With recent research being conducted in categorizing and analyzing the human microbiome, evidence has now linked the human microbiome to a range of diseases. Dysbiosis of the human gut microbiome exists in colon cancer, obesity, and Clostridium difficile infections. The use of fecal microbiota transplants has been proven effective in treating recurrent C.difficile infections by restoring gut microbiota. More needs to be done to establish fecal microbiota transplants procedures, effectiveness, and safety. Once established, fecal microbiota transplants may play a role in modulating other diseases linked to human gut microbiome dysbiosis.

Background and Aim: Acute kidney injury (AKI) is used as a marker of severity in Clostridium difficile infection (CDI) patients. We estimated the true effect of AKI in inpatient mortality of CDI patients, as there are no large-scale, population-based, propensity-matched studies evaluating AKI's effect in this patient cohort. Methods: A retrospective observational study utilizing the National Inpatient Sample from years 2003 to 2012, including all adults with CDI, excluding cases missing data on age, inpatient mortality or gender. Trends and CDI-related complications as mortality predictors were assessed using survey-weighted multivariable regression. We estimated AKI's independent effect by propensity-matching, post-stratifying by chronic kidney disease status, allowing for multiple comorbidity adjustment. Results: A total of 2 859 599 patients with CDI were included, of which 896 122 (31.3%) had principal diagnosis of CDI. AKI prevalence was 22%. Mortality rate was 8.4%, while among AKI patients was higher (18.2%). In multivariable regression, AKI was associated with higher mortality (odds ratio [OR] = 3.16, 95% confidence interval [CI]: 3.02–3.30; P < 0.001), while after propensity matching, AKI increased mortality by 86% (OR = 1.86, 95% CI: 1.79–1.94; P < 0.001). CDI incidence increased by 1.8, together with the rate of AKI (12.6% in 2003 to 28.8% in 2012, P-trend < 0.001). Despite increasing hospitalizations, mortality over the study period decreased to 7.2% (2012) from 9.0% (2003); P-trend < 0.001. Conclusion: Hospital admissions of patients with CDI and concomitant AKI are increasing, but their inpatient mortality has improved over the study period. AKI is a significant contributor to mortality, independently of other comorbidities, complications, and hospital characteristics, emphasizing the need for early diagnosis and aggressive management in such patients.

Controlling norovirus transmission in units with immunocompromised patients is challenging. We present a cluster of norovirus cases that occurred on a stem-cell transplant unit and the prevention efforts that were implemented to limit the outbreak. Protocols developed to control this cluster may provide a model for other facilities. / National Institutes of Health / Revisión por pares

The recent study by Argamany et al1 concluded that the incidence and hospital mortality for Clostridium difficile infection (CDI) differed between major regions of the United States and across different seasonal times of the year. However, these conclusions were not supported by the data in their study because the authors based them exclusively on statistical significance without considering the effect size of their findings. The effect sizes of region and season on CDI were very low or near zero, contradicting their conclusion, as subsequently explained.

Selenoproteins are unique proteins in which selenocysteine is inserted into the polypeptide chain by highly specialized translational machinery. They exist within all three kingdoms of life. The functions of these proteins in biology are still being defined. In particular, the importance of selenoproteins in pathogenic microorganisms has received little attention. We first established that a nosocomial pathogen, Clostridium difficile, utilizes a selenoenzyme dependent pathway for energy metabolism. Following this initial characterization, we demonstrate that this pathway is linked to production of toxins by this organism. Finally, we show that interruption of selenium metabolism is a viable pathway for development of antimicrobials against this, and other selenoprotein dependent pathogens. We investigated whether Stickland reactions (paired amino acid fermentation) might be at the heart of C. difficile's bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases; glycine and D-proline reductase. These selenoenzymes were expressed upon addition of the corresponding Stickland acceptor (glycine, proline or hydroxyproline). Purification of the selenoenzyme D-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys containing) proteins. D-proline reductase utilized only D-proline but not L-hydroxyproline, even in the presence of an expressed and purified proline racemase. The enzyme was found to be independent of divalent cations, and zinc was a potent inhibitor. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of D-proline reductase may differ significantly from similar enzymes from non-pathogenic species. C. difficile pathogenesis is due to the production of toxins, A and B, members of the large clostridial cytotoxin family. Previous studies have shown that toxin production by this organism is influenced by the composition of the growth medium. We examined the impact of Stickland acceptor amino acids (Stickland acceptors; glycine, proline and hydroxyproline) on growth kinetics and yield, protein synthesis, toxin production and gene expression. Although addition of Stickland acceptors moderately increases growth yield and total protein synthesis, there does not appear to be a clear impact on entry into stationary phase. Glycine dramatically increases the amount of toxin released into the growth medium. Conversely, the addition of hydroxyproline suppresses toxin production. We examine possible mechanisms of regulation and demonstrate that CodY, a regulator of toxin gene transcription does not appear to mediate this effect. Given the importance of selenium dependent Stickland reactions to C. difficile growth and toxin production we aimed to examine the efficacy of blocking such pathways as a means of antimicrobial development. Selenide is the only known substrate for selenophosphate synthetase, the first enzyme involved in the specific incorporation of selenium into selenoproteins. We have identified a stable complex formed upon reaction of auranofin (a gold containing drug) with selenide in vitro. Auranofin potently inhibits the growth of C. difficile but does not similarly affect other clostridia that do not utilize selenoproteins to obtain energy. Moreover, auranofin inhibits the incorporation of radioisotope selenium (75Se) in selenoproteins in both E. coli, the prokaryotic model for selenoprotein synthesis, and C. difficile without impacting total protein synthesis. Auranofin blocks the uptake of selenium and results in the accumulation of the auranofin-selenide adduct in the culture medium. Addition of selenium in the form of selenite or L-selenocysteine to the growth media significantly reduces the inhibitory action of auranofin on the growth of C. difficile. Based on these results, we propose that formation of this complex and the subsequent deficiency in available selenium for selenoprotein synthesis is the mechanism by which auranofin inhibits C. difficile growth. The antimicrobial potential of blocking selenium metabolism is further demonstrated in the dental pathogen Treponema denticola. We show that auranofin blocks the growth this organism which also participates in Stickland fermentation. In addition, we provide evidence that the antimicrobial action of stannous salts against T. denticola is also mediated through inhibition of the metabolism of selenium. These studies clearly show that, at least in a subset of microbes that use selenium for the synthesis of selenoproteins, the need for this metalloid can be a useful target for future antimicrobial development.

Introduction: Les infections nosocomiales à Clostridium difficile (C. difficile) représentent un problème majeur de santé publique. La surveillance provinciale québécoise a identifié des facteurs de risque non-modifiables de l’infection(ex.: mission de l’hôpital, nombre de lits), ne permettant toutefois pas de cibler une intervention. Objectifs: Cette étude a pour but: 1)d’identifier les facteurs de risque et/ou protecteurs modifiables associés au taux d’incidence d’infection à C. difficile dans les hôpitaux québécois; 2)d’évaluer si les facteurs sont les mêmes selon le type de mission de l’hôpital (universitaire ou non-universitaire); 3)d’expliquer l’association entre le type de mission et le taux d’incidence de l’infection. Méthodologie: Les facteurs de risque et/ou protecteurs ont été recueillis à l’aide d’un questionnaire sur les mesures de prévention et contrôle des infections à C. difficile dans les hôpitaux(INSPQ 2009-2010). La mesure de résultat est le taux d’incidence de l’infection(2009-2010). Nous avons utilisé des modèles de Poisson multivariés prédictifs pour répondre aux deux premiers objectifs et un modèle explicatif ajusté pour différents facteurs confondants pour répondre au troisième objectif. Résultats: Les facteurs associés à un taux d’incidence plus élevé dans le modèle prédictif pour tous les hôpitaux sont: hôpitaux des grands centres vs régions éloignées(Québec:RTI=2,06, valeur-p=0,0001; Montréal:RTI=1,38, valeur-p=0,005); héberger &lt; 100% des patients symptomatiques d’infection à C. difficile en chambre privée vs 100%(80%-99%: RTI=1,33, valeur-p=0,01; &lt; 80%:RTI=1,84, valeur-p&lt; 0,0001); désinfecter la salle de bain des patients non-infectés 2fois/jour vs 1fois/jour(RTI=1,52, valeur-p=0,02); avoir un plan d’action lors d’éclosion n’incluant pas la surveillance des antibiotiques vs avoir un plan incluant la surveillance(RTI=1,36, valeur-p=0,004). L’hébergement des patients symptomatiques en chambre privée est le seul facteur commun qui ressort dans les modèles spécifiques aux hôpitaux universitaires et non-universitaires. Conclusion: Nous avons identifié des facteurs associés à l’infection sur lesquels il est possible d’intervenir, tant au niveau de la configuration de l’hôpital que des mesures qui y sont appliquées. / Background: Nosocomial Clostridium difficile (C. difficile) infection is a major public health concern. Quebec’s C. difficile provincial surveillance identified risk factors of the infection, such as teaching vocation and number of beds, which are unmodifiable. Thus these factors do not help targeting interventions to reduce the incidence of C. difficile infection. Objectives: This study aims to: 1)identify modifiable risk and/or protective factors associated with the incidence rate of C. difficile infection in Quebec hospitals; 2)evaluate if the risk and/or protective factors are the same according to hospital mission(teaching or non-teaching); 3)explain the association between teaching vocation and higher incidence rate of C. difficile infection. Method: Risk and/or protective factors were collected using a survey on preventive and control measures of C. difficile infection(INSPQ 2009-2010). The outcome is the incidence rate of the infection in Quebec hospitals(2009-2010). We used predictive multivariate Poisson models to answer the first two objectives and an explanatory model adjusted for various confounding factors to answer the third objective. Findings: Factors associated with a higher incidence rate in the multivariate predictive model for all hospitals were: hospitals in large urban centers vs remote regions(Quebec: IRR= 2.06, p-value=0.0001; Montreal: IRR=1.38, p-value=0.005); accommodate &lt; 100% of symptomatic patients with C. difficile infection in private room vs 100%(80%-99%: IRR=1.33, p-value=0.01; &lt; 80%: IRR=1.84, p-value&lt; 0.0001); disinfect washroom of uninfected patients 2 times/day vs 1 time/day(IRR=1.52, p-value=0.02); have an action plan during an outbreak that doesn’t include antibiotic stewardship vs have an action plan during an outbreak that includes antibiotic stewardship(IRR=1.36, p-value=0.004). Accommodation of all symptomatic patients with C. difficile infection in private rooms is the only common risk factor between the models for teaching and non-teaching hospitals. Conclusion: We identified factors associated to the infection on which it is possible to act, whether it is on hospital setting or measures applied in hospitals.

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Clostridium difficile est une bactérie qui, avec l’appauvrissement de la flore intestinale causée par des antibiotiques, produit deux toxines qui altèrent la paroi intestinale et provoquent ainsi des diarrhées pouvant entraîner la mort. Les premiers traitements à base de vancomycine, ont fait apparaître des souches résistantes. Depuis 2012, Santé Canada a approuvé un seul nouvel antibiotique, la fidaxomicine ou tiacumicine B. Ce traitement est plus efficace pour lutter contre C. difficile que la vancomycine. Le macrocycle de la tiacumicine B, un macrolactone, est fabriqué par une bactérie du sol et les enzymes qui agissent dans la voie de biosynthèse sont connues mais leur ordre d’action ne l’est pas encore. Pour cela, il est nécessaire de connaître les substrats de chacune de ces enzymes. Afin de mieux comprendre la biosynthèse de la tiacumicine B, ce mémoire présente la détermination de la structure de TiaP1, un cytochrome P450 qui hydroxyle le carbone 18 du macrocycle. La structure de TiaP2, qui hydroxyle le carbone 20, étant déterminée, l’objectif était de prédire l’identité de son substrat à l’aide de l’arrimage moléculaire. Les résultats appuient l’hypothèse que TiaP2 agirait avant TiaP1 sur le macrocycle. D’après les simulations d’arrimage moléculaire, nous prédisons que la glycosyltransférase TiaG1 (non étudiée ici) ajouterait un résidu de sucre au macrolactone avant que TiaP2 n’hydroxyle le carbone 20. / Clostridium difficile is a bacterium that, with the depletion of gut flora during antibiotic treatment, produces two toxins that alter the intestinal wall causing potentially deadly diarrhea. The first treatments were based on vancomycin and resistance by C. difficile developed rapidly. In 2012, Health Canada approved a new antibiotic, fidaxomicin, which is also named tiacumicin B. This treatment is the most effective treatment against C. difficile. The macrocycle of tiacumicin B, a macrolactone, is produced by a soil bacterium and enzymes that act in the biosynthetic pathway are known. However, their order of action is not established. To know their order of action, it is necessary to know the substrates. This thesis presents the determination of TiaP1 structure, a cytochrome P450 that hydroxylates carbon 18 of the macrocycle of tiacumicin B. The structure of TiaP2, a P450 hydroxylating carbon 20, was already determined, and we attempted to predict its substrate by using a molecular docking approach. Our results confirm that TiaP2 hydroxylates before TiaP1. According to molecular docking results, we predict that the glycosyltransferase TiaG1 (not studied here) adds a sugar on the macrolactone of tiacumicin B before TiaP2 hydroxylates the carbon 20.

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