance. Also we tried to explore if there were certain lessons to be learned regarding effective information activities, and to identify any rooms for improvements.  Methods: 21 Pharmaceutical committees in Sweden were sent 5 questions via email to answer regarding antibiotic communication in the county. This to then analyze the responses using qualitative thematic analysis. Results: 18 counties responded to the questions whereas three themes and one sub-theme were identified; National regulation and surveillance tools are important for counties, subtheme, frequency of information sharing regarding antibiotic prescription, the COVID-19 pandemic’s impact on information activity and Patient’s awareness – an area to focus on. Conclusion: In Sweden, the counties have an important role to relay information, update on guidelines and follow up on antibiotic prescription at prescriber level. The Covid-19 pandemic has increased the knowledge that physical meetings are more effective to convey information. There is also a need to educate the general public though counties are focused on this aspect, majority of counties still need to bring their attention to this particular area.

Doctor of Philosophy / Department of Entomology / Ludek Zurek / Enterococci are a group of commensal bacteria that are important nosocomial pathogens. They are abundant in human sewage and wastewater treatment facilities (WWTF). This study focused on the role of house flies, Musca domestica, in the ecology of enterococci at WWTF in both field and laboratory experiments. The first study objective focused on sampling and characterizing enterococci from house flies and wastewater sludge from four WWTF in northeastern Kansas. Enterococci were quantified, identified, and screened for antibiotic resistance and virulence traits, and genotyped. The profiles of enterococci (spp. diversity, antibiotic resistance and virulence) from WWTF sludge and the house flies were similar, indicating that the flies successfully acquired the bacteria from the WWTF substrate. Enterococci with the greatest amount of antibiotic resistant and virulence traits originated from the WWTF that processed meat waste from a commercial sausage plant. Genotyping of E. faecalis revealed clonal matches from sludge and house flies. The second study objective involved tracking the fate of E. faecalis in the digestive tract of house flies in laboratory assays. Colony forming unit (CFU) counts were highest in the midgut at 1 h and declined during the first 24 h. In the labellum, foregut and hindgut, E. faecalis concentrations were more variable but were overall higher after 24 h. Observations from CFU counts and visualizations under a dissecting microscope revealed that E. faecalis peaked in the crop after 48 h suggesting active proliferation in this region. The third objective of the study involved tracking the emergence of calyptrate muscoid flies from stockpiled biosolid cake at one of the four WWTF. Traps were employed at the site for a total of 47 weeks, totaling 386 trap-weeks. A total of 11,349 calyptrate muscoid flies were identified with the two most common species being stable flies (Stomoxys calcitrans) (9,016, 80.2%) and house flies (2022, 18.0%). Numbers of stable flies and house flies peaked around mid-July each year and a second, smaller peak was observed for stable flies 5-8 weeks later. Estimated annual emergence of stable flies was 551,404 and for house flies 109,188.

Doctor of Philosophy / Department of Grain Science and Industry / Subramanyam Bhadriraju / Ludek Zurek / Feed samples and live stored-product insects from feed mills and swine farms were collected and cultured for Enterococcus spp. The mean concentration of enterococci in insect and feed were 2.7 ± 0.5 × 101 cfu/insect and 6.3 ± 0.7 × 103 cfu/g respectively. A total of 362 isolates of enterococci collected from 89 feed samples and 228 stored-product insects were identified to the species level using PCR. These isolates were represented by Enterococcus casseliflavus (53.0%), E. gallinarum (20.4%), E. faecium (16.2%), E. hirae (5.2%), and E. faecalis (5.0%). Enterococci were phenotypically resistant to tetracycline (48.0%), erythromycin (14.3%), streptomycin (16.8%), kanamycin (12.1%), ciprofloxacin (11.0%), ampicillin (3.3%), and chloramphenicol (1.1%). All isolates were susceptible to vancomycin and gentamicin. Tetracycline resistance was encoded by tetM (50.0%), tetO (15.1%), tetK (0.5%), tetS (0.2%) and other unknown tetracycline determinants. Enterococci carried virulence genes including gelatinase (gelE; 21.5%), an enterococcus surface protein (esp; 1.9%), and cytolysin (cylA; 2.2%). An aggregation substance (asa1) gene was detected in 61.0% of E. faecalis isolates. Fifty perncet of E. faecalis isolates were phenotipically tested positive for aggregation substances. Enterococci with cylA genes were hemolytic (52.0%) and with gelE genes were gelatinolytic (18.5%). The ermB gene, encoding erythromycin resistance was detected in 8.8% of the total isolates. The Tn916/1545 family of conjugative transposons was detected in 10.7% of the isolates. Laboratory experiments showed that adults of the red flour beetle, Tribolium castaneum (Herbst), fed on poultry and cattle feeds inoculated with E. faecalis OG1RF:pCF10, were able to successfully acquire enterococci and contaminate sterile poultry and cattle feeds. To assess the potential of horizontal gene transfer, conjugation assays were carried out with E. faecalis using a donor (wild strains) and recipient (E. faecalis OG1SSP) in ratio of 1:10. Only one isolate (1 out of 18 E. faecalis) could transfer tetM to a recipient using broth mating. However, filter mating assay, followed by PCR confirmation revealed that 89.0% (16 out of 18 E. faecalis) of isolates could transfer tetM to E. faecalis. Transfer ratios of transconjugant per recipients ranged from 2.6 × 10-4 to 1 × 10-9. In summary, feed (52.0%) and stored-product insects (41.6%) collected from feed mills and swine farms carried antibiotic-resistant and potentially virulent enterococci. Our study showed that T. castaneum, a pest commonly associated with feed, served as a potential vector for enterococci in the feed environment. Conjugation assays followed by PCR confirmed presence of the tetM gene on a mobile genetic element(s) such as Tn916 and may be horizontally transferred to other Enterococcus species and to other bacteria of clinical significance.

The crucial role of antibiotics in modern medicine, in curing infections and enabling advanced medical procedures, is being threatened by the increasing frequency of resistant bacteria. Better understanding of the forces selecting resistance mutations could help develop strategies to optimize the use of antibiotics and slow the spread of resistance. Resistance to ciprofloxacin, a clinically important antibiotic, almost always involves target mutations in DNA gyrase and Topoisomerase IV. Because ciprofloxacin is a substrate of the AcrAB-TolC efflux pump, mutations causing pump up-regulation are also common. Studying the role of efflux pump-regulatory mutations in the development of ciprofloxacin resistance, we found a strong bias against gene-inactivating mutations in marR and acrR in clinical isolates. MIC and fitness measurements revealed that amino acid substitutions conferred smaller susceptibility reductions and smaller fitness costs than gene-inactivating mutations, suggesting that resistance mutations in clinical isolates are selected for high fitness rather than high resistance (Paper I and II). We asked whether the high fitness costs of marR-inactivating mutations could be ameliorated without affecting the resistance phenotype. Multiple independent lineages were experimentally evolved to select for improved growth fitness. Whole genome sequencing revealed mutations affecting marA, lon and arcA as potential compensatory pathways. For the marA and lon mutations the improved growth rate was associated with an increased susceptibility (arcA is being investigated). (Paper III). An evolution experiment selecting for ciprofloxacin resistance revealed upon whole genome sequencing the expected mutations in drug target and efflux-regulatory genes, but also in genes encoding aminoacyl-tRNA synthetases. We investigated two independently selected leuS mutations, and concluded that they contributed to ciprofloxacin resistance by activating the stringent response that in turn caused up-regulation of genes involved in efflux. However, these leuS mutations incur a high fitness cost (Paper IV). To summarize, the research findings in this thesis suggest that the potential ciprofloxacin resistome may include more genes than previously thought, but a strong selection for high fitness selectively purifies many resistance mutations from clinical isolates. In conclusion, selection for high relative fitness constrains the spectrum of mutations that survive and get fixed in clinical populations of bacteria.

UA Open Access Publishing Fund / Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections.

O reconhecimento da resistência antimicrobiana como um fenômeno emergente em saúde pública, tem constituído um problema em nível mundial. O abuso na utilização de antibióticos na medicina humana e veterinária, e na agricultura, tem originado incremento na diversidade de micro-organismos resistentes, refletindo em falha terapêutica. Os mecanismos de resistência a antibióticos em micro-organismos são mediados principalmente por genes adquiridos de DNA exógeno. A dinâmica da transferência horizontal é realizada por meio de elementos genéticos móveis que carregam genes de resistência. A ampla distribuição deste tipo de estruturas, como o elemento SXT, isolado inicialmente em V. cholerae, tem contribuído para a disseminação de complexos específicos clonais em determinadas áreas geográficas. Este estudo pioneiro no Brasil pesquisou a presença de elementos SXT, em espécies bacterianas do grupo das gama proteobactérias em espécies ambientais, determinou suas características estruturais e funcionais, incluindo genes de resistência a antibióticos, bem como a sensibilidade aos antibióticos dentre os isolados bacterianos que os abrigam. O resultado foi a classificação de 43 elementos SXT obtidos no Brasil, através da comparação com aqueles descritos na literatura. Dentre os elementos SXT obtidos, quatro são albergados por Morganella morganii, fato inédito na literatura. O conhecimento da evolução bacteriana constitui importante ferramenta para estabelecer estratégias eficazes de controle e tratamento de infecções, sem aumentar a pressão seletiva sobre os micro-organismos, bem como instrumento preciso e de grande importância para subsidiar estudos epidemiológicos. / Recognition of antimicrobial resistance as an emerging phenomenon in public health has been a problem worldwide. The abuse in the use of antibiotics in human and veterinary medicine, and agriculture, has caused an increase in the diversity of resistant microorganisms, reflecting in treatment failure. The mechanisms of antibiotic resistance in microorganisms are primarily mediated by genes acquired from exogenous DNA. The dynamics of the horizontal transfer is performed by mobile genetic elements which carry resistance genes. The wide distribution of these structures, such as the SXT element originally isolated from V. cholerae, has contributed to the spread of specific clonal complexes in certain geographical areas. This pioneering study in Brazil researched the presence of SXT elements in the group of bacterial species in environmental gamma-proteobacteria species, determined their structural and functional characteristics, including genes for resistance to antibiotics and the antibiotic susceptibility among bacterial isolates that harbor them. The result was the classification of 43 SXT elements found in Brazil, by comparison with those found in the literature. Among the SXT elements found, four are sheltered by Morganella morganii, unprecedented in the literature. Knowledge of bacterial evolution is an important to establish effective strategies to control and treat infections without increasing the selective pressure on microorganisms, as well as a precise instrument and very important tool to support epidemiological studies.

O objetivo deste estudo foi caracterizar bactérias multirresistentes (MDR) isoladas de cavalos saudáveis (fezes) e doentes no Brasil e na França. De março de 2012 a dezembro de 2014, amostras clínicas coletadas de cavalos saudáveis e doentes no Brasil foram selecionadas para pesquisa da presença de bactérias MDR. A investigação sobre as amostras franceses foi restria a isolados de Escherichia coli (EC) recuperados a partir de amostras clínicas coletadas entre 2014 e 2015. Nos cavalos brasileiros, a análise de amostras de fezes de animais saudáveis revelou a presença de clones de EC não relacionados pertencentes aos filogrupos A, D ou B2 que carreavam genes como: blaCTX-M-1, blaCMY-2, qnr- e genes add-tipo (amino-transferases); enquanto que nos cavalos doentes foram encontradas EC, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa e Serratia marcescens carreando genes blaCTX-M-15, blaCTX-M-1, rmtD 16S rRNA metilase, qnr-tipo, aac(6´)-Ib-cr e aad-tipo. Nos cavalos doentes franceses de EC MDR foram positivas para CTX-M-1, seguido de M-2- e M-9. Estes resultados destacam a importância de cavalos como um novo reservatório de bactérias MDR. / The aim of this study was to characterize multidrug-resistant (MDR) bacteria isolated from healthy and infected horses in Brazil and France. From March 2012 to December 2014, clinical samples collected from healthy and infected horses, in Brazil, were screened for the presence of MDR bacteria. Investigation on French isolates was restricted to E. coli strains recovered from clinical samples collected between 2014 and 2015. In Brazilian horses, the analysis of fecal samples from healthy animals revealed the presence of clonally unrelated A, D or B2 phylogroups of E. coli strains carrying blaCTX-M-1, blaCMY-2, qnr- and aminoglycoside adenyl transferase (aad)-type genes, whereas in infected horses, E. coli, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens isolates carrying blaCTX-M-15, blaCTX-M-1, rmtD 16S rRNA methylase, qnr-type, aac(6´)-Ib-cr and aad-type genes. In French infected horses, most MDR E. coli isolates were positive for CTX-M-1-, followed by CTX-M-2- and CTX-M-9-type extended-spectrum beta-lactamases. These results highlight the importance of horses as a new reservoir of MDR bacteria.

Clostridium difficile is the most common antibiotic related diarrhéa disease in Sweden. New recommendations from the Swedish public health authority and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) had led to that a more advanced diagnostic algorithm is of priority. Hence this study, whose purpose was to investigate whether the performance of the rapid test C. diff Quik Chek Complete® could enable the introduction of a new diagnostic algorithm for detection of toxin-forming C. difficile in laboratory medicine in Sundsvall, according to these new recommendations. In the study 119 patient stool-samples were analysed with both GeneXpert and C. diff Quik Chek Complete® and these two combined fulfils these new recommendations of detecting toxin A and B from toxigenic C. difficile together with the enzyme Glutamate Dehydrogenase (GDH) which is produced by all C. difficile stems. The results shows that C. diff Quik Chek Complete® is well matched with GeneXpert and that most of the samples would come to be answered immediately after analysis with C. diff Quik Chek Complete®. The laboratory will save both time and money to establish C. diff Quik Chek Complete® in their algorithm for diagnosing C. difficile infection.

D’après un rapport de l’OMS, la tuberculose reste en 2015 l’une des 10 premières causes de décès à l’échelle mondiale. De ce fait, en matière de santé, éradiquer la maladie à l’horizon 2030 est un des objectifs majeurs fixés par les Nations Unies. La bactérie responsable de cette infection, Mycobacterium tuberculosis, est un pathogène obligatoire dont l’origine et l’évolution sont intrinsèquement liées à celles de son hôte principal, Homo sapiens. En effet, les souches actuelles de tuberculose présentent, tout comme l’homme, une forte structure phylogénétique, trace de leur origine géographique. Les pays pauvres et en développement sont les plus touchés par l’épidémie globale, favorisée par des systèmes de santé défaillants et une haute prévalence du VIH. Les pays occidentaux ne sont pas épargnés, menacés par l’émergence de souches de plus en plus résistantes aux antibiotiques provenant en grande partie de l’ex URSS. Au cours de cette thèse, j’analyse l’histoire évolutive, la propagation et l’acquisition de résistances aux antibiotiques de plusieurs épidémies de tuberculose en me basant sur des données génétiques et génomiques. Dans un premier temps je m’intéresse aux effets d’une campagne nationale de traitements en Asie Centrale sur le développement de souches multi-résistantes et met également en lumière le rôle clef de certaines mutations dans le succès des clones présentés. Ainsi cette campagne a été partiellement mise en échec par la présence de souches pré-résistantes, grâce à la survenue de mutations avant même la mise en place des traitements antibiotiques. Par la suite je me suis focalisé sur un clade particulier de souches multi-résistantes, le clone Russe W148. Je présente sa dispersion géographique et temporelle à travers l’Eurasie et démontre l’importance des mutations compensatoires dans son succès épidémique. De plus, la tuberculose ne touche pas seulement les hommes mais infecte également plusieurs autres mammifères. Afin d’appréhender les contraintes adaptatives accompagnants ces changements d’hôtes, j’ai effectué divers tests de sélection dans le but d’identifier les gènes impliqués. Pour finir, nous avons développé un indice souche spécifique, permettant de mesurer le succès épidémique de celles-ci à un niveau individuel. Dans le cadre d’études épidémiologiques, cette mesure peut être croisée avec des informations sur le patient, la souche ou même socio-économiques. / According to a 2015 WHO report, tuberculosis remains one of the top 10 causes of death worldwide. Despite considerable efforts by the United Nations to eradicate the disease by 2030, a global TB epidemic still persists. Its causative agent, the bacterium Mycobacterium tuberculosis, an obligate pathogen, has been plaguing humanity since it originated, and has coevolved with its main host, Homo sapiens, over thousands of years. Contemporary tuberculosis strains exhibit a structured phylogeographic pattern, carrying the genetic print of their geographic origin. The Koch bacillus infects and kills in large numbers, in poor and developing countries, where fragile health care systems, combined with high HIV prevalence, facilitate epidemic spread. In western countries, the major current threats are the multiplication and propagation of antibiotic resistant strains (MDR/XDR) coming predominantly from former Soviet republics. In this thesis, I unravel the evolutionary history, propagation, and acquisition of drug resistance-conferring mutations in different settings, by implementing multiple genetic and genomic data sets. First, focusing on Central Asia, using whole genome sequencing and Bayesian statistics, I assess the effects of a treatment campaign on the development of MDR strains and highlight key mutations in successful strains. More importantly, the success of DOTs campaigns was compromised by the genetic make-up of these outbreak clades (pre-treatment low frequency resistance SNPs). Special attention was also given to a particular outbreak of MDR strains, i.e. the Russian W148 clone. I present its westward spatial and temporal propagation at a continental scale during the last century, and underline the key contribution of compensatory mutations in its epidemic success. However, tuberculosis does not only infect humans, but also has experienced successive mammalian host jumps. To decipher the adaptive constraints accompanying such secondary events, a systemic gene screen with selection signature-detecting algorithms was implemented to identify putative targets during diversifying selection. Finally, novel mathematical tools and indices that reflect the epidemicity of a strain were developed, jumping from a population-driven approach to a strain specific one, with broader epidemiological applications. This allows us to correlate strain fitness with patient, lineage, and socio-economic information.