Clostridium difficile is the leading cause of death from gastrointestinal infections in Canada. Current antiobiotic treatment is non-ideal due to the high incidence of relapse and the rise in hyper-virulent antibiotic-resistant strains. Surface layer proteins (SLPs) cover the entire bacterial surface and mediate adherence to host cells. Passive and active immunization against SLPs greatly enhances survival in hamsters, suggesting that antibody-mediated bacterial neutralization may be an effective alternative therapeutic strategy. Using a recombinant-antibody phage display library, and SLPs from strain QCD 32g58 as bait antigen, we isolated and extensively characterized 11 SLP-specific recombinant single-domain antibodies (sdAbs), in terms of affinity and specificity, intrinsic stability, and ability to inhibit cell motility. Several sdAbs exhibit promising characteristics for a potential oral therapeutic based on their high affinity, high thermal stability, and resistance to pepsin digestion. Our study provides the basis of a proof-of-principle model with which to develop specific, broadly neutralizing and intrinsically stable antibodies for the oral therapy of C. difficile infections, as an alternative to conventional antibiotic treatment.

Clostridium difficile is the leading cause of death from gastrointestinal infections in Canada. Current antiobiotic treatment is non-ideal due to the high incidence of relapse and the rise in hyper-virulent antibiotic-resistant strains. Surface layer proteins (SLPs) cover the entire bacterial surface and mediate adherence to host cells. Passive and active immunization against SLPs greatly enhances survival in hamsters, suggesting that antibody-mediated bacterial neutralization may be an effective alternative therapeutic strategy. Using a recombinant-antibody phage display library, and SLPs from strain QCD 32g58 as bait antigen, we isolated and extensively characterized 11 SLP-specific recombinant single-domain antibodies (sdAbs), in terms of affinity and specificity, intrinsic stability, and ability to inhibit cell motility. Several sdAbs exhibit promising characteristics for a potential oral therapeutic based on their high affinity, high thermal stability, and resistance to pepsin digestion. Our study provides the basis of a proof-of-principle model with which to develop specific, broadly neutralizing and intrinsically stable antibodies for the oral therapy of C. difficile infections, as an alternative to conventional antibiotic treatment.

Clostridium difficile is the leading cause of death from gastrointestinal infections in Canada. Current antiobiotic treatment is non-ideal due to the high incidence of relapse and the rise in hyper-virulent antibiotic-resistant strains. Surface layer proteins (SLPs) cover the entire bacterial surface and mediate adherence to host cells. Passive and active immunization against SLPs greatly enhances survival in hamsters, suggesting that antibody-mediated bacterial neutralization may be an effective alternative therapeutic strategy. Using a recombinant-antibody phage display library, and SLPs from strain QCD 32g58 as bait antigen, we isolated and extensively characterized 11 SLP-specific recombinant single-domain antibodies (sdAbs), in terms of affinity and specificity, intrinsic stability, and ability to inhibit cell motility. Several sdAbs exhibit promising characteristics for a potential oral therapeutic based on their high affinity, high thermal stability, and resistance to pepsin digestion. Our study provides the basis of a proof-of-principle model with which to develop specific, broadly neutralizing and intrinsically stable antibodies for the oral therapy of C. difficile infections, as an alternative to conventional antibiotic treatment.

Clostridium difficile is the most common bacterial cause of infectious diarrhoea in healthcare environments and in 2014 was responsible for 13,785 infections in the UK. C. difficile infection (CDI) is spread via the faecal-oral route and by contact with contaminated surfaces. However, despite the healthcare concerns no tests are available to validate if sufficient cleaning has been conducted. In addition, Polymerase Chain Reaction (PCR) and Enzyme Immunoassays (EIAs)-based tests used to diagnose CDI lack sensitivity and specificity and hence false negative results are commonly obtained. To overcome these concerns the aim of the PhD research has been to develop the first diagnostic test that exploits the specific interactions of C. difficile bacteriophages (phages), viruses that specifically infect and kill C. difficile. In order to develop a C. difficile phage-based test, first suitable phages that can be used for the test were identified and this was conducted by screening 35 different C. difficile phages against 160 clinically relevant C. difficile isolates. Five phages were found to infect the most number of isolates and were investigated further to identify whether a phage-based diagnostic could be developed based on phages binding (adsorption) to different C. difficile subgroups. However, for all five phages, adsorption rates were not consistently high for C. difficile subgroups in comparison to other common bacteria found in similar locations to C. difficile. Therefore, to increase specificity of the phage-based diagnostic test a new approach was taken by tagging two phages with luminescence luxAB genes (reporter phages), which would be expressed once C. difficile cells were infected with the phages. To design the C. difficile reporter phages, non-essential phage genes were replaced with the luxAB genes, but this study revealed mutagenesis of C. difficile was troublesome and extensive optimisation was required. In addition, once the reporter phages had successfully been constructed the luxAB genes were unstable within the phage genome and were lost during phage replication. Despite extensive optimisation and due to time constrains the luxAB genes were not stabilised within the phages but future work will focus on stabilising the genes.

Clostridium difficile infection (CDI) is a major healthcare-associated disease with high recurrence rates. Host colonization is critical for the infectious process, both in first episodes and in recurrent disease, with biofilm formation playing a key role. The ability of C. difficile to form a biofilm on abiotic surfaces is established, but has not yet been confirmed in the intestinal tract. Here, four different isolates of C. difficile, which are in vitro biofilm producers, were studied for their ability to colonize germ-free mice. The level of colonization achieved was similar for all isolates in the different parts of the murine gastrointestinal tract, but pathogen burden was higher in the cecum and colon. Confocal laser scanning microscopy revealed that C. difficile bacteria were distributed heterogeneously over the intestinal tissue, without contact with epithelial cells. The R20291 strain, which belongs to the Ribotype 027 lineage, displayed a unique behavior compared to the other strains by forming numerous aggregates. By immunochemistry analyses, we showed that bacteria were localized inside and outside the mucus layer, irrespective of the strains tested. Most bacteria were entrapped in 3-D structures overlaying the mucus layer. For the R20291 strain, the cell-wall associated polysaccharide PS-II was detected in large amounts in the 3-D structure. As this component has been detected in the extrapolymeric matrix of in vitro C. difficile biofilms, our data suggest strongly that at least the R20291 strain is organized in the mono-associated mouse model in glycan-rich biofilm architecture, which sustainably maintains bacteria outside the mucus layer.

Clostridium difficile infection (CDI) continues to possess a significant disease burden in the United States (US) as well as all over the world. Given the increase in severity and recurrence rate, the decrease in cure rate, and the fact that the virulent ribotype 027 strain remains one of the most commonly identified strains in the US, the Infectious Diseases Society of America (IDSA) published a clinical practice guideline in February 2018 moving away from metronidazole as the first-line treatment for initial CDI and recommending either oral vancomycin or fidaxomicin. The aim of this study is to evaluate the clinical data available comparing the efficacy of primary treatment of CDI between those two antibiotics. We performed a PubMed, PubMed Central, and ScienceDirect database search without restriction to regions, publication types, or languages. A comprehensive literature search was performed from January 1, 1980 up to March 20, 2018. We used the following keywords in different combinations: Clostridium difficile, Clostridium difficile infection, CDI, C. diff, C. difficile, fidaxomicin, vancomycin, pseudomembranous colitis, and antibiotic-associated colitis. The search was limited to human studies. Data were independently extracted by two reviewers with disagreements resolved by a third author. We pooled an odds ratio (OR) on two primary outcomes: Clinical cure rate and rate of recurrence during the follow-up period. The computer search was also supplemented with manual searches by the authors of the retrieved review articles and primary studies. The search phrase "((Clostridium difficile) AND vancomycin) AND fidaxomicin" had the highest yield results. We identified four observational studies with a total of 2,303 patients with CDI that met our inclusion criteria. Compared with vancomycin, fidaxomicin use was associated with a significantly lower recurrence of CDI with a pooled OR of 0.47 (95% confidence interval (CI), 0.37 - 0.60, I2 = 0). On the other hand, there was no significant association of fidaxomicin use with CDI cure rate compared to vancomycin with a pooled OR of 1.22 (95% CI, 0.93 - 1.60, I2 = 0). In light of the recently updated clinical practice guidelines by the IDSA, our review suggests that fidaxomicin has a more sustained clinical response with a statistically significant lower recurrence rate. Although fidaxomicin appears to be the better drug with statistical significance, its cost-effectiveness continues to be an ongoing controversy. More randomized clinical trials are needed to shed light on this matter to assess if there is any clinical significance in fidaxomicin superiority.

In dieser Arbeit wurden 104 stationär isolierte Stämme mit 90 ambulant isolierten Stämmen hinsichtlich ihrer Antibiotikaresistenz und ihrer Ribotypenzugehörigkeit verglichen. Die Proben stammten aus den Jahren 2011 bis 2019, wobei keine stationären Isolate aus dem Jahr 2014 aufzufinden waren, woraufhin aus Gründen der Vergleichbarkeit auch auf ambulante Proben aus diesem Jahr verzichtet wurde. Unter den Isolaten gab es 90 Paare, die sich in Geschlecht, Toxinaktivität, Patientenalter und Datum des Auftretens der Infektion vergleichen ließen. Die 14 übrigen stationären Proben flossen ebenfalls in die Auswertung ein. Folgendes wurde beobachtet: - Die Mehrheit (70%) aller untersuchten Isolate stammte von Patienten, deren Alter zum Erkrankungszeitpunkt zwischen 60 und 90 Jahren lag. - Frauen (57%) waren etwas häufiger betroffen als Männer (43%). Es wurde die minimale Hemmkonzentration (MHK) für die Antibiotika Vancomycin, Metronidazol, Moxifloxacin und Tigecyclin mittels E- Test- Streifen ermittelt sowie die MHK für die Antibiotika Rifaximin und Fidaxomicin mittels Agardilution. Zudem wurde der Ribotyp jedes Stammes anhand der PCR- Ribotypisierung bestimmt. Aus den Untersuchungen ergaben sich die folgenden Ergebnisse: - Im ambulanten Bereich war der RT027 mit 21% am häufigsten vertreten, gefolgt vom RT078 (18%), RT001 (11%) und RT014 (10%). Auch im stationären Bereich war der RT027 mit 30% am häufigsten vertreten, gefolgt vom RT014 (18%), RT001 (12%) und RT078 (8%). - Die Isolate zeigten eine sehr hohe Sensibilität gegenüber Vancomycin (100% der Isolate sensibel), Tigecyclin (97% der Isolate sensibel) und Fidaxomicin (92% der Stämme mit einer MHK</= 0,125 mg/l). - Gegenüber Metronidazol zeigten 22% eine MHK > 2 mg/l. Vor allem die Ribotypen 027 (63% resistent) und 001 (35% resistent) zeigten eine hohe Resistenzlage. Die Ribotypen 078 und 014 waren zu 100% Metronidazol- sensibel. - Gegenüber Moxifloxacin waren 43% der Isolate mit einer MHK > 4 mg/l resistent. Auch hier zeigten die Ribotypen 027 (88% resistent) und 001 (95% resistent) eine hohe Resistenzlage, wohingegen 70% der RT078 und 86% der RT014 Moxifloxa-cin- sensibel waren. - Unter Ausschluss des RT027 waren 95% der Stämme sensibel gegenüber Rifaximin. Von den RT027- Isolaten hingegen waren nur 8% sensibel. Gerade hier empfiehlt sich der Ausschluss eines RT027 vor Beginn einer Rifaximintherapie. Unter Zusammenschau der Eigenschaften der Patientenproben und den ermittelten Resistenzen und Ripotypen können zudem folgende Aussagen aufgestellt werden: - Das Durchschnittsalter der Patienten mit einer RT001-, einer RT027- und CD12- Infektion war im stationären Bereich höher; das der Patienten mit einer RT078-, RT014- und CD6- Infektion war im ambulanten Bereich höher. - Die Resistenzlage im Jahr 2012 war für die Antibiotika Metronidazol, Moxifloxacin und Rifaximin im Vergleich zu den anderen Jahren deutlich erhöht. Es konnte in den letzten drei Untersuchungsjahren eine leicht abnehmende Tendenz Rifaximin- resistenter Stämme beobachtet werden. Für alle anderen Antibiotika konnte weder eine Zu- noch eine Abnahme des Auftretens resistenter Stämme nachgewiesen werden. - In den letzten beiden Untersuchungsjahren nahm der Anteil der RT027- Isolate ab, wohingegen seit 2013 eine stete Zunahme des Auftretens des RT078 zu beobachten war. - Das durchschnittliche Alter der Patienten mit einer RT027- Infektion lag 5,1 J. über dem Gesamtdurchschnitt. Vancomycin und Metronidazol gelten schon lange als Mittel der Wahl zur Therapie einer CDAD. Sie gelten als wirksam und vergleichsweise preiswert. In der Kosten- Nutzen- Abwägung wurden auch die vier anderen Antibiotika berücksichtigt. Es ergaben sich die folgenden Ergebnisse: - Unter der Voraussetzung, dass der Ribotyp des zu therapierenden C. difficile- Stammes nicht bekannt ist, bleibt Vancomycin sowohl bei milden als auch bei schweren Verläufen ein günstiges Mittel der Wahl. - Vor Therapie mit dem Antibiotikum Metronidazol ist der Ausschluss eines RT027 sinnvoll. Handelt es sich nicht um diesen Ribotyp, hat auch Metronidazol mit großer Wahrscheinlichkeit eine gute Wirksamkeit und kann Vancomycin möglicherweise vorgezogen werden (preiswerter, keine Züchtung Vancomycin- resistenter Stämme). - Fidaxomicin hat eine sehr gute Wirksamkeit gegenüber den häufigsten Ribotypen. Derzeit ist eine Fidaxomicintherapie etwa zehnmal teurer als eine Therapie mit Vancomycin. - Wenn ein RT027- Ausschluss erfolgt ist, kann eine Therapie mit Rifaximin erwogen werden (günstiger als Vancomycin, gute Verträglichkeit aufgrund lokaler Wirkung). Die Arbeit hat gezeigt, dass es sowohl im ambulanten als auch im stationären Bereich eindeutig Korrelate zwischen der Ribotypenzugehörigkeit und dem Vorliegen von Antibiotikaresistenzen gibt. Bereits nach Ausschluss der Ribotypen 027 und 001 können Therapieempfehlungen angepasst werden. Teilweise ändert sich die Therapieoption auch unter Berücksichtigung des Ortes der Probengewinnung. In schwierigen Fällen ist eine Therapie nach Ribotypisierung durchaus sinnvoll, da durch eine gezielte Antibiose Rezidive und Resistenz-bildungen vermieden werden. Zusammenfassend zeigt sich, dass flächendeckenden Surveillanceprogrammen, insbesondere dem Mapping virulenter Keime, eine große Bedeutung zugesprochen werden kann. Ihr Fortbestehen und der weitere Ausbau dienen der sinnvollen Therapie des Einzelnen, aber auch der Vorbeugung schwerwiegender Krankheitsausbrüche und Epidemien.:Abkürzungsverzeichnis 1 EINLEITUNG 1.1 Eigenschaften und Epidemiologie von Clostridium difficile 1.2 Vorkommen und Übertragungswege 1.3 Klinik, Verlaufsformen und Risikofaktoren 1.3.1 Pathogenese 1.4 Diagnostik einer CDI 1.5 Therapie und Prophylaxe 1.6 Antibiotika und Resistenzen 1.6.1 Vancomycin 1.6.2 Metronidazol 1.6.3 Moxifloxacin 1.6.4 Tigecyclin 1.6.5 Rifaximin 1.6.6 Fidaxomicin 1.7 Typisierungsmethoden 1.8 PCR- Ribotypisierung 1.9 Eigenschaften der Ribotypen 027, 078, 001 und 014 1.10 Entwicklung der letzten Jahre 2 AUFGABENSTELLUNG 3 MATERIALIEN 3.1 ständig eingesetzte Materialien 3.2 Identifizierung von C. difficile aus Stuhlproben und Isolation 3.3 Beimpfen der Agarplatten mit C. difficile und Bebrütung 3.4 Legen der E- Test- Streifen und Ansetzen der DNA 3.5 Agardilution 3.6 PCR- Ribotypisierung 4 METHODEN 4.1 Patientenstämme 4.1.1 Herkunft der Patientenstämme 4.1.2 Identifizierung von C. difficile aus Stuhlproben und ihre Isolation 4.1.3 Verwendete Isolate 4.2 Beimpfen der Agarplatten mit C. difficile und Bebrütung 4.3 Legen der E-Test- Streifen und Ansetzen der DNA 4.4 Agardilution 4.5 PCR- Ribotypisierung 4.5.1 Polymerase- Kettenreaktion 4.5.2 Ribotypisierung 4.6 Allgemeine Aspekte 5 ERGEBNISSE 5.1 Auswahl der Patientenproben 5.1.1 Toxineigenschaften 5.1.2 Verteilung im Hinblick auf das Patientengeschlecht und das Labor 5.1.3 Verteilung im Hinblick auf das Patientenalter 5.2 Antibiotika- Sensibilität 5.2.1 Klinische Grenzwerte nach EUCAST 5.2.2 Antibiotikaresistenzen der einzelnen Stämme unter Berücksichtigung ihrer Herkunft 5.2.3 Resistenzkombinationen der einzelnen Stämme und ihre Ribotypen 5.3 Ribotypisierung 5.3.1 Isolate des RT027 genauer betrachtet 5.3.2 Isolate des RT078 genauer betrachtet 5.3.3 Isolate des RT001 genauer betrachtet 5.3.4 Isolate des RT014 genauer betrachtet 5.3.5 Diversität der Ribotypen im zeitlichen Verlauf 5.4 Antibiotikasensibilität und Ribotypen im Zusammenhang betrachtet 5.4.1 Vancomycin 5.4.2 Metronidazol 5.4.3 Moxifloxacin 5.4.4 Tigecyclin 5.4.5 Fidaxomicin 5.4.6 Rifaximin 6 DISKUSSION 6.1 Patientenproben, Anzucht, Wachstum 6.2 Toxin- negative Isolate 6.3 Antimikrobielle Empfindlichkeit 6.4 Genotypische Charakteristika 6.5 Antimikrobielle Empfindlichkeit und genotypische Charakteristika im Zusammenhang, Therapieempfehlungen 6.6 Finanzieller Aspekt einer Therapie 7 ZUSAMMENFASSUNG 8 LITERATURVERZEICHNIS 9 SELBSTSTÄNDIGKEITSERKLÄRUNG 9.1 Erklärung über die eigenständige Abfassung der Arbeit 10 CURRICULUM VITAE 11 DANKSAGUNG

Clostridium difficile, the causative agent of C. difficile infection (CDI), causes hundreds of thousands of hospital-acquired infections in the United States and Canada annually. Furthermore, the prevalence and severity of CDI has been on the rise in developed countries, especially with the appearance of “hypervirulent” strains. Detection of CDI is thus of great importance. Traditional detection methods can be time consuming or lack the desired sensitivity. On the other hand, aptamers pose great prospects as diagnostic and therapeutic agents. Aptamers are nucleic acid ligands with molecular recognition capabilities rivaling those of antibodies. They are obtained by a process of in vitro selection known as systematic evolution of ligands by exponential enrichment (SELEX). However, the current approach may result in aptamers that experience non-specific binding in complex or biological samples. Here, we propose a multiple domain selection (MDS) method for aptamer isolation. This method utilizes independent selections on separate components of a single target in order to obtain uniquely specific aptamers. The aptamers can then be unified into a heterobivalent construct able to recognize two sites on one target. We hypothesize the combined aptamer would result in greater affinity and specificity for the target, resulting in greatly increased aptamer utility in current and future applications. In the current study, we have cloned and purified full length C. difficile DnaK as well as the N-terminal domain (NTD) and C-terminal domain (CTD) of the protein. MDS was performed on each target and the resulting aptamers were combined into a heterobivalent construct. The construct resulted in an approximately 100-fold affinity increase relative to the single aptamer for DnaK, and could detect much smaller quantities of target. Although it experienced low level recognition of high concentrations of purified E. coli DnaK, there was no detectable non-specific binding in several biological samples. / Thesis / Master of Science (MSc)

Drug resistant strains of Clostridium difficile are a major health concern with over 3 million cases costing over 1 billion $ per year in the United-States. The diseases associated with these bacteria (CDAD) are toxin-mediated which offers a mean of treating and lessening the severity of CDAD symptoms. Toxin inactivation via antibodies therapy can drastically reduce CDAD morbidity and this project was aiming at investigating the large-scale production and recovery of a novel llama single domain antibody (pSJF2H-ToxA5.1) in recombinant Escherichia coli TG1 targeting C. difficile enterotoxin A (TcdA). In order to achieve these objectives, the project was divided into four segments: 1) ToxA5.1 being an intracellular recombinant protein, obtaining a high biomass production was the first step towards large-scale production. To achieve HCDC, effects of initial glucose concentration and pH-stat feeding strategy were studied; 2) Upon achieving HCDC, effects of parameters such as temperature, induction timing and media supplementation with complex nitrogen sources were investigated; 3) Once large-scale production of ToxA5.1 was obtained, the recombinant protein needed to be recovered and a selective cell lysis scheme where synergistic lysis effects of Triton X-100 and temperature were studied. And finally 4) Single-step purification using nickel nanoparticles (NNP) synthesized via a modified polyol method was studied. Combining the HCDC strategy with a temperature shift and yeast extract addition at the time of induction, ToxA5.1 concentration of 127 mg/L was obtained. Synergistic and selective cell lysis using Triton X-100 and temperature was achieved where 95% of the available ToxA5.1 was recovered and still functional while ToxA5.1 fraction in the resulting lysate increased to 27% in the cell lysate. Single-step purification was achieved using the synthesized NNP which proved to be highly selective and could be used up to five times. Diameter of the NNP synthesized was controlled by using various concentration of ranging from 131 ± 80 nm to 47 ± 20 nm. Using experimental data from binding isotherm, the ToxA5.1-NNP system was modeled.

Anaerobe opportunist Clostridium difficile causes the majority of hospital-acquired antibiotic-associated diarrhea. Infections can be severe because of its ability to withstand many antibiotics, to sporulate and to produce toxins (A, B and binary).     In Sundsvall Hospital C. difficile is detected with real-time PCR, which targets the sequences of toxin B, binary toxin and a regulatory gene deletion seen in the virulent ribotype 027. All positive samples are stored frozen for one month, available for further analysis or outbreak investigation. The aim of this study was to investigate if temperature and transportation time may affect the viability of C. difficile, and the PCR-result.     Frozen feces samples were cultivated, identified with MALDI-TOF and analyzed with real-time PCR after at least one month of storage. To simulate the effect of transportation time, samples were stored at 4-8°C for three and seven days before cultivation and identification. Controls were cultivated after freezing for comparison.     Ninety percent of the frozen samples contained viable C. difficile. Discrepancies between PCR-results were found for two of the oldest samples collected (six months), which turned negative. Fresh samples showed lower amount of viable C. difficile after three days (50 %) than after seven days (60 %) of storage, perhaps because of competition with other bacteria and sporulation. The frozen control group contained a higher viable amount, 75 %. The results indicate that C. difficile tolerates to be stored at low temperatures as practiced today at the laboratory. Transportation time seem to affect the outcome of cultivation, but not the PCR-result.